Biotechnology for Sustainability: Achievements, Challenges and Perspectives

Biotechnology for Sustainability 

Achievements, Challenges and Perspectives 

Editors 

Subhash Bhore, 

K. Marimuthu & 

M. Ravichandran



Editors 

Subhash Bhore, K. Marimuthu & M. Ravichandran 

2017 

1



Subhash Bhore, K. Marimuthu & M. Ravichandran (Editors) 

Published by AIMST University 

2017 

ISBN: 978-967-14475-3-6 (Print version) 

eISBN: 978-967-14475-2-9 (e-Book version) 

2

Published by 

AIMST University 

Printed by 

AIMST University 

Copyright 

© 2017 by the authors; Licensee, Editors; AIMST University, 

Malaysia. This book is an open access book distributed under 

the terms and conditions of the Creative Commons Attribution 

(CC-BY) license (http://creativecommons.org/licenses/by/4.0/). 

CC BY license is applied which allows users to download, copy, reuse and distribute 

articles and or data provided the original article and book is fully cited. This open 

access aims to maximize the visibility of articles, reviews and or perspectives, much of 

which is in the interest of national, regional and global community. 

Disclaimer: The information provided in this book is designed to highlight the views, 

perspectives, achievements and or research findings of respective contributors. While 

the best efforts have been used in preparing this book, Editors and or Publisher make 

no representations or warranties of any kind and assume no liabilities of any kind with 

respect to the accuracy or completeness of the contents and specifically disclaim any 

implied warranties. Neither the Editors nor Publisher of this book shall be held liable or 

responsible to any person or entity with respect to any loss or incidental or 

consequential damages caused, or alleged to have been caused, directly or indirectly, 

by the information highlighted herein. Readers should be aware that the information 

provided in this book may change. 

All articles and or reviews published in this book are deemed to reflect the individual 

views of respective authors and not the official points of view, either of the Editors or of 

the Publisher. 

Cover image: A diagram showing the 17 Sustainable Development Goals (Credit: 

www.un.org/) 

Edited by 

Dr. Subhash J. Bhore (Senior Associate Professor) 1 , 

Dr. K. Marimuthu (Professor) 1, 2 , and 

M. Ravichandran (Senior Professor) 1, 2 

Address for Correspondence: 

1 Department of Biotechnology, Faculty of Applied Sciences, AIMST University, 

Bedong-Semeling Road, 08100 Bedong, Kedah Darul Aman, Malaysia; Telephone 

No.: +604 429 8176; e-mail: subhash@aimst.edu.my / subhashbhore@gmail.com 

2 Chancellery, AIMST University, Bedong-Semeling Road, 08100 Bedong, Kedah Darul 

Aman, Malaysia; Tel. No.: +604 429 1054 /8103; e-mail: marimuthu@aimst.edu.my / 

ravichandran@aimst.edu.my 

Edition 

First; July 18, 2017 

3

Dedication 

This book is dedicated to all researchers working in 

various domains of biotechnology and to all 

stakeholders those are working for the global 

sustainable development to improve the health of the 

people and planet. 

4

Preface 

World Environment Day (WED) is a biggest global annual event celebrated each 

year on June 5 to create the positive awareness to preserve the environment and planet 

earth. This year, the theme for WED-2017 was “Connecting people to nature”. Our 

environment should be healthy for our growth, development and to achieve the sustainable 

development goals (SDGs) adopted by the international community to transform the world. 

Most recently, António Guterres (United Nations Secretary General) precisely 

highlighted that “Without a healthy environment we cannot end poverty or build prosperity. 

We all have a role to play in protecting our only home: we can use less plastic, drive less, 

waste less food and teach each other to care”. In fact, to achieve the SDGs by protecting 

environment, everyone needs to do their part. 

We strongly believe that biotechnology can play an important role directly or 

indirectly in achieving various SDGs. Hence, we had decided to publish a book, 

“Biotechnology for Sustainability” to commemorate the WED and to highlight the 

achievements, challenges and perspectives in various domains of the biotechnology. In 

response to our call for articles, we had received 50 manuscripts. The selected articles 

published in this book are highlighting various issues, achievements, challenges and 

perspectives for the viable development and sustainability. The World Commission on the 

Environment and Development defined sustainability as the “development that meets the 

needs of the present without compromising the ability of future generations to meet their 

own needs”. The United Nations recent estimate suggest that the world’s food supply needs 

to be doubled by the year 2050 to keep up with the growing demand. To achieve this is a 

huge challenge; because, the amount of arable land is continuously decreasing as a result of 

rising urbanization, saline soils and desertification. Biotechnologists (and plant breeders) 

around the world are working persistently to produce crops which will boost the food 

production to meet the growing demand. Genetically engineered crop varieties do offer 

many promising possibilities to boost nutritive value of the food, sustain farming on 

marginal lands, and to minimize the loss by creating pests and disease resistant varieties. 

The articles published in this book are going to be useful in creating awareness 

about the environmental issues, natural resources, biodiversity conservation, sustainable 

development and various biotechnological approaches that could be used to alleviate the 

respective challenges. 

We would like to express our sincere gratitude and thanks to Dato' Seri Utama Dr. 

S. Samy Vellu, Chancellor and Chairman, AIMST University for his support in publishing 

this book. 

We wish to thank all contributing authors for making a common cause with us. This 

book publication project could not have been completed without the courteous cooperation 

of the authors to highlight achievements, challenges and or perspectives in using 

biotechnological approaches for the sustainability. 

We are confident that this book will serve as a reference to various researchers, 

scientists, academicians and graduate students involved in biodiversity conservation, 

environmental protection and various fields of biology and biotechnology. 

It is hoped that a prudent use of biotechnology in the biodiversity conservation, 

environmental protection, and production of more and better quality of food, fiber, fuel and 

drugs will contribute in accomplishing SDGs and to promote peace in the world. 

Subhash J. Bhore 

K. Marimuthu 

M. Ravichandran 

ISBN: 978-967-14475-3-6 ; eISBN: 978-967-14475-2-9 

i

Contents 

Preface ................................................................................................................................ i 

Contents ............................................................................................................................ ii 

Plant Tissue Culture for Sustainability 

C. K. John ....................................................................................................................... 1 

Traditional Medicine of the Tribes in Tamil Nadu and Its Sustainable 

Use through Biotechnology 

Valli Gurusamy, Kavitha Valampuri John, Usha Raja Nanthini 

Ayyakkanu, Ramani Bai Ravichandran ......................................................................... 14 

Vermitechnology – An Eco-Biological Tool for Sustainable 

Environment 

Mahaly Moorthi, Koilpathu Senthil Kumar Abbiramy, Arumugam Senthil 

Kumar and Karupannan Nagarajan................................................................................ 41 

Role of Biotechnology in Food Authentication 

Shobana Manoharan, Raghavan Kuppu and Ramesh Uthandakalaipandian ................... 51 

Management Strategies against Tiny Tigers for Sustainable 

Development of Agriculture 

Viswa Venkat Gantait ................................................................................................... 58 

Designing Greener Pharmaceuticals and Practicing Green Health Is 

Required for Sustainability 

Sridevi Chigurupati, Jahidul Islam Mohammad, Kesavanarayanan 

Krishnan Selvarajan, Saraswati Simansalam, Shantini Vijayabalan and 

Subhash Janardhan Bhore ............................................................................................. 68 

Clonal Propagation of a High Value Multipurpose Timberline Tree 

Species Quercus semecarpifolia Sm. of West Himalaya, India 

Aseesh Pandey and Sushma Tamta ............................................................................... 79 

Spent Mushroom Substrate of Hypsizygus ulmarius: A Novel 

Multifunctional Constituent for Mycorestoration and Mycoremediation 

Padmavathi Tallapragada and Ranjini Ramesh .............................................................. 88 

Biotechnology for Sustainability of Forests 

Kumud Dubey and Kesheo Prasad Dubey ................................................................... 104 

Biotechnological Approaches for Conservation and Sustainable Supply 

of Medicinal Plants 

Sagar Satish Datir and Subhash Janardhan Bhore ........................................................ 117 

ISBN: 978-967-14475-3-6 ; eISBN: 978-967-14475-2-9 

ii

Making Himalayas Sustainable: Opportunities and Challenges in 

Indian Himalayan Region 

Harsh Kumar Chauhan and Anil Kumar Bisht ............................................................. 129 

Natural Polyphenols and Its Potential in Preventing Diseases Related 

To Oxidative Stress as an Alternative Green Nutraceutical Approach 

Sreenivasan Sasidharan, Shanmugapriy, Subramanion Lachumy Jothy, 

Mei Li Ng, Nowroji Kavitha, Chew Ai Lan, Khoo Boon Yin, 

Soundararajan Vijayarathna, Leow Chiuan Herng and Chern Ein Oon ........................ 141 

A Review on Green Synthesis of Nanoparticles and Its Antimicrobial 

Properties 

Karthika Arumugam and Naresh Kumar Sharma ......................................................... 171 

Production of Secondary Metabolites Using a Biotechnological 

Approach 

Produtur Chandramati Shankar and Senthilkumar Rajagopal ....................................... 187 

Potential of Marine Algae Derived Extracts as a Natural Biostimulant 

to Enhance Plant Growth and Crop Productivity 

Lakkakula Satish* and Manikandan Ramesh ............................................................... 200 

Biotransformation of Various Wastes into a Nutrient Rich Organic 

Biofertilizer - a Sustainable Approach towards Cleaner Environment 

Geetha Karuppasamy, Michael Antony D’Couto, Sangeetha Baskaran and 

Anant Achary.............................................................................................................. 212 

Bacterial Endophytes as Biofertilizers and Biocontrol Agents for 

Sustainable Agriculture 

Amrutha V. Audipudi, Bhaskar V. Chakicherla and Shubhash Janardhan 

Bhore .......................................................................................................................... 223 

Microbial Metabolic Engineering: A Key Technology to Deal with 

Global Climate and Environmental Challenges 

Meerza Abdul Razak, Pathan Shajahan Begum and Senthilkumar 

Rajagopal .................................................................................................................... 248 

Biodiesel Production for Sustainability: An Overview 

R. Meena Devi, R. Subadevi and M. Sivakumar .......................................................... 262 

In vitro Cell Bioassays in Pollution Assessment 

Narayanan Kannan, Poorani Krishnan and Ahmad Zaharin Aris ................................. 274 

Lipopeptide Biosurfactants from Bioagent, Bacillus as a Weapon for 

Plant Disease Management 

Sampath Ramyabharathi, Balaraman Meena, Lingan Rajendran and 

Thiruvengadam Raguchander ...................................................................................... 287 

ISBN: 978-967-14475-3-6 ; eISBN: 978-967-14475-2-9 

iii

Biotechnology as a Tool for Conservation and Sustainable Utilization of 

Plant and Seaweed Genetic Resources of Tropical Bay Islands, India 

Pooja Bohra, Ajit Arun Waman and Anuraj Anirudhan ............................................... 295 

Plantibodies for Global Health: Challenges and Perspectives 

Prasad Minakshi, Basanti Brar, Manimegalai Jyothi, Ikbal, Koushlesh 

Ranjan, Upendra Pradeep Lambe and Gaya Prasad ..................................................... 305 

Renewable Energy from Agro-industrial Processing Wastes: An 

Overview 

Sudhanshu S. Behera, Ramesh C. Ray and S. Ramachandran ..................................... 322 

Mitigation of Climatic Change by Organic Agriculture 

Mohan Mani, Manohar Murugan, Ganesh Punamalai and Vijayalakshmi 

Ganesan Singaravelu ................................................................................................... 336 

Application of Anti-vibrio and Anti-quorum Sensing Technology for 

Sustainable Development in Shrimp Aquaculture 

Ramesh Kandasamy, Amutha Raju and Manohar Murugan ......................................... 344 

Promiscuous Rhizobia: A Potential Tool to Enhance Agricultural Crops 

Productivity 

Ikbal, Prasad Minakshi, Basanti Brar, Upendera Praddep Lambe, 

Manimegalai Jyothi, Koushlesh Ranjan, Deepika, Virendra Sikka and 

Gaya Prasad ................................................................................................................ 358 

Organic Farming and Halalan Toyyiban Foods: An Attempt to Relate 

Them 

Quamrul Hasan and Zakirah Othman .......................................................................... 376 

Biotechnological Approaches: Sustaining Sugarcane Productivity and 

Yield 

Ashutosh Kumar Mall and Varucha Misra .................................................................. 386 

Bioremediation: A Biotechnology Tool for Sustainability 

Niharika Chandra, Ankita Srivastava, Swati Srivastava, Shailesh Kumar 

Mishra and Sunil Kumar ............................................................................................. 398 

Sea Urchin - A New Potential Marine Bio-resource for Human Health 

M. Aminur Rahman, Fatimah Md. Yusoff, Kasi Marimuthu and Yuji 

Arakaki ....................................................................................................................... 417 

Marine Pollution and Its Impacts on Living Organisms 

Thavasimuthu Citarasu and Mariavincent Michael Babu ............................................. 444 

Ecology, Distribution and Diversity of Bioluminescent Bacteria in Palk 

Strait, Southeast Coast of India 

Srinivasan Rajendran, Ganapathy selvam Govindarasu and Govindasamy 

Chinnavenkataraman................................................................................................... 456 

ISBN: 978-967-14475-3-6 ; eISBN: 978-967-14475-2-9 

iv

Synthesis of Biocompatible Silver Nanoparticles Using Green Alga 

(Ulva reticulata) Extract 

Ganapathy selvam Govindarasu, Srinivasan Rajendran and Sivakumar 

Kathiresan................................................................................................................... 475 

Diversity and Ethno-Botanical Potential of Tree Plants of Katarniaghat 

Wildlife Sanctuary, Bahraich (UP) India: An Overview 

Tej Pratap Mall ........................................................................................................... 486 

Free Radical Scavenging Potential and Anticancer Activity of Primula 

denticulata Sm. from North-Western Himalayas 

Bilal Ahmad Wani, Mohammed Latif Khan and Bashir Ahmad Ganai ........................ 512 

Panchakavya: Organic Fertilizer and Its Stimulatory Effect on the Seed 

Germination of Abelmoschus esculentus and Solanum melongena 

V. Ramya and S. Karpagam ........................................................................................ 525 

Increasing Human Interference in Katarniaghat Wildlife Sanctuary 

Shiv Pratap Singh ....................................................................................................... 534 

ISBN: 978-967-14475-3-6 ; eISBN: 978-967-14475-2-9 

v


Achievements, Challenges and Perspectives Biotech Sustainability (2017), P1-13 


C. K. John* 

Plant Tissue Culture Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha 

Road, Pune 411008, India;*Correspondence: ck.john@ncl.res.in; Tel.: +91-9822531551 

Abstract: The United Nations has placed great emphasis on sustainability. Three of the 

most important requirements of sustainable development are: eradicating extreme poverty 

and hunger, protecting the environment, and conserving biodiversity. Because of human 

activities the stable functioning of earth‛s life support system – which includes the atmosphere, 

oceans, forests, waterways, biodiversity and biogeochemical cycles, is at risk. 

One of the major contributing factors is the large scale destruction of natural forests. Deforestation 

had many adverse effects; most importantly, the effects on climate, environment, 

and biodiversity. The three pillars of sustainable development are: sustainable agriculture, 

conserving biodiversity, and protecting the environment through reversing the effects of 

deforestation by large scale afforestation. Plant Tissue Culture can greatly contribute in all 

the three. 

Keywords: Afforestation; biodiversity conservation; micropropagation; plant tissue culture; 

sustainable agriculture 

1. Introduction 

The United Nations Summits and 

Commission Reports from the 1987 

Brundtland Commission (World Commission 

on Environment and Development) 

report onwards have placed added 

emphasis on sustainability of all development 

efforts. Three of the most important 

requirements are: 1. Eradicating 

extreme poverty and hunger, 2. Protecting 

the environment, and 3. Conserving 

biodiversity. To eradicate extreme 

poverty and hunger two things are essential: 

first, sustainable agriculture which 

makes food available/affordable and second, 

creation of jobs which translates to 

purchasing power. One of the major factors 

in protecting the environment is reversing 

the loss of natural forests. Conserving 

biodiversity is of great relevance 

now than ever before for the reason that 

our world is fast changing. To have crop 

varieties suitable for this changing environment 

is to preserve as much natural 

variation in plant varieties as possible. 

Plant tissue culture can contribute to all 

the three. In this paper I will elaborate on 

how Plant Tissue Culture, my area of research, 

can contribute to Sustainable Agriculture, 

Protecting Forests, and Conserving 

Biodiversity. 

2. Sustainable development 

In 1987 it was the Brundtland 

Commission (World Commission on Environment 

and Development) report “Our 

Common Future” which brought the concept 

of “Sustainable Development” into 

common use. The World Commission on 

Environment and Development was set up 

by the UN General Assembly in 1983. 

Brundtland Commission Report defined 

Sustainable Development as “Development 

that meets the needs of the present 

without compromising the ability of the 

future generations to meet their own 

needs”. According to the Brundtland 

Commission Report, the needs, in particu- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 1

Biotech Sustainability (2017) 


lar the essential needs of the world‟s poor, 

to which overriding priority should be 

given, and the limitations imposed by the 

State of Technology and Social organization 

on the Environment‟s ability to meet 

present and future needs should be addressed. 

The Brundtland Commission Report 

emphasized the need to integrate 

economic and ecological factors in decision-making 

at all levels for sustainable 

development. These factors include, reviving 

growth, changing quality of 

growth, meeting essential needs for jobs, 

food, energy, water and sanitation, ensuring 

the resource base, reorienting technology 

and managing risks. In its broadest 

sense, the strategy for sustainable development 

aims to promote harmony among 

people and between human beings and 

environment. 

In 1992, at the Earth Summit (Rio, 

1992) there was consensus that environment, 

and economic and social development 

cannot be considered in isolation, 

and in addition to treaties and agreements 

on climate change, biological diversity, 

deforestation, and desertification, the Rio 

Declaration contains fundamental principles 

on which nations can base their future 

decisions and policies, considering 

the environmental implications of socioeconomic 

development. 

In 2000 the Millennium Summit 

of the United Nations, following the 

adoption of the United Nations Millennium 

Declaration, established the eight Millennium 

Development Goals (MDGs) to 

be achieved by the year 2015. The MDGs 

are: 1. to eradicate extreme poverty and 

hunger, 2. to achieve universal primary 

education, 3. to promote gender equality 

and empower women, 4. to reduce 

child mortality, 5. to improve 

maternal health, 6. to combat 

HIV/AIDS, malaria, and other diseases, 

7. to ensure environmental sustainability, 

and 8. to develop a global partnership 

for development. In the present context 

Goal 7: Ensuring environmental sustainability 

is very important. Two of the important 

targets of MDG 7 are: Integrating 

John 

the principles of sustainable development 

into country policies and programs, 

reversing loss of environmental resources, 

and reducing biodiversity loss. 

In 2012, the United Nations 

Rio+20 summit in Brazil committed governments 

to create a set of “Sustainable 

Development Goals” (SDGs). On September 

25 th 2015, countries adopted a set 

of goals to end poverty, protect the planet, 

and ensure prosperity for all as part of 

a 2030 Sustainable Development Agenda. 

Each goal has specific targets to be 

achieved in 15 years. The 17 Sustainable 

Development Goals (SDGs), otherwise 

known as the Global Goals, are a universal 

call for action to end poverty, 

protect the planet and ensure that all 

people enjoy peace and prosperity always. 

The goals are interconnected. 

The key to success on one will involve 

tackling issues associated with another. 

The SDGs work in the spirit of 

partnership and pragmatism, to make 

the right choices now to improve life, 

in a sustainable way, for future generations. 

They provide clear guidelines 

and targets for all countries to adopt in 

accordance with their own priorities 

and the environmental challenges of 

the world at large. The SDGs are an 

inclusive agenda. They tackle the root 

causes of poverty and unite all nations 

together to make a positive change for 

both people and planet (UNDP).The 

15 th SDG of UN relates to Life on land, 

and involves protecting, restoring and 

promoting sustainable use of terrestrial 

ecosystems, sustainably managing forests, 

combating desertification, and halting 

and reversing land degradation and 

halting biodiversity loss. 

The stable functioning of Earth‛s 

life support system – which includes the 

atmosphere, oceans, forests, waterways, 

biodiversity and biogeochemical cycles, is 

a prerequisite for future human development. 

However, as per recent research 

findings this functioning is at risk (Rockström 

et al., 2009). Further human pressure 

may lead to large-scale, abrupt, and 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 2



potentially irreversible changes to Earth‛s 

life support system (Lenton 2011; Barnosky 

et al., 2012). Likely impacts on 

humanity include: diminishing food production, 

water shortages, extreme weather, 

ocean acidification, deteriorating ecosystems, 

and sea-level rise. In this backdrop 

Griggs et al. (2013) suggested that 

we redefine sustainable development as 

“Development that meets the needs of the 

present while safeguarding Earth‟s lifesupport 

system, on which the welfare of 

current and future generations depends.” 

Without economic, technological, and 

societal transformations, chances of largescale 

humanitarian crises exist. Such crises 

could undermine any gains made by 

meeting the MDGs. A re-evaluation of the 

relationship between people and planet is 

necessary (Griggs et al., 2014). 

3. Three pillars of sustainable development 

In the second half of the 20 th century 

there was intensification of agriculture 

in most parts of the world. Intensive 

agriculture involved: (i) expanding farm 

lands, by removing natural forests, (ii) 

better irrigation, by constructing big 

dams, which again submerged vast forests 

in their catchment areas (iii) use of chemical 

fertilizers and pesticides, to produce 

high yields. Destruction of natural forests 

had many adverse effects; most importantly, 

the effects on climate, environment, 

and biodiversity. Extensive use 

of chemical fertilizers and pesticides also 

had their own adverse effects. Excessive 

use of chemical fertilizers has resulted in 

nitrate accumulation, increased soil salinity, 

and water eutrophication. High use of 

pesticides has resulted in development of 

resistance in many pest species. In recent 

years there is much concern about environmental 

contamination by fertilizers 

and pesticides. 

Sustainable Development, that 

meets the needs of the present while safeguarding 

Earth‟s life-support system, on 

which the welfare of current and future 

John 

generations depends, stands on three pillars: 

i. Sustainable agriculture 

ii. Conserving biodiversity 

iii. Protecting the environment 

Increasing food production must 

involve, developing/ introducing better 

(efficient, high yielding, insect-pest resistant) 

varieties of crop plants, conserving 

biodiversity, and protecting environment. 

Plant Tissue Culture can greatly 

contribute in all these. 

4. Plant tissue culture 

Plant tissue culture is the aseptic 

growing of whole plants or parts (cells, 

tissues/ organs) in/ on defined (synthetic) 

nutrient media under controlled (environmental) 

conditions (temperature, light, 

humidity). Usually in glass vessels (test 

tubes, conical flasks, jam bottles etc.) - 

for a review see John et al. (1997). 

Plant tissue culture is based on 

cellular „totipotency‟, the inherent potential 

of a plant cell to regenerate a whole 

plant. Unlike animal cells, most plant 

cells retain the capacity to regenerate the 

whole organism even after undergoing the 

final differentiation. In plants, as long as 

the cells have an intact membrane system 

and a viable nucleus, even highly mature 

and differentiated cells retain the ability 

to regenerate to a meristematic state. 

Though initially, in the first two decades 

of the 20 th Century progress was slow, 

standardization of universal plant tissue 

culture media - White‟s (White, 1933), 

Gamborg‟s (Gamborg et al., 1975) and 

MS (Murashige and Skoog, 1963) 

changed the scene. Plant tissue culture 

media contain minerals, growth factors 

and a carbon source (usually sucrose). 

Controlled environmental factors are light 

(intensity and length – photoperiod), temperature, 

relative humidity. On/ in a custom 

standardized medium, and controlled 

environmental conditions, the explant 

(starting plant material) - usually young, 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 3



undifferentiated tissue, regenerate into 

whole plants. 

4.1. Types of cultures 

Different types of cultures are 

possible: (i) culture of whole plants, (ii) 

embryo culture (embryo rescue), (iii) organ 

culture (shoot tip culture, root culture, 

leaf culture, anther culture etc.), (iv) callus 

culture, (v) cell suspension and single 

cell culture, (vi) protoplast culture. 

4.2. Callus culture 

Callus is an amorphous mass produced 

by cell proliferation, occurring in 

an unorganized manner. In nature it is a 

wound response, or a plant reaction to the 

presence of micro-organisms, insects, or 

to some kind of stress. Under in vitro 

conditions callusing is a response to endogenous 

or exogenous growth regulators. 

The potential for callus formation is 

dependent on the tissue (explant) type. 

Meristematic tissues are more suitable for 

callus induction than mature tissues. Callus 

cultures can be maintained for long by 

sub-culturing the primary callus (callus 

established originally from the explant), 

at periodic intervals. 

4.3. Somaclonal variations 

Long term callus cultures can 

however, suffer from spontaneously arising 

genetic variations, reflected in the 

phenotype of plants regenerated from 

such calli. These variations are known as 

somaclonal variations. Somaclonal variations 

are reported in many species. The 

basis of somaclonal variations is not well 

understood. Chromosomal rearrangements, 

activation of endogenous transposons, 

and changes in the status of DNA 

methylation, are considered to be the contributing 

factors. 

John 

4.4. Suspension cultures 

Culture of unorganized plant cells, 

as single cells/ cell aggregates, in liquid 

medium. Friable callus when cultured in 

agitated liquid medium, the cells separate 

and form a suspension of single cells/ aggregates 

of few cells. These cells/ cell aggregates 

grow/ divide/ separate as a result 

of agitation, and can be continually maintained 

in this state. Growth of cells in 

suspension culture can be more easily 

manipulated in liquid medium than on 

semi-solid medium. Slowly agitating the 

liquid medium on a rotary shaker is necessary 

for the growth of the cultures, 

which can be sub-cultured. Growth in 

single isolated cells can be induced by 

culturing them in hanging drops in microchambers. 

Suspension cultures are useful 

in plant production by somatic embryogenesis 

form single cells. In regeneration 

of plants from callus established on semisolid 

media from small cell aggregates, 

and for the production of secondary metabolites. 

Suspension cultures can also be 

initiated from tissue other than callus 

(Geile and Wagner, 1980). 

4.5. Protoplast cultures 

Protoplasts are plant cells without 

cell walls. In 1882, Klercker isolated protoplasts 

mechanically for the first time. 

The yield of protoplasts was very low. In 

1960, Cocking using enzymes for the first 

time could isolate protoplasts in large 

numbers. Protoplasts can be isolated from 

different plant parts, or from tissues already 

in culture. Enzymatic isolation is 

now the most commonly used method. A 

combination of these two can also be 

used. 

One of the important applications 

of protoplasts is in somatic hybridization. 

Many agents like NaNO 3 (Power et al., 

1990), a higher pH, and a higher concentration 

of calcium ions in the medium 

(Melchers and Labib, 1974), polyethylene 

glycol (Kao and Michayuluk, 1974; 

Wallin et al., 1974), and a high strength 

electric field (Zimmermann and Scheurich, 

1981), are used for obtaining fusion 

between protoplasts. Protoplasts, when 

placed in appropriate media regenerate 

cell walls and form calli, from which 

plants can be regenerated. Protoplasts are 

used for producing somatic hybrids (parasexual 

hybrids), for genetic manipulation, 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 4



and for basic studies on a variety of aspects. 

Regenerating plants from protoplasts 

is difficult in some species. Conventional 

hybridization depends on affinity 

of gametes. Wide crosses are not possible 

because of well-established cross 

breeding barriers. Protoplast fusion makes 

such hybridizations possible. 

4.6. Anther (isolated microspore) cultures 

Guha and Maheshwari (1964) obtained 

haploid embryos, directly from anther 

cultures of Datura innoxia. The 

origin of these embryos was traced to the 

pollen grains. The potential of anther culture 

for obtaining haploid plants, and 

from them by chromosome doubling of 

homozygous diploid plants was apparent. 

In 1974, Nitsch had reported regeneration 

of haploids and homozygous diploids by 

chromosome doubling, from isolated microspore 

culture (Nitsch, 1974a; 1974b). 

Culturing the microspores along with anther 

wall is essential for success. In isolated 

microspores, pollen embryogenesis 

is induced only rarely. 

This technique has great potential 

in plant breeding. Normally it takes selfing 

for many generations to obtain homozygosity 

in parental lines required in 

breeding programmes. This time can be 

considerably reduced by haploid culture 

techniques. 

4.7. Meristem culture and shoot tip culture 

When growing points (meristems) 

of shoots are cultured they continue their 

organized growth. The shoots/multiple 

shoots produced can be rooted to produce 

plantlets. This capacity has practical application 

and economic significance for 

plant propagation. 

Culture of the meristemic zones 

/extreme shoot tip is known as meristem 

culture, and culture of small segments (5- 

10 mm in size) from the shoot tip is 

known as shoot tip culture. It was known 

that meristems of virus infected roots are 

free of the pathogen (White, 1933; 1934). 

Limasset and Cornuet (1949) found that 

John 

shoot tips of virus infected plants are also 

virus-free. Morel and Martin (1950; 1955) 

could produce healthy plants from virusfree 

plants through shoot-tip culture from 

infected mother plants. This is possible 

because the pathogen concentration is not 

uniform in the infected plants, and apical 

buds of rapidly growing shoots are often 

not invaded by the virus. Morel (1960) 

used shoot apices of orchids to obtain 

their rapid clonal multiplication. Shoot tip 

culture has two important practical applications: 

(i) virus eradication and (ii) micro-propogation. 

These developments 

were followed by in vitro propagation of 

plants from shoot tip culture. Initially 

most of the species micropropogated were 

herbaceous (Morel, 1964; Murashige, 

1974). Now methods are available for the 

micropropagation of a large number of 

species belonging to a wide range of plant 

groups. 

4.8. Embryo culture 

Very young to mature embryos 

can be cultured in vitro. Embryo culture is 

one of the oldest applications of plant tissue 

culture in plant breeding. It has many 

practical applications, and very useful in 

obtaining hybrid plants from crosses in 

which post-zygotic incompatibility exists. 

In post zygotic incompatibility, fertilization 

and zygote formation occur on cross 

pollination. The zygote grows, but is not 

accepted by the endosperm. This results 

in embryo abortion at some stage of development 

before maturing of the seed. In 

such instances when the ovary/ ovule/ 

embryo with a part of the maternal tissue 

is excised and cultured on a suitable medium 

and under optimum culture conditions, 

it matures to produce a seedling. 

This procedure is hence called embryo 

rescue. Sharma et al. (1980) obtained few 

hybrids between Solanum melongena and 

S. khasianum by this method. Embryo 

culture is useful also in overcoming seed 

dormancy and for obtaining seed germination 

in some vegetatively propagating 

species in which seeds are produced but 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 5



normally do not germinate (e.g. some 

wild bananas). 

4.9. Invitro pollination and fertilization 

Pre-zygotic incompatibility is one 

of the major limitations in obtaining hybridization 

between many plant species 

and varieties. In pre-zygotic incompatibility, 

the zygote is not formed on cross pollination. 

The pollen either do not germinate 

on the stigma of the female parent or 

the pollen tube gets arrested at some point 

of its growth on the stigma/ in the style. 

A variety of methods used in vivo to 

overcome this barrier. 

Kanta et al. (1962) developed an 

in vitro technique for overcoming prezygotic 

incompatibility. In this method, 

the mature/nearly mature ovaries/ovules 

are cultured on suitable media and pollinated 

in vitro with cross pollen to obtain 

hybrids (Zenktler, 1980; Raghavan, 

1990). 

4.10. Root cultures 

Tip portions from primary and 

secondary roots of many plants can be 

cultured. In 1922, Kotte and Robbins independently 

postulated that true in vitro 

cultures could be raised from meristematic 

cells from root tips and shoot tips. 

Kotte (1922) could cultivate root tips of 

pea and maize in nutrient media for long, 

but no sub-culturing were done. Robbins 

(1922) could subculture his maize root 

cultures. White (1934) obtained unlimited 

growth of tomato roots, using the 

same medium as Robbins (1922), with 

yeast extract. Root cultures are useful in: 

(i) secondary metabolite production, and 

(ii) in basic studies on nematode infections, 

mycorrhizal associations, and root 

nodulation by Rhizobium bacteria. 

4.11. Organogenesis 

Organogenesis is the process of 

initiation and development of a structure 

that shows natural organ form and/or 

function. It is the ability of nonmeristematic 

plant tissues to form various 

organs de novo; the production of roots, 

John 

shoots or leaves. These organs may arise 

out of pre-existing meristems or out of 

differentiated cells. Indirect pathway includes 

a callus stage. 

Direct pathway bypasses a callus 

stage. The cells in the explant act as direct 

precursors of a new primordium, an organ 

or a part in its most rudimentary form or 

stage of development. 

4.12. Somatic embryogenesis 

In plants, embryo-like structures 

can be generated from non-germ cells 

(somatic cells), by circumventing the 

process of normal fertilization. As somatic 

embryos are formed without fertilization 

event, they are genetically identical 

to the parent tissue, and are therefore 

clones. 

Somatic embryogenesis may be 

direct or indirect. Indirect somatic embryogenesis 

involves a callus phase prior to 

embryo production. Direct somatic embryogenesis 

involves production of embryos 

from organized tissue without an 

intervening callus phase. Irrespective of 

the mode of production, anatomical and 

physiological features of somatic embryos 

are highly comparable to zygotic embryos. 

The morphological and temporal developments 

of somatic embryos are very 

similar to that of zygotic embryos. They 

both proceed through a series of distinct 

stages, namely, globular, heart, torpedo 

and cotyledon or plantlet stages for dicotyledons, 

and globular, elongated, scutellar 

and coleoptilar stages for monocotyledons. 

These stages typically span a period 

of several days. In dicots initially small 

globular embryos form which undergo 

isodiametric growth and establish bilateral 

symmetry. In monocots, especially in 

grasses, the transition from globular stage 

follows a series of events occurring simultaneously; 

such as the development of 

scutellum, initiation of the coleoptilar 

notch, tissue differentiation with the development 

of embryogenic vascular system 

and accumulation of intracellular 

storage substances. Somatic embryogenesis 

is used for: large-scale clonal propaga- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 6



tion of elite cultivars, as an alternative to 

conventional Micropropagation, producing 

synthetic (artificial) seeds. Indirect 

somatic embryogenesis (via callus) or 

secondary embryogenesis is used in gene 

transfer. Somatic embryogenesis also offers 

potential model for the study of molecular, 

regulatory and morphogenetic 

events in plant embryogenesis. 

4.13. Micropropagation 

Micropropagation is the tissue culture 

method of clonal propagation of 

plants. Plant tissue culture is rapidly becoming 

a commercial method for propagating 

difficult-to-propagate plants, new 

cultivars (selections, hybrids, transgenic), 

rare/endangered species. Micropropagation 

is usually achieved by the release 

(from dormancy), and growth of preexisting 

(axillary/ lateral) meristems in 

the initial culture. This is followed by repeated 

enhanced formation of axillary 

shoots by sub-culture on medium supplemented 

with plant growth regulators. The 

shoots produced are rooted either in vitro 

or ex vitro (out of culture). 

There are many advantages of Micropropagation. 

Shoot production is reliable 

and consistent. Multiplication rates 

can be three-fold to eight-fold a month. 

Plants produced via shoot culture are usually 

true-to-type and uniform. Allows 

propagation of rare/ endangered/ hybrid/ 

induced mutant/ genetically transformed 

plants. There also are few disadvantages. 

PGRs do not release apical dominance in 

all species. There may be a difference in 

results between juvenile and mature tissue 

of perennial species; shoot cultures may 

require a reversion to juvenility. Rooting 

of the micro-shoots may be difficult. Getting 

uniform shoot production in vitro, 

which is very important in commercial 

operations, may not be possible in some 

instances. The procedure is relatively labor 

intensive, with high upfront costs to 

get started. 

4.13. 1. Applications of micropropagation 

John 

Rapid and large-scale clonal (genetically 

uniform) propagation of plants 

(micropropagation) may allow faster production 

of plants that are slow to propagate 

in vivo. 

The time required for bulking-up 

of new cultivars before they are commercially 

introduced can be drastically decreased. 

Storage of germplasm, e.g. Cryopreservation. 

4.13.2. The process of micropropagation 

a. A small piece of the plant to be cloned 

(the explant) is removed from a 

healthy, well-maintained stock plant 

and surface sterilized (explant varies 

with species, but shoot tips, leaves, 

stem pieces, lateral buds, and young 

flowers or floral parts are used). 

b. Surface sterilized explants are rinsed 

with sterile water, and placed aseptically 

in/ on specially formulated and sterilized 

medium in culture vessels. 

c. The explant may proliferate directly by 

enhanced lateral branching, or the tissue 

may undergo a certain period of 

unorganized growth (callus) prior to 

shoot differentiation. 

d. The growth of the cultures is principally 

determined by the plant growth regulator 

(PGR) content of the culture 

medium (the auxin and cytokinin alone 

or in combination and concentration/s). 

Most cultures are established within 4 

to 12 weeks depending on the species/ 

cultivar. 

e. A proliferating shoot culture can be 

sub-cultured to produce divisions 

which will multiply rapidly. 

f. Rate of multiplication vary and are affected 

by many factors. Production of 

thousands, and in some cases millions 

of plants a year from a single explant 

has been demonstrated 

5. Role of plant tissue culture in sustainable 

agriculture 

Sustainable agriculture requires 

efficient, biotic and abiotic stress resistant 

crop varieties. Germplasm collection and 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 7



storage, simple crop improvement methods 

such as selection and bulking by rapid 

and large scale cloning by micropropagation 

can be of great use. Plant tissue culture 

techniques such as: anther culture, 

dihaploid production, embryo rescue, and 

in vitro pollination and fertilization can be 

very useful in developing crop varieties 

through hybridization. Callus culture, cell 

suspension culture, organogenesis and 

somatic embryogenesis are essential for 

crop improvement through transgenics 

(various genetic engineering techniques). 

6. Role of plant tissue culture in conserving 

biodiversity 

6.1. Biodiversity 

"Biological diversity means the 

variability among living organisms from 

all sources including, inter alia, terrestrial, 

marine and other aquatic ecosystems and 

the ecological complexes of which they 

are part; this includes diversity within 

species, between species and of ecosystems.” 

- Definition of Biodiversity by 

CBD. 

6.2. Importance of biodiversity 

Biodiversity is essential to: (i) ensure 

the production of food, fibre, fuel, 

fodder, etc., (ii) maintain other ecosystem 

services, (iii) allow adaptation to changing 

conditions - including climate change, 

and (iv) sustain rural peoples' livelihoods 

(Convention of Biological Diversity). 

John 

6.3. Threats to biodiversity 

Biodiversity is under serious 

threat as a result of human activities. 

The main dangers worldwide are: (i) Invasion 

by alien species, (ii) Environmental 

degradation, (iii) Climate change and 

global warming, (iv) Urbanization and 

habitat conversion, (v) Population growth 

and ever-increasing demand for resources, 

(vi) Unsustainable over-exploitation of 

natural resources. 

Agriculture contributes significantly 

to conservation and sustainable use 

of biodiversity. However, it is also a major 

driver of loss of biodiversity. This puts 

in jeopardy the sustainability of agriculture 

and ecosystem services and their 

ability to adapt to changing conditions. 

This also poses serious threat to food and 

livelihood security. 

6.4. Plant tissue culture methods for conserving 

biodiversity 

Plant Tissue Culture offers novel 

options for collection, multiplication and 

medium/ long-term ex situ conservation 

of plant biodiversity. By plant cell, tissue, 

and organ culture techniques, rapid and 

large scale multiplication and season independent 

production of planting material 

is possible. This has helped in the conservation 

of many endangered species. Medium-term 

conservation is achieved by 

slow growing cultures. Cryopreservation 

(at −196 °C, in liquid nitrogen) allows the 

safe and cost-effective long-term conservation. 

6.5. In vitro collection 

Potential advantages of in vitro 

methods are: (i) Less space requirement, 

(ii) Pathogen-fee plants, (iii) No need for 

transfer (under storage conditions), (iv) 

Stored cultures can be used as stock for 

vegetative preservation, and (v) International 

exchange of plant material made 

easy because, no use of soil, and no pathogens. 

Basic goals of an in vitro storage 

system are: to maintain genetic stability, 

to keep in indefinite storage without loss 

of viability, and most importantly, to be 

economical. 

Three types of Plant Tissue Culture systems 

are available. They are: (i) Normal 

growth, (ii) Slow growth, and (iii) Cryopreservation. 

6.5.1. Normal growth 

Normal Growth is achieved either 

on semi solid media or in liquid media. 

Normal growth is similar to multiplication 

stage in micro-propagation, and requires 

frequent sub-culture. Considered as genetically 

stabile when achieved through di- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 8



rect organogenesis from apical buds / axillary 

buds as explants. 

6.5.2. Slow growth storage 

By deliberate slowing down of 

growth cultures can be stored at least for 

6 months and maximum up to 6 years 

without sub-culturing. There are many 

ways to achieve slow growth. First, by 

manipulating storage temperature (cold 

storage at 1-9°C) and light (low light intensity). 

Second, increasing osmotic potential 

of the media [by using osmotically 

active compounds such as sucrose (at 

higher ~6%), mannitol etc.]. Third, by 

addition of inhibitors or retardants, for 

e.g. mineral oil overlay (callus), reduced 

oxygen tension etc. 

Plant Growth Retardants are 

chemicals that slow cell division and 

elongation in shoots. They cause plants to 

be shorter and more compact, interrupt 

cell division, stem elongation, and inflorescence 

/ flower formation. But roots 

continue to grow. Plant growth retardants 

may reduce the natural Gibberellic acid, 

or may produce more ethylene. 

6.5.3. Cold storage 

Storage at non-freezing temps, 

from 1-9° C dependent on species. Storage 

of shoot cultures (stage I or II) works 

well for strawberries, grapes, may be for 

many more spp. Transferred to fresh medium 

every 6 months/ annually/ or longer 

periods basis. Advantages of cold storage 

are: (i) simple, (ii) high rates of survival, 

and (iii) useful in micropropagation (especially 

in periods of low demand). The 

disadvantages are: (i) may not be suitable 

for some tropical, subtropical species because 

of susceptibility to cold injury, (ii) 

requires refrigeration, which is more expensive 

than storage at ultra-low temperatures 

(in cryopreservation). An alternative 

to cold storage is the use of a medium 

with reduced nutrients and lacking sucrose 

(as reported in coffee). 

6.5.4. Cryopreservation 

John 

Cryopreservation is the storage of 

living tissues at ultra-low temperatures 

(˗196°C). It is useful in conservation of 

plant germplasm of vegetatively propagated 

species, recalcitrant seed species 

(coconut palm etc.), conservation of tissue 

with specific characteristics, cell lines 

producing secondary metabolites, genetically 

transformed tissues, tissues competent 

to transformation/ mutagenesis, pathogen 

(virus) eradicated tissue for future 

multiplication (as is done in Banana). 

Cryopreservation procedures are 

available only for limited number of plant 

species. Each species/ variety/ tissue type, 

needs standardization for: explant size 

and type, water content, and natural freezing 

resistance. Most studies on cryopreservation 

of plants involve only one or a 

few genotypes. Only few plant 

germplasm collections stored in liquid 

nitrogen currently exist (with a relatively 

limited number of accessions). 

7. Role of plant tissue culture in protecting 

the environment 

Forests are complex ecosystems, 

predominantly composed of trees and 

shrubs, and usually have closed canopies. 

There is nearly 4 billion hectares of forest 

in the world (this is about 30% of the total 

land cover). Depending on the physical, 

geographical, climatic and ecological factors, 

there are different types of forests 

like evergreen forest (mainly composed of 

evergreen tree species) and deciduous 

forest (mainly composed of deciduous 

tree species). India‟s recorded forest area 

is 76.52 million hectares. This is 23.28% 

of the country‟s total geographical area. 

Over 90% of the forest area is under government 

ownership and is managed by the 

forest departments of the state governments 

(State of Forests Report, 2009). 

Forests are important both economically 

and ecologically, and render many services 

to the life support system of earth. 

Forests are the primary source of 

wood. Wood is used to fulfil three basic 

needs: (i) energy, (ii) construction materi- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 9



al, and (iii) industrial raw material. About 

2 billion people in the developing world 

are dependent on forests for their basic 

energy needs (fuel for cooking food). Until 

recently wood has been the chief construction 

material. High strength to 

weight ratio and availability in many 

kinds, ease of cutting and shaping with 

simple tools, and insulation to heat, make 

wood an ideal construction material for 

many purposes. Major industrial uses of 

wood are: (i) paper and pulp, (ii) rayon, 

and (iii) plywood. Besides wood, forests 

are a source of a variety of non-wood forest 

products (NWFPs). Thus forests contribute 

greatly to the economy. Around 

1.6 billion people depend on forests for 

their livelihood. This includes some 70 

million indigenous people. 

Forests play an important role in 

maintaining ecological balance. Forests 

are atmospheric filters. They are the major 

suppliers of oxygen. In photosynthesis 

they fix atmospheric carbon dioxide into 

carbohydrates, sugars, proteins, and many 

forms of biomass, thus playing a significant 

part in the global carbon cycle. Forests 

contribute large quantities of moisture 

to the atmosphere, thus regulating 

climate. Forests also conserve soil and 

water resources. 

The term forest implies „natural 

vegetation‟ of the area, existing from 

thousands of years and supporting a variety 

of biodiversity. More than half of the 

known terrestrial plant and animal species 

live in forests (Millennium Ecosystem 

Assessment, 2005). The forest ecosystem 

has two components - biotic and abiotic. 

The living component includes plants 

(trees, shrubs, herbs etc.), animals and 

microorganisms. Forests are home to 

more than 80 per cent of all terrestrial 

species of animals, plants and insects. 

Globally, deforestation is the major cause 

of loss of biological diversity, and is a 

matter of great concern (Laurance, 2007). 

Worldwide, the area of natural 

forests decreases by some 13 million ha 

annually (this is about 3% of the total forest 

area). This loss of forest area is mostly 

John 

due to conversion to agriculture land and 

urbanization. Deforestation has severe 

consequences for the environment and 

climate. More than 2000 times the total 

energy consumption of the world population, 

of solar energy reaches the earth‟s 

surface. Because of deforestation, not only 

this natural source of energy is wasted, 

but also has a serious negative impact on 

the environment, by way of surface heating, 

and desertification. 

In the tropical and sub-tropical regions 

of the world, receiving about 600 

mm rainfall and above, plantation forestry 

of economically important tree species 

(say teak for timber and eucalypts for 

pulp) can take away pressure for forestry 

resources from natural forests and can add 

to the forest cover. For this, large numbers 

(in millions) of plating material of 

superior varieties (fast growing, better 

adapted, disease and insect-pest resistant 

etc.) are necessary. But forest tree species 

are difficult to breed, because of their 

long generation cycles, highly heterozygous 

natural populations, openly cross 

pollinated nature, and lack of knowledge 

about their genetics. 

Clonal propagation of „superior‟ 

genotypes (identified for desirable traits) 

through tissue culture has been used very 

profitably in case of many tree species. 

Eucalypts have been multiplied and used 

for plantation forestry for a long time 

(FAO Report, 1981). Eucalyptus wood 

from plantation forestry has been used as 

timber, industrial raw material and fuel. 

Plantation forestry using eucalypts may 

not be suitable for some places because of 

high water demand. Teak (Tectona grandis 

Linn. f.) is another success story. In 

early 1980s, scientists from CSIR- 

National Chemical Laboratory, Pune for 

the first time regenerated complete plantlets 

from an 80 year old „elite‟ tree (Gupta 

et al., 1983). Poplars (Populus spp.) are 

another tree species which clonally propagated 

through plant tissue culture techniques, 

and widely cultivated in plantation 

forestry in many parts of the world. 

In India, poplars (Populus spp.) are the 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 10



most popular tree species in agro-forestry 

production systems. Poplars are usually 

intercropped with agricultural crops like 

wheat, rice and sugar cane. Poplars are 

well known for their fast growth, outstanding 

properties and quick and high 

financial returns. Timber from poplars 

often forms the backbone of match, paper, 

sports goods, plywood, and composite 

board industries. 

References 

Barnosky, A. D., Hadly, E. A., Bascompte, 

J., Berlow, E. L., Brown, J. 

H., Fortelius, M., Getz, F. M., 

Harte, J., Hastings, A., Marquet, P. 

A., Martinez, M. D., Mooers, A., 

Roopnarine, P., Vermeij, G., Williams, 

J. W., Gillespie, R., Kitzes, 

J., Marshall, C., Matzke, N., Mindell, 

D. P., Revilla, E. and Smith, A. 

B. (2012). Approaching a state shift 

in Earth‟s biosphere. Nature 486, 52- 

58. 

Cocking E. C. (1960). A method for the 

isolation of plant protoplasts and 

vacuoles. Nature 187, 962-63. 

FAO (1999). State of world‟s forests 

1999, United Nations Food and Agricultural 

Organization, Rome, 154 pp. 

FAO (2007). State of world‟s forests 

2007, ftpfao.org/docrep/fao/009. 

United Nations Food and Agricultural 

Organization, Rome. 

Gamborg, O. L., Shyluk, J. and Kartha, 

K. K. (1975). Factors affecting 

the isolation and callus formation in 

protoplasts from shoot apices of Pisum 

sativum L. Plant Sci. Lett. 4, 

285-92. 

Griggs, D., Stafford-Smith, M., 

Gaffney, O., Rockström, J., Öhman, 

M. C., Shyamsundar, P., 

Steffen, W., Glaser, G., Kanie, N. 

and Noble, I. (2013). Sustainable 

development goals for people and 

planet. Nature 495, 305-7. 

Griggs, D., Stafford Smith, M., Rockström, 

J., Öhman, M. C., Gaffney, 

O., Gisbert Glaser, G., Noble, I., 

John 

Steffen, W. and Shyamsundar, P. 

(2014). An integrated framework for 

sustainable development goals. Ecology 

and Society 19, 49. 

Guha, S. and Maheshwari, P. (1964). In 

vitro production of embryos from anthers 

of Datura. Nature 204, 497-98. 

Gupta, P. K., Mehta, U. and Mascarenhas, 

A. F. (1983). A tissue culture 

method for rapid multiplication of 

Eucalyptus camaldulensis. Plant Cell 

Rep. 2, 296-99. 

Hartley, M. J. (2002). Rationale and 

methods for conserving biodiversity 

in plantation forests. Forest Ecology 

and Management 155, 81–95. 

John, C. K., Nadgauda, R. S. and Mascarenhas, 

A. F. (1997). Tissue Culture 

of Economic Plants, Center for 

S&T of Non-Aligned and other Developing 

Countries, New Delhi, and 

Commonwealth Science Council, 

London. pp. 3-34. 

Kanta, K., Rangaswamy, N.S. and Maheshwari, 

P. (1962). Test tube fertilization 

in a flowering plant. Nature 

194, 1214-1217. 

Kao, K. N. and Michayluk, M. R. 

(1974). A method for high frequency 

intergeneric fusion of plant protoplasts. 

Planta 115, 355-67. 

Kotte, W. (1922). KulturversuchemitisoliertenWurzelspitzen. 

Beitr. Z. 

Allgem. Bot. 2, 413-434. 

Klercker, I. A. F. (1892). Eine method 

zuIsolierunglebenderProtoplasten. 

Oefvers K. Vetensk. Akad.Foerh. 9, 

463-471. 

Laurance, W. F. (2007). Have we overstated 

the tropical biodiversity crisis? 

Trends Eco. Evol. 22, 65-70. 

Lenton, T. M. (2011). Beyond 2°C: redefining 

dangerous climate change for 

physical systems. Wiley Interdisciplinary 

Reviews: Climate Change 2(3), 

451-461. 

Limasset, P. and Cornuet, P. (1949). 

Recherche du virus de la mosaique 

du Tabacdans les meristemes des 

plantesinfectes. C. R. Ac. Sc. 228, 

1971-72. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 11



John 

Melchchers, G. and Labib, G. (1974). 

Somatic hybridization in plants by 

fusion of protoplasts - I. Selection of 

light resistant hybrids of a haploid 

light sensitive varieties of tobacco. 

Mol. Gen. Genet. 135, 277-94. 

Millenium Ecosystem Assessment 

(2005). Ecosystem and human wellbeing: 

current state and trends. Findings 

of the conditions and trends 

working group, In: Hassan, R., 

Scholes, R. and Ash, N. (eds.) Millenium 

ecosystem assessment series. 

Island Press, Washington, U.S.A. 

Morel, G. (1950). Sur la culture des tissue 

de deux Monocotyledones. C. R. 

Ac. Sc. 230, 1099-1101. 

Morel, G. (1960). Producing virus free 

cymbidium.Bulletin of American Orchid 

Society 29, 495-497. 

Morel, G. (1964). Action d 

l‟acidepantotheninesur la croissance 

des tissues d‟Aubepinecultivesin 

vitro. C. R. Ac. Sc. 243, 166-168. 

Morel, G. and Martin, C. (1950). Guerison 

de pommes de terreatteintesd‟unemaladie 

a virus. C. R. Ac. 

Sc. 235, 1324-25. 

Morel, G. and Martin, C. (1955). Guerison 

de pommes de terreatteintes de 

maladies a virus. C. R. Ac. Agri. 41, 

470-75. 

Murashige, T. (1974). Plant propagation 

through tissue cultures. Ann. Rev. 

Plant Physiol., 24, 135-65. 

Murashige, T. And Skoog, F. (1963). A 

revised medium for rapid growth and 

bioasays with tobacco tissue cultures. 

Physiol Plant. 15, 473-497. 

Nitsch, C. (1974a). La culture de pollen 

isolesurmihensynthetique. C. R. Ac. 

Sc. 278, 1031-34. 

Nitsch, C. (1974b). Pollen culture – a 

new technique for mass production of 

haplid and homozygous plants. In: 

Kasha, K. J. Ed. Haploids in Higher 

Plants – Advances and Potential. 

University of Guelph, Guelph, Sweden. 

pp. 123-35. 

Power, J. B., Cummins, S. E., and 

Cocking, E. C. (1990). Fusion of isolated 

plant protoplasts. Nature 225, 

1016-17. 

Reid, W. V. and Miller, K. R. (1989). 

Keeping options alive: the scientific 

basis for conserving biodiversity. 

World Resources Institute, Washington, 

DC. 128 p. 

State of Forests Report (2009). Ministry 

of environment and forests, Government 

of India, New Delhi. 

Raghavan, V. (1980). Embryo culture, 

In: Vasil, I. K. Ed. Perspectives in 

plant cell and tissue culture, Int. Rev. 

Cytology Suppl. 11B, Academic 

Press, New York, USA, pp. 209-240. 

Robbins, W. J. (1922). Effect of autolyzed 

yeast and peptone on the 

growth of excised corn root tips in 

the dark. Bot. Gaz. 74, 59-79. 

Rockström, J. et al. (2009). A safe operating 

space for humanity. Nature 461, 

472–475. 

Sharma, D. R., Chowdhury, J. B., Ahuja, 

U., and Dhankhar, B. S. (1980). 

Interspecific hybridization in genus 

Solanum. A cross between S. 

melongena and S. khasianum through 

embryo culture. Zeitschrift fur Pflanzenzuchtung, 

85(3), 248-253. 

Shindell, D. Kuylenstierna, J. C. I., 

Vignati, E., van Dingenen, 

R., Amann, M., Klimont, Z., Anenberg, 

S. C., Muller, N., Janssens- 

Maenhout, G.,Raes, F., Schwartz, 

J., Faluvegi, G., Pozzoli, L., Kupiainen, 

K., Höglund-Isaksson, 

L., Emberson, L., Streets, 

D., Ramanathan, V., Hicks, K., 

Kim Oanh, N. T., Milly, 

G., Williams, M., Demkine, V. and 

Fowler, D. (2012). Simultaneously 

mitigating near-term climate change 

and improving human health and 

food security. Science 335, 183–189. 

UNEP (1994). Convention on biological 

diversity, UNEP/CBD, Switzerland, 

November 1994, 34 p. 

United Nations (2005). 2005 world 

summit outcome [adoption resolution, 

60th session].United Nations, 

New York, New York, USA. [online] 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 12



URL: http://www.who.int/hiv/univers 

alaccess2010/worldsummit.pdf 

United Nations (2012). The future we 

want: outcome document adopted at 

Rio+20. United Nations, New York, 

New York, USA. [online] 

URL: http://www.un.org/futurewewa 

nt 

United Nations (2012a). The millennium 

development goals report 2012. United 

Nations, New York, New York, 

USA. 

[online] 

URL: http://www.un.org/millennium 

goals/pdf/MDG%20Report%202012. 

pdf 

United Nations (2013). A new global 

partnership: eradicate poverty and 

transform economies through sustainable 

development. The report of 

the High-Level Panel of eminent persons 

on the post-2015 development 

agenda.United Nations, New York, 

New 

York, 

USA. http://www.post2015hlp.org/w 

p-content/uploads/2013/05/UN- 

Report.pdf 

United Nations Open Working Group 

(UN OWG) (2013). Interim progress 

report to UN General Assembly. 

United Nations, New York, New 

York, USA. [online] 

URL: http://sustainabledevelopment. 

un.org/content/documents/1927interi 

mreport.pdf 

United Nations Open Working Group 

(UN OWG) (2014). Outcome document 

- Open Working Group on Sustainable 

Development Goals (19th 

July 2014).United Nations, New 

York, New York, USA. [online] 

URL: http://sustainabledevelopment. 

un.org/content/documents/1579SDGs 

%20Proposal.pdf 

United Nations Sustainable Development 

Solutions Network (UN 

John 

SDSN) (2013). An action agenda for 

sustainable development. Report for 

the UN Secretary-General. Sustainable 

Development Solutions Network, 

New York, New York, USA. [online] 

URL: http://unsdsn.org/resources/pub 

lications/an-action-agenda-forsustainable-development/ 

United Nations Environment Programme 

(UNEP) (2013). Embedding 

the environment in sustainable development 

goals. UNEP Post-2015 Discussion 

Paper 1.UNEP, Nairobi, 

Kenya. 

[online] 

URL: http://www.unep.org/pdf/embe 

dding-environments-in-SDGs-v2.pdf 

Wallin, A., Glimelius, K. G. and Eriksson, 

T. (1974). The induction of aggregation 

and fusion of Daucus carota 

protoplasts by polyethylene glycol. 

Zeitschrift fur Planzenphysiol. 74, 

64-80 

White, P. R. (1933). Plant tissue culture: 

Results of preliminary experiments 

on the culturing of isolated stem tips 

of Stellaria media. Protoplasma 19, 

97-116. 

White, P. R. (1934). Multiplication of the 

viruses of tobacco and ancuba mosaic 

in growing excised tomato roots. 

Phytopathol. 24, 1003-11. 

White, P. R. (1933). Potentially unlimited 

growth of excised tomato root tip 

in a liquid medium. Plant Physiol, 9, 

585-600. 

Zenktler, O. M. (1980). Intra-ovarian 

and in vitro pollination. In: Vasil, I. 

K. Ed. Perspectives in plant cell and 

tissue culture, Int. Rev. Cytology 

Suppl. 11B, Academic Press, New 

York, USA, pp. 137-156. 

Zimmerman, U. and Scheurich, P. 

(1981). High frequency fusion of 

plant protoplasts by electric fields. 

Planta 151, 26-32. 

© 2017 by the author. Licensee, Editors and AIMST University, Malaysia. 

This article is an open access article distributed under the terms and 

conditions of the Creative Commons Attribution (CC BY) license 

(http://creativecommons.org/licenses/by/4.0/). 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 13



Traditional Medicine of the Tribes in Tamil Nadu and Its 

Sustainable Use through Biotechnology 

Valli Gurusamy 1 , Kavitha Valampuri John 2 , Usha Raja Nanthini Ayyakkanu 2 , 

Ramani Bai Ravichandran 3, * 

1 Vice Chancellor, Mother Teresa Women’s University; 2 Department of Biotechnology, 

Mother Teresa Women’s University; 3 Department of Zoology, University of Madras, India; 

*Correspondence: rramani8@hotmail.com; Tel: +91 9444020828 

Abstract: India is a land of mega biodiversity representing about 7% of the world‟s flora 

and 6.5 per cent of world‟s fauna. Tamil Nadu, one of the southern most states of India, is 

rich in forest cover and cultural diversity. Genomic evidence supports the peopling of Tamil 

Nadu from the 1 st wave of migration of humans from the „Out of Africa‟ exodus and points 

out that the tribes of state were among the earliest settlers in the region. The tribal population 

of Tamil Nadu represents 1.02% of the total population of the state. Living in close association 

with the forest, they have accumulated a treasure trove of ethno botanical 

knowledge in the form of traditional medicine. The future of sustainable use of renewable 

forest product lies with the molecular tools of Biotechnology. We present here an analysis 

of the documented literature of the medicinal plants used by the tribes of Tamil Nadu for 

treatment of common disorders. We also present the challenges and prospects within the 

scope of Biotechnology to ensure sustainable use of traditional medicine for the betterment 

of mankind and environment. 

Keywords: Biotechnology; sustainable; tribes; traditional medicine; Tamil Nadu. 


India is a land of enormous cultural, 

linguistic and religious diversity presumably 

because of Man‟s long stay, for 

the past 50-70,000 years in this continent. 

This is an outcome of various migrations 

that took place into India, serving as a 

major corridor for the dispersal of modern 

humans out of Africa (Cann, 2001). Archaeological 

evidences indicated that the 

Indian subcontinent was peopled by various 

migrations since Palaeolithic (300- 

400,000 BCE), starting with the Late 

Pleistocene (Misra, 2001). „The Castes 

and Tribes of Southern India‟ was an attempt 

to catalogue these populations 

(Thurston, 1909).The knowledge of the 

medicinal value of plants, animals and 

other substances and their uses goes back 

to the time of the earliest settlers probably 

along with their 1 st wave of migration out 

of Africa. Traditional Medicine is the sum 

total of long-standing information on the 

knowledge, skills, and health practices 

based on the theories, beliefs, and experiences 

indigenous to different cultures or 

local communities. Traditional medicine 

incorporates plant, animal and mineral 

based medicines and encompasses spiritual 

therapies, manual techniques and 

exercises which can be applied singularly 

or in combination for the maintenance of 

health through the prevention, diagnosis, 

improvement or treatment of physical and 

mental illness. Traditional knowledge has 

been well preserved and orally passed 

from one generation to the next in the 

form of stories, legends, folklore, rituals, 

songs, art, and even laws. Since there is 

no written script the exchange of knowhow 

between diverse communities is a 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 14


Traditional Medicine of the Tribes in Tamil Nadu… 

process of evolution through trial and error 

which makes documentation and record-keeping 

almost impossible. This process 

of exchange and assimilation is continuous, 

and today there is a growing 

awareness among the medical community 

about the intrinsic value of traditional 

medicine, and as a result in India Ayurveda, 

Unani and Siddha have entered the 

mainstream to compliment biomedicine. 

Contemporary Indian society faces the 

challenge of integrating the best of the 

different healing traditions to provide a 

holistic health care. 

2. Traditional knowledge 

Even before classical medical 

knowledge of ancient India was codified 

into the canonical texts of Ayurveda in 

the 6 th century BC, there were abundant 

sources of medical knowhow in the subcontinent 

from prehistoric times. Traditional 

healers can be either folk or tribal 

healers and have worked in intimate relation 

with their environment. Traditional 

healing ranges from simple home remedies 

related to nutrition and treatment for 

minor illnesses, to more sophisticated 

procedures such as midwifery, bone setting, 

blood-letting (therapeutic phlebotomy) 

and treatment of snake bites and 

mental disorders. Some healing practices 

were considered to be sacred and were 

associated with rituals that helped safeguard 

them for there is a substantial overlap 

between healing plants and sacred 

plants. Categories of traditional healers 

are traditionally trained healers, old individuals 

of the community, educated individuals 

acquiring certain knowledge from 

their predecessors, ancient inscriptions in 

the form of copper plate/palm leaf writings, 

old and recent publications in regional 

language. 

3. Indian ethnobotany 

India is one of the richest countries 

in the world not only in biodiversity 

but also in different ethnic groups of an- 

Gurusamy et al. 

cient lineage from the 1 st wave of the „out 

of Africa‟ exodus and thus in vast ethnobotanical 

knowledge. According to the 

2011 census, 104 million tribal people 

speaking over 227 linguistic groups inhabit 

varied geographic and climatic 

zones of the Indian subcontinent. Ethnomedicine 

includes plants, animal products 

and minerals used by tribal communities 

of a particular region or country for medicinal 

purposes other than those mentioned 

in classical streams of the respective 

cultures. Tribal people have been using 

a large number of wild plants as documented 

by ethnobotanical investigations. 

The application of most of the plants recorded 

are either lesser known or hitherto 

unknown to the outside world. India has 

been the country most concerned about 

the conservation of its medicinal plants. 

There are over 45,000 species of vascular 

plants reported from India of which as 

many as 15,000 may be used medicinally. 

The folk medicine system of India use 

about 5,000 plant species with about 

25,000 formulation, whereas the tribal 

medicine involves the use of over 8,000 

plant species with about 1,75,000 specific 

preparations (Pushpangadan and George, 

2010). More than 90% of the raw material 

for traditional medicine comes from wild 

harvesting as this the common method 

used for collecting them (Tandon, 1996; 

Gupta 1998; Ved et al., 1998). About 71 

medicinal plant species are classified as 

“rare”, and of this 92% are in active trade, 

and 74% are traded nationally. It has been 

estimated that between 4,000 and 10,000 

medicinal plant species in India face extinction 

in the local, regional and national 

levels (Hamilton, 2004). In an effort to 

create leadership in affordable and holistic 

health care, India is committed to 

promoting traditional medicines like 

Ayurveda which remained untapped due 

to inadequate scientific scrutiny. Steps 

are being taken to bring in regulatory 

amendments in research and effective enforcement 

for integration of quality products, 

practices and practitioners into the 

AYUSH (Ayurveda, Yoga and Naturopa- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 15



thy, Unani, Siddha and Homoeopathy) 

system at central and state level. 

4. Traditional knowledge of the tribes 

of Tamil Nadu 

According to the 2001 census, 

tribal population in Tamil Nadu is 6, 

51,321 which constitute 1.02% of the total 

population. There are 36 tribes and sub 

tribes in Tamil Nadu. Out of the 36 

Scheduled Tribe communities in the 

state, about six tribal populations Todas, 

Kotas, Krumbas, Paniyas, Irulas, Kattunayakas 

have been classified as primitive 

tribes with incredibly high anthropological 

significance. The primitive tribes 

occupy the length and breadth of the Nilgiri 

district in the Western Ghats of Tamil 

Nadu. One sixth of the land mass of Tamil 

Nadu is covered by forests. The tribes 

of Tamil Nadu live in and around the reserved 

forests and have gained immense 

knowledge on the use of forest produce to 

treat common disorders. 

Todas: They are called by other 

names like Tudas, Thuduvans, and Todar 

(Kavitha V.J., 2008). They are professional 

pastoralists and dairy men, a purely 

pastoral economy in India today, living in 

the higher altitudes in the traditional 

houses called “Munds” that are half barrel 

shaped and are vegetarians. Their dialect 

is independent form of Dravidian Tamil- 

Malayalam. They are fair skinned and 

wear ornaments and their dress is akin to 

the Roman „toga‟. They have two exogamous 

divisions called Tarthar and Teivali. 

There are five socially distinguishable 

sects (clans) such as Pelki, Pekkan, 

Kuttan, Kenna and Jodi (Rajan and Sethuraman, 

1993). Their population was 1,600 

in the 2001 census. 

Kotas: Their other names are Koter, 

Kothewars, and Kohatur. The Kotas 

are musicians and excellent craftsmen 

having mastery over ironworking. They 

are light skinned, with copper hair. They 

speak the “Kota” a Dravidain language. 

Their distribution in the Nilgiri district is 

confined only to seven villages inhabiting 


in moderate altitude of the district namely: 

New Kotagiri (Aggal), Kil-Kotagiri, 

Kundah, Kallimalai, Gudalur, Trichigadi 

and Sholur Kokal.Their settlement is 

called the “Kokkal” with linear row of 

houses in streets, „Keri‟. Each village has 

three Keri known as Kizhkeri, Nadukeri 

and Melkeri. Keri, clan, exogamy is noteworthy 

among Kotas (Kavitha V.J., 

2008). Their chief diety is Kambattrayan. 

Their population was 1,894 in 2001 census. 

Irulas: They are also called as Iuvan,Villiar. 

The Irulas are distributed in 

the lower altitudes of the Nilgiri hills (district). 

They are negrotoid in appearance 

whose chief occupation is as plantation 

labourers in the estates. Their settlements 

are called “Aral”. Their dialect is Tamil 

mixed with Malayalam. Their community 

is divided into seven exogamous clans 

(Kuems): Kupper, Sambe, Kalkatti, Kurunagar, 

Devanan, Peradar and Punger 

(Rajan and Sethuraman, 1991). They are 

basically hunter gatherers. Their population 

was 6,700 in 2001 census. 

Kurumbas: The Kurumbas practice 

hunting food gathering economy, 

well-versed in honey collection techniques. 

They are plain dwelling people 

living in the interior forests of the district. 

Their staple foods are wild tubers (Dioscorea 

bulbosa), wild fruits and other 

minor forests produces. Their settlements 

are called “Mottam”. They are dark 

skinned and speak the Kurumba dialect. 

Kurumbas are a heterogenous population 

having divisions such as Halu Kurumbas, 

Betta Kurumbas, Mullu Kurumbas, Jess 

kurumbas and Urali Kurumbas. Their 

population was 6,872 in the 2001 census. 

Paniyas:The Paniyas are negrotoid 

people living in bamboo huts at the 

junction of Kerala and Tamil Nadu border. 

They work as labourers with Wayanad 

Chettis though they were basically 

hunter gatherers. Their settlements are 

called “Paddi”. They possess excellent 

skills in the art of fishing by employing 

certain plant parts like bark of Eugenia sp. 

and leaves of Aibizzia sp. as stupefying 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 16




agents. They speak the Paniya dialect, 

practice Illam, patilineage, and their inheritance 

is by “Marumakkathayam”. 

They numbered 6,700 in 2001 census. 

Kattunayakas: They are also 

called as Shola Nayakans, Jenu or Teen 

Kurumbas. They are another group of forest 

dwellers who are nomadic in nature, 

their staple foods are honey, wild fruits 

and tubers. Their settlements are called 

“Paadi”. They are short and black with 

protruding forehead. They have curly hair 

and speak Kannada language. Eating bison 

flesh is a cultural taboo with them. 

The social customs and religious practices 

of Kattunayakas are akin to Kurumbas in 

many respects. They population was 

1,425 in the 2001 census. 

Paliyars: They are found in the 

hilly regions of Madurai, Dindigul, Theni, 

Tirunelveli and Virudhunagar districts. It 

is believed that Paliyars are indigenous 

people of Palani hills of Kodaikanal and 

speak Tamil. Physically they are similar 

to the Semong of Malaya and other Indian 

tribal communities. They can be grouped 

into three categories based on their life 

styles, namely, nomadic, semi nomadic 

and settled. Nomadic Paliyars don't build 

houses; they live temporarily in rock 

caves called 'Pudai'. 

Ethnobotanical traditional 

knowledge for the tribes of Tamil Nadu 

was retrieved using Pubmed and Google 

using the keywords ethnomedicne, tribes, 

Tamil Nadu. The ethnobotanical data presented 

here included knowledge form 

seven tribal groups of Irula, Paniya,Kurumba, 

Kota, Thoda, Kattunayakkans 

and Paliyars (Table 1). These published 

research articles were then analysed 

manually to ascertain the traditional 

knowledge of these communities related 

to the study area and the usage pattern of 

the medicinal plant species. The data was 

further analysed using graphical representations 

for summarizing and interpreting 

for the major families of plant species 

represented, part of the plant used and 

cure for disorder from the traditional medicinal 

knowledge of the tribes of Tamil 

Nadu. 

5. Ethnomedical wealth of tribes of 

Tamil Nadu 

A total of 229 medicinal plants 

used by the tribes of Tamil Nadu belonging 

to 79 families for the treatment of 

more than 40 disorders were documented. 

The percent representation of the families 

of plants used as medicine by the tribes of 

Tamil Nadu is represented in Figure 1. 

Euphorbiaceae is the largest family represented 

by 18 species at 12% followed by 

Fabaceae by 14 species (9%), Lamaceae 

by 13 species (8%), Asteraceae by 11 

species (7%), Solaneaceae and Rutaceae 

by 10 species each (6%) and Ascelpiadacea 

by 8 species(5%). This data marks a 

direction for scientific researchers as to 

which of the families to search for to 

identify bio active compounds. 

Of the primary parts of the plant 

used the leaves formed 40% of usage in 

traditional medicine followed by the root 

and bark at 11%, whole plant and fruit at 

8%, stem at 7% and seed at 5% (Figure 

2). It is of utmost importance to see this 

data in the light of the major families represented 

for medicinal use by the tribes of 

Tamil Nadu. It is also important to test 

these bioactive compounds from different 

parts of the plant as a combination and to 

use bioactive compounds different families 

in conjunction for any particular disorder. 

The data when analysed for major 

remedies against diseases it revealed that 

the tribes of Tamil Nadu had traditional 

remedies for wounds (31) and skin problems 

(29), followed by stomach aches 

(17), diahorrea and headache (13), Cold, 

cough, fever (12) and rheumatic diseases, 

gastric disorders and toothache (9). It is 

interesting to note that the ethnomedicine 

of the tribals has 9 remedies for women to 

ease labour pain and 3 to induce lactation 

(Figure 3). Tribal ethnomedicine also has 

remedies for diabetes and jaundice (5). 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 17




Anacardiaceae 

2% 

Fabaceae 

9% 

Lamaceae 

8% 

Asteraceae 

7% 

Solanaceae 

6% 

Aplaceae 

2% 

Euphorbiaceae 

12% 

Rutaceae 

6% 

Boraginaceae 

2% Meliaceae 

2% 

Asclepiadaceae 

5% 

Menispermaceae 

2% 

Mimosaceae 

2% 

Myrataceae 

2% 

Pandanaceae 

2% 

Piperaceae 

2% 

Combretaceae 

3% 

Convolvulaceae 

3% 

Malvaceae 

3% 

Zingiberaceae 

3% 

Sapindaceae 

3% 

Verbenaceae 

3% 

Amaranthaceae 

4% 

Caesalpiniacea 

4% 

Acanthaceae 

4% 

Figure 1: Percent representation of major plant families used by Tamil Nadu tribes as medicine. 

Branches Heartwood 

0% 0% 

Nuts 

0% 

Resin Fruits 

0% 1% 

Flowers 

1% 

Rhizome 

2% 

Tuber 

2% Latex 

3% 

Seed 

5% 

Leaves 

40% 

Stem 

7% 

Fruit 

8% 

Whole Plant 

8% 

Root 

11% 

Bark 

11% 

Figure 2: Parts of the Plant used in traditional healing among the tribes of Tamil Nadu. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 18




Table 1: Ethnomedical traditional knowledge of the tribes in Tamil Nadu 

No. Botanical Name Local name Part Used Medicinal uses Tribe Reference 

Acanthaceae 

1 Adhatoda zeylanica Medicus Adathodai Leaves Asthma, Cold, fever PL e 

Adhatoda zeylanica Medicus Auduthoda Root, bark, Cough, asthma ,eye pain IR g 

flowers 

2 Andrographis lineata Wallich ex Siriyanangai Leaves Cough, diabetes, scorpion and PL e 

Nees 

snake bite 

3 Andrographis paniculata (Burm.f.) Periyanangai or Leaves Scorpion sting 

PL e 

Wall. ex Nees 

Nilavembu 

and snakebites, menorrhagia 

4 Asystasia gangetica Valukai keerai Leaves Appetite PL e 

5 Blepharis maderaspatensis (L.) Roth. Vettukaaya pachilai Leaves Fractured bones, Cuts PL e 

6 Phlebophyllus kunthianum Nees Kurinji chedi Leaves Nervous disorder PL e 

Alangiaceae 

7 Alangium salvifolium (L.f.) Wangerin Alinji Fruit Eye infections PL e 

Amaranthaceae 

8 Achyaranthes aspera L. Uthrunk Leaves Cuts, wounds and sores KO b 

Achyranthes aspea L. Cherukadalai Whole Plant Sprain ached in the Joints KT b 

Achyranthes aspera L Nayuruvi Leaves Diverticulosis & Diverticulitis KU i 

Achyranthes aspera L. Nayurvi Geeda Whole Plant Ease child birth and labour KU b 

pain 

Achyranthes aspera L. leaves New born babies, lactation IR g 

9 Achyranthes bidentata Blume, Kithoop Leaves Rapid healing of wounds KU i 

10 Achyranthus bidentata Blume Naiyur Leaves Skin disorders including scabies 

KO a 

11 Aerva lanata (L.) Juss.ex Schult Kannupila & Pannaipoo 

leaves New born babies, lactation IR g 

Celosia argentea L. 

12 Alternanthera sessilis (L.) Nilakirai Leaves Diarrhoea KU i 

Alternanthera sessilis (L.) Nilakirai Leaves Roughage KU i 

13 Amaranthus gangeticus L. Mulai keerai Whole plant Good digestion,Constipation, KU i 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 19




Table 1: Continued… 

Anacardiaceae 

14 Anacardium occidentale Bark, Fruit Fever, warts 

IR, c 

latex 

PA 

15 Mangifera indica Maavae pattae Bark Stomach pain IR, c 

PA 

16 Spondias pinnata Kattu Maa Bark Acute diarrhoea IR, c 

PA 

Annonaceae 

17 Annona squanmosa L. Seetha mara Seeds Vermifuge PA b 

Apiaceae 

18 Centella asiatica (L) Urban Vallarai Whole Plant Refrigerant TH b 

Centella asiatica (L) Urban Gottala Whole Plant Toothache KT b 

Centella asiatica (Linn.) Urban. Kidth Kot Leaves Stomach problems Cools the KO a 

body 

19 Coriandrum sativum Linn. Kothumull Leaves Refrigerant and diuretic KO a 

Aplaceae 

20 Buplerum wightii P.K. Mukherjee, Malai seragam Root Easy delivery IR-S d 

21 Centella asiatica (L) Urban, Kutheraikokku Leaves Digestive agent, blood circulation 

IR-S d 

22 Heracleum ceylanicum Gardner ex C.B. Poonaikal sedi Leaves Insect allergy IR-S d 

Clarke 

Apocymeeae 

23 Rauvolfia serpentine (L.) Benth. ex Kurz Chivanamelpodi Root Stomachache KT f 

24 Alstonia scholaris (L.) R.Br. Paalooram pattai Stem Lactation PL e 

Araceae 

25 Acorus calamus L. Vasambu Rhizome Speech PL e 

26 Colocasia esculenta (L.) Schott Kattu shembu Tuber Worms KT f 

Colocasia esculenta (L.) Schott. Chembu Leaves & 

Rhizome 

Small red colour boils appearing 

on the skin 

KU h 

Arecaceae 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 20





27 Phoenix sylvestris (L.) Roxb. Injai Stem Easy Delivery IR g 

Aristolochiaceae 

28 Aristolochia indica L. Perumanthikodi Root Fever IR g 

29 Aristolochia tagala Cham. Modhalaikodi. Leaves Diarrhea and vomiting IR-S d 

Asclepiadaceae 

30 Ceropegia candelabrum L. Perun kodi Leaves Headache PL e 

31 Cryptolepis buchananii Roem & Paalkodi/Karunkodi Latex Wound PL e 

Schul 

32 Gymnema hirsutum W&A Sakarasedi Leaves Diabetes KU i 

33 Gymnema sylvestre (Retz.) R. Br. Ex Sirukurinjan Leaves Diabetes and nervous disorder PL e 

Gymnema sylvestre R.Br. 

Sirukurinjan & ha- 

Leaves Diabetes IR g 

karikolli 

34 Hemidesmus indicus H.f. Nannari Whole 

Plant, Root, 

leaves 

Fever, menorrhagia, stomachache 

35 Holostemma ada-kodien Schult. Ada kizhangu Tuber Fever KT f 

36 Pergularia daemia (Fors.) Chio Veli parutthi Leaves Headache PL e 

37 Tylophora indica (Burm. f.) Merr. Nangilai Leaves, Snakebite PL e 

Root 

Asparagaceae 

38 Asparagus racemosus Willd. Ammaikodi Tuber Stomachache KT f 

Asteraceae 

39 Adenostemma lavenia (L.) Kuntze Kasirukai Leaves Skin diseases IR-S d 

40 Ageratum conyzoides L. Nasar soppu Leaves Cough and cold KU b 

Ageratum conyzoides Linn. Pugudu thalai Leaves Wound KO a 

41 Artemisia nilagarica (C. B Clarke) 

Pamp. 

Manikoland 

Leaves & 

stem 

Removing worms from 

wounds both in humans and 

animals 

42 Artemisia parviflora Buch – ham. Ex Railpundu Leaves Headache IR-S d 

Roxb. 

43 Bidens pilosa L. Katu kunni Leaves White patches on the legs KU h 

PL 

KU 

e 

i 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 21





44 Blumea lacera (Burm.f.) DC. Navakkarandai Leaves Improving vision KT f 

45 Chromolaena odorata (L.) King & Robins. 

Vettukkaya pacha Leaves Cuts and wounds KT f 

46 Elephantopus scaber L. Anashovadi Root Stomach ache KT f 

47 Siegesbeckia orientalis Linn. Potaz Leaves Skin rashes, insect bites and KO a 

allergies 

Sigesbeckia orientalis L. Nadukadachi Leaves Wounds and parasitic skin KU h 

problems 

48 Sonchus oleraceus L. Kaalaadi pachilai Leaves Wound PL e 

49 Tridax procumbens L. Vettukayapoondu leaves Wounds to stop bleeding IR g 

Tridax procumbens L. Vettukkaya thalai Leaves Sores KT f 

Balanophoraceae 

50 Balanophora fungosa Fors and Fors. Vaer chedi Whole Plant Skin disease PL e 

Berberidaceae 

51 Mahonia leschenaultii (Wight & Mullu kadambu Bark Skin disease PL e 

Arn.) Tak. ex Gamble 

52 Berberis tinctoria Lesch Jakkala Leaves & 

stem 

Bignoniaceae 

53 Radermackera xylocarpa (Roxb.) K. 

Schum. 

Dysentery, Bloating of stomach 

Vadencarni Stem Fever KT f 

Bischofiaceae 

54 Bischofia javanica Blume. Romaviruksha patta Bark Nervous disorder, hair growth PL e 

Boraginaceae 

55 Carmona retusa (Vahl) Masam. Kurangu vetthilai Leaves Fertility PL e 

56 Nasturtium indicum (L.) DC. Kadge Root Portion 

Ear diseases KU i 

57 Trichodesma zeylanica R.Br Jalke maram Root Round patches appearing on KU h 

the skin 

Burseraceae 

58 Boswellia serrata Roxb. Ex Colebr. Kungiliyam Resin Cold PL e 

KU 

i 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 22





Caesalpiniacea 

59 Caesalipinia bonduc (L) Roxb. Porumaielai Root, seeds Gastric disorders Increase 

body weight 

IR-S 

60 Bauhinia racemosa Mara avara Bark Boils IR, 

61 Cassia auriculata L. Avarai Fruit, 

leaves, 

flowers 

62 Cassia fistula Gaggai pattai, Bark 

Konnai mara 

c 

PA 

Skin and scalp IR g 

Sudden “sicknesses” , diarrhoea 

and stomach pain 

Cassia fistula L. Konnei Stem bark Stomach ache KT f 

63 Pterolobium hexapetalum Kari indu Leaves Ease delivery pain PL e 

64 Tamarindus indica Puli Fruit Nursing mothers, eczema IR, c 

PA 

Caparidaceae 

65 Capparis sepiaria L. Thoratti Root Wounds and scratches IR g 

Capparis zeylanica L. Adandai leaves Increase appetite IR g 

66 Celome monophylla L. Kadugu sedi Leaves Earache IR-S d 

Caricaceae 

67 Carica papaya L. Poppilli Poppilli mara Fruit Indigestion and Constipation KU i 

Caryophyllaceae 

68 Drymaria cordata (L.) Roem. & 

Schult. 

IR, 

PA 

Kodi charai Leaves Heel cracks PL e 

Celastraceae 

69 Celastrus paniculatus Willd. Valulurai Root Body pain KT f 

Chenopodiaceae 

70 Chenopodium ambrosioides L. Jaregida Whole plant Intestinal cramps KU h 

Colchicaceae 

71 Gloriosa superba L. Kodanki kizhangu. Tuber Sleeping tablet IR-S d 

Combretaceae 

d 

c 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 23





72 Pterocarpus marsupium Roxb. Vengai Stem Rheumatic pain KT f 

73 Terminalia bellirica (Gaertn.) Roxb. Tani Bark Body pain KT f 

74 Terminalia chebula Retz. Kadukai Fruit Muscular dislocation KT f 

Terminalia chebula Retz. Kadukkai maram Leaves Cold, Cough, Fever, stomach- 

PL e 

ache 

75 Terminalia crenulata Heyne ex Roth Karimathi Bark Internal bleeding KT f 

Compositae 

76 Bidens pilosa L., Katu Katu kunni Leaves White patches on the legs KU i 

Convolvulaceae 

77 Aroyreia hirsute Wight & Arn. Meenidal Leaves Male Child KO a 

78 Ipomoea alba L. Velutha Leaves Skin diseases KU h 

79 Melothria maderaspatana Cogn. Solapushni kai Stem Prolonged cough KU i 

80 Trichosanthes cucumerina L. Peyppadal Fruit Headaches IR g 

Dioscoreaceae 

81 Dioscorea oppositifolia L. var. 

tomentosa. 

Ebenaceae 

82 Diospyros ferrea (Wild.) Bahk. Var. buxifolia 

Nurulai/Valli kilangu 

Rhizome Stomacache PL e 

Veeraii Fruit Bood circulation IR g 

Ericaceae 

83 Gaultheria fragrantissima Wall. Ameerpan Leaves Headache Relieve body 

sprains and pains 

Erthoroxylaceae 

84 Erythroxyium monogynum Roxb. Jeevadalli maram Stem Bark Acute skin disease KU b 

Erythroxylum monogynum Jeevathalimara Bark Scabies IR, c 

PA 

Euphorbiaceae 

85 Acalypha fruticosa Forsskal. Chinni chedi Leaves Dysentery, skin disease PL e 

86 Acalypha indica L. Kuppaimeni leaves Ear pain, snake bite and scabies 

IR g 

KO 

a 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 24





87 Acalypha paniculata Miq. Paruva thazhai Leaves Pimples, stomachache PL e 

88 Breynia rhamnoides, Muell Poolan Root & White patches on the skin all KU h 

Leaves over the body 

89 Euphorbia antiquorum L. Sathura kalli Latex Body pain PL e 

90 Euphorbia hirta L. Ammanpatcharisi Leaves & Pimples KU h 

Latex 

91 Euphorbia rothiana Spreng, Kopot Latex Sores, grow hairs, insect repellant 

KU i 

92 Excoecaria agallocha L. Thillai Latex Antiseptic IR g 

93 Excoecaria crenulata L. Vellai thillai Stem Skin disease PL e 

94 Jatropha curcas L. Kaatu amanku Leaves Headache KT f 

95 Jatropha tanjorensis Ellis & Saroja Katamanukku Latex Antiseptic IR g 

96 Mallotus philippensis Chaneri mara Bark Stomach pain and diarrhoea IR, c 

PA 

97 Phyllanthus amarus Schum. & Thonn. Kila nelli Whole plant Jaundice KT f 

Phyllanthus amarus Schum. & Thonn. Kizhanelli leaves Jaundice IR g 

98 Phyllanthus emblica L. Nelli Fruit Stomachache KT f 

99 Ricinus communis Kottamuthu Bark Quick delivery, sprains, IR, c 

breathing 

problems 

PA 

100 Ricinus communis Linn. Amanaku Leaves, Headache KO a 

Seeds 

101 Securinega virosa (Willd.) Baill. Pula Root Joint pain KT f 

102 Euphorbia rothiana Spreng. Kapsi Leaves Sudden sickness and giddiness KO a 

Fabaceae 

103 Acacia nilotica (L.) Willd. ex. Del. Karuvelam leaves Dysentery, burns or scalds IR g 

104 Albizia lebbeck (L.) Benth. Vagai seeds Lesions of lepers IR g 

105 Caesalpinia bonduc (L.) Roxb. Kazhchikai seeds Hydrocele IR g 

106 Canavalia lineata (Thunb.) DC. Kozhiavarai seeds Several Disorders, General 

health 

IR g 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 25





107 Clitoria ternatea L. Sangu pushpam Infection of eyes, headache, IR g 

snake bites 

108 Dalbergia sissoides Veetimara Stem Acute diarrhoea IR, c 

PA 

109 Flemingia strobilifera (L.) R. Br. ex Ait. Kaduthuvarai Whole plant Mental disorders KT f 

110 Macrotyloma uniflorum (Lam.) verdc Kollu Seeds Abortifacient PA b 

111 Millettia splendens Manalikodi Stem Burns and scalds , acute diarrhoea 

IR, c 

PA 

112 Mucuna pruriens (L.) DC. Poonikali seeds Several Disorders, General IR g 

health 

113 Pongamia pinnata (L.) Pierre Pongan seeds Rheumatic disease IR g 

114 Pterocarpus marsupium Pennae pattae Bark Abortifacient IR, c 

PA 

115 Shuteria vestita W&A Kadu belaga Leaves Boils appearing on the skin KU h 

116 Tephrosia purpurea (L.) Pers. Averi Headaches IR g 

Gentianaceae 

117 Enicostema axillare (Lam.) Raynal Vellarugu Root Toothaches IR g 

Hypoxidaceae 

118 Curculigo orchioides Gaertn. Nelapanai Rhizome Snake bite IR-S d 

Labiatae 

119 Coleus malabaricus Periya tulasi Leaves Asthma KU i 

120 Plectranthus nilghericus Benth. Sone gida Whole Plant Minor wounds KU i 

Lamaceae 

121 Geniosporum tenuiflorum (L.) Merr. Nilathulasi Whole plant Catfish bites IR g 

122 Leucas aspera (Willd.) Link Thumbai Root Tooth brush, resistant to snake IR g 

poison 

123 Anisochilus carnosus (L.f.) Wallich. Saetthupun thazhai Leaves Skin disease PL e 

124 Anisomeles malabarica (L.) R. Br. Ex. Paei miratti Stem Wound PL e 

Sims 

125 Coleus parviflorus Benth Nila Tuber Itching, boils on the skin KU h 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 26





126 Leucas biflora (Vahl.)R.Br. Kaduthumbae Whole plant Skin irritations KU h 

127 Leucas indica (L.) R. Br. ex Vatke Mosappullu Leaves Cough KT f 

128 Ocimum basilicum Var purpurasens Katu thulasi Whole plant Skin inflammations after the KU h 

insect bites 

129 Plectranthus coleoides Benth. Mudupattan or Leaves Cold, delivery pain, hair PL e 

Omavalli chedi 

growth, wounds 

130 Plectranthus malabaricus (Benth.) R.H. Ellamabai Leaves Heart attack IR-S d 

Willemse 

131 Prunella vulgaris Linn. Kadthur Root Refrigerant Haematanic KO a 

132 Aloe vera (L.) Burm.f Sotru Kattrazhai Leaves Hair and skin Diseases KU h 

133 Asparagus racemosus Willd. Thanneer vittan Leaves Heel cracks PL e 

kilangu 

Lobeliaceae 

134 Lobelia heyneana Roem. & Schult. Upperi chedi Leaves, Skin disease PL e 

Flowers 

135 Lobelia leschenaultiana (Presl) Skottsb. Bombari thalai. Whole Plant Sickness in cattle KO a 

Loganiaceae 

136 Strychnos nux-vomica Yetti Bark Acute stomach pain IR, 

PA 

c 

Lythraceae 

137 Lagestroemia microcarpa Wight Tindiyam Bark Burns KT f 

Malvaceae 

138 Hibiscus rosa sinensis L. Chembarathi Flowers Strenthening hair KU h 

139 Malvatrum coromandelianum (L) Garke Kalakenikai Root Stomach pain IR-S d 

140 Sida acuta Burm f. Pilla valatthi chedi. Leaves Dandruffs , strengthening hair PL e 

141 Sida rhombifolia L. Kal gadale Leaves Wounds KU i 

Sida rhombifolia L. Chitra mutti Root Rheumatic pain KT f 

142 Side cordifolia L. Arathae Leaves Snakebite PA b 

Meliaceae 

143 Azadirachta indica Veppamaram Stem Toothache, post-natal complications 

IR, 

PA 

c 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 27





144 Azadirachta indica A. Juss. Veppam leaves Stomach worms IR g 

145 Cipadessa baccifera (Roth.) Miq. Pulipancheddi Leaves Unconsciousness and anaemia KT f 

Cipadessa baccifera (Roth.) Miq. Pulippan chedi Leaves Diarrhoea PL e 

Cipadessa baccifera (Roth.) Miq. Marundha soppu Leaves Rheumatism IR-S d 

Menispermaceae 

146 Cissampelos pareira L. var. hirsuta Urikkakodi Tuber Snakebite KT f 

(Ham. ex DC.) Forman 

147 Cissampelos pureira, L. Koodibatale Leaves Headache, fever, burning sensation 

KU i 

in chest 

148 Cyclea peltata (Lam.) Hook. f. & Thomson 

Para Tuber Body pain KT f 

Cyclea peltata (Lam.) Hook.f. Sethari Kodi Leaves Cough, cold and body pain IR-S d 

149 Tinospora cordifolia (Wild) Miers ex Amrithavalli Leaves White rashes appearing on the KU h 

Hook.F. & Thoms 

body 

Mimosaceae 

150 Acacia caesia (L.) Willd. Nanjupattai Bark Wound PL e 

Acacia caesia (L.) Willd. Kari Indu Stem bark Body pain KT f 

151 Acacia leucophloea (Roxb.) Willd. Sarayapattai maram Bark Cuts PL e 

152 Mimosa pudica L. (Mimosaceae) Thotalvadi Whole plant Body pain KT f 

Moraceae 

153 Ficus infectoria Roxb Selakai Fruit Food KU i 

154 Ficus racemosa L Athikai Fruit Eye sight KU i 

Moringaceae 

155 Moringa concanensis Nimmo Kattu murukka Bark Abortifacient IR, 

PA 

156 Moringa oleifera Murunga Bark Dog or scorpion bite IR, 

PA 

Musaceae 

157 Musa paradisiaca Vazhai Stem Acute diarrhoea IR, 

PA 

c 

c 

c 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 28





Myrataceae 

158 Eucalyptus polybractea R. T Baker Karpura mara Leaves & 

bark 

Round patches between fingers 

159 Psidium guajava L. Koyyapazham Fruit Gastric troubles and ulcers in 

stomach 

Psidium guajava L. Koyyapazham Fruit & Anti-dysenteric and Antidiar- 

160 Syzygium cumini (L.) Skeels Naval Pazham, 

Leaves 

Stem Bark, 

Fruit & 

Seeds 

rhoeal 

Sore throat, dysentery, ulcers, 

purifying blood, antidiarrhoeal 

and anti diabetic 

Syzygium cumini (L.) Skeels Naval bark Diarrhoea IR g 

Nyctaginaceae 

161 Mirabilis jalapa L. Thottanembi Root , 

Leaves 

Cuts and wounds KO b 

Orchidaceae 

162 Cymbidium aloifolium (L.) Sw. Ottai Root Ear pain IR g 

163 Malaxis densiflora (A. Rich.) Kuntze Kuntze, Nelnethch Leaves Wounds KU i 

Oxalidaceae 

164 Oxalis corniculata L. Pulichen segae Whole Plant Febrifuge PA b 

Oxalis corniculata L. Puliyankeerai Leaves Vomiting and headache IR-S d 

Oxalis corniculata Linn. Pulch Leaves Anti-emetic Restorative tonic 

after child birth 

KO 

Pandanaceae 

165 Pandanus odoratissimus Kaithae Stem Fracture IR, 

PA 

166 Passiflora calcarata Mast Potul Leaves Skin diseases KU h 

167 Passiflora foetida L. Narati chedi Whole Plant Arthritic problems KU b 

Periplocaceae 

168 Hemidesmus indicus (L.) R. Br. Nannari Root Mouth ulcers KT f 

Piperaceae 

169 Piper betle L. Thabulam Whole plant Cuts and wounds KT f 

KU 

PA 

KU 

KU 

h 

b 

i 

i 

a 

c 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 29





170 Piper brachystachyum Wall., Kadu kurumulaku Fruit Cough KU i 

171 Piper nigrum L. Milagu Seeds Throat infection PL e 

Plantaginaceae 

172 Plantago erosa Wall. Kalthal Leaves Wounds as a antiseptic KO a 

173 Plantago lanceolata L. Neela kare Leaves Boils on the legs KU h 

Plumbaginaceae 

174 Plumbago zeylanica L. Chitthira moolam Root Stomachache PL e 

Plumbago zeylanica L. Cithiramalliver. Root Insect bite IR-S d 

Poaceae 

175 Cymbopogon citratus L. Karppura pul Root Pimples KU h 

176 Cynodon dactylon (L.) Pers. Arugampul Branches Body coolant IR g 

Polygonaceae 

177 Rumex nepalensis Spreng. Gundott or Sukkutu Root Refrigerant and laxative KO a 

Rumex nepalensis spreng. 

Keerai 

Kekal Ott, Gund 

Ott 

Root Jaundice KO b 

Proaceae 

178 Cynodon dactylon (Linn.) Pers. Nagirki Leaves Relief from sudden sickness KO a 

Ranunculaceae 

179 Clematis gauriana Roxb. Meenae Leaves & 

stem 

Wounds & skin Diseases KU h 

Clematis gouriana Roxb. Ex. DC. Attumeesai chedi Leaves Skin disease PL e 

Rhamnaceae 

180 Ziziphus mauritiana Yelluchi maram Bark Gastric disturbance IR, 

PA 

c 

Ziziphus mauritiana Lam. Elanthai Bark Old wounds IR g 

Ziziphus mauritiana Lam. Ilantha Whole Plant Mouth freshener KT f 

Rubiaceae 

181 Catunaregam spinosa (Thunb.) 

Tirvengadum 

Madukarei Root Ulcers KT f 

182 Rubia cordifolia L. Sappli Koth Stem Restorative, jaundice KO b 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 30





Rubia cordifolia L. Kalutharupan chedi Leaves Heel cracks PL e 

Rubia cordifolia L. Periya nangai. Leaves Cough, cold and nervous disorders 

IR-S d 

Rubia cordifolia Linn. Maral Leaves Injuries caused by fire KU i 

Rubia cordifolla L. Muthang Root Dysmenorrhoea KT b 

Rutaceae 

183 Citrus aurantium L. Eravae kai Fruit Digestion, Hemorrhoids KU i 

184 Clausena dentata (Willd.) Roem. Anai thazhai Leaves Wound PL e 

185 Glycosmis mauritiana (Lam.) Yaich. Panasedi Leaves Headache IR-S d 

186 Glycosmis pentaphylla (retz.) DC. Eruputtal Whole Plant Stomach ache and abdominal KT b 

discomfort 

187 Murraya koenigii L. Karivepilla Leaves Skin inflammations KU h 

188 Murraya paniculata Chedichi Bark Toothache IR, c 

PA 

189 Naringi crenulata (Roxb) Nicolson Naivalampattai Bark General health IR-S d 

190 Ruta chalepensi L. Aruvatha Geeda Leaves Infant convulsions KO b 

191 Ruta graveolens L. Aruvadam Leaves Skin diseases KU h 

Ruta graveolens L. Arubathansedi Leaves Diarrhea, stomach pain and IR-S d 

vomiting 

192 Toddalia asiatica (L.) Lam. Surai leaves, Fuit Fever, headache IR g 

Toddalia asiatica (L.) Lam. Kindu mullu Leaves, Stomachache, toothache PL e 

stem, Root 

bark 

Toddalia asiatica (Linn). Lam. Vaseri Leaves , Vermifuge KO a 

Seeds 

Salvadoraceae 

193 Azima tetracantha Lam. Sankan leaves Fever IR g 

194 Salvadora persica L. Vagai Fruit, Root Rheumatic pains, tooth brush IR g 

Santalaceae 

195 Santalum album Santhana mara Heartwood Refrigerant, skin diseases IR, 

PA 

c 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 31





Santalum album L. Sanddanamara Seed Skin troubles, refrigerant KU i 

196 Thesium wightianum Wall. ex Wight Anaikchi Whole Plant Cheek to prevent bulging IR-S d 

Sapindaceae 

197 Cardiospermum halicacabum L. Mudakkathan whole plant Rheumatoid arthritis IR g 

Cardiospermum halicacabum L. Poovanthi Nuts Body wash IR g 

198 Dodonaea angustifolia L.f. Marundha soppu Leaves Rheumatism IR-S d 

199 Dodonaea viscose (Linn.) Jacq. Vilari thalai Leaves Wounds and injuries, joint KO a 

sprains and bone fracture 

200 Dodonea viscosa Linn. Manantha Leaves Fracture KU i 

201 Schleichera oleosa Jagada mara Bark Abortifacient IR, c 

PA 

Sapotaceae 

202 Manilkara hexandra (Roxb.) Dubard Pala maram Latex Toothaches IR g 

203 Tinospora cordifolia (Willd.) Miers Seenthil stem Many Disorders IR g 

Simaroubaceae 

204 Ailanthus excelsa Roxb. Pekalathi Bark, leaves After child birth IR g 

Solanaceae 

205 Datura stramnium L. Umbathi Leaves Inflamed wound and sores KO b 

Datura stramonium L. Yemmuth Leaves & Piles KU i 

Fruit 

206 Physalis peruviana L. Urechithuvar Leaves Wound KU i 

207 Solanum anguivi Lam. Kandan kathiri Fruit, leaves Colds, coughs 

IR g 

and fever, intestinal worms 

208 Solanum denticulatum Periya midinje Whole Plant Migraine KU i 

209 Solanum erianthum D.Don Malai sundai Fruit Toothache PL e 

210 Solanum indicum Linn. Sunda maram Root & Toothache and snakebite KU i 

Leaves 

211 Solanum nigrum L. Mana thakkali Leaves Ulcer, wound PL e 

Solanum nigrum Linn. Ikki sop Leaves Stomach disorders Skin rashes KO a 

212 Solanum sisymbrifolium Limk. Vadadana Seeds Vermifuge KO a 

213 Solanum surattrense Burm. F Kandankathiri Fruit Toothache PL e 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 32





214 Solanum trilobatum L. Thoodhuvalai Leaves Asthma, Cold PL e 

Tamaricaceae 

215 Tamarix indica willd Kattuchaukku leaves Laxative IR g 

Thunberigiacea 

216 Thunbergia fragrans Roxb. Kakka Valli Root Snake-bite KT b 

Tiliaceae 

217 Grewia aspera Roxb Dadchi maram Bark Diarrhoea KU i 

218 Grewia tiliifolia Vahl. Unu Root bark Swellings KT f 

Ulmaceae 

219 Holoptelea integrifolia Vellaya Bark Swellings IR, c 

PA 

Umbelliferae 

220 Centella asiatica (L.) Urban. Vallarai Leaves Jaundice PL e 

Verbenaceae 

221 Gmelina arborea Roxb Perungilai/ 

Root bark Piles PL e 

Kumilamaram 

222 Gmelina asiatica L. Kumalai Nochi Fruit Bathing IR g 

223 Lantana camara L. Unni chedi Whole plant Cuts and wounds KT f 

Lantana camara L. Thusik Leaves Gum bleeding and tooth-ache KO b 

Lantana camera L. Parale gida Flowers Skin inflammations KU h 

224 Tectona grandis Thekku Bark Constipation IR, c 

PA 

Tectona grandis F. Thekku Bark Ease child birth and labour KU b 

pain 

Tectona grandis L. Thekku Bark Ease child birth and labour PA b 

pain 

Tectona grandis L.f. Thekku Stem Stomach ache and dysentery KT f 

225 Vitex negundo L. Nochhi Leaves Rejunvating skin KU h 

Vitex negundo L. Kumalai Nochi leaves Repel mosquitoes, body pain IR g 

Vitex negundo L. Notchi Fruit Cold, Cough, Fever, headache PL e 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 33





Zingiberaceae 

226 Alpinia calcarata Rosc. Arathi poo Rhizome Immunity PL e 

227 Costus speciosus (J. Koen.) Smith. Koshtam Leaves Diabetes PL e 

228 Curcuma longa L. Manjal Rhizome Itching, glowing skin KU h 

Curcuma longa L. Manjal Rhizome Scorpion bite KT f 

229 Elatteria cardamomum (L.) Maton. Yelakkai Fruit Stomachache PL e 

KU = Kurumba, IR = Irula, KT = Kota, TH = Thoda, PA = Paniya, PL = Paliyar, KT = Kattunayakas. a, (Rajan and Sethuraman, 1991); b, (Rajan 

et al., 1997); c, (Rajan et al., 2001); d, (Murugesan et al., 2005); e, (Ignacimuthu et al., 2006); f, (Udayan et al., 2007); g, (Ragupathy 

and Newmaster, 2009); h, (Deepak et al., 2014a); I, (Deepak et al., 2014b) 

Thus, the medicinal uses of the tribal traditional medicines depict 

their medical history through the ages. They have tried and found 

cure to their major health problems such as wounds and skin problems. 

This data also throws light on a cultural aspect of their life 

style, mainly that they held their women in high esteem for they 

have painstakingly developed remedies form medicinal plants to alleviate 

labour pains and post-partum medical care. 

6. Towards sustainable local production of traditional medicines 

Sustainable development denotes a development that meets the 

needs of the present without compromising the ability of future generations 

to meet their own needs. It encompasses two key concepts, 

the concept of needs, in particular meeting the essential needs of the 

poor and the idea of limitations that is the environment‟s ability to 

meet these needs. Thus sustainable development seeks to relieve 

poverty, create equitable standards of living, satisfy the basic needs 

of all peoples, and establish sustainable political practices, while ensuring 

that there are no irreversible damages to natural resources and 

nature. 

The tropical countries are gifted with vast resources of medicinal 

plants and the recent global renaissance in traditional medicines has 

created a large market for herbal products that can be exploited by 

these countries they meet up to quality and safety specifications. 

Population explosion, incidence of side effects of synthetic medicines 

and our inability to provide modern medicines to a vast section 

of the population living in rural and remote areas of the country due 

to non-availability, in accessibility and unaffordability have been the 

prime reasons for the growing popularity of alternative medicines 

amongst rural and remote population, and neo-rich people in the developed 

countries. Access to quality health care is an enormous public 

health global issue at the scientific, clinical, economic, political 

and policy levels. It is one aspect of the "great divide" that exists 

between and within every country in the world; it is the difference in 

access to health care between the rich and the poor. The very basis 

for promoting the local production of herbal remedies is to provide 

cost effective medicines to populations who cannot afford costly 

medicines. The World Health Organization (WHO) has been repeatedly 

stressing that the goal of „health for all‟ cannot be accomplished 

without herbal medicines. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 34




Nervous disorder 

Hair 

Burns 

Antiseptic 

Lactation 

Heel cracks 

Fracture 

Eye infections 

Boils 

Asthma 

Vermifuge 

Ear pain 

Abortifacient 

Snake bite 

Jaundice 

Diabetes 

Body pain 

Refrigerant 

Fever 

Toothache 

Gastric disorders 

Labour pain 

Rheumatic disease 

Cold, Cough, Fever 

Headache 

Diahorrea 

Stomacache 

Skin Problems 

Wound 

0 5 10 15 20 25 30 35 

Figure 3: Traditional Medicine of the Tribes of Tamil Nadu in the treatment of common ailments. 

7. Strategies for promoting sustainable 

production of traditional medicines 

The first step in promoting sustainable 

production of traditional medicine is developing 

a standardized mode of production 

which will meet the standards of quality, 

efficacy and safety as defined in the 

WHO guidelines. Thus bringing these 

plants into large scale cultivation ensures 

that endangered plant are protected by cultivation 

and by involving the indigenous 

people over-exploitation will be avoided 

while adding to income for the indigenous 

tribes. Majority of the plants are still gathered 

and collected from the wild and relatively 

few are cultivated in farmlands. 

World Wildlife fund report (2004) reported 

that 20% of the medical plants worldwide 

are in the treat of disappearing (Pan 

et al., 2013). A paradigm shift from wild 

collection to structured cultivation of medicinally 

important plants will further ensure 

the purity, authenticity and sustainable 

supply of raw drugs. Suitable infrastructure 

like production equipment, potable 

water, storage facilities and postharvest 

quality monitoring and marketing 

are very critical. Trained man power with 

an appropriate background in pharmaceu- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 35



tical sciences in bioprocess technology is 

an integral part of the quality of the finished 

product (Cordell and Colvard, 2012). 

The concept of sustainability directs us to 

shift form non-renewable chemical drug 

discovery programs to natural renewable 

sources (Cordell, 2011). Sustainable production 

of traditional medicine will require 

an integrated approach encompassing the 

different disciples of science like ethnobatany, 

chemistry, biomedicine, mathematics 

and physics. Traditional medicine has 

still not come to the forefront because of 

the following challenges faced by the 

pharmaceutical companies in providing the 

capital investment in developing and marketing 

them namely, collection of plants is 

a time consuming process and requiring 

extensive negotiations related to access, 

insufficient documentation and lack of 

ethno medical knowledge, lack of institutional 

and financial support, limited availability 

of trained man power, low yield, 

long discovery process and expensive synthesis, 

lack of scientific validation of the 

quality, safety and efficacy of traditional 

formulations, lack of appropriate technology 

for post-harvest and pre-processing 

purposes, low market value for traditional 

medicinal plants, lack of methodologies 

for the preservation of medicinal extracts 

for extended shelf life. 

8. Biotechnology: challenges and prospects 

for the sustainable use of traditional 

medicine 

Biotechnology with its robust tools 

and state of art technology will play a crucial 

role in the sustainable use of traditional 

medicinal drugs in future. Although the 

use of transgenic plants is a debatable for 

the preservation of biodiversity, genetic 

engineering will play an important role in 

saving medicinal plants, which are rare or 

endangered (Cordell, 2011). Ethnomedical 

information about biological evaluation of 

plant extracts and their constituents, the 

chemistry of natural sources, and the clinical 

evaluation of plant extracts are still not 


accessible globally. Biotechnological aspect 

of herbal remedies involves extraction 

of plant‟s active ingredients and formulation 

into final product. First, it must be 

established unequivocally that the source 

of the plant material is authentic. A plant 

extract usually contains hundreds of active 

ingredients and in some cases the bioactive 

compound is usually not known. Bioactivity 

guided processes are then used to separate 

the active compound. This is further 

tested on in-vitro or in-vivo models for 

their functional efficacy. There are many 

disease conditions for which such biological 

models are not easily available, or if 

even available, would be beyond the 

means of many researchers. Therefore a 

more practical and cost effective way is to 

use total extracts of traditional medicinal 

plants for which abundant ethnomedical 

evidence exits. The mode of formulation 

can then be based on the traditional methods 

used to prepare that particular remedy, 

taking steps to establish safety through in 

vivo and in vitro studies followed by appropriate 

pilot clinical trials for cytotoxic, 

mutagenic and therapeutic perspectives. 

The formulation should be free of potentially 

toxic insecticides, pesticides and 

heavy metals. The formulation must be 

evaluated for microbial contamination both 

fungal and bacterial, and radiation contamination 

during the stages of processing of 

the material (Tan et al., 2004). Quality 

control of the product is then done by conducting 

accelerated stability tests as well 

as on-shelf stability tests. 

The pharmacological approach in 

developing a drug includes bringing as 

much chemical diversity as possible to the 

biological screening interface but with no 

consideration given to the origin of the 

plant derived materials, chemo-diversity, 

functional diversity of the constituents, 

ethno-medical association of the plant or 

known or novel active constituent of the 

plant extracts (Tan et al., 2004). The other 

challenge is that the active constituent is 

often extracted and analyzed only at a single 

point in time, ignoring daily metabolic 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 36



flux, seasonal variation in enzyme activities, 

and the biosynthetic genes which are 

present, but not fully functional. Methodologies 

that are able to characterise the 

majority of the constituents without individual 

isolation of active constituents need 

to be developed to help validation and 

standardization. India has a vast and diverse 

wealth of traditional medicinal 

knowledge that can be used for bioprospecting 

to benefit both the country and 

the indigenous people. 

9. Bioprospecting of traditional medicine 

to combat disease 

The search for new drugs is the 

vast opportunity in the ambit of Biotechnology 

given the vast majority of diseases 

encountered today and improved health 

care services. 


There are three main types of 

search strategies: biorational, chemorational 

and random approaches. An anti- 

HIV bioactive compound Conocurvone 

was discovered as a result of random approach 

of screening strategies. Drugs discovered 

using bio-rational approaches 

were artemisinin, morphine, quinine, and 

ephedrine. Bio-rational approach is mostly 

guided by the ethnomedical information 

generated from the traditional medicines 

and the most effective approach to date. 

These medicinal plants contain reservoir of 

etho-medical and ethno-botanical traditional 

knowledge, which is an important 

guide to discovery of many new drug lead 

molecules (Table 2). As there are many 

existing and emerging diseases that cannot 

be treated by the current plethora of drugs 

and the additional burden of increasing 

Table 2: Bioactive compounds from medicinal plants and their clinical uses 

SN Bioactive compound Species Clinical Uses 

1 Mevastatin & lovastatin Penicillin spp. 

Cholesterol 

lowering 

2 Ivermectins Streptomycetes spp. 

Anthelmintic and antiparasitic 

3 Reserpine Rauwolfia serpentine Antihypertensive 

4 Ephedrine Ephedra sinca Antiasthma 

5 Atropine Belladonna Anticholinergic 

6 Teprotide Bothrops jaracaca Cardiovascular diseases 

7 

Vincristine and 

vinblastine 

Catharanthus roseus 

Anti-cancer drug 

8 Paclitaxel Taxus brevifolia Anti-cancer drug 

9 Camptothecian Camtotheca acuminate Anti-cancer drug 

10 Podophylotoxin Podophylum peltatum Skin Cancer 

11 Bryostatin-1 Bugula neritina 

12 Cyclosporins and rapamycin Penicillium notatum 

Ovarian 

carcinoma and non- 

Hodgkin‟s‟ lymphoma 

Antimicrobial and antiplasmodial 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 37



drug resistant pathogens, the need for the 

hour is the development of new arsenal of 

drugs to combat them based on the traditional 

knowledge. 

10. Biotechnological tools to be exploited 

for sustainable production of traditional 

medicines 

Biotechnology produces a vast array of 

tools for the successful discovery and validation 

of traditional medical drugs. These 

tools have been developed in the 80‟s and 

90‟s and today there is vast improvement 

in their technological aspects after subsequent 

modification of the initial technology. 

Each technique has its own pros and 

cons and care needs to exercise in the employment 

of a suitable technology to provide 

an appropriate traditional medicine 

based drug. Techniques like in vitro regeneration 

through mciropropagation, callus 

mediated organogenesis, somatic embryogenesis, 

cryopreservation, production of 

secondary metabolites and genetic transformation 

holds a tremendous potential for 

the production of high quality plant based 

medicines mainly because of the multiplication 

rate, pathogen free material, plant 

preservation and regeneration success to 

yield bioactive compound that ensures reduced 

costs compared to the natural synthesis 

by the plants. 

11. Intellectual property rights (IPR) for 

sustainable use of traditional medicine 

It is very vital to consider intellectual 

property rights of the local people 

when the ethnomedical documentation is 

done for traditional medical knowledge. 

The economic implication of the eventual 

commercial production of standardized 

traditional medicinal drug should include 

the welfare of the people from whom the 

traditional knowledge was documented. 

Biopiracy becomes an ever looming issue 

when large pharmaceutical companies confiscate 

medicinal plants to make new 


drugs, the costs escalate and these drugs 

will not be available to the local people 

because of the costs which is also a critical 

an ethical issue (Pan et al., 2013). 

11.1 The Kani model of benefit sharing of 

traditional medicine 

The Kanis inhabit the forests of the 

Thiruvananthapuram district of Kerala and 

Thirunelveli district of Tamil Nadu in 

southwestern India. In 1987, scientists 

from Tropical Botanic Garden and Research 

Institute (TBGRI) while collecting 

ethnomedical data form Kani tribal people 

discovered that the tender fruits of Arogyappacha 

(Trichopus zeylanicus subsp. 

travancoricus) have anti-ageing, antidepressant 

and anti-fatigue property. This 

paved a way for the scientists at TBGRI to 

develop a scientifically validated and 

standardised herbal drug called Jeevani, a 

formulation consisting of four ingredients 

and Arogyappacha was one of the constituents. 

Jeevani has been found to be therapeutically 

effective having anti-fatigue and 

immuno-enhancing properties and it has 

also shown good hepato-protective and 

anti-stress properties. Subsequently, 

TBGRI decided to share 50% of the licence 

fee and royalty with the Kani people 

to encourage an equitable sharing of the 

benefits arising from the utilisation of such 

knowledge, innovations and practices as 

stated in the mandate of Article 8(j) of the 

Convention on Biological Diversity 

(CBD). This is considered to be one of the 

first models for benefit sharing in the 

world, which is popularly known as the 

TBGRI Model for Benefit Sharing. 

12. Conclusion 

Sustainable local production of traditional 

medicines requires an enabling 

environment and effective partnerships 

between traditional health practitioners, 

researchers, public and the private sector. 

There is a strong need for indexing eco and 

ethno information of medicinal plants, sustainable 

cultivation of the traditional medi- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 38



cal plants, protecting the intellectual property 

rights of the indigenous people and 

using the various tools of Biotechnology in 

formulating a superior product that is cost 

effect to the very same indigenous people. 

Recent innovations in Biotechnology have 

impacted the use of molecular tools for 

sustainable use of a renewable natural resource 

for the betterment of India and its 

indigenous communities. Modern tools of 

Genomics can be applied to traditional 

medicine without the need for transgenics. 

Protection of intellectual property rights of 

the traditional medicinal knowledge of indigenous 

people in the form of benefit 

sharing and bioprospecting the vast biodiversity 

available in the country is the need 

of the hour to march India in the field of 

bio pharmaceuticals as a global leader. It is 

now is the hands of young researchers to 

work under well placed regulatory framework 

in converting traditional medicinal 

knowledge into a safe and novel bio drug 

for the treatment of existing and emerging 

disorders. 

Acknowledgements 

The authors gratefully 

acknowledge the DST NRDMS (2016 to 

2018) project to Ramani Bai, R. We are 

also thankful for the UGC MRP (2015- 

2018) project to Kavitha, V.J. We are 

grateful to all the tribes of Tamil Nadu for 

their vast knowledge in Traditional Medicine. 

References 


Cann, R.L. (2001). Genetic clues to dispersal 

of human populations: Retracing 

the past from the present. Science, 

291, 1742-1748. 

Cordell, G. A. and Colvard, M. D. 

(2012). Natural products and traditional 

medicine: turning on a paradigm. 

Journal of Natural Products, 3 

(75), 514-525. 

Cordell, G.A. and Colvard, M.D. (2005). 

Some thoughts on the future of ethnopharmacology. 

Journal of Ethnopharmacology, 

1-2, 5-14. 

Cordell, G.A. (2011). Sustainable medicines 

and global health care. Planta 

Medica., 11 (77), 1129-1138. 

Deepak, P. and Gopal, G.V. (2014). Nilgiris: 

A Medicinal Reservoir. The 

Pharma Innovation Journal, 3(8), 

73-79. 

Deepak, P and Gopal, G.V. (2014). Ethnomedicinal 

practices of Kurumba 

tribes of Nilgiri District, Tamil Nadu, 

India, in treating skin diseases. 

Global J Res. Med. Plants & Indigen. 

Med., 3 (1), 8-16. 

Fabricant, D.S. and Farnsworth, N.R. 

(2001). The value of plants used in 

traditional medicine for drug discovery. 

Environmental Health Perspectives, 

1 (109), 69-75. 

Gupta, A. Vats, S. K. Lal, B. (1998). 

Curr Sci. 75, 565. 

Hamilton, A. C. (2004).Biodivers Conserv., 

13, 1477. 

Ignacimuthu, S. Ayyanar, M. Sivaraman, 

S.K. (2006). Ethnobotanical 

investigations among tribes in Madurai 

District of Tamil Nadu (India).Journal 

of Ethnobiology and 

Ethnomedicine, 2, 25. 

Kavitha, V.J. (2008). Studies on the genomic 

diversity of Southern Indian 

breeding isolates.Ph.D Thesis.Madurai 

Kamaraj University, 

Tamil Nadu, India. 

Misra, V.N. (2001). Prehistoric colonisation 

of India.J Biosci, 26,491-531. 

Murugesan, M. Balasubramaniam, V. 

and Arthi, H. (2005).Ethno Medical 

Knowledge of Plants Used By Irula 

Tribes, Chengal Combai, The Nilgiris, 

Tamilnadu.Ancient Science of 

Life, 24(4),179-182. 

Pan, S.Y. Zhou, S.F. Gao, S.H. et al., 

(2013). New Perspectives on How to 

Discover Drugs from Herbal Medicines: 

CAM's Outstanding Contribution 

to Modern Therapeutics. Evidence-Based 

Complementary and Alternative 

Medicine. URL: 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 39



http://dx.doi.org/10.1155/2013/6273 

75. 

Pushpangadan, P. and George, V. 

(2010). Ethnomedical practices of rural 

and tribal populations of India 

with special reference to mother and 

child care. Indian journal of Traditional 

Knowledge, 9 (1), 9-17. 

Ragupathy, S. and Newmaster, S.G. 

(2009). Valorizing the 'Irulas'traditional 

knowledge of medicinal plants 

in the Kodiakkarai Reserve Forest, 

India.Journal of Ethnobiology and 

Ethnomedicine, 5,10. 

Rajan, S. and Sethuraman, M. (1991). 

Plants Used In Folk Medicine by the 

Kotas of Nilgiri District, Tamil Nadu. 

Ancient Science of Life, 4, 223- 

230. 

Rajan, S. and Sethuraman, M. (1993). 

In-digenous folk practices Among 

Nilgiri Irulas. International J. Indigenous 

knowledge and development 

monitor (Netherland), 1(3), 19-20. 

Rajan, S. Baburaj, D.S. Sethuraman, M. 

Parimala, S. (2001). Stem and stem 

bark used medicinally by the Tribals 

Irulas and Paniyas of Nilgiri District, 


Tamil Nadu. Journal of Natural 

Remedies, 1, (49-54). 

Sethuraman, M. and Suresh, D.B. 

(1997).Plants from the Traditional 

Medical System of the Nilgiri 

Tribes.Ancient Science of Life, 26(4), 

1742-1748. 

Tan, B.H. Bay, B.H. Zhu, Y.Z. (2004). 

Plants in Drug Discovery – Creating 

a New Vision‟. In: Novel Compounds 

from Natural Products in the 

New Millennium. Cordell G. A. 

(eds). World Scientific Publishing, 

Singapore, pp. 1-19. 

Tandon, V. (1996).Med Plant Conserv.Newslett, 

pp 2, 12. 

Thurston, E. (1909). The Castes and 

Tribes of Southern India.7 volumes. 

Madras. 

Udayan, P.S. Tushar, K. V.George. S. 

Balachandran, I. (2007). Ethtnomedicinal 

information from Kattunayakas 

tribes of Mudumalai wildlife 

sanctury, Nilgiris district,Tamilnadu. 

Indian J traditional knowledge, 6 (4), 

574-78. 

Ved, D. K. Anjna, M. Shankar, D. 

(1998). Curr Sci., 75, 341. 

© 2017 by the authors. Licensee, Editors and AIMST University, Malaysia. 




ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 40



Vermitechnology – An Eco-Biological Tool for 

Sustainable Environment 

Mahaly Moorthi 1 , Koilpathu Senthil Kumar Abbiramy 1, *, Arumugam Senthil Kumar 2 

and Karupannan Nagarajan 3 

1 PG and Research Department of Zoology & Wildlife Biology, A.V.C. College (Autonomous), 

Mannampandal - 609305, Mayiladuthurai, Nagapattinam District, Tamilnadu, India; 

2 PG and Research Department of Zoology, Chikkaiah Naicker College, Erode – 638 

004, Tamilnadu, India; 3 PG and Research Department of Zoology, Sri Vasavi College, 

Erode – 638 316, Tamilnadu, India; *Correspondance: moorthideksha@gmail.com / ksabbiramy@gmail.com; 

Tel; +91-8526385977 

Abstract: The word vermi, typically indicates earthworms. Vermitechnology is a simple 

process, which uses earthworms to produce earthworms, good quality compost (vermicompost) 

through organic waste recycling and other products involving earthworms. This technology 

is inevitable in managing biodegradable wastes, biomass or organic material that 

can be degraded or composted thus contributing to the environment indirectly. Solid waste 

management through Vermitechnology contributes more for the sustainability of the environment. 

The major components of Vermitechnology can be considered as Vermiculture 

(mass production of earthworms), Vermicomposting (production of vermicompost) and 

Vermiwash (the extract of vermicompost). In 1996, „Vermitech‟, the vermicomposting 

technique was developed by Mr. A. Thimmaiah at the Indian Agricultural Research Institute 

(IARI), New Delhi, India. As IARI is also known as „Pusa Institute‟, this innovative 

technology was dedicated to the institute and named „Pusa Vermitech‟. „Pusa Vermitech‟ 

was developed to provide a simple solution to poor farmers. This method has now become 

popular in Bhutan, Costa Rica, India, Italy, Nepal and Sri Lanka. It appears that Vermitechnology 

is going to play an important role for the sustainability of agriculture and environment. 

This chapter is highlighting the Vermitechnology, as an eco-biological tool for 

the sustainable environment. 

Keywords: Solid waste management; vermicomposting techniques; vermiculture; vermitechnology; 

vermiwash 


The advent of organic farming has 

made farmers innovative and nature 

friendly. Vermitechnology, an effective 

replacement for chemical input is the 

most sought after due to its costeffectiveness 

and quality of enriching the 

soil. Vermicompost is becoming the principal 

manure for crops in the field of organic 

farming. The market crisis for agricultural 

products has also contributed to 

the popularity of vermicomposting. The 

manure comes in handy especially for 

small-holders who do not have the money 

to buy expensive, chemical fertilizers. 

The manure is used rampantly for all sorts 

of crops. The biggest beneficiaries are 

women who have formed themselves into 

NGO-trained Self-Help Groups (SHGs). 

These women can easily prepare the 

Vermitech products in their backyard and 

sell the excess one left after their own use 

to neighbouring estates or farmers. The 

NGO-run institutes like these are assisted 

by the local bodies like grama pancha- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 41


Vermitechnology – An Eco-Biological Tool for Sustainable… 

Moorthi et al. 

yats, zilla panchayats, which organize 

seminars and teach farmers the methods 

of Vermitechnology. Government agricultural 

departments also buy earthworms in 

bulk from these SHGs to conduct their 

own projects on vermiculture. In fact, the 

Supreme Court‟s ruling criteria is that to 

decide on metro status would be the particular 

district‟s level of participation in 

organic farming. 

But the low sense of awareness 

still remains a problem even within the 

municipalities that have undergone seminars 

on Vermitechnology. Neither the 

Boards nor the local bodies have ideas on 

the perspectives of Vermitechnology. 

Technical expertise alone does not make 

organic farming. Unless there is a uniformity 

of procedure, the advantages of 

organic farming – low cost, more soil fertility 

and eco-friendliness - will not come 

through. 

After all, by preparing Vermitech 

products, the farmer is making the soil 

healthy. In turn, he‟s supplying healthy 

crops into the market. The content of organic 

carbon, the index for the presence 

of humus in the soil, is in high Ranges. So 

the farmers must contribute considerably 

more towards ecology and food production. 

He deserves all the support he can 

get. With the right financial support from 

the Government and a more organized 

network of cultural units, Vermitech 

products, as a form of enrichment can 

generate a steady source of income for the 

impoverished folk of agricultural areas. 

There are numerous sources of 

waste produced in India where degradable 

organic matter is either partially or fully 

generated. Solid waste consists of the discarded 

portion of the households, dead 

animals, trade, commercial, agricultural 

and industrial waste and other large waste 

like debris from construction site, furniture 

etc. Solid wastes are generally categorized 

as domestic, industrial and hazardous 

or biomedical waste. Studies were 

made on some solid wastes like sewage 

sledges and solids from waste water 

(Mitchell et al., 1980); wastes from processed 

potato; wastes from supermarkets 

and restaurants; wastes from poultry, 

pigs, cattles, sheeps, goats, horses (Edwards 

and Bater, 1992) as well as horticultural 

residues from dead plants and 

spent wastes from mushroom industry 

(Edwards,1988). 

The degradable organic matter 

from these wastes when dumped in open 

undergoes either aerobic or anaerobic 

degradation. These un-engineered 

dumpsites permit fine organic matter to 

become mixed with percolating water to 

form leachate. The potential for this 

leachate to pollute adjoining water and 

soil is high. India where a lot of solid organic 

waste is available in different sectors 

with no dearth of manpower, the environmentally 

acceptable Vermitechnology 

using earthworms can very well be 

adopted for converting waste into wealth. 

Considerable work has been carried out 

on vermicomposting of various organic 

materials and it has been established that 

epigeic forms of earth-worms can hasten 

the composting process to a significant 

extent, with production of a better quality 

of composts as compared with those prepared 

through traditional methods. The 

viability of using earthworms as a treatment 

or management technique for numerous 

organic waste streams has been 

investigated by a number of workers 

(Logsdon, 1994; Madan, 1988; Singh, 

2002). Similarly a number of industrial 

wastes have been vermicomposted and 

turned into nutrient rich manure 

(Sundaravadivel, 1995). Hand et al. 

(1988) defined vermicomposting as a low 

cost technology system for the processing 

or treatment of organic wastes. 

A growing awareness of some of 

the adverse economic and environmental 

impacts of agrochemicals in crop production 

has stimulated greater interest in the 

utilization of organic amendments such as 

compost or vermicompost for crop production 

(Follet, 1981). Therefore, the sustainability 

has to be restored by some 

means of regular food security. Utilization 

of earthworms may be an answer as 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 42



an ecologically sound, economically viable 

and socially acceptable technology. 

The present chapter reviews on various 

aspects involved in Vermitechnology and 

thus managing the organic waste leading 

to a sustainable environment. 

2. Vermiculture 


The process of culturing of earthworms 

using scientific methods is known 

as Vermiculture. Earthworms are known 

as biological indicators of soil health. In 

soil earthworms‟ activity and their casting 

support a lot amount of microbial populations. 

Microbial populations such as bacteria, 

fungi, Actinomycetes and protozoans 

grow well, also insects like spiders, 

millipedes, and other nematodes that are 

essential for sustaining the soil fertility 

grow well. Presence of soil biome enriches 

the soil fertility. Thus earthworms 

which form the base for the survival of 

other organisms can be cultured artificially 

and used for many purposes. The ultimate 

goal of this technology is the betterment 

of soil fertility and health of human 

beings. 

In recent years considerable attention 

has been focused upon the potential 

role of intensive earthworm culture, or 

vermiculture. It is now accepted that the 

economic value of vermiculture lies in (i) 

reduction of noxious qualities associated 

with organic wastes, e.g. elimination of 

smell; (ii) generation of a useful compost; 

and (iii) production of earthworm biomass. 

Various Vermiculture systems, 

which have been designed primarily for 

biological waste control, are producing 

earthworms in large quantities. This chapter 

covers the production of the earthworms 

which can be used as a source of 

food, primary proteins, and as drugs. 

Now-a-days, people are very 

much eager in culturing earthworms as a 

part time business, as a source of income. 

The culturing of worms becomes a promising 

business because of its need in 

enormous amount in organic farming, in 

big municipalities for the treatment of solid 

wastes (John Paul et al., 2011), as a 

source of live feed in poultry and aquaculture 

industries. Worms have a number 

of other possible uses on farms, including 

value as a high quality animal feed. The 

earthworms are also used as bait in 

freshwater sport fishing. They are also 

sold to small scale business people who 

maintain garden at home and to nurseries 

that do organic gardening and composting 

and sell saplings. Also there are demands 

for the pure form of vermicastings in various 

places. 

2.1. Methods for Vermiculture 

Vermiculture is the culture of 

earthworms. The goal is to continually 

increase the number of worms in order to 

obtain a sustainable harvest. The worms 

are either used to expand a vermicomposting 

operation or sold to customers 

who use them for the same or other purposes. 

If the goal is to produce vermicompost 

then we want to have maximum 

worm population density all of the 

time. If the goal is to produce worms then 

we keep the population density low 

enough that reproductive rates are optimized. 

Vermiculture as a business must 

be started in a small scale units. After 

learning the technique of culturing, one 

can start his business at large scale. Culturing 

of earthworms needs minimum requirements 

and care on a regular schedule. 

Culturing can be done by two methods. 

i. Container method. 

ii. Tank method. 

The first method consists of culturing 

earthworms in containers made of 

plastic and the medium or their bedding 

(commonly called as vermibeds) can be 

done in it. Containers in the shape of rectangular 

boxes or tubes of one foot width, 

3 feet length and 2 feet height can be utilized. 

The shape of the containers can 

change according to the availability in the 

area of culturing. These containers with 

vermibeds must be placed in a proper environment. 

Either it can be placed in separate 

thatched roof shed if economically 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 43



viable or can be placed in spaces available 

inside the house itself. Thus depending 

on the economical status, the place 

can be selected. The conditions in maintaining 

the vermibeds are, direct sunlight 

must be avoided and it must be safe from 

insects and rodents. 

The second method of culturing 

earthworms is by tank method. By this 

way multiplication of earthworms can be 

done in large scale. For this method, cement 

tanks of dimensions one meter 

width × one meter height × 3-5 meter 

length can be constructed. These tanks 

must be constructed inside a thatched roof 

shed so that direct sunlight and rain can 

be avoided. The shed can be of any dimensions 

according to the land available. 

Thus the length of the tank and the no of 

tanks to be constructed can also be done 

accordingly. The shed must be constructed 

in East-West direction length wise to 

avoid direct sunlight and preferably open 

from all sides with unpaved floor with 

raised ground (atleast 6 inches) to protect 

the area from flooding during the rains. 

2.1.1. Vermibed preparation 

The vermibed for both the methods 

can be prepared using any kind of degradable, 

non-toxic organic waste like 

leaf litters, agricultural wastes (Suthar, 

2010), shredded newspaper (Updegraff, 

1971), cardboard, etc... can be utilized as 

medium. Wastes from fruit and vegetable 

market such as potato peels, onion peels, 

cabbage leaves, carrot and radish leaves, 

lettuce leaves, moldy bread can also be 

used (Suthar, 2009). Though organic 

wastes serve as food for earthworms, they 

can‟t be directly implemented for vermiculture. 

In other words, the worms 

can‟t eat the organic matter directly. Thus 

it must be subjected to partial decomposition 

and then used as a medium for vermibed. 

The organic matters must be 

shredded and alternate layers of organic 

matter and mixture of cow dung is spread 

on the floor of shed constructed for vermiculture. 

Before the implementation of 


organic matter for partial decomposition, 

care must be taken that, both the organic 

matter and the cow dung are shad dried. 

This is because; wet organic matter and 

cow dung may contain the cyst and larvae 

of other microorganisms and insects. So if 

implemented wet, they may grow along 

with earthworms and do hindrance. 

The alternate layers of organic 

matter and cow dung are arranged in the 

form of heap and 50% moisture must be 

maintained. Cover the heap with thatched 

coconut leaves. This set up must be kept 

for atleast 30 days. The organic matter, 

along with cow dung mixture must be 

turned over with a spade once in a week. 

The microorganisms which are present in 

the cow dung slowly degrade the organic 

matter. After 40 days the process of partial 

decomposition will be finished and 

now it can be used as a medium for vermibed. 

In the containers, the vermibed 

must be prepared by mixing the partially 

decomposed organic waste and cow dung 

(shade dried and powdered) in 1: 1 ratio. 

This ratio is recommended by many scientists 

and has been proved in many literatures 

also. Nearly 50% of moisture must 

be maintained in the bed. Earthworms can 

be introduced after two days. Leave the 

setup for 45 days. The worms eat the degraded 

organic matter and convert them 

into vermicastings. At the same time they 

also reproduce and increase in number. 

Vermibed for tank method can also 

be prepared in the same way but the 

partially degraded waste and the cow 

dung must be added in large quantities 

into the tank. Earthworms can also be released 

in large quantities. Water must be 

sprinkled on the vermibed once or twice 

in a week according to the humidity prevailing 

in the atmosphere. Temperature of 

about 37 degree Celsius must be maintained. 

If the climate is hot, wet jute bags 

can be screened on the sides of the tank. 

By the tank method, earthworms can the 

reproduced 300 times greater within 45 

days or 60 days (depending upon the 

worms cultured). The reproducing capaci- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 44



ty of exotic worms is greater than the indigenous 

worms. It is said that eight red 

worms multiply into 1,500 redworms (Eisenia 

fetida) in six months. These earthworms 

can be used to prepare vermicompost 

or for other purposes or can be sold. 

The vermibeds must be covered 

with a Jute mat to protect earthworms 

from birds and insects. Water is sprinkled 

on the vermibeds daily according to requirement 

and season to keep them moist. 

The waste is turned upside down once in 

a week. The appearance of black granular 

crumbly powder on top of vermibeds indicates 

the casts of earthworm. Addition 

of more amount of cow dung ensures the 

fecundity rate of earthworms. 

2.1.2. Harvesting of earthworms 

The vermibed after 45 days is 

converted into vermicastings. This indicates 

that all the organic matter along 

with cow dung is eaten up by the worms 

and converted into vermicastings. Now 

there is no food available for the worms. 

The content in the container or in the tank 

must be removed and new beds must be 

replaced. Before the replacement, the 

worms must be harvested. The very recent 

and new technique followed to harvest 

earthworms is using “fresh cow dung 

balls”. Fresh cow dung attracts the earthworms 

very much. Thus balls of fresh 

cow dung, of 15 to 20 cm in diameter are 

placed inside the vermibeds. For container 

method, one ball is enough while for 

tank method, four to six balls can be used 

to harvest earthworms. 

As soon as the cow dung balls are 

placed, the earthworms start migrating 

into the fresh cow dung balls. The cow 

dung balls were kept for 6 to 8 hours, after 

which are removed. Thus the earthworms 

can be harvested and used as inoculums 

for the new vermibed. The vermicastings 

in the container or the tank is 

removed and made into heap. This heap is 

left undisturbed for a day. Next day, the 

vermicastings are removed slowly from 

the outer side with the help of spade, 

sieved, and packed as biofertilizer. The 


small worms (juveniles) present segregate 

in the centre of the heap and can be collected. 

2.2. Application of earthworms 

Adult earthworms can be sold as 

fish bait and the young ones can be used 

as inoculums for fresh bedding. Many 

industries like aquaculture and Poultry 

will buy earthworms for using it as live 

feed. Business people who are interested 

in constructing vermicomposting units 

(house hold, small scale or large scale 

unit) will be purchasing the earthworms. 

Earthworms can be sold to new business 

people who are about to start their vermiculture 

or composting unit. Worms are 

also bought by academic institutions for 

research purposes. Earthworms can be 

sold to wholesalers who then resell the 

worms to bait shops, home and organic 

nurseries, and other users. 

3. Vermicomposting 

Earthworms facilitate the stabilization 

of organic wastes because their activity 

maintains aerobic conditions and 

ingested solids are converted into discrete 

odourless casts (Edwards, 1988). Thus the 

end product of vermicomposting is referred 

to as "vermicasting" or vermicompost. 

This is a nutrient rich organic substance 

that can be added to soil to increase 

its organic matter content and 

available nutrients. Vermicomposting is 

getting enormous importance in the amelioration 

of severe problems associated 

with the disposal of large quantities of 

organic wastes (John Paul, 2005). Earthworms 

feed on organic matter, in which 

5-10% is taken as food intake for their 

growth and the rest is excreted. Exotic 

earthworms like Red worms (Eisenia fetida) 

and African worm (Eudrilus eugeniae) 

and Indigenous species like Lampito 

mauritti and Perianyx excavatus are 

proved to be effectively in vermicomposting 

(Karmegam and Daniel, 2009, Kaur, 

et al., 2010). Vermicomposting can be 

done in three ways. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 45



i. Container method 

ii. Tank method 

iii. Pit method 

For all the three methods, the preprocessing 

of organic matter, i.e. the partial 

decomposition of organic matter is 

essential and it can be done as said above 

for vermiculture. 

3.1. Container method 

This method of composting is 

normally followed in small scale vermicomposting 

units or in houses where 

vermicomposting is done. The containers 

and the vermibed preparations can be followed 

as said for vermiculture. The only 

difference is the ratio of organic matter 

and cow dung. For vermiculture, the ratio 

of 1: 1 must be maintained in order to increase 

the fecundity rate. Because, the 

main aim of vermiculture is production of 

earthworms. While during vermicomposting, 

the ratio can be altered as 2: 1 or 

even to 3: 1 according to the availability 

of cow dung. An advantage of container 

method over traditional composting, is 

that they can be kept inside the composting 

shed during the winter, thus allowing 

this process to be done all over the year. 

3.2. Tank method 

Same as for vermiculture, the tank 

can be constructed and the process of 

vermicomposting can be done. Thus this 

method counts for large scale of vermicomposting. 

The only difference in 

both is, production of large amount of 

earthworms is the aim in vermiculture 

while production of large amount of vermicompost 

is the aim in vermicomposting. 

The same type of shed can be constructed 

for this purpose. The size can according 

to the availability of organic 

waste (the shed can be constructed in the 

same way as said for vermiculture). 

3.3. Pit method 

A pit of approximately 

4m×6m×4m (breath× length× depth) must 

be constructed. Vermibed is actually constructed 

with a layer of loamy soil placed 


at the bottom, about 15 to 20 cm thick 

above a thin layer (5 cm) of broken bricks 

and coarse sand. Earthworms are introduced 

into the loamy soil, which the 

worms will inhabit as their home. 150 

earthworms may be introduced into a 

compost pit of about 2m x 1m x 0.75m, 

with a vermibed of about 15 to 20 cm 

thick. Handful lumps of fresh cattle dung 

are then placed at random over the vermibed. 

The compost pit is then layered to 

about 5 cm of dry leaves or preferably 

chopped hay/straw or agricultural waste 

biomass or any non-toxic organic waste. 

This layer of organic waste and cow dung 

must be repeated till the top of the surface. 

For the next 30 days the pit is kept 

moist by watering it whenever necessary. 

The bed should neither be dry or soggy. 

The pit may then be covered with coconut 

or Palmyra leaves or an old jute (gunny) 

bag to discourage birds. Plastic sheets on 

the bed are not recommended as they trap 

heat. All these organic wastes can be 

turned over or mixed periodically with a 

pick axe or a spade. If the weather is very 

dry it should be dampened periodically. 

Red worms and African worms consume 

large amounts of organic matter and 

hence they are recommended for composting. 

Though exotic earthworm species 

are used in pit method, the indigenous 

worms as invade the pit and do their 

role. 

3.4. Precautions 

Vermicomposting unit or pit should 

be protected from direct sun light. To 

maintain moisture level, spray water on 

the composting unit as and when required. 

Large amounts of waste can cause 

odours and attract dogs or rodents, ant, rat 

and bird. Thus, preventive measures 

should be taken like net or anti-insect 

propellants to avoid them. 

3.5. Harvesting Vermicompost 

Vermicompost can be harvested following 

the same method as that of vermiculture, 

using fresh cow dung balls and 

heap method. Some consumers are picky 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 46



in getting the pure form of vermicompost, 

i.e. the vermicasts, which are collected on 

the top of vermibed. At that case, vermicasts 

can be collected with a spatula 

once in a week and can be packed and 

sold. The rate of pure form of vermicastings 

is also higher when compared to 

the vermicompost. When large amount of 

compost are to be harvested, for example 

the compost obtained from tank method 

or from pit method, first the worms are 

harvested with the help of fresh cow dung 

balls, then the whole material is moved to 

a plain area or on a plastic sheet and made 

into a single heap and is exposed to light, 

scooped, sieved and packed for sales. 

Earthworms or undecomposed materials 

if seen any, are collected and returned to 

the compost pile. 

For faster rate of harvesting vermicompost, 

the original heap is better divided 

into several smaller pyramids. To 

enhance the earthworm movement towards 

the centre of the heap, ball of raw 

or fresh cow dung can be placed at the 

centre. Always earthworms have high affinity 

towards fresh cow dung. Thus after 

few hours the worms can be easily separated 

and the compost can be harvested 

and packed. 

3.6. Advantages of vermicompost 

Vermicompost is rich in all essential 

plant nutrients, hence provide excellent 

effect on overall plant growth, 

encourages the growth of new 

Shoots / leaves 

Vermicompost is free flowing, easy 

to apply, handle and store and does 

not have bad odour. 

It improves soil structure, texture, 

aeration, and water holding capacity. 

Vermicompost is rich in beneficial 

microorganisms which fix the Nitrogen 

and Phosphorous in the soil. 

Vermicompost may contain earthworm 

cocoons from which juveniles 

may come and increases in population 

in the soil where applied. 

 

 

 

 

 


It neutralizes the soil acidity or alkalinity. 

Vermicompost is free from pathogens, 

toxic elements, weed seeds 

etc. 

Vermicompost minimizes the incidence 

of pest and diseases. 

It contains valuable vitamins, enzymes 

and hormones like auxins, 

gibberellins etc. 

The nutrients available in vermicompost 

(in general) are Organic 

carbon (9.5 – 17.98%), Nitrogen 

(0.5 – 1.50%), Phosphorous (0.1 – 

0.30%), Potassium (0.15 – 0.56%), 

Sodium (0.06 – 0.30%), Calcium 

and Magnesium (22.67 to 47.60 

meq/100g), Copper (2 – 9.50 mg 

kg-1), Iron (2 – 9.30 mg kg-1), Zinc 

(5.70 – 11.50 mg kg-1) and Sulphur 

(128 – 548 mg kg-1). 

3.7. Storing and packing of vermicompost 

Watering is stopped for atleast 5 

days at this stage. The first lot of Vermicompost 

is ready for harvesting after 2- 

2 ½ months and the subsequent lots can 

be harvested after every 6 weeks of loading. 

The vermibed is loaded for the next 

treatment cycle. The harvested vermicompost 

should be stored in dark, cool 

place. It should have minimum 40% 

moisture. Sunlight should not fall over the 

composted material. It will lead to loss of 

moisture and nutrient content. It is advocated 

that the harvested composted material 

is openly stored rather than packed in 

over sac. Packing can be done at the time 

of selling. If it is stored in open place, periodical 

sprinkling of water may be done 

to maintain moisture level and also to 

maintain beneficial microbial population. 

If the necessity comes to store the material, 

laminated over sac is used for packing. 

This will minimize the moisture evaporation 

loss. Vermicompost can be stored for 

one year without loss of its quality, if the 

moisture is maintained at 40% level. 

4. Vermiwash 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 47



Vermiwash is the liquid biofertilizer 

collected after the passage of 

water through a column of worms. It is 

very useful as a foliar spray. It is a collection 

of excretory products and excess secretions 

of earthworms along with micronutrients 

from soil organic molecules. 

Vermiwash units can be set up in a plastic 

or iron barrel of 200 litre capacity for a 

large scale production. While for production 

in small scale, a plastic bucket of 15 

litre capacity is enough. 


4.1. Method of preparation 

The holding unit, i.e. the plastic 

barrel must be taken and fixed in a stand 

or on a high platform. A hole is drilled on 

one side at the bottom and a vertical limb 

of a T joint tube is attached in a way that 

half to one inch of the tube projects inside 

the barrel. A tap is attached to the end of 

the horizontal limb and the other end is 

closed with a dummy nut. The whole set 

up is mounted on a suitable pedestal. 

Keeping the tap open, a layer of broken 

bricks or pebbles is filled up to 25-30cm 

inside the barrel. Water is made to flow 

through this layer, followed by 20-30 cm 

layer of coarse sand. This forms the basic 

filtering unit. Over this a 60-75 cm layer 

of good loamy soil along with organic 

waste and cow dung is kept moistened. In 

this layer earthworms like, E. fetida, E. 

eugeniae and lumbricus terrestris may be 

introduced. Cattle dung pats and hay are 

placed on top of this layer of soil for 

mulching purpose. The unit is moistened 

every day. For two days after the introduction 

of earthworms the tap must be 

kept closed. After two days, open the tap, 

about 30 – 40 litres of vermiwash drains 

out. 

Similar setup can also be made in 

bucket for production in small scale. The 

layer of organic matter here last for 30-45 

cm thickness and the mulching of 2-3 cm 

thicknesses must be spread to prevent 

evaporation. Spray water regularly for 7-8 

days for both of the setup so that 60% of 

moisture is maintained. Introduce 1000- 

2000 numbers of earthworms for the barrel 

setup while 100-200 earthworms is 

enough for a bucket. Keep a pot at the 

bottom of the stop cork of the bucket so 

that waterfalls drop by drop. Every day 

30-40 litres in barrel and about 3-4 liters 

of vermiwash in the bucket setup can be 

collected. 

As the tap is closed and water is 

sprinkled on top of the unit, the water 

slowly percolates through the compost 

carrying with it nutrients through the filter 

unit. When the tap is opened after two 

days, vermiwash is collected, which is 

sprayed on plants as a foliar spray. The 

vermicasts formed on the surface of the 

unit may also be collected periodically. 

4.2. Application 

The vermiwash may be diluted 

with water in 1:1 ratio or it may be diluted 

with 10 per cent cow‟s urine, which is 

an effective growth tonic and pesticide. 

Mix 1 liter of vermiwash with 7-10 liters 

of water and spray the solution on the leaf 

(upper lower side) in the evening at the 

growing crop. Mix 1 liter of vermiwash 

with 1 litre of cow urine and then add 10 

liters of water and mix thoroughly and 

keep it over night before spraying. 50-60 

litres of such solution can be sprayed in 

one hectare of land to control various 

crop diseases. 

5. Perspectives 

As a processing system, the vermicomposting 

of organic waste is very 

simple. Worms ingest the waste material - 

break it up in their rudimentary gizzards, 

consume the digestible, putrefiable portion 

and then excrete a stable, humus-like 

material that can be immediately marketed 

and has a variety of documented benefits 

to the consumer. Vermitechnology is 

a promising technique that has shown its 

potential in certain challenging areas like 

augmentation of food production, waste 

recycling, management of solid wastes 

etc. In most of the countries, soil pollution 

is increasing due to accumulation of 

organic wastes and on the other side there 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 48



is shortage of organic manure. Organic 

waste could be converted into vermicompost 

using Vermitechnology to increase 

the fertility and productivity of the agricultural 

land and to produce nutritive and 

safe food. Hence we need to use and 

promote this ecofriendly technology for 

the sustainability of agriculture and environment. 

References 

Edwards, C. A. (1988). Breakdown of 

animal, industrial and organic 

wastes by earthworms. In: 

Earthworms in Waste and Environment 

Management. Edwards 

CA, Newhauser EF (Eds). SPB 

Academic Publishing, The 

Hague. pp. 21 – 31. 

Edwards, C. A. and Bater, J. E. (1992). 

The use of earthworms in environmental 

Management, soil biol. 

Biochemical Journal 24 (12), 

pp.1683-1689. 

Hand, P., Hayes, W. A., Satchell, J. E., 

Frankland, J. C., Edwards, C. A. 

and Neuhauser, E. F. (1988). The 

vermicomposting of cow slurry. 

Pedobiologia 31, 49-63. 

Follet, R., Donahue, R. and Murphy, L. 

(1981). Soil and Soil Amendments. 

Prentice- hall. Inc., New Jersey. 

John Paul, J.A. (2005). Municipal solid 

waste generation, characterization, 

microbial activity, vermicomposting 

and management 

in Dindigul Town. Ph.D. Thesis. 

The Gandhigram Rural Institute – 

Deemed University, Gandhigram, 

Tamil Nadu, India. 

John Paul., Karmegam, N. and Daniel, 

T. (2011). Municipal solid waste 

(MSW) vermicomposting with an 

epigeic earthworm, Perionyx 

ceylanensis Mich. Bioresource 

Technology 102, 6769–6773. 

Karmegam, N. and Daniel, T. (2009). 

Investigating efficiency of Lampito 

mauritii (Kinberg) and Perionyx 

ceylanensis Michaelsen for 


vermicomposting of different 

types of organic substrates. Environmentalist 

29, 287–300. 

Kaur, A., Singh, J., Vig, A.P., Dhaliwal, 

S.S., and Rup, P.J. (2010). Cocomposting 

with and without Eisenia 

fetida for conversion of 

toxic paper mill sludge to a soil 

conditioner. Bioresource Technology 

101, 8192–8198. 

Logsdon, G. (1994). Worldwide progress 

in vermicomposting. Biocycle 

35(10),63-5. 

Madan, M., Sharma, S., Bisaria, R. and 

Bhamidimarri, R. (1988). Recycling 

of organic wastes through 

vermicomposting and mushroom 

cultivation. Alternative waste 

treatment systems 132-141. 

Mitchell, M. J., Hornor, S. G. and 

Abrams, B. I. (1980). Decomposition 

of sewage sludge in drying 

beds and the potential role of the 

earthworm, Eisenia fetida. Journal 

of Environmental Qualilty 9, 

373-378. 

Singh, A. and Sharma, S. (2002). Composting 

of a crop residue through 

treatment with microorganisms 

and subsequent vermicomposting. 

Bioresource Technology 

85:107-11. 

Sundaravadivel, S. and Ismail, S. A. 

(1995). Efficacy of a biological 

filter unit in the treatment of distillery 

effluents. Journal of Ecotoxicology 

and Environmental 

Monitoring 5(2), 125-9. 

Suthar, S. (2009). Vermicomposting of 

vegetable-market solid waste using 

Eisenia fetida: Impact of 

bulking material on earthworm 

growth and decomposition rate. 

Ecological Engineering 35, 914– 

920. 

Suthar, S. (2010). Recycling of agroindustrial 

sludge through vermitechnology. 

Ecological Engineering 

36, 1028–1036. 

Updegraff, D.M. (1971). Utilization of 

cellulose from waste paper by 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 49




Myrothecium verrucaria. Biotechnology 

and Bioengineering 

13, 77–97. 


This article is an open access article distributed under the terms 

and conditions of the Creative Commons Attribution (CC BY) license 


ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 50




Shobana Manoharan 1 , Raghavan Kuppu 1 and Ramesh Uthandakalaipandian 2, * 

1 Department of Molecular Biology, School of Biological Sciences, Madurai Kamaraj University, 

Madurai - 625021, Tamil Nadu, India; 2 Assistant Professor, Department of Molecular 

Biology, School of Biological Sciences, Madurai Kamaraj University, Madurai - 

625021, Tamil Nadu, India; *Correspondence: ramesh.biological@mkuniversity.org; Tel: 

+91-9489014892 

Abstract: Food is the most essential component of life for survival. The quality of food 

products is essentially to be authenticated as they are closely related to the health of human 

beings. “Foodomics”, is a concept to utilize technology for improvement of food and nutrition. 

Food products are authenticated in benefit of both consumers as well as commercial 

traders. Food authentication is done by analytical techniques like chromatography, Fourier 

Transform Infrared Spectroscopy (FTIR), Nuclear Magnetic Resonance Spectroscopy 

(NMR), Gas Chromatography - Mass Spectroscopy (GC-MS) and Liquid Chromatography 

- Mass Spectroscopy (LC-MS). Plant or animal based food products are widely authenticated 

for their origin, species nomenclature using DNA barcodes - a genomics based approach. 

The protein molecules of food products serve as key molecules for authentication using 

proteomics approach such as 2-dimensional gel electrophoresis and 2-dimensional difference 

gel electrophoresis (DIGE). Thus, food authentication is mandatory to substantiate the 

geographical origin, species nomenclature, food composition, genetic modification of a 

food product that reaches to the hands of consumers in the food markets. Thus, the development 

of rapid, novel food authentication methods to validate sea food products, Genetically 

Modified (GM) food products based on biotechnological approaches helps to provide 

quality assured food products to the human community. This review briefs about the various 

techniques employed in food authentication, their advantages and applications in food 

biology. 

Keywords: Analytical techniques; food authentication; genomics; proteomics 


Nowadays wide range of food 

products are available from various countries 

to the consumers but the quality of 

the food consumed is of great concern as 

they are directly linked to human health. 

“Foodomics” is a new term coined at the 

International Conference, Cesena (2009), 

Italy (foodomics.eu) which deals with the 

application of the recent „omics‟ technology 

to promote the field of food and nutrition. 

Foodomics is thus defined as, “a 

new approach to food and nutrition that 

studies the food domain as a whole with 

the nutrition domain to reach the main 

objective, the optimization of human 

health and well-being” (Capozzi and Bordoni, 

2013). According to Hippocrates 

„Let food be thy medicine and medicine 

be thy food‟; hence, the ratification of 

food components becomes vital to shield 

human health. Development of quality 

assurance methods to authenticate the 

food products based on their geographical 

origin, composition, originality, certification 

of their species in case of animal or 

plant derived products are of prodigious 

attention from both consumer and commercial 

trader‟s opinion. The need for 

food authentication and its application 

differs from country to country. For ex- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 51



ample, geographic origin of the food is a 

principal authenticating criterion to be 

mandatorily confirmed in Europe, numerous 

schemes for food quality analysis like 

PDO (Protected Designation of Origin), 

PGI (Protected Geographical Indication) 

and TSG (Traditional Specialities Guaranteed) 

are also followed by them in order 

to look after the conventional production 

methods (Drivelos and Georgiou, 2012). 

There are a huge number of valid reports 

till date stating about the adulterants, substitutes 

and confused species in herbal 

formulations as like the recounted instances 

of Chinese herbs that caused severe 

intoxications and even deaths due to 

adulterants or substitutes (But, 1994). 

Thus, food authentication is indispensable, 

in earlier days it was carried out 

based on analytical chemistry techniques. 

Conversely, in recent days it is accomplished 

by various other methods like genomics, 

proteomics and metabolomics for 

the reason that food authentication being 

a multi-disciplinary research that utilizes 

data from biology, chemistry, chemometrics 

and bioinformatics (Ibanez et al., 

2013; Moore et al., 2012). 

2. Food authentication 

Shobana et al. 

Food labelling is a common practice 

to be followed in order to approve the 

originality of food products bought in 

hand to the consumers. Food authentication 

is the process of confirmation a food 

product for its originality as described in 

their labels (Figure 1). This process has 

grabbed wide attention because of the upsurging 

perception among the people 

about food quality and safety (Danezis et 

al., 2016). Food authentication is mainly 

done at the following circumstances, 

i. Validation of imported traditional 

foods, 

ii. Identification of Genetically Modified 

Products (GMPs) from unauthorised 

GMP‟s, 

iii. Identification on adulterants or 

substitutes, 

iv. Corroboration of medicinally important 

plant species that may contain 

confused species, 

v. Verification of Geographical 

origin of plant or animal derived 

food products (Figure 2). 

3. Chemometric methods 

Chemometric methods comprises 

of chromatographic and spectroscopic 

methods. Chromatographic analysis sepa- 

Food Labelling 

Food Authentication 

Food Composition Database 

Figure 1: Important steps involved in developing a database for food industry. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 52




Food Product 

Food 

Authentication 

Chromatographic 

techniques 

Adulteration 

Species 

Confusion 

DNA Barcodes 

Proteome 

analysis 

Figure 2: Importance of food authentication. 

rates analogous chemical compounds in 

food products. Food is composed of proteins, 

lipids, carbohydrates, phytochemicals 

and many other small molecules such 

as food additives, colourants and preservatives. 

In general, these compounds are 

chemically distinct based on their molecular 

weight, polarity, charge etc., Development 

of fingerprint pattern using chromatography 

(High Performance Liquid 

Chromatography - HPLC) to differentiate 

food free from adulterants or constituents 

succours in food labelling (Cserhati et al., 

2005; Reinholds et al., 2015; Georgiou 

and Danezis, 2015). 

Fourier Transform Infrared Spectroscopy 

(FTIR) analysis can be done for 

the functional assessment of nominal frequency 

wave number obtained from the 

metabolites studied. They identify the 

presence of fatty acids and other metabolites 

of the food and help in development 

of the FTIR fingerprint region which can 

serve as a unique, preliminary tool to indicate 

the nutritional index of the food. 

Further analysis of metal concentrations 

can be done using Atomic Absorption 

Spectra (AAS) or Inductively Coupled 

Plasma Mass Spectroscopy (ICP - MS). 

Fatty acids and Protein analysis using LC 

- MS and GC - MS techniques respectively 

facilitate the identification of individual 

molecular components from which unique 

marker compounds for a food may be 

identified. Nuclear Magnetic Resonance 

Spectroscopy (NMR) analysis is a fast 

and more reliable technique to profile the 

complete metabolome of a food. The 

peaks pertaining to the functional groups 

are analysed and unique peaks are used 

for differentiating the food in terms of 

quality (Cozzolino, 2012; Longobardi et 

al., 2013; Hohmann et al., 2015). Proton 

Transfer Reaction Mass Spectroscopy are 

used for analysis of volatile organic compounds, 

reports based on these techniques 

distinguish organically grown tomatoes 

from conventional ones (Hohmann et al., 

2015). In few instances, highly complex 

food products are difficult to be differentiated 

using HPLC, FTIR or NMR at such 

rationale Gas Chromatography (GC) or 

Liquid Chromatography (LC) coupled to 

Mass Spectrometry (MS), have flourished 

as better food authentication tools (Figure 

3). Few applications of chemometric 

methods are sated below, 

i. Authentication of wild, farmed 

fish food species (Capuano et 

al., 2012), 

ii. Fatty acid content confirmation 

in animal and plant ex 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 53




HPLC 

Barcode 

FTIR 

Food 

Authentication 

DGGE 

NMR 

GC-MS 

Figure 3: Techniques involved in food authentication. 

iii. 

tracted oils (Yang et al., 

2013), 

Validation of mineral content 

in eggs (Giannenas et al., 

2009). 

4. Genomic methods 

This approach involves the use of 

genetic material, Deoxyribonucleic Acids 

(DNA) either as complete genome for 

evaluation or amplification of signature 

DNA sequences to authenticate a food 

product. Molecular techniques such as 

Random Amplified Polymorphic DNA 

(RAPD), Restriction Fragment Length 

Polymorphism (RFLP), Inter Simple Sequence 

Repeats (ISSR), Simple Sequence 

Repeats (SSR), Sequence Characterized 

Amplified Region (SCAR) use the genome 

variation in certification of food 

products (Ali et al., 2014). Meta DNA 

barcoding, de novo sequencing and Next 

Generation Sequencing are some of the 

techniques that lead to the high throughput 

sequencing of entire genome. Denaturing 

Gradient Gel Electrophoresis 

(DGGE) is used in microbial food authorization 

such as cheese, curd, probiotic yoghurts 

and other fermented dairy products 

(Arcuri et al., 2013). Commercial herbal 

formulations are mostly in processed or 

powdered form, in those cases mini DNA 

barcodes are employed for authentication. 

And most of the food products of animal 

and plant origin are in processed forms 

where the food source cannot be verified 

by other chemometric methods and in 

such circumstances DNA barcodes play a 

vital role in validation of plant or animal 

species used in manufacturing of a food 

product. DNA barcodes are short, conserved 

sequences that are employed in 

molecular taxonomy classification of 

plants and animal‟s species. Cytochrome 

Oxidase I [CO I] gene of mitochondrial 

(mt) DNA is a universally acceded molecular 

marker used for DNA barcoding 

in animals (Hebert et al., 2003). In plants, 

maturase K (mat K) and RubisCO L 

(Ribulose 1, 5 bisphosphate carboxylase / 

oxygenase) gene abet in barcoding (Janzen 

et al., 2009). 

5. Proteomic methods 

Food proteomics research helps in 

analysis of individual marker protein 

components from mixture of proteins using 

Polyacrylamide gel electrophoresis 

(1D or 2D) along with LC - MS for protein 

characterisation. Characterisation of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 54




Table 1: Applications of food authentication 

No Food Product Authentication Method Source 

1. Olive Oil GC - MS Yang et al., 2013 

2. Eggs ICP - MS Giannenas et al., 2009 

3. Salmon 

1 H NMR Capuano et al., 2012 

4. Tomatoes Proton Mass Spectroscopy Hohmann et al., 2015 

5. Italian Sweet Cherry NMR Longobardi et al., 2013 

6. Nigella sativa seed oil FTIR Nurrulhidayah et al., 2011 

7. Cheese 16s rDNA PCR and DGGE Arcuri et al., 2013 

genetically modified products, soy proteins 

in foods and dairy products are done 

based on proteomic analysis (Gallardo et 

al., 2013). This proteomics based approach 

is of wide importance and applications 

in food industry as they accurately 

pin point the difference between original 

and adulterated food products. 

6. Immunological methods 

Immunological methods rely on 

the binding specificity of the antibodies 

designed to an atypical antigen i.e., allergens, 

toxins etc. in food by use of Enzyme 

Linked Immuno Sorbent Assay 

(ELISA) (Asensio et al., 2008). For instance, 

they are used to spot the presence 

of potential fish allergen parvalbumin 

(Gajewski and Hsieh, 2009) in seafood 

industry. These techniques rely on specificity 

and also have an added advantage 

of validating a large number of samples 

with high precision in a short span of 

time. 


Food authentication not only ensures 

food quality but also assures human 

health. Emergence of new research fields 

such as transgenics, foodomics, nutrigenomics 

has resulted in wide range of 

neoteric products that need to be validated 

for substitutes prior to their distribution in 

commercial markets for edible purpose. 

Some of the practical applications of food 

authentication based on the abovementioned 

techniques are tabulated in Table 

1. Construction of food authentication 

databases will be fruitful for high 

throughput validation of food products. 

Acknowledgement 

Authors are thankful to the Educational 

fellowship, UGC-NON-NET 

Scheme. Authors are also thankful to the 

State-of-art infrastructure facility provided 

by CEGS, School of Biological Sciences, 

Madurai Kamaraj University, India. 

References 

Ali, M. E., Razzak, M. A., and Hamid, 

S. B. A. (2014). Multiplex PCR in 

species authentication: probability 

and prospects - a review. Food Analytical 

Methods, 7(10), 1933-1949. 

Arcuri, E. F., El Sheikha, A. F., Rychlik, 

T., Piro Metayer, I., and Montet, 

D. (2013). Determination of 

cheese origin by using 16S rDNA 

fingerprinting of bacteria communities 

by PCR–DGGE: Preliminary 

application to traditional Minas 

cheese. Food Control, 30(1), 1-6. 

Asensio, L., Gonzalez, I., Garcia, T., 

and Martin, R. (2008). Determination 

of food authenticity by enzymelinked 

immunosorbent assay (ELI- 

SA). Food Control, 19(1), 1-8. 

But P. P. H. (1994). Herbal poisoning 

caused by adulterants or erroneous 

substitutes. Journal of Tropical 

Medicine and Hygiene, 97,371–374. 

Capozzi, F., and Bordoni, A. (2013). 

Foodomics: a new comprehensive 

approach to food and nutrition. 

Genes and nutrition, 8(1), 1-4. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 55




Capuano, E., Lommen, A., Heenan, S., 

de la Dura, A., Rozijn, M., and 

van Ruth, S. (2012). Wild salmon 

authenticity can be predicted by 1H‐ 

NMR spectroscopy. Lipid Technology, 

24(11), 251-253. 

Cozzolino, D. (2012). Recent trends on 

the use of infrared spectroscopy to 

trace and authenticate natural and 

agricultural food products. Applied 

Spectroscopy Reviews, 47(7), 518- 

530. 

Cserhati, T., Forgacs, E., Deyl, Z., and 

Miksik, I. (2005). Chromatography 

in authenticity and traceability tests 

of vegetable oils and dairy products: 

a review. Biomedical Chromatography, 

19(3), 183-190. 

Danezis, G. P., Tsagkaris, A. S., Camin, 

F., Brusic, V., and Georgiou, C. A. 

(2016). Food authentication: techniques, 

trends and emerging approaches. 

TrAC Trends in Analytical 

Chemistry, 85, 123-132. 

Drivelos, S. A., and Georgiou, C. A. 

(2012). Multi-element and multiisotope-ratio 

analysis to determine 

the geographical origin of foods in 

the European Union. Trends in Analytical 

Chemistry, 40, 38-51. 

Gajewski, K. G., and Hsieh, Y. H. P. 

(2009). Monoclonal antibody specific 

to a major fish allergen: parvalbumin. 

Journal of Food Protection, 

72(4), 818-825. 

Gallardo, J. M., Ortea, I., and Carrera, 

M. (2013). Proteomics and its applications 

for food authentication and 

food-technology research. Trends in 

Analytical Chemistry, 52, 135-141. 

Georgiou, C. A., and Danezis, G. P. 

(2015). Elemental and isotopic mass 

spectrometry. Comprehensive Analytical 

Chemistry, 131-143. 

Giannenas, I., Nisianakis, P., Gavriil, 

A., Kontopidis, G., and Kyriazakis, 

I. (2009). Trace mineral content 

of conventional, organic and 

courtyard eggs analysed by inductively 

coupled plasma mass spectrometry 

(ICP-MS). Food Chemistry, 

114(2), 706-711. 

Hebert, P. D., Cywinska, A., and Ball, 

S. L. (2003). Biological identifications 

through DNA barcodes. 

Proceedings of the Royal Society 

of London B: Biological Sciences, 

270(1512), 313-321. 

Hohmann, M., Monakhova, Y., Erich, 

S., Christoph, N., Wachter, H., 

and Holzgrabe, U. (2015). Differentiation 

of organically and conventionally 

grown tomatoes by chemometric 

analysis of combined data 

from proton nuclear magnetic resonance 

and mid-infrared spectroscopy 

and stable isotope analysis. 

Journal of Agricultural and 

Food Chemistry, 63(43), 9666-9675. 

Ibanez, C., Garcia Canas, V., Valdes, 

A., and Simo, C. (2013). Novel 

MS-based approaches and applications 

in food metabolomics. Trends 

in Analytical Chemistry, 52, 100- 

111. 

Janzen, D. H., Hallwachs, W., Blandin, 

P., Burns, J. M., Cadiou, J., Chacon, 

I., and Franclemont, J. G. 

(2009). Integration of DNA barcoding 

into an ongoing inventory of 

complex tropical biodiversity. 

Molecular Ecology Resources, 

9(s1), 1-26. 

Longobardi, F., Ventrella, A., Bianco, 

A., Catucci, L., Cafagna, I., Gallo, 

V., and Agostiano, A. (2013). Nontargeted 

1 H NMR fingerprinting 

and multivariate statistical analyses 

for the characterisation of the geographical 

origin of Italian sweet 

cherries. Food Chemistry, 141(3), 

3028-3033. 

Moore, J. C., Spink, J., and Lipp, M. 

(2012). Development and application 

of a database of food ingredient 

fraud and economically motivated 

adulteration from 1980 to 

2010. Journal of Food Science, 

77(4), R118-R126. 

Nurrulhidayah, A. F., Man, Y. B., Al- 

Kahtani, H. A., and Rohman, A. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 56



(2011). Application of FTIR spectroscopy 

coupled with chemometrics 

for authentication of Nigella sativa 

seed oil. Journal of Spectroscopy, 

25(5), 243-250. 

Reinholds, I., Bartkevics, V., Silvis, I. 

C., van Ruth, S. M., and Esslinger, 

S. (2015). Analytical techniques 

combined with chemometrics for 

authentication and determination of 


contaminants in condiments: A review. 

Journal of Food Composition 

and Analysis, 44, 56-72. 

Yang, Y., Ferro, M. D., Cavaco, I., and 

Liang, Y. (2013). Detection and 

identification of extra virgin olive 

oil adulteration by GC-MS combined 

with chemometrics. Journal 

of Agricultural and Food Chemistry, 

61(15), 3693-3702. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 57



Management Strategies against Tiny Tigers for 

Sustainable Development of Agriculture 

Viswa Venkat Gantait* 

Zoological Survey of India, M-Block, New Alipore, Kolkata-700053, West Bengal, India; 

*Correspondence: v.gantait@rediffmail.com; Tel: 09433463555 

Abstract: Amongst different pests of agriculture, the plant parasitic nematodes are considered 

the worst one because of devastations they cause to the crops. Due to their microscopic 

nature, they are the hidden enemies of soil and crop damages caused by them have not been 

fully formulated. For sustainable development of agriculture and to stop the yield loses 

causes by these tiny pests of crops, potential control measures should be explored and 

adopted against them. By following various physical, chemical, biological and botanical 

methods, some of cultural practices and also by regulatory methods nematode infestation in 

agricultural fields must be stopped or checked at certain level. This will help to enhance 

crop production, which will ultimately be helpful to improve the gross national product 

(GNP) of country and agricultural sustainability. This article provides an overview of management 

strategies which could be used against plant parasitic nematodes to boost the sustainable 

development of agriculture. 

Keywords: Agriculture; control measure; plant parasitic nematode; sustainable development 


1.1. What are tiny tigers? 

Tiny tigers! How funny the term 

is? Tiger is one of the most ferocious carnivores 

of the world. Not a joke, the nematodes 

are now treated as tiny tigers in 

crop fields. Even, these are more harmful 

than tigers, as far as agriculture is concerned. 

They represent one of the most 

abundant groups and probably the second 

largest one in the animal kingdom, immediately 

behind the arthropods (Hugot et 

al., 2001). 

Nematodes represent sharply differentiated 

primitive group of invertebrates, 

commonly known as round 

worms, thread worms or eelworms, distinctly 

different from segmented worms 

like earthworms and flatworms. Simply, 

these are also called ‘nemas’. These are 

multicellular, vermiform, triploblastic, 

pseudocoelomate, bilaterally symmetrical, 

unsegmented animal, possessed between 

the phylum Platyhelminthes and Annelida 

in the animal kingdom. They generally 

have a cylindrical body while a few may 

be fusiform, saccate or kidney-shaped. 

Those are characterized by having a body 

cavity, complete digestive tract, well developed 

reproductive system, excretory 

and nervous system; but lacking circulatory 

and respiratory system. Most of them 

are microscopic in size, but may be seen 

with naked eyes. 

1.2. Where we can find them? 

Nematodes are highly diverse in 

their habitats ranging from Himalayan 

peak to the sea floor, from Arctic to Antarctic 

(Lal, 1998). They can withstand 

extreme adverse environmental conditions 

and may be found in Polar Regions 

to tropics. They occur almost everywhere 

on the earth like in ocean, river, lake, 

pond, estuary, island, soil, hill and rocks, 

desert hot springs etc. Interestingly, their 

habitat is unsurpassed by any other meta- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 58


Management Strategies against Tiny Tigers… 

zoan invertebrate group, because they are 

found in all types of habitats (Bohra and 

Baqri, 1997). They are so numerous that 

if everything on the earth were to disappear 

except the nematodes, the outlines of 

habitats would still be dimly visible; the 

mountains, lakes and oceans, the plants 

and the animals would all be outlined by 

the nematodes present (Cobb, 1914). 

Based on the habitat, the total 

nematode population can be categorized 

into three groups: marine, animalparasitic, 

soil and freshwater nematodes 

(Ayoub, 1980). The marine nematodes 

constitute about 50% of the total nema 

population, where as animal-parasitic 

nematodes make up only 15% of the 

known nematode species. The soil and 

freshwater nematodes can be spitted into 

two finer divisions: free-living and plantparasitic. 

Free living nematodes comprising 

about 25% of the total nematode population. 

The plant-parasitic nematodes 

constitute only 10% of the total nematodes 

(Figure 1). 

According to Crofton (1966) most 

of the soil holds about 90% nematodes at 

top six inches. (Nicholas, 1984) opined 

that the soil nematodes are usually most 

abundant near the soil surface, with the 

majority within the top 10 cm, though 

some may be found much deeper. He also 

stated that, populations are much denser 

in the zones, rich in organic matter or 

Gantait 

where fine plant roots are concentrated; 

adequate soil moisture and oxygen also 

favours dense populations of nematodes. 

1.3. What they feed? 

Nematodes are microphagous, 

mycetophagous, saprophytic, phytoparasitic, 

predatory, carnivorous and even 

cannibalistic in nature. Most of them feed 

on bacteria, fungi, other microorganisms 

and decaying matter. They can parasitize 

plants, different animals including man 

and even other nematodes also. Depending 

on the diverse diets Yeates et al. 

(1993) categorized nematodes into eight 

feeding groups: plant feeders, algal feeders, 

hyphal feeders, substrate ingesters, 

unicellular eukaryote feeders, bacterial 

feeders, predators and omnivorous. 

2. Important roles in agricultural aspects 

Plant parasitic and soil-inhabiting 

nematodes have been known for their virulence 

causing significant loss to agricultural 

and horticultural crops (Bohra and 

Baqri, 2004). Several species have come 

to be recognized as useful predators in the 

control of different insects and nematodes 

also. The possibility of using nematodes 

for control of different plant parasitic 

nematodes was first suggested by Cobb 

(1917). The role of predatory nematodes 

Figure 1: Pie diagram 

showing the different 

groups of nematodes. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 59



in biological control of plant parasitic 

nematodes was described by Jairajpuri et 

al. (1990). They regulate microbial biomass 

and nitrogen mineralization by killing 

and feeding on nematodes and other 

microorganisms that pass from bottom to 

top tropic levels in an ecosystem (Wardle 

and Yeates, 1993). Some species are 

known to play a vector role in transmitting 

so many soil-borne bacterial, fungal 

as well as viral pathogens to their hosts 

(Jairajpuri and Ahmad, 1992). The innocuous 

nematodes, those have no concern to 

the farmer or gardener; those feed on bacteria, 

fungi, algae and even other nematodes, 

play an important role in controlling 

soil nutrient cycling (Tahseen, 2006). 

Each species possibly plays a significant 

role in the ecosystem inhabits and undoubtedly 

has a major role in maintaining 

the natural ecological balance. Nematodes 

are intimately involved in many parts of 

the soil ecosystem, so they can be used as 

bioindicators of sustainability for soils. 

Because of their ubiquity and diversity, 

nematodes are used in measuring the impact 

of various perturbations on ecosystems, 

such as pollution, organic enrichment 

and physical disturbance (Tahseen, 

2006). 

2.1. How they depress crop yields 

Plant parasitic nematodes depress 

crop yields by the following important 

ways. 

They feed on plant parts and deprive 

the host of its nutrients. 

During feeding they cause mechanical 

injury to different parts of plants 

and their feeding sites serve as entrypoints 

of other pathogenic fungi and 

bacteria. 

Act as vectors of different fungal, 

bacterial and viral pathogens of 

plants. 

They can secret various enzymes in 

the plant tissues during feeding and 

the effected plants show abnormal 

growth responses to these secretions/excretions 

like hypertrophy. 

 

Gantait 

They can also act as modifiers of 

host substrates and render them more 

susceptible to other plant pathogens. 

3. Important symptoms of their attack 

Being protected under soil and 

having microscopic size, nematodes are 

practically the hidden enemies of crops. 

Symptoms of their attacking are not striking 

in most cases and therefore overlooked. 

The important symptoms may be 

tabulated as follows (Table 1). 

4. Common names of some important 

nematodes 

The plant parasitic nematodes belonging 

to the order Dorylaimida are migratory 

root ectoparasitic in nature. The 

genera, Trichodorus and Paratrichodorus 

under the family Trichodoridae browse 

along the root surface of plant. On the 

other hand, genera like Longidorus, Paralongidorus 

and Xiphinema feed for 

longer period at specific sites of deeper 

root tissues. Apart from causing direct 

minor or major root damages, these 

nematodes are of great economic importance 

as vectors of nearly 22 soilborne 

plant viruses. All the Tylenchids 

i.e. the nematodes belonging to the order 

Tylenchida are totally plant parasites. 

They are ectoparasitic, semi-endoparasitic 

or endoparasitic in nature. Most of the 

endoparasitic nematodes form root-galls 

and leaf-galls of plants. The common 

names of some important plant parasitic 

nematodes are as follows. 

5. Management strategies 

For sustainable development of 

agriculture, the adverse effects of nematodes 

have to be eradicated or minimized. 

For this purpose different control 

measures should be adopted against them. 

The first and foremost effort is to reduce 

increasing populations of these hidden 

enemies of crops, under the soil. The di- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 60



Table 1: Symptoms of nematode infestation 

Symptoms 

Produced by above-ground feeders 

Crinkled and distorted stems and foliages 

Dead and devitalized buds 

Leaf lesions 

Leaf and seed galls 

Necrosis and discoloration 

Produced by below-ground feeders 

Poor growth, stunting patchiness, discolored 

foliage, wilting etc. 

Root galls 

Root lesions 

Reduced root system 

Rots of fleshy plants 

rect and indirect benefits to control these 

tiny tigers of agricultural fields improved 

plant health, thereby reducing their 

changes of suffering from nematode diseases 

and also increased the plants ability 

to withstand adverse growing conditions, 

resulted improvement of crop production. 

Principal strategies for nematode management 

are cultural practices, physical, 

biological, botanical, chemical and regulatory 

methods. 

6. Cultural Practices 

To control nematode pests, cultural 

practices are the most effective and 

economical means which can be achieved 

by crop rotation, fallowing, deep summer 

plaughing, water flooding, growing antagonistic 

or trap crops, removal of infected 

plant debris, selection of healthy 

planting materials, application of organic 

manures and fertilizers etc. 

Some species of nematodes are 

able to feed and multiply on certain crops, 

but not on others. The crop plants on 

which they cannot feed and abundantly 

reproduce are called non-host. By crop 

rotation system, the crops to be grown in 

between the susceptible host crops should 

be immune or resistant to nematodes or at 

least non-host plants which help to eradicate 

or minimize the nematodes problems. 

Caused by the species 

Anguina spp. 

Aphelenchoides spp. 

Aphelenchoides besseyi 

Anguina spp. 

Rhadinaphelenchus spp. 

Most of the species 

Gantait 

Meloidogyne spp., Nacobbus spp., Ditylenchus 

spp., Xiphinema spp. etc. 

Pratylenchus spp., Radopholus spp. 

Trichodorus spp., Belonolaimus spp. 

Ditylenchus spp. 

Fallowing is a very common practice 

and cheaper way to minimize the 

nematode problems by keeping the lands 

from all vegetations for a certain period. 

Complete fallow without allowing any 

plant or weed to grow invariably ensures 

the parasitic nematodes will have no host 

to feed. Thus, those are deprived of food 

and killed by the solar heat and soil desiccation. 

Deep summer ploughing involves 

exposure of soils to solar heat and desiccation, 

helps to kill the nematode pests 

along with other pathogens. For enhancement 

of the efficacy of solar heating, 

polyethylene mulching of moist soil 

during hottest period is being advocated. 

It considered as a very effective and good 

control measure against nematode pests 

and can be used with other cultural practices 

like crop rotation, green manuring, 

inter cropping as well as with nematicides 

etc. (Mathur et al., 1987). 

In the field where there is enormous 

availability of water and nematode 

infested area is uniformly leveled, flooding 

can be adopted as a routine practice 

and very effective measure. Under submerged 

condition, chemicals lethal like 

hydrogen sulphide and ammonia to these 

noxious pests are released. Asphyxiation 

and microbial decomposition products 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 61



Gantait 

due to anaerobic condition help to kill the 

nematodes. 

The crops like mustard, marigold, 

neem etc. are treated as antagonistic 

crops, those have chemicals or alkaloids 

as root exudates which repeal or suppress 

the plant parasitic nematodes. These 

plants can be grown along with main crop 

or may be included in crop rotation. 

The basic concept with trap cropping 

system is that two crops are grown in 

the field, out of which one crop is highly 

susceptible to nematode. When nematodes 

attack such crop, very carefully it 

should be remove and destroyed or burnt 

totally. Thus, the main crop escapes from 

nematode infestation. Cowpea is a very 

good example of such susceptible crop for 

destroying root-knot nematodes. 

Early detection of infected plants, 

immediate removal and destruction of 

those helps to reduce the spreading of 

nematodes in the field. Selection of nematode-free 

healthy planting materials or 

plantation of nematode-resistant varieties 

is also a very effective method to avoid 

nematode infestation in field. 

Soil treatments with organic manures, 

green crop residues or green leaf 

manures, farm yard or poultry manures, 

oil cakes, and oils of neem, karanja, castor 

etc. significantly checked nematode 

population. The use of such materials also 

encourages the development of nematodeantagonistic 

microbes and predacious 

nematodes also those help to control 

nematode infestation in agricultural 

fields. 

6.1. Physical methods 

The hot water treatment, hot water 

drenching, rabbing with slow burning materials, 

soil solarization, electrical soil 

heating, washing and cleaning of seed etc. 

are the most effective physical methods to 

control nematodes infestations to crops. 

For denamatization, rhizomes, 

bulbs, corns, tubers and fleshy roots of 

plantations and also other planting materials 

are submerged into hot water for certain 

periods. Prior to plating the seed materials 

like banana corns, onion bulbs, tuber 

seeds and roots of seeding can be 

dipped in 5-55°C hot water for about 10 

minutes. 

The most primitive way of heating 

the soil on large scale is by putting a fire 

over it. Burning a layer of dry leaves on 

soil killed nematode pests. Rabbing of 

bajara husk, paddy husk, wheat straw etc. 

become most effective against nematodes. 

Burning materials outside and incorporating 

ash into the soil surface satisfaction 

nematode kill. 

Soil solarization is the most recent 

method for control of plant parasitic nematodes 

(Sharma & Trivedi, 1991). The 

technique consists of covering the moist 

soil with a good quantity, clear and transparent 

plastic film during the period of 

intense sunshine and increasing substantial 

soil temperature over the nonsolarized 

soil. Increased soil temperature 

coupled with soil moisture invariably result 

the significant reduction in population 

densities of phytoparasitic nematodes. 

The nematode pests of crops are 

effectively controlled by this method but 

it is restricted to summer season and only 

in the tropical and subtropical region of 

the world. 

Careful washing of tubers, bulb 

and other planting materials prevent this 

nematode infestation in new planting 

fields. Modern mechanical seed cleaning 

methods have been developed to remove 

the seed galls to form normal healthy 

seeds. Sanitation, the use of clean tools 

and equipments in field also prevent nematodes 

infestation. Soil amendments and 

frequent irrigation can also help to reduce 

nematode-damage of crops. 

6.2. Biological methods 

Biological control method of 

nematodes include the use of predaceous 

and parasitic organisms such as fungi, 

bacteria, protozoans, viruses, nematodes, 

tardigrades, collembolans, mites etc, even 

antagonistic higher plants also. This 

method, in fact, should be considered a 

skillful manipulation of the biosphere 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 62



against nematodes pest of agricultural 

fields for achieving maximum benefits. 

There are three types of components of 

biological control of nematodes. 

Natural: Where the agent are already 

present at levels to be sufficient for 

suppression of nematode development. 

Induced: The agents are already 

present in the soil and only their activities 

are stimulated by modifying the environment 

or by applying inciters. 

Introduced: The agents are applied 

by man from outside. 

There are more than 50 species of 

predaceous fungi which have the capacity 

to kill nematodes in agricultural field 

(Jain, 2003). These fungi capture nematodes 

by traps, mechanical traps and constricting 

rings. 

There are several reports of bacteria, 

present inside the nematode body. 

Pasteuria penetrans has been described 

as potential biological agent against nematodes. 

They prevent reproduction and 

eventually kill the root-knot nematodes 

and many other species. Some rhizospheric 

bacteria like Azotobactor chroococcum, 

Azospirilum lipoferum, and some Pseudomonas 

spp. have found to be promising 

in reducing nematode population. Rootknot 

nematode larvae infected with viruses 

were observed to exhibit sluggishness. 

6.3. Botanical methods 

Due to their facile biodegradability, 

selective toxicity only to target pests, 

Gantait 

safety to non-target organisms and the 

environment as a whole and renewable 

nature, the botanical pesticides offer alternate 

strategy to the prevalence use of 

synthetic nematicides (Mishra, 1998). Indiscriminate 

use of chemical pesticides to 

control nematode pest in agriculture give 

rise to serious problems like food contamination, 

adverse effects on non-target organisms 

and environment, as well as development 

of pesticidal resistance in 

many nematode pests. For this reason, the 

use of bio-pesticides of botanical origin 

for the management of plant parasitic 

nematodes has been increased presently. 

Different parts of botanicals directly, the 

extracts of botanical parts or the product 

of botanicals are used for nematode management. 

Parts of different plants having 

nematicidal value are used directly 

against phytonematodes, infesting various 

crops (Table 2). Chopped leaves of pineapple, 

karanja and neem leaves etc. could 

be significantly reduced the root-knot, 

reniform and other nematodes also. Various 

parts of Crotolaria, marigold, Kentucky 

blue grass etc. in powdered form 

also reduced nematode population. 

Chopped castor leaves, Subabool leaves 

prevent gall nematodes. Chopped shots of 

latex-bearing plants significantly suppressed 

the population build up of reniform 

and root-knot nematodes. 

Table 2: Common names of some important phytonematodes 

No. Genera/Species Common names 

1. A. fragariae Spring dwarf nematode 

2. Anguina spp. Seed gall, Leaf gall nematodes 

3. Anguina tritici Ear-cockle nematode, Wheat gall nematode. 

4. Aphelenchoides besseyi Rice white tip nematode, White tip nematode 

5. Aphelenchoides ritzemabosi Chrysanthemum foliar nematode 

6. Aphelenchoides spp. Bud and leaf nematodes, foliar nematodes 

7. Belonolaimus spp. Sting nematodes 

8. Belonololaimus gracilis Pine sting nematode 

9. Cacopaurus spp. Sessile nematodes 

10. Criconema spp. Spine nematodes 

11. Criconemoides citri Citrus ring nematode 

12. Criconemoides spp. Ring nematodes 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 63



Gantait 


13. Ditylenchus angustus Rice nematode 

14. Ditylenchus destructor Potato root nematode, Potato tuber nematode, 

Iris nematode 

15. Ditylenchus dipsaci Stem nematode, Tulip root nematode, Bulb 

nematode 

16. Ditylenchus myceliophagus Mushroom spawn nematode 

17. Dolichodorus spp. Awl nematodes 

18. Dorylaimus spp. Spear nematodes 

19. Globodera rostochiensis Golden nematode of potato 

20. Globodera spp. Cyst nematode 

21. Helicotylenchus spp. Spiral nematodes 

22. Hemicriconemoides spp. Sheathoid nematodes 

23. Hemicycliophora spp. Sheath nematodes 

24. Heterodera avenae Great root nematode, Cereal nematodes 

25. Heterodera cruciferae Cabbage cyst nematode 

26. Heterodera glycines Soybean cyst nematode 

27. Heterodera goettingiana Pea cyst nematode, Pea root nematode, Alfalfa 

root nematode 

28. Heterodera schachtii Sugar beet nematode 

29. Heterodera spp. Cyst-forming nematodes 

30. Hirschmanniella oryzae Rice root nematode 

31. Hoplolaimus spp. Lance nematode, Spear nematode 

32. Longidorus spp. Needle nematode 

33. Meloidodera spp. Cystoid nematode 

34. Meloidogyne arenaria Peanut root knot nematode 

35. Meloidogyne brevicauda Indian root knot nematode 

36. Meloidogyne exigua Coffee root knot nematode, Brazilian root knot 

nematode 

37. Meloidogyne incognita Southern root knot nematode 

38. Meloidogyne javanica Javanese root knot nematode 

39. Meloidogyne spp. Root knot nematodes, Root-gall nematodes 

40. Nacobbus spp. False root knot nematodes 

41. Paratylenchus spp. Pin nematodes 

42. Pratylenchus spp. Root lesions nematodes, Meadow nematodes 

43. Radopholus similis Burrowing nematode 

44. Rhadinaphelenchus cocophilus Coconut palm nematode, Red ring nematode 

45. Rotylenchulus reniformis Reniform nematode 

46. Rotylenchus spp. Spiral nematodes 

47. Trichodorus spp. Stubby root nematodes 

48. Tylenchorhynchus claytoni Stunt nematode, Teaselate stylet nematode 

49. Tylenchorhynchus martini Sugarcane stylet nematode 

50. Tylenchorhynchus spp. Stunt nematode, Stylet nematode. 

51. Tylenchulus semipenetrans Citrus root nematode 

52. Xiphenema spp. Dagger nematodes 

Certain botanicals in the form of aqueous 

extracts of various parts have great potential 

against nematodes, the aqueous extracts 

of fresh neem leaves; fruit skin of 

Citrus reticulata Blanco and Momordica 

charantia L.; leaves of Ageratum conizoides 

L., Anacardium occidentale L., 

Argemone mexicana L., Datura stramonium 

L. etc.; aqueous root extract of Ocimum 

sanctum L.; seed extracts of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 64



Gantait 

Vernonia anthelmintica Wild, Holarrhea 

antidysenterica Wall; bulb extracts of Allium 

sativum L. and many other plant extracts 

have potentiality to prevent nematode 

infestation in agricultural fields. 

Different plant products like oil 

seed cakes, oils, seeds, and various other 

formulations are extensively used for the 

management of plant parasitic nematodes. 

6.4. Chemical methods 

The chemicals those are used for 

controlling nematodes are the nematicides. 

These are the soil fumigants, applied 

to the soil and diffuse through the 

soil as gas and acted against nematodes. 

The use of nematicides for the management 

of plant parasitic nematodes in agriculture 

becomes essential when other 

methods are unable to protect the crops 

from these pests, or spreading of nematodes 

is so high in the field. Before planting, 

the nematicidal application in the 

field in proper doses resulted in nematode-free 

rhizosphere, healthy root system, 

efficient use of minerals, moisture 

and also reduces the chances of invasion 

of other harmful soil microorganism. 

Kuhn (1881) first used chemical (CS 2 ) 

against Heterodera schachtii in Germany. 

The discovery of DD-mixture in 1943, 

EDB in 1945 and DBCP in 1954 played 

remarkable role in demonstrating the 

nematode damage and crop loses. The use 

of methylisothiocyanate, precursor compounds 

like daromet, methamsodium, methylisothiocyanate 

mixture like vorlex etc. 

also help in controlling nematodes. The 

non-volatile nematicides like fensulphothion, 

aldicarb, carbofuran, ethoprop 

etc. are also very promisible nematicides. 

But the use of nematicides is a 

costly proposition and creates toxic hazards 

and environmental pollution. Few of 

them like DBCP, MBr, aldicarb etc. have 

been banned already. The use of nematicides 

is not so popular in agriculture except 

in few cases where drastic spreading 

of nematodes occurs in the field. 

6.5. Regulatory methods 

Quarantine principles are traditionally 

employed to restrict the movement 

of infected plant materials and contaminated 

soil into a state or country. 

Many serious plant parasitic nematodes 

spread from one country to another and 

from one state to other. The potato cyst 

nematode, Globodera rostochiensis 

spread from Peru to almost whole of Europe 

and UK through seed potatoes and 

gunny bags. The stem and bulb nematode, 

Ditylenchus dipsaci got introduced in 

southern parts of Sweden also through 

seeds. For this reason, plant quarantine 

has been introduced at state, national and 

international levels as a legal restriction to 

check the spreading of nematode pest. 

Regulatory control of pests and diseases 

is the legal enforcement of measures to 

prevent them from spreading. Strict regulations 

have been made against G. rostochiensis 

and Rhadinaphelenchus cocophilus, 

the red ring nematode of coconut. 

Domestic quarantine regulations have also 

been imposed to restrict the movement 

of potato to prevent the spread of potato 

cyst nematode from Tamil Nadu to other 

states in India. 

7. Conclusion 

The plant parasitic nematodes are 

undoubtedly the most widespread and insidious 

pests of crops. The management 

practices against these hidden enemies of 

agriculture to be adopted depend upon the 

degree of infection, relative value of the 

crop, filed size, level of capital investment, 

practicability and feasibility of the 

control strategy. The cultural practices are 

simple and effective methods of nematode 

pest control, adopted by the farmers. 

Physical methods are also simple and 

popular for management of nematode infestation. 

The biological and botanical 

methods are eco-friendly rather than others. 

Though chemical methods may create 

health hazards and causes environmental 

pollution but for urgent need and to check 

severe attack by serious nematode pests, 

and when other control measures are not 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 65



so fruitful, the chemical methods may be 

adopted against these noxious pests of 

various crops. For sustainable development 

of agriculture, a combination of different 

management systems integrated in 

the correct manner can help to manage 

the nematode problems. 


I am thankful to Dr. Kailash 

Chandra, Director, Zoological Survey of 

India, Kolkata for providing facilities and 

encouragement to prepare this article. 

References 

Ayoub, S. M. (1980). Plant Nematology: 

An Agricultural Training Aid. Nema 

Aid Publication. USA. pp. 195. 

Bohra, P. and Baqri, Q. H. (1997). Plant 

and soil nematodes. State fauna 

Series 6; Fauna of Delhi, 

Zoological Survey of India 75-108. 

Bohra, P. and Baqri, Q. H. (2004). State 

Fauna Series 8, Fauna of Gujarat 

(Part-2). Zoological Survey of India 

355-400. 

Cobb, N. A. (1914). North American 

free-living fresh water nematodes. 

Transactions of the American Microscopical 

Society 33, 35-100. 

Cobb, N. A. (1917). The mononchs 

(Mononchus, Bastian): a genus of 

free-living nematodes. Soil Science 

3, 431-486. 

Crofton, H. D. (1966). Nematodes. 

Hutchinson University Library, 

London. pp. 160. 

Hugot, J. P., Baujard, P. and Morand, 

S. (2001). Biodiversity in helminthes 

and nematodes as a field of 

study: an overview. Nematology 3 

(3), 199-208. 

Jain, R. K. (2003). Integrated pest 

mengement for plant parasitic 

nematodes. In: Nematode management 

in plants. Trivedi, P. C. (ed.). 

Scientific Publishers (INDIA), 

Jodhpur. pp. 293-304. 

Gantait 

Jairajpuri, M. S. and Ahmad, W. 

(1992). Dorylaimida: Free-living, 

Predaceous and Plant parasitic 

Nematodes. Oxford and IBH Publishing 

Company Private Limited, 

New Delhi. pp. 458. 

Jairajpuri, M. S., Alam, M. M. and 

Ahmad, I. (1990). Nematode 

biocontrol: Aspects and Prospects. 

CBS Publishers and Distributors, 

Delhi, India. pp. 155. 

Kuhn, I. (1881). Die Ergebnisse der 

versuche zur Ermittelung der ursach 

der Ruben mudigkeit und zur 

Erforschung der Natur der 

nematode. Ber physiol. Lab. Univ. 

Halle 3, 1-53. 

Lal, A. (1998). Application of computers 

in nematode identification. In: Recent 

Advances in Plant Nematology. 

Trivedi, P. C. (ed.). CBS Publishers 

and Distributors, Daryaganj, New 

Delhi, pp. 107-114. 

Mathur, B. N., Handa, D. K. and 

Swarup, G. (1987). Effect of deep 

summer ploughings on the cereal cyst 

nematodes, Heterodera avenae and 

yield of wheat in Rajasthan, India. Indian 

Journal of Nematology 17, 292- 

295. 

Mishra, S. D. (1998). Botanicals in the 

management of plant parasitic nematodes. 

In: Recent advances in plant 

nematology. Trivedi, P. C. (ed.). 

CBS Publishers and Distributors, 

New Delhi. pp. 226-246. 

Nicholas, W. L. (1984). The biology of 

free-living nematodes. Clarendon 

Press, Oxford, Second Edition. pp. 

251. 

Sharma, R. and Trivedi, P. C. (1991). 

Nematicidal properties of some leaf 

extracts against Meloidogyne incognita. 

Journal of Phytopathology Research 

4 (2), 131-137. 

Tahseen, Q. (2006). The non parasitic 

nematodes. In: Plant Nematology in 

India. Mohilal, N. and Gambhir, R. 

K. (eds.). Parasitology Laboratory, 

Dept. of Life sciences, Manipur 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 66



University, Manipur, India. pp. 159- 

177. 

Wardle, D. A. and Yeates, G. W. 

(1993). The dual importance of 

competition and predation as regulatory 

forces in terrestrial ecosystem: 

evidence from decomposer food 

webs. Oecologia 93, 303-306. 

Gantait 

Yeates, G. W., Bongers, T., De Goede, 

R. G. M., Freckman, D. W. and 

Georgieva, S. S. (1993). Feeding 

habits in soil nematode families and 

genera: An outline for soil ecologists. 

Journal of Nematology 25, 

315-331. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 67



Designing Greener Pharmaceuticals and Practicing 

Green Health Is Required for Sustainability 

Sridevi Chigurupati 1, *, Jahidul Islam Mohammad 2 , Kesavanarayanan Krishnan Selvarajan 

3 , Saraswati Simansalam 4 , Shantini Vijayabalan 1 , Subhash Janardhan Bhore 5 

1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, AIMST University, 

Semeling, 08100, Bedong, Kedah, Malaysia; 2 Department of Pharmacology, Faculty of 

Medicine, CUCMS, Cyberjaya, Selangor, 63000, Malaysia; 3 Department of Pharmacology 

and Toxicology, College of Pharmacy, University of Hail, Hail, Kingdom of 

Saudi Arabia; 4 Department of Clinical Pharmacy, Faculty of Pharmacy, AIMST University, 

Semeling, 08100, Bedong, Kedah, Malaysia; 5 Department of Biotechnology, 

Faculty of Applied Sciences, AIMST University, Semeling, 08100, Bedong, Kedah, Malaysia; 

*Correspondence: sridevi_ch@aimst.edu.my; Tel: +6-04-429-8000 extn., 1284 

Abstract: The global demand for drugs is increasing day-to-day and in the same fashion 

pharmaceutical effluents released from industry adversely affect the environment and human 

health. Discharge of pharmaceutical effluents either directly or indirectly into the environment 

results in substantial pollution. Disposal of such toxic effluents into the environment 

also affects the ecosystems either by direct or indirect pathway. Several researchers 

from environmental biotechnology domain have reported the presence of pharmaceuticals 

in water and soil. To minimize the environmental pollution and to develop the sustainable 

solutions, various methods to convert pharmaceutical effluents into non-toxic and biodegradable 

organic matter has been proposed and discussed by the scientific community. This 

chapter discusses the fate of pharmaceutical waste in the environment, its impact on human 

health, methods adopted to treat effluents before its disposal into the environment, QSAR 

studies in the design of biodegradable agents and future research directions to protect the 

environment for sustainability. 

Keywords: Bioaccumulation; biodegradation; environment; green pharmaceuticals; pharmaceutical 

effluents 


In recent years, improper disposal 

of chemical waste has greatly affected 

both environment and human health. Disposal 

of chemical waste products as such 

into the environment can accumulate and 

affect both the biotic and abiotic factors 

of ecosystems. Hence, the fate of chemicals 

or pharmaceuticals after its disposal 

into the environment should be considered 

by the health care industry and regulatory 

agencies. Ecotoxicological studies 

of chemicals, drugs, chemical intermediates, 

drug metabolites and pharmaceutical 

waste should be performed by the manufacturer 

in order to know their fate in the 

environment. Toxic concentrations of these 

chemical wastes get accumulated in 

seawater, wastewater, sewage sludge’s, 

water bodies and in the living organisms 

that are part of affected ecosystems. Environmental 

pollution caused by humans 

can adversely affect the wellbeing of both 

living and non-living things of ecosystem. 

The health of both animals and humans 

are affected when food materials contaminated 

with the toxic substances are con- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 68


Designing Greener Pharmaceuticals and Practicing Green… 

Chigurupati et al. 

sumed. Constant pollution of the environment 

has disturbed several species as 

the toxic effluents can either destroy them 

or affect their reproductive life cycle 

permanently (Goudie, 2006; Chigurupati 

et.al., 2016). 

Recently, the concerns about environmental 

pollution on endocrine- related 

disease and disorders have been identified. 

Some environmental pollutants (natural 

and anthropogenic) interfere with 

endocrine system and their hormones, 

which can affect both animal and human 

health. These chemicals are called as endocrine 

disruptors. Proper disposal methods 

of chemical wastes should be considered 

to minimize their adverse effects on 

environment. To minimize the risk of 

environmental pollution, biodegradation 

is the method adopted by several chemicals 

and pharmaceutical industries before 

a chemical or pharmaceutical waste has 

been released into the environment. Biodegradation 

is a process whereby the 

complex organic compounds are deteriorated 

aerobically by microbes into simple 

organic matter, for instance CO 2 , H 2 O, 

NH 3 , and CH 4 etc. (OECD Statistics 

Directorate, 2002). 

This chapter highlights the fate 

and impact of pharmaceutical waste in the 

environment and on human health, respectively. 

Various methods used to treat 

effluents, the role of QSAR studies in biodegradation 

and the possible research 

directions to protect the environment 

from toxic effluents are also discussed. 

2. Pharmaceutical biowaste and its 

environmental impact 

In general, man-made biowaste 

causes the environmental pollution and 

can pose a threat to the public health. 

Biowaste originates from various sources 

as stated below (Rand-Weaver et al., 

2013): 

• Formulation and industrial set up. 

• Management and packing of hazardous 

chemicals along with godowns, 

warehouses or tanks used 

in fuel and chemical industries. 

• Carriage i.e. air, road, rail, pipelines, 

and water. 

• Impurities emission such as Nitrogen 

dioxide, Carbon monoxide, 

particulate matter (≤10μm), 

total suspended particulate matter, 

Sulphur dioxide and volatile organic 

compounds such as acetonitrile, 

dichloromethane, ethylene 

glycol, methanol, N,N dimethyl 

formamide and toluene. 

• Various types of effluents (may be 

lethal in nature) those are not effectively 

biodegraded. The effluents 

those are able to go straight 

towards oceans, lakes, rivers, 

streams or other bodies of water. 

The discharge because of overflow, 

including storm water runoffs, 

could likewise be a potential 

risk. 

Figure 1 depicts that how drug 

metabolites or drugs reaches water bodies 

and results in contamination of environment. 

In addition to health benefits, 

pharmaceuticals (drugs, chemicals, pills, 

medicine, sedatives, stimulants, narcotics 

etc.) are also known to cause various 

types of damage and or pollution. The 

risks from the pharmaceuticals could be 

categorized as (Callejaet al., 1994): 

• Ecotoxicological damage elicited to 

the environment. 

• Malignant- contribute to the causality 

of cancer. 

• Persistent-remain hazardous for a long 

time. 

• Bio-accumulative–gathers as it make 

its way up the food chain. 

• Disastrous due to a catastrophe, mishap, 

calamity or grave manifestation 

in any area 

Seepage of the chemicals or 

pharmaceuticals to the environment is 

common from the usage of diagnostics, 

antiseptics and individual care products. 

The pharmaceuticals, their metabolites 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 69




Figure 1: A diagrammatic sketch showing imaginable sources and paths for the pharmaceutical 

deposits to reach into the water bodies including our drinking water. 

and transformation products if not eradicated 

during sewage handling then they 

may come in the aquatic environment and 

in the long run can reach the drinking water 

bodies and may come to our homes 

through water supply (see Figure 1). The 

active amalgams pharmaceuticals or its 

derivatives could enter into environment 

by various routes or non-point sources; 

for instance waste, sewage treatment 

plants (STPs) and landfill effluent or from 

animals treatment facilities (Calamari et 

al., 2003). 

Pharmaceuticals, particularly 

hormones were the primary focus of research 

and public consciousness in the 

1970s. The hormones do not (bio) degrade 

completely. However, the concept 

produced enthusiasm in 1980s. Other 

constituents like heavy metals, aromatic 

polycyclic hydrocarbons, pesticides and 

detergents were also the issue of widespread 

examination during 1980s (Jones 

et al., 2008). 

Deliberate research on contamination 

of environment by various pharmaceuticals 

is undertaken by some research 

teams in few countries. However, the 

presence of approximately 160 various 

drugs and or various pharmaceuticals 

chemical associates is reported in STPs 

effluent and in ground and surface water 

bodies (WHO, 2011). Even in drinking 

water samples, some Active Pharmaceutical 

Ingredients (APIs) were found (WHO, 

2011). They are additionally distinguished 

in the freezing environment. Contrasted 

with the free water, phase analysis 

of APIs is difficult in bio solids and sewage 

sludge for proper comprehension 

(Daughton and Ternes, 1999). 

Medications are useful in aquaculture, 

livestock farming, and veterinary 

and in other sectors of agriculture. However, 

improper practices results in contamination 

of environment; for instance, 

seepage of growth promoters are found in 

contaminated soil. Various types of chemicals 

used in agriculture and those become 

source of environmental contamination or 

biowaste can have an adverse impact on 

human health (Table 1). Veterinary antibiotics 

could end up in the groundwater 

or in the soil. By runoff, it may be cleared 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 70




Table 1: Different types of wastes and their impact on human health (Ferrari et al., 2003; 

Tauxe-Wuersch et al., 2005; Isidori et al., 2006; Khanal et al., 2006) 

No Type of waste Effect on humans 

1. Nuclear waste Hair: The losing of hair occurs with radiation exposure at 200 

rems. 

Brain: Brain cells do not replicate, they damaged directly at 

the exposure is 5,000 rems. 

Thyroid: The thyroid gland is vulnerable to radioactive iodine. 

In adequate amounts, radioactive iodine can destroy all or part 

of the thyroid. 

Blood System: The blood's lymphocyte cell count will be reduced 

once a person is exposed to around 100 rems, leaving 

the victim more susceptible to infection. This is often referred 

to as mild radiation sickness. 

Heart: Immediate damage to small blood vessels and perhaps 

cause heart failure and death directly by intense exposure to 

radioactive material at 1,000 to 5,000 rems. 

Gastrointestinal Tract: Exposure to 200 rems or more radiation 

harm to the intestinal tract lining will cause nausea, 

bloody vomiting and diarrhea. The radiation will begin to destroy 

the cells in the body that goes division rapidly. These including 

blood, GI tract, reproductive and hair cells, and harms 

their DNA and RNA of living cells. 

Reproductive Tract: Since reproductive tract cells multiply 

rapidly, these areas of the body can be damaged at rem levels 

as low as 200. Long-term, some radiation sickness victims will 

become sterile. 

2. Environmental 

waste 

Interaction of humans to agrichemicals is common and results 

in acute and chronic health threats, together with acute and 

chronic neurotoxicity (fumigants, insecticides and fungicides,), 

lung chemical burns (anhydrous ammonia), newborn 

methemoglobinemia and also impairment (paraquat). 

3. Agrichemicals Soil pollution effects causes leukemia and it is danger for 

young children as it can cause developmental damage to the 

brain furthermore it illustrated that mercury in soil increases 

the risk of neuromuscular blockage, causes headaches, kidney 

failure, depression of the central nervous system, eye irritation 

and skin rash, nausea and fatigue. Soil contamination closely 

related to air and water pollution, so numerous things come out 

as similar as caused by water and air pollution. 

into surface water bodies. Wide variety of 

indications of various active ingredients 

in liquid state and in the soil has been established. 

The primary reviews that have 

researched the transfer and the related 

risks have been published only recently 

(Corcoran et al., 2015). 

Antibiotics transpire naturally into 

the soils and the resistance against these 

antibiotics influences the inhabitant’s dynamics 

in soils. The profusion of these 

natural antibiotics was less and appeared 

as restricted to the adjacent surroundings 

(Kümmerer, 2004). The composition of 

the soil-lodging species is found to be influenced 

by antimicrobial agents. Antibiotics 

in the soil appear to support fungal 

development. The circumstances in the 

areas beneath fish farms are critical due to 

more concentrations of antimicrobial 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 71




agents as a result of seepage. Some antimicrobial 

agents are responsible to diminish 

the number of microscopic organisms 

around aquaculture facilities or where the 

seepage of antimicrobial agents was noticeable 

(Oliveira et al., 1995). The degree 

of the impact of ciprofloxacin on microbial 

salt marsh groups was contrarily 

related to the level of sorption to the sediments. 

Even though ciprofloxacin is a 

wide-spectrum antibiotic, its influence on 

sediment microbial organisms was selective 

and seemed to favor Gram-negative 

and sulfate-reducing bacteria. (Sengupta 

et al., 2013). This type of the environmental 

contamination can cause the imbalance 

in ecosystems. 

The impacts of oxytetracycline in 

environmentally applicable concentrations 

on enchytraeids, springtails and 

earthworms have been analyzed. Neither 

one of the antibiotics demonstrated any 

harmfulness against the organisms under 

scrutiny. However, the capability of a pollutant 

to accumulate in organisms must be 

considered seriously. Antibiotics that are 

inadequately water soluble, particularly if 

the bio-concentration factor is between 

500 and 1000 or if the octanol/water distribution 

coefficient crosses the value of 

1000, have a tendency to get accumulated 

in organisms. The substance enrichment 

in organisms has been proved for a few 

antibiotics, e.g., sulphadimethoxine (Call 

et al., 2013). 

3. Biodegradation of pharmaceutical 

products 

Globally, the production of pharmaceuticals 

continues to grow year by 

year, and with its environmental concerns 

pertaining to not just production, but also 

consumer waste and disposal (Wu et al., 

2010). The drugs (pharmaceuticals) consumed 

are eventually either excreted by 

humans and animals and or disposed 

which causes environment pollution. 

Most of the pharmaceuticals produced, 

finally make their way to the ecosystems 

and causes environmental pollution as 

well as affects the flora and fauna at various 

extents. Hence, the dumping of pharmaceuticals 

into the environment is now 

recognized as a serious issue (Boxall, 

2004). 

There are many techniques and or 

processes utilized to overcome this type 

of pollution problem. Few of them are - 

(i) Thermal process; (ii) Chemical process; 

(iii) Irradiation process; (iv) Biological 

process (biodegradation); and (v) 

Mechanical process. 

Among the five above stated processes 

used to minimize the waste or pollution, 

biodegradation techniques (biological 

process) are considered as greener 

technique/technology that involves the 

use of various biological processes to 

overcome the adverse effects of pharmaceutical 

effluents into the environment. 

Biodegradation is a complex and natural 

decomposition process of organic substances 

with the help of biochemical reactions 

of microbes (OECD Statistics 

Directorate, 2002). Although, pharmaceuticals 

signify a minor fraction of all chemicals 

that are dumped into the environment, 

exceptional care must be taken 

about their presence in the environment. 

Because, they are ubiquitous and disseminate 

easily; they act on biological systems; 

they show a lot of side effects in 

non-targeted bodies; and they are known 

to cause chronic toxicity even at low concentrations 

(Enick and Moore, 2007). 

As stated by Jones et al. (2005), 

the occurrence of pharmaceutical contaminants 

in the environment was reported 

first time in late 1970’s (Jones et al, 

2005). The major source of pharmaceuticals 

in the environment is due to their improper 

disposal and leaching from landfill 

sites to natural water (Jones et al., 2004) 

and most often the drugs enter into sewages 

and culminate in water sources. 

There is no any guarantee that the pharmaceutical 

drugs and their effluents are 

removed by merely dumping in sewage 

(Boxall, 2004). Moreover, many times, it 

is proven that the water waste treatment 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 72




plants are inefficient in managing the 

pharmaceutical effluents (Ternes, 1998). 

The main biodegradation techniques 

are - (i) aerobic biodegradation; 

(ii) anaerobic biodegradation, and (iii) 

biodegradation in activated sludge and 

pure culture. 

3.1. Biodegradation by Aerobes 

Aerobic biodegradation is the 

breakdown of organic substances by microorganisms 

in the presences of oxygen. 

The microorganisms used in the process 

require oxygen for growth; hence these 

microbes are called as aerobes. Aerobic 

degradation of hydrocarbons is an example 

of this biological process. Aromatic 

hydrocarbons could be converted into 

natural intermediates; for instance, protocatechuate 

and catechol (Figure 2) 

(Boxall, 2004). Gram negative bacteria 

that possess plasmids produce enzymes 

needed for the aromatic compounds degradation. 

This is possible because of hydroxylation 

reaction which introduces a 

hydroxyl group (-OH) into substrate, in 

this case hydrocarbons (Chatterji, 2003). 

3.2. Biodegradation by anaerobes 

Anaerobic biodegradation is the 

breakdown of organic substances by using 

microbes in the absences of oxygen. 

When the anaerobes are predominant over 

the aerobic microbes then anaerobic biodegradation 

is preferred. It is broadly 

used to treat biodegradable waste and 

wastewater sludge since it gives mass and 

volume reduction. There are four key biological 

and chemical reactions involved in 

anaerobic biodegradation namely, hydrolysis, 

acidogenesis, acetogenesis, and 

methanogenesis (Figure 3). 

3.3. Biodegradation in activated sludge 

and pure culture 

Activated sludge treatment is the 

most well-known process for the 

wastewater treatment. Two disposal 

forms act simultaneously in the activated 

sludge, either the biodegradation by the 

microbes or sorption to solids. 

Example: Compounds like 

mefenamic acid and ibuprofen, the greater 

part of the expulsion is because of biodegradation 

(Jones et al., 2007). The simpler 

a molecule, the higher chance for biodegradation 

to occur (Jones et al., 2004). 

During biodegradation, the xenobiotic 

compounds can be changed in three diverse 

ways namely, hydrophobic, hydrophilic 

and or mineralization transformation. 

Carbamazepine (CBZ) is one of 

the pharmaceuticals with the lowest removal 

efficiency when treated with activated 

sludge treatment (Ternes, 1998). 

Subsequently, CBZ is not influenced either 

by sorption or by the microbes. Most 

positive outcomes with respect to the biodegradation 

of CBZ are by a pure culture. 

4. QSAR studies in biodegradation 

Persistent, bioaccumulative, and 

toxic chemicals show high lipid solubility 

and low water solubility which lead to 

high potential for bioaccumulation. The 

European REACH regulation is the first 

to work on quantitative structure–activity 

relationship (QSAR) models for biodegradation. 

REACH inspires the use of substitutes 

to animal testing which includes 

predictions from QSAR models. QSAR 

Model mainly involves three types of data 

generation - Persistence data generation; 

biodegradation data generation; and toxicity 

data generation. For estimating biodegradation, 

there are few important data 

bases such as Syracuse BIODEG, BIO- 

DEG database, BIOLOG Database, MITI 

Database, and ESIS Database. 

The partially observed persistence; bioaccumulation 

and toxicity data, the expensive 

testing composed with the regulatory 

limitations and the international encouragement 

to minimize animal models motivates 

a greater dependence on QSAR 

models in the biodegradation assessment. 

Several QSAR biodegradation models 

have been established for selected groups 

of structurally similar compounds like: 

specific number of alcohols, chlorophenols, 

n-alkyl phthalates, chloroanisoles, 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 73




Figure 2: A diagrammatic sketch showing degradation of natural aromatic and some xenobiotic 

compounds. 

Figure 3: Schematic diagram showing anaerobic biodegradation of xenobiotics. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 74




meta-substituted anilines and parasubstituted 

phenols etc. Most of the 

Quantitative structure- biodegradation 

relationships (QSBRs) depend on the octanol/water 

partition coefficients, alkaline 

hydrolysis rate constants, van der Waals 

radii and also on molecular connectivity 

indices. Generally, the association between 

physicochemical properties or molecular 

descriptors and biodegradation 

rates were satisfactory; but, generally these 

models have not been used much. Because, 

use of these models is limited to 

the specific classes of chemicals for 

which these models were created. Hence, 

these models are unsuitable to predict biodegradation 

rates for chemicals outside 

of those classes (Howard et al., 1992; 

Dimitrov et al., 2005). 

Therefore, in recent years a very 

rigorous development of new and better 

qualitative and quantitative biodegradability 

models by the usage of new and 

well developed statistical and computational 

methods are developed. Weighted 

molecular fragments are used as model 

descriptors with an idea that molecular 

fragments may have an attractive or hindering 

effect on biodegradability. Numerous 

statistical techniques have been used 

in determining weights: linear and nonlinear 

regression modelling partial least 

square (PLS) and neural networks. The 

results of QSBR studies are strongly influenced 

by the way the molecule is 

fragmented. To overcome this, the Multi- 

CASE approach has been established to 

generate all possible fragments of the 

molecules and to subsequently select the 

statistically most significant ones to suit 

proper results. These fragments are then 

used to develop regression models between 

screened fragments and the final 

results (Banerjee et al., 1984; Niemi et 

al., 1987). 

5. Test guidelines for degradation and 

bioaccumulation studies 

Biodegradability test is performed 

to evaluate the fate of a chemical substance 

in the environment. The information 

obtained from the degradability 

test is mainly required for its hazard or 

risk assessment in aquatic environment. 

The waste water get in contact with the 

sewage treatment microbes and chemical 

substances enter into the oceanic environment. 

The process whereby the chemical 

substance gets bioaccumulated in an 

(aquatic) organism is known as bioaccumulation 

The bioaccumulation degree of a 

chemical substance at a given period is 

the collective data of the competing processes 

of uptake, distribution, transformation 

and excretion. The information on 

degradation and accumulation could be 

obtained by performing appropriate 

standardized tests recommended by the 

OECD (Table 2). 

6. Malaysian government regulations 

on pharmaceutical pollution 

In 2005, the Ministry of Natural 

Resources and Environment (MNRE) has 

published guidelines on the disposal of 

clinical and pharmaceutical wastes. Any 

waste listed in the First Schedule of the 

Environmental Quality (Scheduled 

Waste) Regulations known as Scheduled 

Waste, this type of waste can only be disposed 

in the predetermined premises and 

waste must be treated before its disposal. 

These wastes may possess either inorganic 

or organic components, e.g. discarded 

drugs comprising psychotropic or dangerous 

substances such as carcinogens, mutagens 

or teratogens (SW 403) (Ministry 

of Natural Resources and Environment, 

2009). 

In 2010, the Pharmaceutical Services 

Division initiated ‘Return Your 

Medicines Programme’ in which patients 

were encouraged to return the expired and 

unused drugs to the pharmacies in the 

government hospitals. The aim of this 

programme was to promote safe disposal 

of medications and mitigate the undesired 

adverse effects of APIs on the environment 

as well as living beings. At the end 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 75




Table 2: OECD test guidelines to assess the environmental fate of chemicals 

(OECD Guidelines for the testing of chemicals - OECD, no date) 

Test No Test Title 

301 Ready Biodegradability 

302A Inherent Biodegradability: Modified SCAS Test 

302B Inherent Biodegradability: Zahn-Wellens/ EVPA Test 

302C Inherent Biodegradability: Modified MITI Test (II) 

303 Simulation Test - Aerobic Sewage Treatment - A: Activated Sludge 

Units; B: Biofilms 

304A Inherent Biodegradability in Soil 

305 Bioaccumulation in Fish: Aqueous and Dietary Exposure 

Bioconcentration: Flow-through Fish Test 

306 Biodegradability in Seawater 

307 Aerobic and Anaerobic Transformation in Soil 

308 Aerobic and Anaerobic Transformation in Aquatic Sediment Systems 

309 Aerobic Mineralization in Surface Water – Simulation Biodegradation 

Test 

310 Ready Biodegradability - CO 2 in sealed vessels (Headspace Test) 

311 Anaerobic Biodegradability of Organic Compounds in Digested Sludge: 

by Measurement of Gas Production 

312 Leaching in Soil Columns 

313 Estimation of Emissions from Preservative - Treated Wood to the Environment 

314 Simulation Tests to Assess the Biodegradability of Chemicals Discharged 

in Wastewater 

315 Bioaccumulation in Sediment-dwelling Benthic Oligochaetes 

316 Phototransformation of Chemicals in Water – Direct Photolysis 

317 Bioaccumulation in Terrestrial Oligochaetes 

of year 2016, the Ministry of Health 

(MoH) had disposed nearly RM 2 million 

worth of medicines, most of which were 

obtained through the ‘Return Your Medicines 

Programme’. The most common 

medicines returned under this programme 

were the ones used for treatment of diabetes, 

hypertension, hyperlipidemia and gastritis. 

The common reason for the patients 

to incomplete the course of medicines 

was a change or discontinuation of a 

treatment (Ministry destroys RM 2 million 

worth of expired meds - Nation | The Star 

Online, 2016). 

7. Concluding remarks 

The pharmaceuticals should be 

designed by using green chemistry techniques; 

so that at the end, products can be 

easily broken down into innocuous degradation 

products and do not persist for 

long time in the environment. The organic 

carcinogenic solvents should be replaced 

with green solvents wherever it is possible. 

Industries should more focus on the 

minimum utilization of atoms for the synthesis 

of pharmaceutical products. 

Biological based products are naturally 

less poisonous and promote the principles 

of green chemistry by expanding prospects 

to develop expected processes exploiting 

renewable resources. Ultimately, 

renewable resources do have a potential 

to produce a significant amount of pharmaceutical 

materials which are currently 

produced using either hazardous and or 

nonrenewable materials. Green chemical 

synthetic procedures should replace the 

conventional procedures of producing 

pharmaceuticals. It is not only essential to 

promote the health of people but also for 

the sustainability of the industry and 

planet. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 76



References 

Banerjee, S., Howard, P. H., Rosenberg, 

A. M., Dombrowski, A. E., 

Sikka, H., and Tullis, D. L. (1984). 

Development of a general kinetic 

model for biodegradation and its application 

to chlorophenols and related 

compounds. Environmental Science 

and Technology, 18(6), 416–422. 

Boxall, A. B. A. (2004). The environmental 

side effects of medication. EMBO 

Reports, 5(12), 1110–1116. 

Calamari, D., Zuccato, E., Castiglioni, 

S., Bagnati, R., and Fanelli, R. 

(2003). Strategic survey of therapeutic 

drugs in the rivers Po and Lambro 

in northern Italy. Environmental Science 

and Technology, 37(7), 1241– 

1248. 

Call, D. R., Matthews, L., Subbiah, M., 

and Liu, J. (2013). Do antibiotic residues 

in soils play a role in amplification 

and transmission of antibiotic resistant 

bacteria in cattle populations? 

Frontiers in Microbiology, 4, 193- 

196. 

Calleja, M. C., Persoone, G., and Geladi, 

P. (1994). Comparative acute toxicity 

of the first 50 multicentre evaluation 

of in vitro cytotoxicity chemicals 

to aquatic non-vertebrates. Archives 

of Environmental Contamination 

and Toxicology, 26(1), 69–78. 

Chatterji, A. K. (2003). Introduction to 

environmental biotechnology. Prentice-Hall 

of India. 

Chigurupati, S., Mohammad, J. I., and 

Krishnan, K. (2016). Focus on Environment. 

Focus on Environment, 1, 

74-88. 

Corcoran, J., Winter, M. J., Lange, A., 

Cumming, R., Owen, S. F., and Tyler, 

C. R. (2015). Effects of the lipid 

regulating drug clofibric acid on 

PPARα-regulated gene transcript levels 

in common carp (Cyprinus carpio) 

at pharmacological and environmental 

exposure levels. Aquatic 

Toxicology, 161, 127–137. 


Daughton, C. G., and Ternes, T. A. 

(1999). Pharmaceuticals and personal 

care products in the environment: 

agents of subtle 

change? Environmental health perspectives, 

107(Suppl 6), 907-938. 

Dimitrov, S., Dimitrova, G., Pavlov, T., 

Dimitrova, N., Patlewicz, G., Niemela, 

J., and Mekenyan, O. (2005). 

A stepwise approach for defining the 

applicability domain of SAR and 

QSAR models. Journal of Chemical 

Information and Modeling, 45(4), 

839–849. 

Enick, O. V, and Moore, M. M. (2007). 

Assessing the assessments: pharmaceuticals 

in the environment. Environmental 

Impact Assessment Review, 

27(8), 707–729. 

Ferrari, B., Paxeus, N., Giudice, R. Lo, 

Pollio, A., and Garric, J. (2003). 

Ecotoxicological impact of pharmaceuticals 

found in treated 

wastewaters: study of carbamazepine, 

clofibric acid, and diclofenac. Ecotoxicology 

and Environmental Safety, 

55(3), 359–370. 

Goudie, A. (2006). The Human Impact 

on the Natural Environment: Past, 

Present, and Future. John Wiley and 

Sons. 

Jones, O. A., Voulvoulis, N., and 

Lester, J. N. (2005). Human pharmaceuticals 

in wastewater treatment 

processes. Critical Reviews in Environmental 

Science and Technology, 

35(4), 401–427. 

Howard, P. H., Stiteler, W. M., Meylan, 

W. M., Hueber, A. E., Beauman, J. 

A., Larosche, M. E., and Boethling, 

R. S. (1992). Predictive model for 

aerobic biodegradability developed 

from a file of evaluated biodegradation 

data. Environmental Toxicology 

and Chemistry, 11(5), 593–603. 

Isidori, M., Nardelli, A., Parrella, A., 

Pascarella, L., and Previtera, L. 

(2006). A multispecies study to assess 

the toxic and genotoxic effect of 

pharmaceuticals: furosemide and its 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 77



photoproduct. Chemosphere, 63(5), 

785–793. 

Jones, O. A. H., Dondero, F., Viarengo, 

A., and Griffin, J. L. (2008). Metabolic 

profiling of Mytilus galloprovincialis 

and its potential applications 

for pollution assessment. Marine 

Ecology Progress Series, 369, 169– 

179. 

Jones, O. A. H., Voulvoulis, N., and 

Lester, J. N. (2004). Potential ecological 

and human health risks associated 

with the presence of pharmaceutically 

active compounds in the 

aquatic environment. Critical Reviews 

in Toxicology, 34(4), 335–350. 

Jones, O. A. H., Voulvoulis, N., and 

Lester, J. N. (2007). The occurrence 

and removal of selected pharmaceutical 

compounds in a sewage treatment 

works utilising activated sludge 

treatment. Environmental Pollution, 

145(3), 738–744. 

Khanal, S. K., Xie, B., Thompson, M. 

L., Sung, S., Ong, S.-K., and Van 

Leeuwen, J. (2006). Fate, transport, 

and biodegradation of natural estrogens 

in the environment and engineered 

systems. Environmental Science 

and Technology, 40(21), 6537– 

6546. 

Kümmerer, K. (2004). Resistance in the 

environment. Journal of Antimicrobial 

Chemotherapy, 54(2), 311–320. 

The Star Online. (2016). Ministry destroys 

RM 2 million worth of expired 

meds – Nation. 

Ministry of Natural Resources and Environment. 

(2009). Guidelines on 

The Handling and Management of 

Clinical Wastes In Malaysia, 1–29. 

Niemi, G. J., Veith, G. D., Regal, R. R., 

and Vaishnav, D. D. (1987). Structural 

features associated with degradable 

and persistent chemicals. 

Environmental Toxicology and 

Chemistry, 6(7), 515–527. 


OECD Statistics Directorate. (2002). 

OECD glossary of statistical terms - 

household definition. 

Oliveira, R. da G. B., Wolters, A. C., 

and Elsas, J. D. van. (1995). Effects 

of antibiotics in soil on the population 

dynamics of transposon Tn5 carrying 

Pseudomonas fluorescens. 

Plant and Soil, 175(2), 323–333. 

Rand-Weaver, M., Margiotta-Casaluci, 

L., Patel, A., Panter, G. H., Owen, 

S. F., and Sumpter, J. P. (2013). 

The read-across hypothesis and environmental 

risk assessment of pharmaceuticals. 

Environmental Science 

and Technology, 47(20), 11384- 

11395. 

Sengupta, S., Chattopadhyay, M. K., 

and Grossart, H. P. (2013). The 

multifaceted roles of antibiotics and 

antibiotic resistance in nature. 

Frontiers in microbiology, 4,1- 

13. 

Tauxe-Wuersch, A., De Alencastro, L. 

F., Grandjean, D., and Tarradellas, 

J. (2005). Occurrence of several 

acidic drugs in sewage treatment 

plants in Switzerland and risk assessment. 

Water Research, 39(9), 

1761–1772. 

Ternes, T. A. (1998). Occurrence of 

drugs in German sewage treatment 

plants and rivers. Water Research, 

32(11), 3245–3260. 

W. H. O. (2011). Pharmaceuticals in 

Drinking-water Public Health and 

Environment Water, Sanitation, Hygiene 

and Health, 

(WHO/HSE/WSH/11.05). 

Wu, M., Atchley, D., Greer, L., 

Janssen, S., Rosenberg, D., and 

Sass, J. (2010). Dosed Without Prescription: 

Preventing Pharmaceutical 

Contamination of Our Nation's 

Drinking Water (Natural Resources 

Defense Council). URL: 

http://docs.nrdc.org/health. 

© 2017 by the authors. Licensee, Editors and AIMST University, Malaysia. This article 

is an open access article distributed under the terms and conditions of the Creative 

Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 78


Achievements, Challenges and Perspectives BiotechSustainability (2017), P79-87 

Clonal Propagation of a High Value Multipurpose 

Timberline Tree Species Quercus semecarpifolia Sm. of 

West Himalaya, India 

Aseesh Pandey 1, 2 and Sushma Tamta 2, * 

1 Govind Ballabh Pant National Institute of Himalayan Environment and Sustainable Development, 

Sikkim Unit Pangthang 737101, East Sikkim, Gangtok, India; 2 Plant Tissue Culture 

Laboratory, Department of Botany, DSB Campus, Kumaun University, Nainital 

263001, Uttarakhand, India; *Correspondence: sushmatamta@gmail.com; Tel: 

+918126966284 

Abstract: Tree species across the alpine timberline are most vulnerable to climate change 

and requires immediate attention for their conservation. Quercus semecarpifolia Sm. forms 

the extensive forests in alpine timberline region and it is among the highly exploited tree 

species of western Himalaya. Attempts were made to develop in vitro clonal propagation 

procedure for Q. semecarpifolia. Nodal explants, derived from a single mature tree growing 

in natural stands, were cultured on different nutrient media for the optimization of nutrient 

medium. Woody plant (WP) medium supplemented with 8.88 µM 6-benzylaminopurine 

(BA) and 0.72 µM Gibberellic acid (GA 3 ) has yield significant shoot multiplication response. 

For root induction a two- step method was applied. Microshoots treated with 100 

µM indole-3-butyric acid (IBA) for 24h, showed the significantly higher rooting response. 

Present study leads a way forward to conservation and sustainable utilization of this high 

value tree species. Moreover, further strengthening and optimization efforts are required to 

develop a low cost procedure for the development of the nursery of clonally propagated Q. 

semecarpifolia plants. Thus, the threat-mitigation strategies can be developed through the 

introduction of these in vitro raised plants into the natural forest. 

Keywords: Clonal propagation; nodal explant; Quercus semecarpifolia; timberline 


Alpine timberline is the most sensitive 

ecotone to changing climate (Smith 

et al., 2009). The regeneration of tree 

species in this region is expected to affect 

adversely in coming years. Ample evidences 

of a typical regeneration of timberline 

tree species are already documented 

by various researchers across timberlines 

of different mountains located in 

diverse geographical provinces (Harsch et 

al., 2009; Walck et al., 2011; Kirdyanov 

et al., 2012). The global mean surface 

temperature has risen by 0.6ºC over the 

last 100 years (Wang et al., 2016), and the 

level of precipitation in Northern Hemisphere 

has increased by approximately 5- 

10% over mid and high latitudes (IPCC, 

2013). Due to diverse climatic conditions, 

topography, and precipitation regimes the 

composition of timberline varied across 

the globe and dominated by different tree 

species. The Himalaya harbors a unique 

mountain system and represents the highest 

alpine timberline of the world. The 

timberline of Indian Himalayan region 

(IHR) is dominated by different tree species 

from east to west. The IHR consists 

of more than 35 species of genus Quercus 

(Singh and Singh 1992). The genus Quercus 

is represented by five evergreen tree 

species (Q. leucotrichophora, Q. floribunda, 

Q. glauca, Q. lanuginosa and Q. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 79


Clonal Propagation of Quercus semecarpifolia … 

semecarpifolia) and one deciduous exotic 

tree species (Q. serrata) in Uttarakhand, 

Western Himalaya, India (Pandey and 

Tamta, 2012). Among these, Q. 

semecarpifolia (Kharsu) possess highest 

elevational range (2500-3300m asl) and 

forms timberline in the region (Singh and 

Singh, 1992). 

The world's highest timberline is 

also utilized by local peoples for their daily 

needs (agricultural tools, fuel, food, 

fodder, spiritual rituals etc.) and livelihoods 

(agriculture, silk worm rearing, 

livestock rearing, etc.). This dependency 

creates enormous pressure on multipurpose 

evergreen tree species like Q. 

semecarpifolia. This species is considered 

as the oldest and overexploited plant of 

sub-alpine zones (Singh et al., 2010). The 

green leaves of Q. semecarpifolia are 

used in tasar silk-worm rearing and livestock 

fodder; bark and galls for tannin; 

dried branches and boles are used in the 

preparation of agricultural implements 

and used as fuel wood (Tamta et al., 

2008; Pandey, 2013). This high magnitude 

of human pressure along with low 

regeneration (Bisht et al., 2012), short 

viability of seeds (Pandey and Tamta, 

2013), and unavailability of a decent seed 

crop every year (Pandey 2013) could be 

precariousfor thenatural regeneration of 

this species in changing climate. Considering 

the role of Q. semecarpifolia in the 

economy as well as ecology of Himalaya, 

immediate attention is required for the 

conservation of this species through sustainable 

utilization of its genetic resources. 

The ex situ conservation through 

plant tissue culture technique is extensively 

used method for the conservation and 

sustainable harnessing of use values of 

threatened and high value woody species 

such as Citrus sinensis (Pandey and Tamta, 

2016); Berberis aristata (Brijwal et al., 

2015); Quercus serrata (Pandey and 

Tamta, 2014); Berberis chitria (Pandey et 

al., 2013); Quercus semecarpifolia (Tamta 

et al., 2008) and other Quercus species 

(Wilhelm, 2000). Based on the source of 

explant, the plant tissue culture technique 

Pandeyand Tamta 

is comprises of various propagation 

methods. However, the clonal propagation 

through nodal explants derived from 

mature trees growing in natural forest is 

tedious, still one of the best method to 

develop true-to-type plants of desired 

quality. In present study attempts were 

made to develop clonal propagation procedure 

for Q. semecarpifolia using nodal 

explant derived from mature elite tree 

growing in Q. semecarpifolia dominated 

forest, west Himalaya India. 

2. Material and methods 

2.1. Plant material 

Nodal explants were collected 

from the mature tree growing in China 

peak forest area (2619m asl; 29 27N and 

79 29E) of district Nainital, Uttarakhand, 

India. Twigs (10-15 cm) were collected 

from different parts of mature trees 

(from stump sprouts, or from juvenile 

parts) and brought to the Plant Tissue 

Culture laboratory, Department of Botany, 

D.S.B. campus Kumaun University, 

Nainital, India in a mini-chillier (-10C; 

Genei, Banglore). In laboratory, the twigs 

were dipped in water and kept in room 

temperature for 1h. Within same day, the 

twigs were defoliated and cut in to small 

segments (1.0-1.5cm). These nodal segments 

having at least one dormant bud 

were used as explant. 

2.2. Chemicals, glassware and culture 

conditions 

Each chemical used during the experiment 

was of analytical grade and purchased 

from Himedia, Pvt. Ltd. Mumbai, 

India except labolene (Qualizen, India) 

and Bavistin (purchased from local market). 

The plant growth regulators were 

procured from Duchefa Biochemie, The 

Netherlands. All the glasswares used during 

the experiment were purchased from 

Borosil India. Cultures were maintained 

at 25±1°C under a 16h/8h light/dark photoperiod 

with an irradiance of 42 μmol 

m −2 s −1 inside and 60 μmol m −2 

s −1 outsidethe culture vessels/ flasks pro- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 80



vided by cool fluorescent tubes (40 W; 

Philips from Saveer Biotech Limited, 

New Delhi, India). 

2.3. Explant preparation 

Nodal explant were initially 

washed in running tap water for 10 min 

and then immersed in detergent solution 

(labolene, 0.1%, v/v; 20 min), and rinsed 

thoroughly with distilled water. Washed 

explants were treated with fungicide (bavistin, 

0.5%, w/v; 30 min) with gentle 

shaking. Those treated explants were introduced 

to laminar air flow cabinet, 

where after 5 rinses of autoclaved double 

distilled water, nodal segments were surface 

disinfected with mercuric chloride 

(HgCl 2, 0.1%, w/v; 10 min). This treatment 

was followed by subsequent 5 rinses 

of autoclaved double distilled water (2 

min each). The exposed ends of nodal 

segments were excised out prior to inoculation. 

To check the contamination nodal 

explants were cultured in WA: water agar 

medium for one week (Figure 1a). 

2.4. Shoot induction and nutrient medium 

selection 

To select the nutrient medium for 

shoot induction and multiplication, contamination-free 

nodal segments were inoculated 

either in full strength MS: Murashige 

and Skoog (1962) or WP: Lloyd 

and McCown (1980) nutrient medium. 

Each medium was supplemented with 6- 

benzyleamenopurene (BA, 4.44 µM) and 

activated charcoal (AC, w/v, 20mg l -1 ) to 

monitor the shoot induction responses. 

2.5. Shoot multiplication 

After 30 day on shoot induction 

media, explants with positive shoot induction 

response were transferred to shoot 

multiplication media (Figure 1). Different 

concentrations of cytokinin (BA, 4.44- 

22.20 μM) alone or in combination with 

IAA (1.43 μM) or GA 3 (0.73-1.44 μM) 

were tested for shoot multiplication responses 

in WP full strength nutrient medium. 

The sucrose concentration was 3.0% 


(w/v) and the media was solidified with 

0.8% agar (w/v) and/or 0.24% clarigel 

(w/v). Besides, casein hydrolysate (CH, 

w/v, 500mg l -1 ) and activated charcoal 

(AC, w/v, 20mg l -1 ) were also incorporated 

in to each shoot multiplication medium. 

Each experiment consisted of 12 

explants and repeated twice. Subculturing 

was carried out at 6 week interval and data 

on shoot number and shoot length were 

recorded during subculture. 

Explant selection experiment was 

carried out in culture vessels (50 ml volume, 

20 ml medium per tube). The contamination-free 

explants were transferred 

to conical flasks (250 ml volume, 100 ml 

medium per flask) for further growth and 

development. WP basal media devoid of 

growth regulators served as control in all 

experiments. All culture vessels and 

flasks were plugged with non-absorbent 

cotton plugs and sterilized at 121°C for 

15 min. The pH was adjusted to 5.7- 5.8 

by 0.1M NaOH or HCl before autoclaving 

at 1.06 kg cm -2 (121°C) for 20 min. 

2.6. Rooting of microshoots 

Microshoots (2.0-3.0 cm, with 

well-developed leaves) were introduced 

to a two step-rooting procedure; as described 

by Pandey and Tamta et al (2012). 

Briefly, during the first step excised microshoots 

were cultured in full strength 

WP media supplemented with different 

concentrations of indole-3-butyric acid 

(IBA 50.0 or 100.0 M) for 24 or 48 h 

and cultures were placed in a dark during 

this step (Table 2). In the second step these 

treated shoots were transferred to PGRfree 

half-strength WP medium, solidified 

with clarigel (0.25%) and exposed to 16 h 

photoperiod. The percentage of root formation, 

lengths of the formed roots and 

length of longest root were recorded after 

4 weeks of incubation in PGR-free halfstrength 

WP medium. 

2.7. Acclimatization 

After 4 week of transfer to PGRfree 

medium, these shoots with roots were 

taken out from the culture flasks and wa- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 81




Figure 1: In vitro propagation of Quercus semecarpifolia. a) Explant inoculation in WA 

medium to check contamination; b) contamination free explants having positive response; 

c-d) Shoot proliferation in WP supplemented with (BA 4.44 M); e) shoot elongation in 

BA+GA 3 (4.44 M +1.45 M); f) shoot multiplication in BA+GA 3 (8.88 M +1.45 M); g) 

root induction in IBA 100 M for 24 h. 

-shed gently with distilled water to remove 

traces of clarigel. After recording of 

data, plantlets were transferred to thermacol 

pots (8 cm width and 10 cm height) 

containing soil and farmyard manure (3:1, 

w/w). These plants were placed inside 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 82



growth chamber under16 h photoperiod 

(60μmol m −2 s −1 ) at 25±2°C temperatures 

and 60% relative humidity. Plants were 

watered on alternate days with 1/4 basal 

WP medium devoid of sucrose and acclimatized 

over a period of 4 week. 

2.8. Statistical analysis 

Experiments were performed in a 

completely randomized design to determine 

the effect of treatments and concentrations 

on plant vigour. Data presented as 

mean values ± standard error (SE) and 

collected from three independent experiments. 

The statistical analyses were performed 

using SPSS (Statistical Package 

for Social Science, version 20). Level of 

significance was determined by analysis 

of variance (ANOVA) and statistical significance 

mean values were grouped by 

using Duncan’s multiple range post hoc 

test (p



BA+GA 3 (8.88+0.72M) (Figure 1e). 

Though the number of shoots were significantly 

(p



used; BA reported to be the best plant 

growth regulator in Quercus shumardii 

for shoot multiplication (Bennett and Davies, 

1986). 20 μM BA was found to be 

best for adventitious shoot induction and 

multiplication of individual shoots of Q. 

semecarpifolia (Tamta et al., 2008). In 

contrast to multiplication from petiolar 

tube of Q. semecarpifolia the higher concentrations 

of BA were not found to be 

the best concentration although it induced 

highest number of shoots/bud but the 

length of shoots was reduced. Shoot multiplication 

was followed by rooting. Earlier 

study in Q. semecarpifolia suggests 

that IBA is the better responded auxin 

(root inducer) than NAA and a two steprooting 

procedure resulted 100.0% rooting 

response, without the formation of basal 

callus (Tamta et al., 2008). In present 

study two step method was used and 

about 88.89percent rooting was recorded 

within 20 days of culture. Similar method 

of rooting has also been reported earlier in 

other Quercus species such as Q. suber 

(Manzanera and Pardos, 1990) and Q. 

leucotrichophora and Q. glauca (Purohit 

et al., 2002b). During acclimatization the 

semi-rooted microshoots were not able to 

survive. The survival rate of microshoots 

was poor due to undeveloped rooting system 

to survive ex vitro conditions. 

5. Conclusion 

Results of present study are encouraging 

and could be useful in developing 

a rapid clonal propagation method for 

this important multipurpose tree species. 

However, the low rate of multiplication 

and poor survival rate of plantlets in the 

soil are the main challenges at the present. 

Therefore, to address the low regeneration 

and to improve survival rate of Q. 

semecarpifolia further research is required. 



The authors express their gratitude 

to G.B. Pant National Institute of Himalayan 

Environment and Sustainable Development, 

Almora, Uttarakhand, India for 

providing financial assistance through 

integrated eco-development research programme 

(IERP) for Indian Himalayan region 

(IHR) during 2009-2011. The head 

department of botany, D.S.B. Campus and 

department of biotechnology, Bhimtal 

Campus of Kumaun University, Nainital, 

Uttarakhand India are highly acknowledged 

for providing necessary facilities 

for experimentation. 

References 

Bennet, L.K. and Davies, F.T. (1986). In 

vitro propagation of Quercus shumardii 

seedlings.Horticulture Science 

21(4), 1045-1047. 

Bisht, H., Prakash, V. and Nautiyal, 

A.R. (2012). Factors Affecting Regeneration 

Potential of Quercus 

semecarpifolia, Smith: A Poor Regenerated 

Oak of Himalayan Timberline.Research 

Journal of Seed 

Science 5, 63-70. doi: 

10.3923/rjss.2012.63.70. 

Bonga, J. and Aderkas, P. (1992).In 

vitro culture in trees, Kluwer Academic 

Publ, Dordrecht-Boston- 

London. pp. 207. 

Brijwal, L., Pandey, A. and Tamta, S. 

(2015). In vitro propagation of the 

endangered species Berberis aristata 

DC. via leaf-derived callus. In 

Vitro Cellular and Developmental 

Biology-Plant 51(6), 637-647. 

Chalupa, V. (1987). Effect of benzylaminopurine 

and thidiazuron on in 

vitro shoot proliferation of Tilia 

cordata Mill., Sorbus aucuparia L. 

and Robinia pseudoacacia L. Biologia 

Plantarum 29, 425-429. 

Harsch, M.A., Hulme, P. 

E., McGlone, M. S. and Duncan, 

R. P. (2009). Are tree lines advancing? 

A global meta-analysis of treeline 

response to climate warming.Ecology 

Letters 12,1040–1049. 

IPCC, (2013). Climate Change 2013: The 

Physical Science Basis. Contribu- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 85




tion of Working Group I to the Fifth 

Assessment Report of the Intergovernmental 

Panel on Climate Change, 

Cambridge, UK. pp. 1535. 

Kirdyanov, A. V., Hagedorn, F., 

Knorre, A. A., Fedotova, E. 

V.,Vaganov, E. A., Naurzbaev, M. 

M., Moiseev, P. A. and Rigling, A. 

(2012). 20th century treeline advance 

and vegetation changes along 

an altitudinal transect in the Putorana 

Mountains, northern Siberia. Boreas 

41, 56–67. 

Linington, I.M. (1991). In vitro propagation 

of Dipterocarpusintricatus. 

Plant Cell Tissue Organ Culture 27, 

81–88. 

Lloyd, G. and McCown, L.B. (1980). 

Commercially feasible micropropagation 

of mountain laurel, Kalmia 

latifolia, by use of shoot-tip culture. 

Comb. Proc. International Plant 

Propagation Society 30, 421-427. 

Manzanera, J.A. and Pardos, J.A. 

(1990). Micropropagation of juvenile 

and adult Quercus suber 

L.Plant Cell Tissue Organ Culture 

21, 1-8. 

Mensuali-Sodi, A., Panizza, M., Serra, 

G. and Tognoni, F. (1993). Involvement 

of activated charcoal in 

the modulation of abiotic and biotic 

ethylene levels in tissue-cultures. 

Scientia Horticulture54, 49–57. 

Murashige, T. and Skoog, F. (1962). A 

revised medium for rapid growth 

and bioassays with tobacco tissue 

cultures.Physiologia Plantarum 15, 

473-497. 

Pan, M. J. and Staden, J. V. (1998). The 

use of charcoal in in vitro culture–A 

review. Plant growth regulation 

26(3), 155-163. 

Pandey, A. and Tamta, S. (2012). Influence 

of kinetin on in vitro rooting 

and survival of banj oak (Quercus 

leucotrichophora L.). African Journal 

of Biotechnology 62 (11), 

12538-12545. 

Pandey, A. (2013). Propagation of two 

species of oaks using in vitro methods. 

Ph.D. thesis submitted to 

Kumaun University Nainital, Uttarakhand, 

India. 

Pandey, A. and Tamta, S. (2013). Effect 

of pre-sowing treatments on seed 

germination in Quercus serrata 

Thunb. andQuercus semecarpifolia 

Sm. International Journal of Biodiversity 

and Conservation 5(12), 

791-795. 

Pandey, A. and Tamta, S. (2014).In 

vitro propagation of the important 

tasar oak (Quercus serrata Thunb.) 

by casein hydrolysate promoted 

high frequency shoot proliferation. 

Journal of sustainable forestry 

33(6), 590-603. 

Pandey, A. and Tamta, S. (2016). Efficient 

micropropagation of Citrus 

sinensis (L.) Osbeck from cotyledonary 

explants suitable for the development 

of commercial variety. African 

Journal of Biotechnology 

15(34), 1806-1812. 

Pandey, A. Brijwal, L. and Tamta, S. 

(2013).In vitro propagation and phytochemical 

assessment of Berberis 

chitria: An important medicinal 

shrub of Kumaun Himalaya, India. 

Journal of Medicinal Plants Research7(15), 

930-937. 

Purohit, V.K., Palni, L.M.S., Nandi, 

S.K. and Rikhari, H.C. (2002a). In 

vitro plant regeneration through cotyledonary 

nodes of Quercus floribunda 

Lindl. ex A. Camus (Tilonj 

oak), a high value tree species of 

central Himalaya. Current Science 

833, 101-104. 

Purohit, V.K., Palni, L.M.S., Nandi, 

S.K., Vyas, P. and Tamta, S. 

(2002b). Somatic embryogenesis in 

Quercus floribunda, an important 

Central Himalayan oak. In: Role of 

plant tissue culture in biodiversity 

conservation and economic development. 

Nandi SK, Palni LMS, 

Kumar A (Ed) Gyanodaya Prakashan, 

Nainital, pp. 41-52. 

Singh, G. and Rawat, G.S. (2010). Is the 

future of oak (Quercus spp.) forest 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 86



safe in the Western Himalaya? Current 

Science 98, 1420-1421. 

Singh, J.S. and Singh, S.P. (1992). Forests 

of Himalaya: Structure, functioning 

and impact of man. 

Gyanodaya Prakashan, Nainital, 

pp.294. 

Smith, W. K., Germino, M. J., Johnson, 

D. M. and Reinhardt, K. (2009). 

The altitude of alpine treeline: a 

bellwether of climate change effects. 

The Botanical Review 75(2), 

163-190. 

Tamta, S., Palni, L.M.S., Purohit, V.K. 

and Nandi, S.K. (2008). In vitro 

propagation of brown oak (Quercus 

semecarpifolia Sm.) from seedling 

explants.In Vitro Cellular and Development 

Biology-Plant44(2),136- 

141. 

Vengadesan, G. and Pijut, P.M. 

(2009).In vitro propagation of 

northern red oak (Quercus rubra 

L.). In Vitro Cellular and Development 

Biology-Plant45, 474-482. 

Vieitez, A. M., Pintos, F., San-Jose, M. 

and Ballester, A. (1993). In vitroshoot 

proliferation determined by 

explant orientation of juvenile and 

mature Quercus rubra L. Tree Physiology 

12, 107-117. 


Vyas, S.C. (1984). Systemic fungicides, 

Tata McGraw hill Publishing Co. 

Ltd. New Delhi. 

Walck, J. L., Hidayati, S. N., Dixon, K. 

W., Thompson, K.E.N. and 

Poschlod, P. (2011). Climate 

change and plant regeneration from 

seed. Global Change Biology 17, 

2145–2161. 

Wang, B., Chen, T., Xu, G., Liu, X., 

Wang, W., Wu, G. and Zhang, Y. 

(2016). Alpine timberline population 

dynamics under climate change: 

a comparison between Qilian juniper 

and Qinghai spruce tree species 

in the middle Qilian Mountains of 

northeast Tibetan Plateau. Boreas. 

10.1111/bor.12161. ISSN 0300- 

9483. 

Wilhelm, E. (2000). Somatic embryogenesis 

in oak (Quercus spp.). In Vitro 

Cellular and Developmental Biology-Plant 

36(5), 349-357. 

Yam, T.Y., Ernst, R., Arditti, J., Nair, 

H. and Weatherhead, M.A. 

(1990). Charcoal in orchid seed 

germination and tissue culture media: 

a review. Lindleyana5, 256– 

265. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 87



Spent Mushroom Substrate of Hypsizygus ulmarius: A 

Novel Multifunctional Constituent for Mycorestoration 

and Mycoremediation 

Padmavathi Tallapragada 1, * and Ranjini Ramesh 2 

1 Department of Microbiology, Centre for Post Graduate Studies, Jain University, 18/3,9 th 

Main, Jayanagar 3 rd Block, Bangalore, India; 2 Department of Environmental Science, 

Mount Carmel College, Autonomous, 58, Palace Road, Vasanthnagar, Bangalore, India; 

*Correspondence: vam2010tpraviju@gmail.com / t.padmavathi@jainuniversity.ac.in; Tel: 

+91 9448533337 

Abstract: ‘Spent Mushroom Substrate’ (SMS) is a composted growing medium that results 

from the mushroom growing process. The spent substrate remains after harvesting the 

mushrooms, which is entangled with innumerable mushroom threads (collectively referred 

as ‘mycelia’), would have been biochemically modified by the mushroom enzymes into a 

simpler and more readily digestible form, which could then be used in ‘mycorestoration’ 

and ‘mycoremediation’. Mushroom mycelia can produce a group of complex extracellular 

enzymes that can degrade and utilize the lignocellulosic wastes found in nature, which also 

reduces their potential for pollution. It has been revealed recently that mushroom mycelia 

can play a significant role in the restoration of damaged environments. Saprotrophic, endophytic, 

mycorrhizal and even parasitic fungi or mushrooms can be used in ‘mycorestoration’, 

which can be performed in four different ways: ‘mycofiltration’ (using mycelia to filter 

contaminated water), ‘mycoforestry’ (using mycelia to restore degraded forests), ‘mycoremediation’ 

(using mycelia to eliminate toxic wastes from soil and water) and ‘mycopesticides’ 

(using mycelia to control insect pests). These methods represent the potential 

to create a clean ecosystem, where no damage will be left after fungal implementation. ‘Applied 

Mushroom Biology’ can not only convert this huge amount of lignocellulosic wastes 

into human food but also can produce notable nutraceutical products, which have several 

health benefits and it is discussed in this chapter. 

Keywords: Applied mushroom biology; Hypsizygus ulmarius; mycoremediation; mycorestoration; 

spent mushroom substrate 


Soils in the tropical regions of the 

world are fragile, contain very less organic 

matter and are prone to severe degradation, 

especially with increased deforestation 

and loss of topsoil. These attributes 

of tropical soils put constraints on foodcrop 

production in these regions of high 

and dense human populations. In the last 

few decades, Green Revolution practices 

like using pesticides, synthetic fertilizers 

and high-yielding varieties have been followed 

to overcome the constraints (Dalgaard 

et al., 2003). With the help of these 

technologies, there has been a world-wide 

doubling of food crop production, but at 

the cost of environmental degradation of 

soil and water quality, reduction in biodiversity 

and suppression of ecosystem 

functions (Vance, 2001). Today, more 

than one billion people lack in food security 

and many village communities in these 

areas are continuously affected by a 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 88


Spent Mushroom Substrate of Hypsizygus ulmarius 

steady reduction of food grains. In addition, 

the increase in industrialization has 

polluted our environment with chemicals 

and toxins of various kinds (Singh et al., 

2011) Most contaminated sites usually 

contain a mixture of non biodegradable 

persistent compounds, which increase the 

difficulties of remediation. This is due to 

the intensification of agriculture, range of 

crops grown and the diversity of manufacturing 

industries. The excess usage of 

chemical fertilizers has also contributed 

to the deterioration of the environment, 

with soil degradation, loss of soil fertility 

and agricultural productivity being the 

main consequences (Khan and Ishaq, 

2011). 

For improving the long-term sustainability 

of industry and agriculture, 

emphasis should be on the holistic management 

of natural resources. Microorganisms 

can control pollution and pests, 

maintain the fertility of soil and enhance 

plant growth, with no major adverse effects 

on the environment or other nontarget 

organisms (Gomathi and Ambikapathy, 

2011). These types of mechanisms 

rely on stimulating the growth of specific 

species of micro-organisms or mixtures of 

microflora native to the contaminated 

sites and are thus, able to remediate the 

area more easily and efficiently (Kumar et 

al., 2010). 

Recent research has favored the 

techniques of ‘bioremediation’ for cleaning 

up the above types of sites, as it is 

both environment-friendly and of relatively 

low-cost (Sasek, 2003). Bioremediation 

is the addition of biological agents, mainly 

microbes like yeast cells, fungi or bacteria 

to detoxify the contaminated soil and 

water. When fungi are specifically used, it 

is known as ‘mycoremediation’. Ligninolytic 

basidiomycete fungi such as Phanerochaete 

chrysosporium, Pleurotus ostreatus, 

Lentinula edodes, etc. are well 

known mycoremediation agents. These 

microbes use the xenobiotics to be degraded 

as nutrients or as sources of energy 

(Tang et al., 2007). Fungi play an important 

role in bioremediation, besides 

Tallapragada and Ramesh 

being utilized extensively in industry, agriculture, 

medicine, food and textile industries 

(Prabhakaran et al., 2011). 

Hypsizygus ulmarius, the ‘Elm 

Oyster Mushroom’ (Figure 1), is a new 

variety of edible mushroom, developed by 

the Indian Institute of Horticultural Research 

(IIHR), Bangalore - 

www.iihr.res.in. It is a type of basidiomycete, 

also known as ‘white rot fungi’, of 

which there are about 1,400 known species. 

It can be commercially cultivated by 

solid-state fermentation method, using 

agricultural wastes such as paddy straw, 

coconut husk, tea and saw dust, among 

others. Mushroom cultivation is environment-friendly, 

in addition to providing a 

cost-effective source of food protein for 

vegetarians and a source of income for 

rural women (Ahmed et al., 2009). Mushrooms 

are a good source of vitamins and 

minerals, while having low content of 

fats, carbohydrates and dietary fiber. With 

their nutritional value, mushrooms can 

reduce malnutrition in the rural poor to a 

large extent, and are also effective in reducing 

the occurrence of life-style diseases 

like hypercholesterolemia, hypertension, 

diabetes and cancer (Alam et al., 

2007). 

Figure 1: Hypsizygus ulmarius: The Elm 

Oytser Mushroom, growing naturally on 

the bark of Elm trees 

(http://www.mushroomexpert.com/hypsiz 

ygus_ulmarius.html) 

‘Spent mushroom substrate’ 

(SMS) is the by-product of mushroom 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 89




cultivation, and contains the fungal mycelium, 

fermented substrate, residues of inorganic 

nutrients and secreted enzymes 

such as ligno-cellulases, proteases and 

peroxidases (Medina et al., 2009). SMS 

is also a rich source of carbon, nitrogen 

and other nutrients, and can be added to 

enhance crop growth and maintain soil 

fertility. It contains a consortium of bacteria 

and fungi which can mediate the formation 

and weathering of soil, nutrient 

and water mobilization, nitrogen fixation 

and denitrification processes. The fungal 

mycelium on the spent substrate is similar 

to ‘Arbuscular mycorrhizal fungi’ – AMF 

(Jonathan et al., 2013). AM fungi especially 

function to mobilize water and 

phosphorus for plants (Manimegalai et 

al., 2011). ). Pleurotus florida is a known 

‘mycorestoration agent’, as it improves 

soil fertility by phosphate solubilization, 

increases aeration and water movements 

through soil and enhances plant growth 

(Kumar et al., 2010). 

White rot fungi are some of nature’s 

most efficient lignin degraders from 

the microbial world, due to their ability to 

produce several kinds of lignin and phenol-degrading 

enzymes such as laccases 

and peroxidases. Laccases are coppercontaining, 

glycosylated polyphenol oxidases. 

Their broad substrate specificity 

increases their significance in industrial 

and biotechnological applications such as 

biomechanical pulping of cellulosic matter, 

bleaching of pulp and degradation of 

dyes, chloro-phenols and a variety of xenobiotic 

and aromatic compounds, mainly 

by reduction of oxygen to water (Patel et 

al., 2008). Extracellular laccase is usually 

secreted into the medium in small quantities. 

Its production is affected by typical 

fermentation factors such as media composition, 

carbon-nitrogen ratio of growth 

media, pH, temperature and diffusion of 

oxygen into the media (Revankar and 

Lele, 2006). Their production is stimulated 

by the addition of inducers such as 

phenolic and aromatic compounds like 

catechol, guaiacol, veratryl alcohol, ferrulic 

acid, 2,6-dimethoxyphenol, etc (Ikehata 

et al., 2004). 

Phenol, also known as carbolic acid, 

is a highly toxic element that is added 

in the manufacture of resins, herbicides 

and various other industrial processes 

(Amara and Salem, 2010). It is one of the 

most persistent chemicals, with high toxicity 

even at low concentrations, and is 

considered a ‘priority pollutant’ under the 

Environment Protection Act, 1986 

(Chandrakant et al., 2006). Phenols can 

be degraded by various white rot fungi 

like P.florida, L.edodes and H.ulmarius 

(Ranjini and Padmavathi, 2013; 2012). 

Pleurotus florida, Pleurotus ostreatus, 

Pleurotus flabellatus and Pleurotus 

sajor-caju have also been studied for 

their potential in dye decolourization 

(Faraco et al., 2009). Azo dyes are added 

in textile, pharmaceutical, cosmetic and 

food industries. After processing, almost 

forty percent of the dye is released into 

wastewater. This affects the aesthetics, 

transparency and oxygen levels of the receiving 

water, making it toxic (Ali et al., 

2008). Even at very low concentrations 

(



of dye-polluted areas is because of their 

ability to penetrate and break the chemical 

structure of the dye molecules, as they 

have long hyphae and also their capability 

to degrade a wide range of dyes, especially 

anthraquinone and triphenylmethane 

dyes (Palmieri et al., 2005). According to 

studies by Kodam et al., (2005), azo dyes 

can be decolorized by white-rot fungi. 

Microbial decolourization uses both oxidative 

and reductive steps, oxidation 

brought about by peroxidases (lignin peroxidase, 

manganese peroxidase, versatile 

peroxidase, etc.) and laccases, usually 

found in these fungi for breaking down 

lignin, the main constituent of woody 

substrates (Gomaere and Govindwar, 

2009). 

The production of ligninolytic enzymes 

by white rot fungi is regulated to a 

great extent by the concentration and carbon-nitrogen 

sources added. Mikiashvili 

et al. (2005) proved this during his research 

on Trametes versicolor. It was observed 

that both the nature (i.e.) organic 

or inorganic source of nitrogen and concentration 

of nitrogen are important factors 

that regulate their secretion. Media 

with high nitrogen content produced 

higher quantity of laccase activity in Lentinus 

edodes, Rigidoporus lignosus and 

Trametes pubescens, while nitrogenlimited 

conditions enhance enzyme production 

in Pycnoporus cinnabarinus, P. 

sanguineus and Phlebia radiata. In some 

cases, high nitrogen content of the substrate 

suppresses enzyme activity. This 

occurs in substrates with high lignin content, 

correlated with a reduction in activity 

of peroxidase or phenol oxidase. However, 

substrates with low lignin content 

degrade well, which is correlated with 

increase of cellulase activity. This shows 

that white-rot fungi are more important in 

high-lignin substrates, where they are the 

primary organisms responsible for the 

secretion of phenol oxidase. This enzyme 

is suppressed by the addition of excess 

nitrogen. In low-lignin substrates, excess 

nitrogen stimulates a wider group of cellulose-degrading 

fungi and bacteria that 


mainly produce cellulases (Singh et al., 

2002). 

Carbon sources also have a regulating 

effect on enzyme secretion by white 

rot fungi. In Phanerochaete chrysosporium, 

ligninolytic genes are triggered by the 

depletion of carbon in the media (Wang et 

al., 2008). In Trametes pubescens, significant 

laccase secretion occurs when glucose 

is reduced to a critical level. Glucose 

and cellobiose are good inducers of laccase, 

while fructose and cellulose inhibit 

it (Bettin et al., 2008). The above observations 

indicate that carbohydrates can 

regulate laccase secretion in white rots. 

The source of carbon used is fungusspecific. 

Other factors like pH, temperature, 

the presence of inducers and inhibitors 

have their own effects on enzyme 

secretion. The effect of extremes of pH 

may be due to the fact that it alters the 

three-dimensional structure of the enzymes. 

Higher temperatures can reduce 

enzyme production by drying of the substrate 

(Patel et al., 2008). Surfactants like 

Tween 20 and Tween 80 are inhibitors, 

while veratryl alcohol, vanillic acid, ferulic 

acid, guaiacol and copper sulphate are 

inducers (Ikehata et al., 2004). 

The science of ‘Applied Mushroom 

Biology can provide solutions to the 

above environmental problems in the following 

ways: 

i. Mushroom cultivation - Production 

of inexpensive food protein (mushrooms) 

using agricultural byproducts 

like paddy straw, coconut 

husk, tea, and saw dust, which also 

generates the spent mushroom substrate 

(SMS). 

ii. Mycorestoration – Addition of the 

above generated spent mushroom 

substrate to improve the fertility of 

marginal and contaminated soils by 

increasing soil aeration and the 

availability of phosphorus and potassium 

to plants. 

iii. Mycoremediation – Uptake and/ or 

degradation of environmental pollutants 

like phenol and dyes using 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 91



fungal enzymes present in the above 

generated spent substrate (Figure 2). 

2. Mycorestoration of soil using spent 

mushroom substrate from mushroom 

cultivation 


Spent mushroom substrate (SMS) 

also known as ‘spent mushroom compost’ 

(SMC), is generated as a by-product after 

the harvest of mushroom crop. Recently, 

it has been proposed to re-name it as ‘post 

mushroom substrate’ because it is not really 

‘spent’ and has many uses remaining. 

The composition of spent mushroom substrate 

varies depending on the type of 

mushroom cultivated and the agricultural 

waste material used as substrate. It is an 

excellent source of humus, although much 

of its nitrogen content is used up by the 

growing mushrooms. Overall, it is a good 

source of the macro nutrients viz., nitrogen, 

phosphorus and potassium, in addition 

to having trace elements. This makes 

it suitable for supporting plant growth 

(Kulshreshtha and Sharma, 2014). This is 

also because it behaves similar to ‘arbuscular 

mycorrhizal fungi’ (AMF), associated 

with plant-growth-promoting rhizobacteria 

(PGPR). The nitrogen content of 

SMS varies from 0.4-13.7% with a C: N 

ratio of 9 to 15: 1. It contains cations like 

K + , Na + , Ca 2+ , Mg 2+ ; and anions like Cl - , 

NO 3 - , SO 4 2- ; all essential for optimal plant 

growth. It improves physical soil properties 

by decreasing its density, surface 

crust formation and diurnal temperature 

changes; in addition to increasing infiltration, 

aeration and water-retaining capacities. 

It maintains a high organic content of 

soil. It can be added singly or as a supplement 

to conventional bio-fertilizers, 

though it functions better in combination 

with other bio-fertilizers (Rinker and Kan 

Zeri, 2004). 

Restoring a degraded or stressed 

soil using mycorrhizae and myco-biofertilizers 

is known as ‘mycorestoration’. 

Humans have the ability to synergize mycorrhizae 

and use them for healing forest 

habitats that have suffered from stress, 

toxic waste or poor nutrition, making 

them critical to our mutual evolutionary 

survival (Stamets, 2006). Most plants also 

have associated with them diverse groups 

of plant-growth-promoting-fungi (PGPF) 

and AMF. 

Can remove 

malnutrition 

Research Process Flow 

Applied Mushroom Biology 

Mushroom Cultivation 

Effective 

SWM 

Food protein 

Nutraceuticals 

Mycoremediation 

Generation of SMS 

Mycorestoration 

Pharmaceuticals 

Medicines 

Food 

Industry 

Uptake of phenol and dyes 

Bioremediation of soil pollutants 

Release of Phosphorus 

Soil fertility improved 

Reduced fertilizer dependence 

7 

Figure 2: The science of applied mushroom biology. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 92



2.1. The mushroom industry and mushroom 

cultivation 

The ever-increasing demand for 

protein-rich vegetarian food and the inefficiency 

of conventional methods have 

resulted in the need to explore alternatives 

for low cost production of protein-rich 

food like mushrooms (Mukherjee and 

Nandi, 2004). The mushroom industry 

has a world production greater than 25 

million tonnes. The largest producer is 

China, which cultivates more than 20 million 

tonnes and account for over 80% of 

the world's mushroom production (Li, 

2012). Research has proved that production 

of 1 kg mushrooms will generate 5 

kg of spent residual material called spent 

mushroom substrate or SMS. An average 

farm discards about 24 tonnes of SMS per 

month (Singh et al., 2011). In Ireland, 

approximately 2,54,000 tonnes of SMS is 

generated each year (Barry et al., 2012) 

and in The Netherlands, more than 

8,00,000 tonnes (Oei and Albert, 2012). 

In some countries, waste management of 

SMS is a major problem faced by farmers 

and the government. The obvious solution 

is to increase the demand for SMS 

through exploration of new applications, 

being recycled and reused. 

The spent substrate is a composted 

organic medium, made from renewable 

agricultural residues such as paddy straw, 

wheat straw, sawdust, sugarcane bagasse, 

hay, poultry manure, ground corncobs, 

cottonseed meal, cocoa shells, gypsum 

and other substances (Jordan et al., 2008). 

Generally, each cultivation cycle lasts for 

5 to 6 months, after which the spent substrate 

would be disposed. In Malaysia, an 

average farm producing 100 tonnes of 

fresh mushrooms per annum generates 

approximately 438 tonnes of SMS. The 

current disposal strategy of SMS in Malaysia 

is by burning, spreading on land, 

burying, composting with animal manure 

or land-filling. 

2.2. Cultivation process for hypsizygus 

ulmarius 


For obtaining the spent mushroom 

substrate (SMS) of H.ulmarius, it is cultivated 

on agricultural wastes such as paddy 

straw and coconut husk (Khan et al., 

2008), by solid-state fermentation method, 

as prescribed by Chang (1999). 

2.2.1. Substrate preparation and sterilization 

Crushed rice straw is used for cultivation. 

Straw is cut to 2-6 cm pieces, 

soaked overnight and autoclaved, at 

121 o C and 15 psi, while coconut husk is 

separated and soaked for 5-6 hours, then 

autoclaved as above. 

2.2.2. Spawn rate 

The quantity of spawn used for 

inoculation is 5% of its total weight (50 

gm spawn for 1 kg substrate). 

2.2.3. Spawning of substrate bag 

The pasteurized substrate is filled 

into transparent perforated polyethylene 

bags; incubated at 23-25 o C for 12 to 14 

days. Mushrooms form around the edges 

of bag perforations and are harvested approximately 

3 to 4 weeks later. 

2.2.4. Spawn broadcasting 

After spawning, the bags are 

moved to a room where temperature is 

around 18–20 o C and relative humidity is 

close to 95-98%. The first 12-21 days are 

completed without artificial lighting. At 

the end of this period, 4 hours light is 

provided daily using fluorescent bulbs. At 

the time of formation of mushrooms, 

fresh air is let in to lower CO 2 levels. 

Studies have shown that paddy 

straw is the preferred substrate for cultivation 

of H.ulmarius. However, coconut 

husk could be used as a supplement to 

enhance stipe length. Increasing stipe 

length can make picking the fruit during 

harvest much easier and thereby, increase 

its competitiveness in the commercial 

market. Rice straw, cotton waste, coir, 

baggase and banana leaves are all also 

considered suitable substrates for growing 

oyster mushrooms (Belewu and Belewu, 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 93



2005). The yield and quality depends on 

the C: N ratio, composition of vitamins, 

phytohormones, macro, and micro elements 

present in the substrate (Adenipekun 

and Gbolagade, 2006). The use 

of these agricultural wastes in mushroom 

cultivation provides a solution for their 

disposal, and effective solid waste management. 

The cultivation of edible mushrooms 

offers one of the most feasible and 

economic methods for environmentfriendly 

bio-conversion and disposal of 

agro-lignocellulose waste (Cohen et al., 

2002). The spent substrate after two or 

three harvests of mushrooms can be used 

for mycorestoration and mycoremediation 

studies. 

2.3. Applications of spent mushroom substrate 

The most significant applications of 

spent mushroom substrate (SMS) is its 

ability to increase and retain the organic 

content of soil or the potting medium (by 

increasing the release of major plant nutrients 

such as phosphorus and potassium); 

it also increases the porosity of soil 

by creating air spaces (due to the threadlike 

nature of fungal mycelia) – 

www.mushroom-sms.com. 

Some of the other applications of 

SMS are as follows: 

Consistency of quantity and quality – 

A consistent amount of SMS can be produced 

annually as mushrooms can be cultivated 

throughout the year with regulated 

temperature and humidity conditions. 

Consistent high quality can also be produced 

by standardizing the process and 

materials used for cultivation. 

High water and nutrient retention 

capacity - Water and nutrient retention 

capacity of the soil increases by addition 

of SMS due to the filamentous nature of 

the mycelia that creates a network, similar 

to ‘mycorrhizal fungi’, which traps the 

water molecules and also the released nutrients. 


There is a reduction or complete absence 

in the growth of weeds- when SMS 

is added to soil. 

SMS is already supplemented with 

nitrogen- along with its innate ability to 

release phosphorus and potassium, it 

makes the soil enriched in all the three 

major plant nutrients – nitrogen, phosphorus 

and potassium. 

There is absence of heavy metals and 

toxins- in soil supplemented with SMS. 

2.4. Use of spent mushroom substrate 

(SMS) of Hypsizygus ulmarius as a 

mycorestoration agent 

The potential of the SMS of H. 

ulmarius has been studied for improving 

soil fertility and plant growth by the release 

of soil phosphorous, improving aeration 

and disease resistance. Phosphorous 

is one of the limiting factors for plant 

growth in most soils. SMS added singly 

increased soil phosphorus; with conventional 

bacterial biofertilizers such as Azotobacter 

sp. and fungal biofertilizers like 

G.intraradices, it enhances soil porosity, 

production of leaves, auxiliary buds and 

flowers; also root biomass, soil carbon 

and nitrogen. Hence, it is more beneficial 

when the SMS of H.ulmarius was used as 

a supplement to conventional fertilizers, 

rather than as a stand-alone bio-fertilizer. 

A lot of literature has indicated that there 

is a stimulatory effect of ‘plant growthpromoting 

Rhizobacteria’ (PGPR) on the 

growth of plants, due to the presence of 

beneficial micro-organisms. However the 

mechanisms of this stimulation have not 

been discussed in detail in most of the 

literature. The modes of action that have 

been studied, are, however, as follows: 

Increased supply of nitrogen to the host 

by microbial nitrogen-fixation; increase in 

supply of phosphorus, sulphur and iron; 

increase in surface area of roots due to 

production of phytohormones and stimulation 

of mutualistic relationships between 

the host plant and other algae and 

fungi (Banerjee, 2006). 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 94



3. Mycoremediation of soil using white 

rot fungi 

Bioremediation mainly depends 

on the ability of microorganisms like bacteria 

and fungi to produce enzymes that 

break down the pollutants to nonhazardous 

products. As bioremediation is 

effective only where environmental conditions 

permit microbial growth and activity, 

its usage often involves the manipulation 

of environmental parameters to 

allow faster microbial growth and degradation 

(Karigar and Rao, 2011). The limitations 

of bacterial growth are due to variations 

in pH, temperature, oxygen, soil 

organic matter, moisture and optimum 

level of nutrients, poor bioavailability of 

contaminants and the presence of toxins 

(Vidali, 2001). In this respect, fungi are 

far better adapted, as they exhibit high 

tolerance toward low pH and drought 

conditions, characteristic of contaminated 

and marginal lands. Most bioremediation 

systems operate under aerobic conditions, 

which is also a condition well suited for 

fungi (Leung, 2004). 

Processes such as formation of insoluble 

metal oxalates, biosorption or 

chelation on the surface of polymers are 

used by fungi for removing pollutants 

from soil and water (Sasek, 2003). Some 

of the white rot fungi produce all the ligninolytic 

enzymes, while others produce 

only one or two of them. Lentinus edodes 

and Pleurotus spp. are fungi with important 

medicinal, biotechnological and 

environmental applications (Elisashvili et 

al., 2008). They are capable of producing 

hydrolytic and oxidative enzymes like 

laccases and peroxidases, which are essential 

in breaking down the lignocellulosic 

biomass into low molecular weight 

compounds that support mycelial growth 

and fruiting (Reddy et al., 2003). 

3.1. Recovery of ligninolytic enzymes 

from spent mushroom substrate of 

white rot fungi 

Enzymes reported in the literature 

are derived mainly from the mycelia of 


macro-fungi grown in submerged fermentation. 

Enzymes can also be extracted 

from ‘solid substrate fermentation technology’. 

Laccase is the most common enzyme 

isolated from the spent mycelium 

substrate (SMS) of Agaricus bisporus 

(Mayolo-Deloisa et al., 2009), Pleurotus 

sajor-caju, P.ostreatus, Lentinus edodes, 

Flammulina velutipes, Hericium erinaceum 

and Hypsizygus ulmarius. However, 

the productivity of lignin peroxidase (per 

microgram of SMS) was found to be the 

highest in the SMS of P.sajor-caju; it was 

twice, 22, 30 and 86-fold higher than that 

of β-glucosidase, laccase, xylanase and 

cellulase, respectively. Certain methods 

for extraction and purification of enzymes 

like laccase are dialysis, ultra-filtration, 

anion-exchange chromatography and gel 

filtration (Quaratino et al., 2007). However, 

most of these experiments have 

been carried out using the fruiting bodies 

or mycelia of mushroom, not the spent 

substrate. The important parameters influencing 

enzyme yield are pH, temperature, 

extraction medium, incubation time, 

inoculum density and nitrogen source. 

3.2. Mycoremediation of phenol using 

spent mushroom substrate 

Phenol, if ingested, inhaled or absorbed 

through the skin, quickly penetrates 

the surface and causes severe irritation 

to the eyes and respiratory tract. It is 

potentially carcinogenic to humans (Muftah 

et al., 2009). The Hazardous Waste 

Management Rules 1989 permits only 5 

kg of phenol per year for disposal. The 

actual quantities disposed are much greater. 

In addition, the present treatment 

methods are chemical intensive and further 

contaminate the environment. This 

has made it imperative that new nonchemical 

methods like mycoremediation 

are devised (Nuhoglu and Yalcin, 2005). 

There is a heightened concern over public 

health and environmental hazards due to 

the presence of organic toxins like phenols 

and dyes in waste water (Eriksson et 

al., 2007). Phenols and azo dyes are well 

known for their bio-recalcitrant nature 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 95



and acute toxicity. They are being continuously 

introduced into ponds, lakes and 

rivers through from chemical and textile 

industries of different scale and category. 

The major sources of phenol pollution 

are waste water coming from paint 

industries, pesticides, resin production 

and petrochemical industries. Phenols are 

considered as primary pollutants under 

directive 80/778/EC, since they are harmful 

to organisms even at very low concentrations 

(Calace et al., 2002). The maximum 

concentration has been set at 0.5 

mg/l for total phenols in drinking water, 

and individual concentration should be 

under 0.1 mg/l. 

The main routes of exposure to 

phenol include breathing contaminated 

air; inhaling cigarette smoke, drinking 

water from contaminated surface or 

groundwater supplies, swallowing or inhaling 

products containing phenol or 

coming into contact with contaminated 

water and products containing phenol 

through bathing (Ahmaruzzaman, 2008). 

The common method to detect intermediate 

products of phenol breakdown 

is extraction by organic solvent after esterification 

or acetylation, followed by 

gas chromatography and mass spectroscopy 

(GC-MS). However, this method 

has two disadvantages: (1) as the intermediates 

are mostly polar compounds, 

they dissolve easily in water and the nonor 

low-polar solvents don’t get extracted 

completely (2) Esterification is suitable 

only for derivatizing acids, while acetylation 

only for hydroxylated compounds. In 

addition, it is not possible to detect these 

intermediates by high-performance liquid 

chromatography (HPLC). This method 

needs not only many calibration standards 

but takes a lot of time as there are several 

unknown compounds in the intermediates, 

and some compounds would have 

the same retention time (Guo et al., 

2006). 

A possible degradation mechanism 

for phenol was given by Devi and 

Rajashekhar (2011). The hydroxyl radicals 

attack the phenol molecule leading to 


the formation of dihydroxy benzene. Further 

degradation proceeds through the 

cleavage of dihydroxy benzene to give 

pent 2-enedioic acid and formaldehyde, in 

addition to benzoquinone and maleic acid. 

The decarboxylation of maleic acid and 

ring opening of hydroquinone result in the 

formation of oxalic acid. Decarboxylation 

of oxalic acid leads to the formation of 

carbon dioxide and water. 

3.2.1. Application of spent mushroom 

substrate of white rot fungi in mycoremediation 

of phenolic compounds 

The enzyme systems of ‘white rot 

fungi’ contain laccase, lignin peroxidase 

and manganese-dependent peroxidases 

which catalyses metabolism of many lignin-like 

structures, for example, PAHs 

and phenols (Eggen and Sasek, 2002). 

Phenol oxidation using SMS from 

A.bisporus was reported and laccase was 

identified as the main enzyme responsible 

(Trejo-Hernandez et al., 2001). Studies 

have also supported the idea of decontaminating 

phenolic compounds using 

SMS from cultivation of edible mushroom. 

3.2.2. Phenol tolerance and degradation 

by spent mushroom substrate of 

Hypsizygus ulmarius 

H.ulmarius tolerated and degraded 

phenol better under carbon and nitrogen 

limiting conditions of the growth medium. 

The optimum carbon sources for its 

growth were glucose, mannitol and cellulose, 

while ammonium nitrate, ammonium 

chloride and sodium nitrate were the 

optimum nitrogen sources. Peroxidase 

and manganese peroxidase were the enzymes 

secreted in maximum quantity during 

phenol degradation, followed by laccase 

(Ranjini and Padmavathi, 2012; 

2013). 

3.3 Mycoremediation of dyes using spent 

mushroom substrate 

The majority of natural dyes are 

produced from plant sources like roots, 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 96



berries, bark, leaves, wood and also fungi 

and lichens. Azo compounds have aryl or 

alkyl functional groups, with vivid colors 

like reds, oranges and yellows. About 

50% of dyes produced in the world are 

azo dyes (Perumal et al., 2007). They are 

extremely recalcitrant and when released 

with the wastewater, remains in the water 

or soil and adversely impacts the photosynthetic 

ability of phytoplanktons in water. 

They interfere with the functioning of 

chlorophyll in the plankton, as they color 

the water and prevent the proper absorption 

of light (Duran and Esposito, 2000). 

Many methods have been tried for achieving 

decolorization of azo dyes in 

wastewater, but most of them like nanofiltration, 

specific coagulation, use of activated 

carbon and multiple effect evaporators 

are very expensive. Bio-treatment 

is a cheaper and environmentally better 

alternative (Olukanni et al., 2006). Degradation 

by micro-organisms utilizes their 

enzymes, mainly laccases and peroxidases 

produced by few bacteria and white 

rot, brown rot and soft fungi (Duran and 

Esposito, 2000). Only these enzymes are 

found to be effective in breaking down 

the high structural integrity and variety of 

azo dyes. Decolourization and/or ‘bioadsorption’ 

of dye-containing wastewater 

by dead or living biological matter (biomass), 

white-rot fungi and other microbial 

cultures are the subject of many studies 

reviewed in several recent papers (Aksu, 

2005). In particular, these studies demonstrate 

that ‘bio-sorbents’ from suitable 

microbial biomass can be used for dye 

decolourization; this is because certain 

dyes have an affinity for binding with 

some microbial species. The use of biomass 

is becoming popular due to its 

availability in large quantities and costeffectiveness. 

It is produced in fermentation 

processes to synthesize antibiotics 

and enzymes. Here, a large amount of byproducts 

are generated, which can be used 

in bio-sorption of pollutants. Aksu and 

Tezer (2005) have shown the uptake of 

588.2 mg of reactive black 5 per g of biomass 

of Rhizopus arrhizus. Chu and 


Chen (2002 a, b) also reported the use of 

biomass for the removal of basic dyes. 

Decolourization by living and 

dead microbial cells involves mechanisms 

such as surface adsorption, ion-exchange, 

complexation (coordination), complexation–chelation 

and micro-precipitation. 

Cell walls consist of polysaccharides, proteins 

and lipids and offer many functional 

groups for the above reactions. Dyes can 

interact with these active groups on the 

surface of the cell. The accumulation of 

dyes by biomass may involve a combination 

of active, metabolism-dependent and 

passive transport mechanisms. It starts 

with diffusion of the adsorbed solute to 

the surface of the microbial cell 

(O’Mahony et al., 2002). 

Laccase oxidizes the phenolic 

group of the azo dye with the participation 

of one electron, generating a phenoxy 

radical and then oxidizes it to a carbonium 

ion. A nucleophilic attack on the phenolic 

ring carbon bearing the azo linkage 

to produce 3-diazenyl-benzenesulfonic 

acid (III) and 1, 2-naphthoquinone then 

takes place (Camarero et al., 2005). Phenolic 

radicals get oxidized further to yield 

oligomers. Under certain conditions, the 

C-C-formed dimers take part in coupling 

reactions to form extended quinines (Zille 

et al., 2005). 

In reactive dyes, the chromophore 

contains a substitute that is activated and 

allows the dye to directly react to the substrate 

surface. They are added for dyeing 

of cotton or flax. 

The spent mushroom substrate 

(SMS) of P.sajor-caju offer an economical 

source of industrially important enzymes 

decolourize dyes (Singh et al., 

2002). Singh et al. (2011) has shown the 

decolorization of eight dyes (viz.) trypan 

blue, amido black, remazol brilliant blue 

R, bromophenol blue, crystal violet, methyl 

green, congo red and methylene blue 

using lignin peroxidase extracted from 5- 

month-aged SMS of P. sajor-caju coupled 

with veratryl alcohol as a redox mediator. 

Further, three azo group dyes, reactive 

black 5, reactive orange 16 and 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 97



disperse blue 79; two anthraquinone 

group dyes, disperse red 60 and disperse 

blue 56 and textile wastewater from all 

the five reactive and disperse dyes were 

also successfully decolourized by crude 

enzymes from SMS of P.sajor-caju. The 

mechanisms of enzymatic dye decolourization 

were most probably due to laccase 

and manganese peroxidase, as a recent 

study showed that under stimulation by 

malachite green, a triphenylmethane dye, 

the laccase and manganese peroxidase 

levels in the enzyme extracts of 

P.ostreatus were increased by 1.4 and 

2.1-fold, respectively (Papinutti and Forchiassin, 

2010). Thus, dyes can be removed, 

degraded and detoxified by enzymatic 

biological processes and also 

physical adsorption using SMS (Gao et 

al., 2011). Use of SMS in bioremediation 

of dyes is both time saving and costeffective. 

3.3.1. Dye degradation using spent mushroom 

substrate (sms) of hypsizygus 

ulmarius 

The SMS of H.ulmarius was effective 

in degrading three categories of 

dyes (viz.) azo (Congo red), heterocyclic 

(Methylene blue) and reactive (Solochrome 

black), with Methylene blue being 

most effectively decolourized, followed 

by Solochrome black and Congo 

red. Here, laccase was the enzyme secreted 

in greater quantity, followed by manganese 

peroxidase. Maximum secretion of 

both enzymes was observed during decolourization 

of Congo red (Ranjini and 

Padmavathi, 2015). 

4. Conclusion 


To conclude the effective management 

of agricultural wastes as a part of 

solid waste management is mushroom 

cultivation which utilizes agricultural 

wastes in an environment-friendly manner 

and solves their disposal problem, as they 

are generated in large amounts each year. 

The fertility of marginal soils increases in 

an environment-friendly manner - mycorestoration. 

Using the spent mushroom 

substrate (SMS), a by-product of its cultivation, 

as a bio-fertilizer, either singly or 

in combination with conventional biofertilizers 

improves soil fertility, especially 

phosphorus, which is mostly insoluble 

and unavailable to plants. Phosphorus is 

one of the three macro nutrients of soil 

that can determine the yield of crops and 

agricultural income to farmers, and reduce 

their dependence on chemical fertilizers. 

Soil contamination by phenolic 

compounds and dyes can be remedied using 

the spent mushroom substrate (SMS) 

of H.ulmarius. Phenolic waste is an integral 

part of biomedical and industrial 

waste. Present treatment methods are 

chemical-intensive and further pollute the 

environment. 

The spent mushroom substrate 

(SMS) of H. ulmarius can also be used to 

decolorize dyes. Dyes color and pollute 

water bodies, kill aquatic organisms by 

increasing toxicity, and known toreduce 

of light and dissolved oxygen in water. 

The present treatment methods are mostly 

physico-chemical in nature. 

References 

Adenipekun, C.O. and Gbolagade, J.S. 

(2006). Nutritional requirements of 

Pleurotus florida (Mont.) Singer, a 

Nigerian mushroom. Pakistan Journal 

of Nutrition 6, 597-600. 

Ahmaruzzaman, Md. (2008). Adsorption 

of phenolic compounds on lowcost 

adsorbents: a review. Advances 

in Colloid and Interface Science 

143, 48-67. 

Ahmed, S.A., Kadam, J.A., Mane, V.P., 

Patil, S.S. and Baig, M.M.V. 

(2009). Biological efficiency and 

nutritional contents of Pleurotus 

florida (Mont.) Singer cultivated on 

different agro-wastes. Nature and 

Science 7, 44-48. 

Aksu, Z. (2005). Application of biosorption 

for the removal of organic pol- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 98




lutants, a review. Process Biochemistry 

40, 997-1026. 

Aksu, Z. and Tezer, S. (2005). Biosorption 

of reactive dyes on the green 

alga Chlorella vulgaris. Process Biochemistry 

40, 1347–1361. 

Alam, N., Hossain, M.S., Khair, A., 

Amin, S.M.R. and Khan, A. 

(2007). Comparative study of oyster 

mushrooms on plasma lipid profile 

of hypercholesterolemic rats. Bangladesh 

Journal of Mushrooms 1, 15- 

22. 

Ali, N., Ikramullah, G., Lutfullah, A., 

Hameed and Ahmed, S. (2008). 

Decolourization of Acid red 151 by 

Aspergillus niger SA1 under different 

physico-chemical conditions. 

World Journal of Microbiology and 

Biotechnology 24, 1099-1105. 

Amara, A.A. and Salem, S.R. (2010). 

Logical and experimental design for 

phenol degradation using immobilized 

Acinetobacter spp. culture. 

IIUM Engineering Journal 11, 89- 

104. 

Banerjee, M.R., Yesmin, L. and Vessey, 

J.K. (2006). Plant Growthpromoting 

Rhizobacteria as Biofertilizers 

and Bio-pesticides. In: Rai, 

M. K. (Ed.), Handbook of Microbial 

Biofertilizers. Food Products Press, 

New York, p. 137-215. 

Barry, J., Doyle, O., Grant, J. and 

Grogan, H. (2012). Supplementary 

of spent mushroom substrate (SMS) 

to improve the structure and productivity 

as a casing material. In: 18th 

Congress of the International Society 

for Mushroom Science. Beijing, 

China, pp. 735–742. 

Belewu, M.A. and Belewu, K.Y. (2005). 

Cultivation of mushroom (Volvariella 

volvacea) on banana leaves. 

African Journal of Biotechnology 4, 

1401-1403. 

Bettin, F., Montanari, Q., Calloni, R., 

Gaio, T.A., Silveira, M.M. and 

Dillon, A.J.P. (2008). Production of 

laccases in submerged process by 

Pleurotus sajor-caju PS-2001 in relation 

to carbon and organic nitrogen 

sources, antifoams and Tween 

80. Journal of Industrial Microbiology 

and Biotechnology 36, 1–9. 

Calace, N., Nardi, E., Petronio, B.M. 

and Pietroletti, M. (2002). Adsorption 

of phenols by paper mill sludges. 

Environmental Pollution 118, 

315-319. 

Camarero, S., Ibarra, D., Martinez, 

M.J. and Angel, T.M. (2005). Lignin-derived 

compounds as efficient 

laccase mediators for decolourization 

of different types of recalcitrant 

dyes. Applied Environmental Microbiology 

1775–1784. 

Chandrakant, K., Aravind, M., 

Manjunath, N. and Dae Jin Yun. 

(2006). Phenol degradation by immobilized 

cells of Arthrobacter 

citreus. Biodegradation 17, 47-55. 

Chang, S. T. 1999. Global impact of edible 

and medicinal mushrooms on 

human welfare in the 21st century: 

non-green revolution. International 

Journal of Medicinal Mushrooms, 1, 

1-7. 

Chu, K.H. and Chen, K.M. (2002) a. 

Reuse of activated sludge biomass: 

I. Removal of basic dyes from 

wastewater by biomass. Process Biochemistry 

37, 595–600. 

Chu, K.H. and Chen, K.M. (2002) b. 

Reuse of activated sludge biomass: 

II. The rate processes for the adsorption 

of basic dyes on biomass. Process 

Biochemistry 37, 1129–1134. 

Cohen, R., Persky, L. and Hadar, Y. 

(2002). Biotechnological applications 

and potential of wood degrading 

mushrooms of the genus Pleurotus. 

Applied Microbiology and Biotechnology 

58, 582–594. 

Dalgaard, T., Hutchings, N.J. and Porter, 

J.R. (2003). Agroecology, scaling 

and interdisciplinarity. Agricultural 

Ecosystems and Environment 

100, 39-51. 

Devi, L.G. and Rajashekhar, K.E. 

(2011). A kinetic model based on 

nonlinear regression analysis is pro- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 99




posed for the degradation of phenol 

under UV/solar light using nitrogen 

doped TiO 2 . J. Molecular Catalysis 

A: Chemical 334, 65-76. 

Duran, N. and Esposito, E. (2000). Potential 

applications of oxidative enzymes 

and enzymatic activity in relation 

to litter composition, N deposition 

and mass loss. Biogeochemistry 

60, 1-24. 

Eggen, T. and Sasek, V. (2002). Use of 

edible and medicinal oyster mushroom 

[Pleurotus ostreatus 

(Jacq.:Fr.) Kumm.] spent compost 

in remediation of chemically polluted 

soils. International Journal of 

Medicinal Mushrooms 4, 255–261. 

Elisashvili, V., Penninckx, M., Kachlishvili, 

E., Tsiklauri, N., Metreveli, 

E., Kharziani, T. and 

Kvesitadze, G. (2008). Lentinus 

edodes and Pleurotus spp. lignocellulolytic 

enzymes activity in submerged 

and solid-state fermentation 

of lignocellulosic wastes of different 

composition. Bioresources Technology 

99, 457-462. 

Eriksson, E., Baun, A., Mikkelsen, P.S. 

and Ledin, A. (2007). Risk assessment 

of xenobiotics in stormwater 

discharged to Harrestup Ao, Denmark. 

Desalination 215, 187-197. 

Faraco, V., Pezzella, C., Miele, A., 

Giardina, P. and Sannia, G. 

(2009). Bioremediation of coloured 

industrial wastewaters by the whiterot 

fungi Phanerochaete chrysosporium 

and Pleurotus ostreatus 

and their enzymes. Biodegradation 

20, 209-220. 

Forgacs, T.C. and Oros, G. (2004). Removal 

of synthetic dyes from waste 

waters: a review. Environment International 

30, 953-971. 

Gadd, G.M. (2001). Fungi in bioremediation. 

Cambridge University 

Press, Cambridge. 

Gao, J.F., Wang, J.H., Wu, X.L., 

Zhang, Q.A. and Peng, Y.Z. 

(2011). Protonated spent mushroom 

substrate as a potential low-cost biosorbent 

for the removal of Reactive 

Red 15 from aqueous solutions. 

Green Environment Bulletin 20, 51– 

62. 

Gomaere, S.S. and Govindwar, S.P. 

(2009). Brevibacillus laterosporus 

MTCC 2298: a potential azo dye 

degrader. Journal of Applied Microbiology 

106, 993-1004. 

Gomathi, S. and Ambikapathy, V. 

(2011). Antagonistic activity of fungi 

against Pythium debaryanum 

(Hesse) isolated from Chilli field 

soil. Advances in Applied Sciences 

Research 2, 291-297. 

Guo, Z., Ma, R. and Li, G. (2006). Degradation 

of phenol by nano materials 

TiO 2 in wastewater. Chemical Engineering 

Journal 119, 55-59. 

http://www.mushroomexpert.com/hyps 

izygus_ulmarius.html 

Ikehata, K., Buchanan, D.I. and Smith, 

D.W. (2004). Recent developments 

in the production of extracellular 

fungal peroxidises and laccases for 

waste treatment. Journal of Environmental 

Engineering and Science 

3, 1-19. 

Inbaraj, B., Selvarani and Sulochana, 

N. (2002). Evaluation of a carbonaceous 

sorbent prepared from pearl 

millet husk for its removal of basic 

dyes. Journal of Scientific and Industrial 

Research 61, 971-978. 

Jonathan, S.G., Oyetunji, O.J., 

Olawuyi, O.J. and Uwukhor, P.O. 

(2013). Application of Pleurotus ostreatus 

SMC as soil conditioner for 

the growth of soybean (Glycine 

max). Academia Arena 5, 55-61. 

Jordan, S.N., Mullen, G.J. and Murphy, 

M.C. (2008). Composition 

variability of spent mushroom compost 

in Ireland. Bioresource Technology 

99, 411–418. 

Karigar, C.S. and Rao, S.S. (2011). 

Role of microbial enzymes in the 

bioremediation of pollutants: A review. 

Enzyme Research Article ID 

805187, doi:10.4061/2011/805187. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 100



Khan, F. and Ishaq. (2011). Chemical 

nutrient analysis of different composts 

(Vermicompost and Pitcompost) 

and their effect on the growth 

of a vegetative crop Pisum sativum. 

Asian Journal of Plant Science and 

Research 1,116-130. 

Khan, M.A., Tania, M., Amin, S.M.R., 

Alam, N. and Udin, M.N. (2008). 

An investigation on the nutritional 

composition of mushrooms (Pleurotus 

florida) cultivated on different 

substrates. Bangladesh Journal of 

Mushrooms 2, 17-23. 

Kodam, K.M., Soojhavon, I., Lokhande, 

P.D. and Gawai, K.R. 

(2005). Microbial degradation of reactive 

azo dyes under aerobic conditions. 

World Journal of Microbiology 

and Biotechnoogy, 21, 367-370. 

Kulshreshtha, S. and Sharma, K. 

(2014). Perspectives of bioremediation 

through mushroom cultivation. 

Journal of Bioremediation and Biodegradation 

5, 5. 

http://dx.doi.org/10.4172/2155- 

6199.1000e154. 

Kumar, A., Bisht, B.S., Joshi, V.D. and 

Dhewa, T. (2010). Review on Bioremediation 

of Polluted Environment: 

A Management Tool. International 

Journal of Environmental 

Science 1, 1079-1093. 

Leung, M. (2004). Bioremediation: techniques 

for cleaning up a mess. Journal 

of Biotechnology, 2, 18–22. 

Li, Y. (2012). Present development situation 

and tendency of edible mushroom 

industry in China. In: 18th 

Congress of the International Society 

for Mushroom Science. Beijing, 

China, pp. 3–9. 

Manikandan, N., Kuzhali, S.S. and 

Kumuthakalavalli, R. (2012). Decolourization 

of textile dye effluent 

using fungal mycoflora isolated 

from spent mushroom substrate 

(SMS). Journal of Microbiology 

and Biotechnology Research 2, 57- 

62. 


Manimegalai, V., Ambikapathy, V. and 

Panneerselvam, A. (2011). Population 

dynamics of soil mycoflora in 

the paddy fields of Thanjavur District, 

Tamilnadu. European Journal 

of Experimental Biology 1, 14-19. 

Mayolo-Deloisa, K., Trejo-Hernández, 

M.D.R. and Rito-Palomares, M. 

(2009). Recovery of laccase from 

the residual compost of Agaricus 

bisporus in aqueous two-phase systems. 

Process Biochemistry 44, 

435–439. 

Medina, E., Paredes, C., Perez-Murcia, 

M.D., Bustamante, M.A. and 

Moral, R. (2009). Spent mushroom 

substrate as component of growing 

media for germination and growth 

of horticultural plants. Bioresource 

Technology 100, 4227-4232. 

Mikiashvili, N., Elisashvili, V., Wasser, 

S. and Nevo, E. (2005). Carbon and 

nitrogen sources influence the ligninolytic 

enzyme activity of 

Trametes versicolor. Biotechnology 

Letters 27, 955-959. 

Muftah, H., El-Naas, Shaheen, A., Al- 

Muhtaseb and Makhlouf, S. 

(2009). Biodegradation of phenol by 

Pseudomonas putida immobilized in 

polyvinyl alcohol (PVA) gel. Journal 

of Hazardous Materials 164, 

720-725. 

Mukherjee, R. and Nandi, B. (2004). 

Improvement of in vitro digestibility 

through biological treatment of water 

hyacinth biomass by two Pleurotus 

spp. International Journal of Biodeterioration 

and Biodegradation 

53, 7–12. 

Nuhoglu, A. and Yalcin, B. (2005). 

Modeling of phenol removal in a 

batch reactor. Process Biochemistry 

40, 1233–1239. 

O’ Mahony, T., Guibal, E. and Tobin, 

J.M. (2002). Reactive dye biosorption 

by Rhizopus arrhizus biomass. 

Enzyme and Microbial Technology 

31, 456–463. 

Oei, P. and Albert, G. (2012). Recycling 

casing soil. In: 18th Congress of the 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 101



International Society for Mushroom 

Science. Beijing, China, pp. 757– 

765. 

Olukanni O.D., Osuntoki, A.A. and 

Gbenle, G.O. (2006). Textile effluent 

biodegradation potentials of textile 

effluent-adapted and nonadapted 

bacteria, African Journal of 


Palmieri, G., Cennamo, G. and Sannia, 

G. (2005). Remazol brilliant blue R 

decolourization by the fungus Pleurotus 

ostreatus and its oxidative enzymes. 

Enzyme and Microbial 

Technology 36, 17-24. 

Papinutti, L. and Forchiassin, F. 

(2010). Adsorption and decolourization 

of dyes using solid residues 

from Pleurotus ostreatus mushroom 

production. Biotechnology and Bioprocess 

Engineering 15, 1102– 

1109. 

Patel, H., Gupte, A. and Gupte, S. 

(2008). Biodegradation of fluoranthene 

by basidiomycetes fungal isolate 

Pleurotus ostreatus HP-1. Applied 

Biochemistry and Biotechnology 

157, 367-376. 

Perumal, S. M., Munuswamy, M., 

Sellamuthu, P.S., Kandasamy, M. 

and Thangavelu, K. P. (2007). Biosorption 

of textile dyes and effluents 

by P.florida and T. hirsuta with 

evaluation of their laccase activity. 

Iraninan Journal of Biotechnology 

5, 114-118. 

Prabhakaran, M., Merinal, S. and 

Paneerselvam, A. (2011). Investigation 

of phylloplane mycoflora 

from some medicinal plants. European 

Jourmal of Experimental Biology 

1, 219-225. 

Quaratino, D., Federici, F., Petruccioli, 

M., Fenice, M. and D'Annibale, A. 

(2007). Production, purification and 

partial characterization of a novel 

laccase from the white-rot fungus 

Panus tigrinus CBS 577.79. Antonie 

Van Leeuwenhoek 91, 57–69. 

R. Ranjini and T. Padmavathi. (2015). 

Decolourization of azo, heterocyclic 


and reactive dyes using spent mycelium 

substrate (SMS) of Hypsizygus 

ulmarius. Journal of Environmental 

Biology, 36, 1083-1088. 

Ranjini R. and Padmavathi T. (2012). 

Phenol tolerance of Pleurotus florida 

under varying conditions of nitrogen 

sufficiency. European Journal 

of Experimental Biology 2, 75- 

82. 

Ranjini, R. and Padmavathi, T. (2013). 

A preliminary assessment of phenol 

tolerance and degradation by spent 

mycelium substrate (SMS) of novel 

edible mushroom Hypsizygus ulmarius. 

Journal of Scientific and 

Industrial Research 72, 767-771. 

Reddy, G.V., Babu, P.R., Komaraiah, 

P., Roy, K.R.R.M. and Kothari, 

I.L. (2003). Utilization of banana 

waste for the production of lignolytic 

and cellulolytic enzymes by solid 

substrate fermentation using two 

Pleurotus species (P ostreatus and 

P. sajor-caju). Process Biochemistry 

38, 1457–1462. 

Revankar, M.S. and Lele, S.S. (2006). 

Enhanced production of laccase using 

a new isolate of white rot fungus 

WR-1. Process Biochemistry 41, 

581-588. 

Rinker, D.L. and Kang, S.W. Zeri. 

(2004). Recycling of oyster mushroom 

substrate. In: Mushroom 

Growers’ Handbook - 1.9, 187-191. 

Sasek, V. (2003). In: Sasek, V., Glaser, 

J.A. and Baveye, P. (ed.) The utilization 

of bioremediation to reduce 

soil contamination: problems and 

solution, Kluwer Ac. Pub., Amsterdam. 

Singh, A.D., Vikineswary, S. and Noorlidah, 

A. (2002). Extraction of enzymes 

from spent compost of Pleurotus 

sajor-caju and its potential use 

for decolourization of synthetic dye. 

Malaysian Journal of Science 21, 9– 

16. 

Singh, A.D., Vikineswary, S., Abdullah, 

N. and Sekaran, M. (2011). Enzymes 

from spent mushroom sub- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 102



strate of Pleurotus sajor-caju for the 

decolourization and detoxification 

of textile dyes. World Journal of 

Microbiology and Biotechnology 

27, 535–545. 

Stamets, P. (2006). Mycelium Running: 

How mushrooms can help save the 

world. Ten Speed Press, New York, 

p.19-29, 69-105,111-113, 151, 196- 

199. 

Tang, C.Y., Criddle, Q.S., Fu, C.S. and 

Leckie, J.O. (2007). Effect of flux 

(trans membrane pressure) and 

membranes properties on fouling 

and rejection of reverse osmosis and 

nanofiltration membranes treating 

perfluorooctane sulfonate containing 

waste water. Journal of Environmental 

Science and Technology 

41, 2008-2014. 

Trejo-Hernandez, M.R., Lopez- 

Munguia, A. and Ramirez, Q.R. 

(2001). Residual compost of Agaricus 

bisporus as a source of crude 

laccase for enzymatic oxidation of 

phenolic compounds. Process Biochemistry 

36, 635–639. 

Vance, C.P. (2001). Symbiotic nitrogen 

fixation and phosphorus acquisition. 


Plant nutrition in a world of declining 

renewable sources. Plant Physiology 

127, 390-397. 

Vidali, M. (2001). Bioremediation. An 

overview. Pure and Applied Chemistry 

73, 1163–1172. 

Wang, P., Hu, X., Cook, S., Begonia, 

M., Lee, K.S. and Hwang, H, M. 

(2008). Effect of culture conditions 

on the production of ligninolytic enzymes 

by white rot fungi Phanerochaete 

chrysosporium (ATCC 

20696) and separation of its lignin 

peroxidase. World Journal of Microbiology 

and Biotechnology 24, 

2205–2212. 

Zille, A., Gornacka, B., Rehorek, A. 

and Paulo, A.C. (2005). Degradation 

of azo dyes by Trametes villosa 

laccase over long periods of oxidative 

conditions. Applied Environmental 

Microbiology 6711–6718. 

Zumriye, G. and Karabayir, G. (2008). 

Comparison of biosorption properties 

of different kinds of fungi for 

removal of Gryfalan Black RL metal-complex 

dye. Bioresource Technology 

99, 16, 7730 - 7741. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 103




Kumud Dubey 1 and Kesheo Prasad Dubey 2, * 

1 Centre for Social Forestry and Eco-Rehabilitation, 3/1, Lajpat Rai Road, Allahabad, 

Uttar Pradesh, India; 2 GM (East) Forest Corporation, Allahabad 24 B Agnipath, Forest 

Corporation, Allahabad, Uttar Pradesh, India; *Correspondence: dkumud@yahoo.com 

/ dkesheo@yahoo.co.in; Tel.: +91 9415536077 

Abstract: Biotechnology has various applications which could be utilized to enhance the 

sustainability in various sectors. Forest Biotechnology was started only during 1990s in 

the earnest sense. It encompasses structural and functional studies of genes and genomes 

(including development and application of genetic molecular markers); various methods 

of vegetative reproduction, and asexual insertion of genes into forest plant species. Traditionally, 

productivity of forests has been improved by introduction of new germplasm 

developed through tree genetics and breeding, as trees are strategically and efficiently 

able to utilize both horizontal as well as vertical space in an optimum way. The application 

of modern biotechnological tools and techniques that span the diverse fields of plant 

biology, genetic transformation and discovery of genes associated with complex multigenic 

traits. Modern innovations have added an exclusively new dimension to forest tree 

improvement programs. However, forest biotechnology is still lagging behind because of 

longer time periods required for planning, investigation and field research, poor juvenilemature 

correlations and multiplicity of selection criteria. But in the present scenario, 

with growing population, economic development, environmental degradation and Climate 

Change, the demand for more biomass production from trees for production of 

miscellaneous forest products and services, renewable energy alternatives to fossil fuels 

and carbon sequestration etc. is ever increasing. Currently, the major challenge is to 

maintain the original characteristics of pristine virgin forests for in situ biodiversity conservation 

for a sound natural genetic foundation. Genetic trees for pest and disease resistance 

will help in enhancing plantation survival, productivity and yield that would 

lead to eco-restoration of native tree species. This chapter highlights various application 

of biotechnology in forestry which could help in boosting sustainability. 

Keywords: Bioreclamation; biotechnology; carbon sequestration; markers; micropropagation; 

phytoremediation 


Over the past few years, techniques 

of cell biology, genetic screening, 

and gene manipulation are being used to 

develop improved plant varieties. The 

term „biotechnology‟ is coined for the 

collective use of these techniques. Biotechnology 

may be defined as “any technological 

application that uses biological 

systems, living organisms, or derivatives 

thereof, to make or modify products or 

processes for specific use” 

(www.biodiv.org; FAO, 2001). It provides 

important tools for the sustainable 

development of agriculture, fisheries and 

forestry and can be of significant help in 

meeting the growing needs of population. 

Rapid advancements have been 

and are being made in the scientific enquiry 

and investigation with regards to 

plant biotechnology throughout the universe 

today. Considerable progress has 

already been achieved in the field of med- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 104



ical biotechnology, animal biotechnology 

and agricultural biotechnology. Research 

and applications of biotechnology in the 

field of forestry are also advancing rapidly. 

The term forest tree biotechnology 

started during 1980s. It involves a broad 

collection of tools for breeding, propagation 

and modern innovations that focus on 

a portion of a biological system (Yanchuk, 

2001). As commonly used, forest 

tree biotechnology encompasses structural 

and functional studies of genes and genomes 

(including development and application 

of genetic markers); various methods 

of vegetative reproduction such as 

micropropagation, tissue culture, and somatic 

embryogenesis and genetic engineering, 

which is the physical manipulation 

and asexual insertion of genes into 

organisms (FAO, 2004). However, forest 

biotechnology is still aged behind because 

of inherent problems related with forestry, 

such as longer time periods for planning 

and investigation, poor juvenilemature 

correlations (i.e. the characteristics features 

of young trees are not necessarily 

accurate indicators of those found in mature 

individuals), the multiplicity of selection 

criteria (e.g. timber quality, quantity, 

fuel wood, medicinal values, fodder etc.). 

Moreover, biotechnology in forestry requires 

collaborative and integrated application 

of knowledge and techniques 

drawn from several diverse disciplines 

like agriculture, silviculture, genetics, microbiology, 

molecular biology, plant 

physiology etc. Biotechnology applications 

in forestry are a growing area of interest. 

Initial applications of forest tree 

biotechnology targeted to improved 

productivity and quality of plantation forests. 

Such use, with appropriate social 

controls, can help to reduce impacts on 

natural forest ecosystems from timber 

harvest-related perturbations (Sedjo, 

2001). Through biotechnology faster 

growing trees can be produced thereby 

decreasing the growth as well as harvesting 

period. Biotechnology can also reduce 

threats to tree health by introducing the 

traits that confer resistance to different 

Dubey and Dubey 

diseases. Improvements through tree biotechnology 

may also improve weed control 

enabling young trees to compete with 

weeds in natural ecosystem. Trees genetically 

engineered for pest resistance may 

promote plantation survival and yield, and 

also lead to restoration of native tree species. 

Other potential benefits include enhancing 

the ability of trees to tolerate abiotic 

stress; restoring contaminated sites 

through phytoremediation; facilitating 

weed control using more environmentally 

benign treatments; producing new industrial 

products; modifying biomass chemistry 

to improve pulp and biofuels production; 

and improving carbon sequestration 

to mitigate greenhouse gas emissions. In 

addition, biotechnology, especially Genetic 

Engineering methods, offers unique 

and important tools to conduct research to 

identify the biological mechanisms for 

control of many ecologically and economically 

significant traits. The application 

of biotechnology offers a great potential 

to hasten the pace of tree improvement 

for desired need and can improve 

the productivity of forest and environment. 

Most of the biotechnologies used 

in forestry today involve vegetative reproduction 

through tissue culture and molecular 

marker applications. However, 

Genetically Engineered Plants are also 

likely to play a major role in forestry. 

2. Application of biotechnology in forestry 

In the present scenario, with growing 

population and economic development, 

the demand of wood products is 

increasing which will increase the demand 

for more biomass production from 

trees in the future for carbon sequestration 

and to meet the demand for forestry 

products and renewable energy alternatives 

to fossil fuels (Scholes and Noble, 

2001). Forestry products are the third 

most valuable commodity after oil and 

gas. Trees supply the bulk of fiber for 

pulp, paper, packaging and building 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 105



needs. Three billion people depend on 

wood for fuel. So we must harvest wood. 

But forests also are an essential component 

of our ecology. It is essential to enhance 

the productivity of plantation forests 

in order to meet the ever increasing 

future world demand for wood and woodbased 

products in a sustainable manner 

that preserves natural forests and biodiversity 

(Scholes and Noble, 2001). The 

major challenge of foresters today is to 

maintain the natural characteristics of forests 

while meeting society‟s need for 

products produced from trees. Implementation 

of improved silvicultural techniques 

and forest management practices 

are being used to manage the forest in 

sustainable manner. Productivity of the 

forest has also been improved by the introduction 

of new germplasm developed 

through genetics and breeding efforts for 

tree species. The application of new biotechnological 

tools and techniques that 

span the fields of plant biology, genetic 

transformation and discovery of genes 

associated with complex multigenic traits 

along with genetic engineering have added 

a new dimension to forest tree improvement 

programs. Significant progress 

has been made during the past few years 

in the area of plant regeneration via organogenesis 

and somatic embryogenesis 

(SE) for economically important tree species. 

These advances have not only 

helped the development of efficient gene 

transfer techniques but also have opened 

up avenues for using new high growth 

performance clonally replicated planting 

stocks in forest plantations. Advancements 

in gene cloning and genomics 

technology in forest trees have enabled 

the discovery and introduction of valueadded 

traits for wood quality and resistance 

to biotic and abiotic stresses into 

improved genotypes. One of the greatest 

challenges today is the ability to extend 

this technology to the most elite 

germplasm, such that it becomes an economically 

feasible means for large-scale 

production and delivery of improved 

planting stock. Commercialization of 


such newly developed planting stocks as 

new varieties generated through clonal 

propagation and advanced breeding programs 

or as transgenic trees with highvalue 

traits, is expected in the near future, 

and these trees will enhance the quality 

and productivity of our plantation forests. 

The application of plant biotechnology 

techniques promises potentially significant 

genetic improvement of forestry species 

and may become very attractive in 

view of several constraints traditionally 

imposed by the long life cycles and physical 

size of trees (Schuch,1991). The potential 

applications of biotechnology in 

forestry may be classified in three broad 

categories: Plant Culture, Plant Protection, 

and Plant Utilization. 

2.1. Plant tissue culture 

Genetic improvement in plantation 

forestry relies significantly on conventional 

breeding techniques which have 

been extensively used to improve various 

characteristics in forest trees such as 

growth and form, volume yield, resistance 

to pathogens and quality of the end product 

(Walter et al., 1998). Traditional 

breeding techniques involve identification 

of superior trees with desired traits and 

selection of the offspring having desired 

traits. It was the major technique used for 

the planting stock improvement in 1970s. 

In the 1990s, biotechnology was introduced 

in forestry in earnest (Sedjo, 2001). 

Forest biotechnology offers new perspectives 

in the genetic improvements of forest 

trees through tissue culture and genetic 

engineering. Plant tissue culture broadly 

refers to the techniques of growing 

plant tissues or parts on a nutrient medium 

containing minerals, sugars, vitamins 

and plant hormones, all under sterilized 

conditions. It is a basic technique to be 

used for multiplying elite clonal 

germplasm or genetically engineered 

plants of forestry species. Plant tissue culture 

of forestry species are aimed to fulfill 

following objectives: 

i. Micro-Propagation: To develop protocol 

for micro propagation of im- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 106



ii. 

iii. 

portant forestry species and improve 

vegetative propagation procedures 

including tissue culture procedures. 

Genetically engineering: To identify, 

characterize and isolate genes of interest 

and genetically engineer tree 

seedlings to acquire desirable traits 

and qualities. 

To develop molecular markers to 

help aid in progeny selection. 

2.1.1. Micro-propagation of forestry species 

In India, forestry biotechnologies 

involve vegetative propagation mostly 

through tissue culture. The method offers 

substantial advantages over plants obtained 

through seed origin. It controls genetic 

diversity, ensures greater uniformity 

and high proportion of non-additive genetic 

variance, eliminates inbreds, provides 

clones of desired traits and helps in 

predicting the yield of plantations. Vegetative 

propagation allows cloning of superior 

lines and prevents the loss of desirable 

traits and the uncertainties associated 

with sexual reproduction. In addition, the 

ability to propagate transformed cells 

(cells to which DNA has been introduced 

via the techniques of biotechnology viz. 

Genetic Transformation) and to regenerate 

plants from cultured cells, micropropagation 

is prerequisite to genetic engineering. 

Micro-propagation is a term used 

here to describe methods of in vitro vegetative 

multiplication including rooted micro-cuttings, 

organogenesis and somatic 

embryogenesis. Micro-propagation is 

aimed at cloning superior individuals or at 

„bulk‟ (in mixture) propagating new 

genotypes with high genetic potential but 

available in limited quantities (such as 

materials obtained by controlled pollination). 

Vegetative propagation bypasses 

the genetic mixing associated with sexual 

reproduction. Researches are mainly focused 

on the improvement of rooting procedures 

for cuttings and micropropagation 

through organogenesis and 

somatic embryogenesis. 


Micropropagation by microcuttings 

consists of mass producing vegetative 

copies of desired genotypes by either 

axillary or adventitious budding. Micropropagation 

by microcuttings is carried 

out on more than twenty species including 

Populus alba, P. deltoides, P. tremula and 

Populus hybrids in Germany and India 

(Cornu, 1994), Spain (Bueno et al., 2003) 

and Lithuania (Kuusiene, 2002); Eucalyptus 

camaldulensis, E. globulus, 

E. grandis, E. nitens, E. tereticornis and 

E. urophylla in South Africa, Spain and 

Portugal (Watt et al., 2003), India (Watt 

et al., 2003; Nadgauda in press), Vietnam 

and Thailand (O. Monteuuis personal observation) 

and Australia; Acacia mangium, 

A. melanoxylon, A. mangium × 

A. auriculiformis in Malaysia and South 

Africa (Galiana et al., 2003; Monteuuis et 

al., 2003; Quoirin, 2003); Tectona grandis 

in India (Bonga and Von Aderkas, 

1992; Nicodemus et al., 2001; Nadgauda, 

in press), Vietnam, Brazil and Indonesia 

(O. Monteuuis personal observation), 

Thailand (Kjaer et al., 2000), Costa Rica 

(Schmincke, 2000), Malaysia (Goh & 

Monteuuis, 2001); Pinus sp. in United 

States (Rahman et al., 2003); Anogeissus 

latifolia and A. pendula in India (Saxena 

and Dhawan, 2001); Gmelina arborea, 

Artocarpus chaplasha, A. heterophyllus, 

Azadirachta indica and Elaeocarpus robustus 

in Bangladesh (Sarker et al., 1997; 

Roy et al., 1998). 

Somatic embryogenesis, or production 

of embryos from somatic cells, is 

in fact a cloning technique, as opposed to 

zygotic embryogenesis, in which germinal 

cells give rise to seedlings that are all 

genetically different. Somatic embryogenesis 

is a type of plant tissue culture 

that starts with a piece of donor plant and 

forms new embryos. In the right culture 

conditions, embryos could be developed 

from the somatic cells. The process of 

somatic embryogenesis derives usually 

from callus formation induced by applying 

cytokinic or auxinic exogenous 

growth regulators to very juvenile plant 

tissues. In the most favourable situations, 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 107



some undifferentiated cells of these calli 

can evolve into somatic embryos characterized 

similarly to zygotic embryos, by a 

shoot–root bipolar structure. This basically 

distinguishes somatic embryogenesis 

from microcuttings consisting first of a 

shoot from which an adventitious root 

must subsequently develop. Somatic embryogenesis 

offers the advantage of rapid 

embryo multiplication in a small space. 

Somatic embryogenesis is of specific interest 

in the long term because it has the 

potential for producing, inexpensively, 

large numbers of clones that can be propagated 

as artificial seeds. In the case of 

conifers, somatic embryogenesis, especially 

when derived from a single cell, 

seems the most suitable regeneration and 

propagation technique. In broad-leaved 

species, vegetative propagation of genetically 

engineered materials is likely to use 

a combination of micropropagation and 

rooted stem cuttings, at least in the beginning. 

The advantages of somatic embryogenesis 

in comparison with micropropagation 

through microcuttings are especially 

with regard to multiplication rate and 

genetic modification applications. Somatic 

embryogenesis is preferred to micro 

propagate conifers (Sutton, 2002; Lelu- 

Walter and Harvengt, 2004). However, 

there are still serious obstacles to largescale 

operational application of somatic 

embryogenesis to forest trees, for example: 

Only few species and within these 

species, only few genotypes can produce 

somatic embryos. 

Success has been obtained with few 

exceptions, mainly with juvenile tissues 

coming for instance from immature 

zygotic embryos. 

There are risks that somaclonal variation 

may decrease the value of the 

genotypes produced by somatic embryogenesis, 

resulting in a considerable 

waste of time, material and money. 

True-to-typeness, particularly, 

may remain a problem for certain 

genotypes and efforts are still needed 

for optimizing this technique to make 


it more reliable, especially when using 

mature selected genotypes. 

Organogenesis or the creation of 

plantlets from tissues such as cotyledons 

is not common and rarely used in forestry. 

Organogenesis and somatic embryogenesis 

have been achieved in a large number 

of woody plant species. However, in a 

majority of tree species, micropropagation 

has been carried out by employing 

juvenile explants, for example 

embryos, cotyledons and shoots tips from 

seedlings. Clonal propagation from mature 

trees, in particular conifers, is still 

very difficult by tissue culture and remains 

a challenging biotechnological 

problem. Micro propagation of commercial 

forest tree species is a major research 

and development goal. 

2.1.2. Genetic engineering 

Future prospects for genetic engineering 

of forest trees are high. Through 

the use of genetic engineering techniques, 

individual genes of interest may be isolated 

from the donor organism and transferred 

to a target microbe, animal, or 

plant cell. Forest trees have generally 

been more difficult to work with mainly 

due to their long generation times and life 

cycles. Through genetic engineering, foreign 

genes can be transferred to a forest 

tree resulting in faster tree improvement 

and unique gene combinations which 

cannot be achieved by traditional tree 

breeding. The successful genetic engineering 

of a forest tree requires four factors: 

i. A desirable gene must be identified 

and isolated from a donor organism. 

ii. A plant regeneration protocol 

from single cells or a small group 

of cells. 

iii. A mechanism for inserting or 

transferring foreign DNA into a 

target cell is required. 

iv. Additional DNA sequences for 

regulating the target gene, e.g. a 

promoter is necessary to cause the 

target gene to function in the 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 108



proper tissue when and where desired. 


Considerable research is focused 

on identifying, characterizing and isolating 

genes in trees that are responsible for 

traits of particular interest, including 

stress responses viz. drought, frost and 

water-logging etc., disease tolerance and 

or resistance growth characteristics, wood 

quality and flowering. The examples of 

traits for which genes are being sought in 

commercial forest trees include lignin 

composition, disease and insect resistance, 

dormancy, cold hardiness and 

growth rate etc. Genes of interest can be 

divided into two categories. First, genes 

those are responsible in governing agronomic 

traits or growth of trees. These 

genes are expected to lower the cost of 

wood. This category includes genes for 

disease and pest resistance or tolerance of 

environmental stresses. Genes that help a 

crop to grow more efficiently, such as 

herbicide tolerance, also fall into this 

group. A second category includes genes 

for value-added traits that improve production 

efficiency, product quality, or 

product value and are expected to increase 

the value of wood or quantity and 

quality of active bio-chemicals. For instance, 

genes for reduced lignin content 

or lignin type which are more easily removed 

during pulping fall into this category. 

Genes responsible for improved fiber 

characteristics would also be included 

here. Several value-added traits are being 

actively studied in order to isolate valuable 

genes. 

Several gene transfer systems are 

available for movement of foreign DNA 

into target plants. The most common 

method uses Agrobacterium tumefaciens. 

This soil-borne bacterium is able to naturally 

genetically engineer plants to create 

an environment in which the bacterium 

can thrive. Molecular biologists have altered 

this organism to insert target genes 

into plants without causing plant disease. 

Other methods for gene transfer in forest 

trees include particle bombardment, electroporation 

and polyethylene glycol. Most 

successful work on genetic transformation 

of forest tree species genomes so far has 

been obtained by using juvenile material, 

for example from an explants produced 

from juvenile tissues which have much 

higher regeneration capacities than older 

material. Successful genome modification 

reports of adult selected plant material are 

very rare, except in poplars precisely because 

of its greater capacity to regenerate. 

Transformation of a number of 

tree species by genetically engineering 

trees has been reported. Populus spp. 

serves as the model for much of the research 

because techniques for genetic engineering 

and micropropagating are relatively 

advanced. However, research in 

this area is still modest because several 

technical problems have to be solved. 

Limitations to the broad use of 

genetic engineering to improve forest 

trees include primarily the following 

facts: 

i. Only a few genes for specific 

traits of commercial interest have 

been identified, 

ii. Culturing cells of many tree spe- 

iii. 

cies is difficult and 

Regenerating whole plants from 

cultured cells of commercial tree 

species has met with only limited 

success. 

Those biochemical activities 

that are specific to woody plants or 

even to single species or varieties of trees, 

such as wood fiber formation and resistance 

(or susceptibility) to specific insects 

or diseases often must be studied in 

the tree species of interest. It is important 

to note, however, that much of the research 

on non-forest plants is relevant to 

commercial forest trees. For example, the 

research underway on cellulose biosynthesis 

in cotton plants is focused on uncovering 

enzymes and genes for which 

similar counterparts can be anticipated in 

trees. The same is true for photosynthesis 

research on spinach. Major research efforts 

are underway in many laboratories 

on the small plant Arabidopsis thaliana 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 109



because it completes its life cycle in 6 

weeks. Funds for much basic biochemical 

research are more effectively spent on 

plants other than forest trees. 

2.1.3. Molecular markers 

A serious problem today is in 

identifying desired progeny before they 

get too old to propagate vegetatively, by 

root cuttings, for example. Markerassisted 

selection (MAS) has given further 

impetus to tree breeding and selection. 

Molecular markers allow rapid identification 

of the gene or genes of interest 

in minute samples. Molecular markers are 

genetically linked to a given allele on a 

given locus and can therefore, be used to 

predict the presence of the allele with 

great accuracy (FAO, 2004). Biochemical 

and molecular markers play a significant 

role in many forest biotechnology activities 

for the selection of desired progeny.Research 

aimed at developing molecular 

markers for screening is accelerating. 

Marker applications for fingerprinting and 

paternity analysis been used for characterization 

of genetic diversity. Isozymes, 

randomly amplified polymorphic DNAs 

(RAPDs) and restriction fragment length 

polymorphisms (RFLPs) have been widely 

used for genetic diversity and mapping 

studies, though the current trend favours 

microsatellites (nuclear and cytoplasmic) 

and AFLPs (amplified fragment length 

polymorphisms). Currently, ESTs (expressed 

sequence tags) and SNPs (single 

nucleotide polymorphisms) represent the 

most active area of marker development 

(FAO, 2004). 

3. Plant protection 

Forest trees are affected by a 

large number of different insect pests and 

diseases, the latter caused by fungi, bacteria 

and viruses. Biotechnology offers a 

powerful approach to mitigating the damage 

caused by insects and diseases as well 

as by environmental stressors. Insects and 

diseases probably cause great damage to 

Indian forests; environmental stressors 


like drought and water logging add to it, 

quite substantially. Shoot borer in Shorea 

robusta and Fusarium wilt of Dalbergia 

sissoo greatly damage these important 

species of timber. Through the application 

of Biotechnology threats to tree health 

can be reduced. Research is showing 

promise in the introduction of traits that 

confer resistance to pests and pathogens 

that weaken or cause heavy mortality of 

trees. Improvements through tree biotechnology 

may also improve weed control 

enabling young trees to get a head 

start over nutrient-robbing competitors. 

The research falls into two categories: (a) 

direct protection through control of insects 

and diseases and (b) indirect protection 

through development of resistant tree 

varieties. 

3.1. Direct protection 

The biotechnology product „„Bt 

toxin” is already being used commercially 

in agriculture and forestry. Produced by 

the bacterium Bacillus thuringiensis, this 

toxin is effective against lepidopterous 

insects. One biological approach is to use 

pheromones to attract the insects to a central 

point for control. Techniques of molecular 

biology apparently have not yet 

been applied to these pests. Direct biocontrol 

of rusts and other fungal, bacterial 

and viral diseases of trees is labour intensive 

and costly at best. 

3.2. Indirect protection 

Trees have a wide variety of natural 

defenses against insects and diseases, 

which is why most microbes, insects and 

viruses do not cause tree damage. Considerable 

research is underway to identify 

genes that confer resistance to fusiform 

rust and white pine blister rust in resistant 

varieties and species of pines. When these 

genes have been identified, they can theoretically 

be inserted into susceptible trees 

to provide resistance. This is fairly longrange 

research. In the near term, the identification 

of markers for these resistance 

genes will allow marker-assisted tree 

breeding and asexual progeny selection to 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 110



hasten the development of resistant lines 

of trees. Stress tolerance genes are also 

being sought. Another biotechnological 

approach to indirectly controlling insects 

and diseases in trees is to insert foreign 

genes that confer resistance. Thus, several 

tree species have been transformed with 

insect-controlling genes such that for Bt 

toxin. 

The production of insect resistant 

plants via genetic engineering has generally 

taken one of two approaches. The 

first approach makes use the Bt toxin derived 

from Bacillus thuringiensis. This 

toxin damages the digestive mechanisms 

of the larvae that feed upon it. The toxin 

specifically affects insects belonging to 

the lepidopteran, dipteran and coleopteran 

orders of insects, which include a number 

of major herbivores of forest tree species. 

The Bt toxin gene was first used to transform 

hybrid poplars by McCown et al., 

(1991) via direct (biolistics) gene transfer. 

The introduction of the Bt toxin gene resulted 

in a significant reduction in forest 

tent caterpillar (Malacosoma disstria) 

survival and growth rates of the gypsy 

moth larvae (Lymantria dispar). Herbicide 

resistant crops have been one of the 

major products of the first generation of 

agricultural biotechnology. They are intended 

to reduce weed control costs, increase 

control flexibility, facilitate the use 

of low-tillage (and thus reduced erosion) 

cropping systems and enable broadspectrum 

and environmentally benign 

herbicides to be more readily employed. 

The first successful transformation of a 

woody species was reported in Populus 

alba × P. grandidentata using Agrobacterium 

tumefaciens (Fillatti et al., 1987). 

Transgenic hybrid poplars, with a reduced 

sensitivity to glyphosate, an extensively 

used broad-spectrum herbicide, were produced. 

4. Plant utilization 

Genetic transformation of tree for 

its commercial end-use can be achieved 

through biotechnology. For example lignin 

composition can be quantitatively and 


qualitatively modified through genetic 

engineering for expected financial gains 

from pulp processing improvements. Lignins, 

which enhance cell wall mechanical 

properties and hardness, are difficult to 

process and are a significant limitation in 

processing wood into paper pulp by 

chemical treatment. Genetic transformation 

to modify lignin characteristics is 

a key research feature on species used in 

the paper industry. The aim is to regulate 

the activity of key enzymes involved in 

the lignin biosynthesis pathway (Jouanin 

et al., 2000; Le et al., 2003). 

5. Benefits of biotechnology in forestry 

Forestry is in the take-of stage today as 

biotechnology is introduced into its several 

operations bringing promising developmental 

changes having tremendous potential. 

The use of biotechnology for tree 

improvement can bring economic, social 

and environmental benefits. Biotechnology 

has many useful applications in forestry 

viz. lignin reduction, fiber modification, 

pest and disease resistance, bio control 

methods against weeds, pests and diseases, 

cellulose content enhancement, development 

of cold and hot tolerant species 

of a desired species, modification of biomass 

chemistry to improve biofuels and 

pulp production, phyto-remediation of 

problem contaminated sites viz. arid, ravine, 

saline and usar sites, improving 

Carbon sequestration potential to mitigate 

greenhouse gas emissions and sterility, 

which is an important factor to prevent 

modified genes from “leaking” into the 

natural environment. The short term economic 

gains from the introduction of biotechnology 

to forestry will be lower costs 

and increased availability of wood and 

wood products at shorter rotation than 

usual. Innovations in forest biotechnology 

have the potential to address important 

environmental issues, including the rehabilitation 

of habitats altered by diseases 

like the Sal Borer Attack in Sal Forests, 

Drying of Sheesham, or invasive exotics. 

Moreover, the increased productivity of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 111



biotechnology driven tree plantations may 

free huge chunks of natural or primary 

forest areas from ever increasing pressures 

to supply industrial wood and thus 

improve their ability to maintain and preserve 

biodiversity. And as trees are genetically 

modified to be able to grow in previously 

unsuitable areas such as arid or 

Usar lands and saline soils the newly created 

forests could not only produce more 

wood but also enhance watershed protection 

and sequester carbon for climate 

change mitigation. 

5.1. Benefits of biotechnology 

5.1.1. Economic benefits 

Introduction of any technology 

for the consumer simply means that relative 

prices of the desired goods fall compared 

to that which would have been in 

the absence of the particular technological 

innovation. In other words, technology 

features increased productivity, that is, 

enhanced output per unit of input. Alternatively, 

technology can be either cost 

(input) reducing or yield (output) enhancing. 

For society, more output for the 

equivalent expenditure of inputs means 

societal increase in efficiency. Incidentally, 

Plantation Forestry has received some 

success in recent decades because of its 

associated cost-reducing technology that 

has given wood from planted forests a 

competitive price advantage over that 

harvested from natural forests. The potential 

applications of immediate interest of 

cost-reducing biotechnology to forestry 

are increased wood production, improved 

tree form and wood quality, enhanced 

survival, quicker growth rates and enhanced 

resistance to insects, diseases and 

herbicides. In addition, production and 

processing costs of wood or chips could 

be reduced as well as financial and environmental 

costs for pulping. 

Paper production, for instance, 

requires fiber with adequate strength to 

allow sheets to be produced on highspeed 

machines, an attribute determined 

by the wood fiber characteristics. Therefore, 

in pulp making for paper production, 


desirable traits would be the easy breakdown 

of wood fibers and the removal of 

lignin during chemical processing. 

Through Biotechnology, the raw material 

for paper production can be customized to 

meet the requirements of producers thereby 

wood values increase to that extent. 

5.1.2. Environmental benefits 

Forestry Biotechnology may also 

be used to create a number of desirable 

environmental outputs (Table 1). 

Table 1: Utilization of biotechnology for 

environmental benefits 

No. Biotechnological 

innovation 

Environmental 

output 

1 Cheaper plantation 

wood substitutes 

for wood 

from natural forests 

Pressure to log 

primary forests 

can be reduced 

2 Trees are genetically 

modified to 

grow in arid, usar 

or saline conditions 

3 Trees are genetically 

modified to 

adapt to traditionally 

unsuitable 

sites 

4 Cold-tolerant 

species of a desired 

genus are 

developed 

Ecological forests 

can be established 

on degraded 

lands 

Carbonsequestrating 

forests 

can be established 

on sites 

previously not 

suitable for forestry 

The altitudinal 

and geographical 

range of desirable 

tree species can 

be extended 

Trees genetically engineered for 

pest and disease resistance may promote 

plantation survival and yield and also lead 

to eco-restoration of native tree species 

like Sal. Biotechnology has the potential 

to enhance the ability of trees to tolerate 

abiotic stresses; restoration of contaminated 

sites through phytoremediation; facilitation 

of weed control using more environmentally 

safe treatments; modification 

of biomass chemistry to improve 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 112




pulp and biofuels production; and improving 

carbon sequestration to mitigate 

greenhouse gas emissions. Biotechnology 

also offers the potential to assist in 

ecosystem restoration and repair. Similarly, 

biotechnology may help deal with invasive 

exotics, which have in many places 

threatened locally and easily available 

indigenous species. Modified tree species 

also prove useful in providing ecological 

and environmental services in areas 

where trees now have difficulty in surviving, 

such as arid or drought-prone areas 

areas and areas with saline soil or frost 

zones and industrial waste effluents viz. 

Dairy waste disposal sites, Alumunium 

waste effluents-Red Mud, Fly Ash Ponds 

in Thermal Power Plants etc.. Another 

contemporary and very important application 

of biotechnology from the present 

Climate Change scenario involves creation 

of biological sinks, a potential tool to 

mitigate the build-up of greenhouse gases 

associated with global warming. Land 

Areas and Wetlands not currently forested 

could grow carbon-sequestering plantations 

of Genetically Modified transgenic 

trees (GM Trees). 

The most threatening cost of Biotechnology 

is the after-effect of transgenic 

plants on the natural ecosystem, when 

there would be genetic exchange(s) between 

domestic and wild populations. In 

cases where plantation tree species are 

exotic, genetic “outcrossing” to the natural 

environment would not be a factor. 

Where genetic exchange could be a problem, 

planting sterile trees or varieties with 

reduced or delayed flowering would lessen 

the likelihood of their “escape” to the 

natural environment. In the case of the 

herbicide-tolerant gene, the consequences 

of release into the wild are probably 

small. Herbicides are unlikely to be applied 

to most of the natural environment, 

and where necessary, other types could be 

used to which the escaped genes do not 

confer tolerance. In the long term, the 

herbicide in question will almost surely 

be replaced periodically in the normal 

course of product change and development. 

Thus, the presence of that modified 

gene in the natural environment appears 

unlikely to constitute any serious environmental 

problem, either short- or longterm. 

For qualitative genes that affect 

tree form or wood fibre characteristics, 

release into the natural environment is 

unlikely to provide a competitive advantage 

in survival and therefore, unlikely 

to have significant or adverse consequences 

on the ecology. However, the 

consequences could be different if a survival 

gene is involved. For example, the 

introduction of Bacillus thuringiensis (Bt) 

gene makes a plant toxic to certain pests. 

The release into the wild of such a gene 

could constitute a major problem if it altered 

the comparative competitive position 

of wild vegetation vis-a-vis those 

pests. However, the seriousness of this 

problem depends on the probability of the 

transfer of a survival gene into the wild, 

the scale of the transfer and the comparative 

change in the competitive ecological 

balance within the natural habitat. Since 

pests adapt via natural selection to modified 

genes, the long-term impact of the 

release of the modified gene into the natural 

environment will be mitigated. Subsequently, 

wild populations would gradually 

become resistant to the Bacillus thuringiensis 

(Bt) gene, thereby undermining its 

long-term effectiveness against those 

pests. 

Transgenic biotechnology has become 

controversial in agriculture. Some 

of those controversies appear to be spilling 

over to forestry too. The controversy 

revolves around a number of issues. One 

such issue involves the effects of biotechnology, 

particularly the introduction of 

transgenic plants on human health. The 

food safety issue is not generally raised 

for plants such as forest trees, which are 

not usually a food source. However, cellulose 

is increasingly being used as filler 

in food products, and the food safety issue 

could become a concern, subsequently to 

be encountered by the forest biotechnologists. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 113



Therefore, the most visible costs 

of forest biotechnology are those associated 

with Modified Genetically Engineered 

(GE) trees which include among 

others, effects of the newly acquired traits 

on forest ecosystem structure and function; 

unintended consequences of inserted 

genes on tree and forest biology; reliability 

of the newly encoded traits to produce 

the desired outcomes; and persistence 

and potential impacts of the introduced 

genes in native populations through 

the dispersal of pollen, seeds or vegetative 

propagules (Frankenhuyzen and 

Beardmore, 2004). Other apparent risks 

from biotechnology are associated with 

loss of genetic diversity from vegetatively 

propagating a small number of highly selected 

varieties. Another serious disadvantage 

of vegetative propagation of 

highly selected varieties may ultimately 

result in vulnerability to insect and microbial 

pests and also to stressful climatic 

events. 


Before taking any new biotechnological 

strides in the forestry sector, owing 

to the complexity of forest ecosystems, 

several ecological benefits, costs 

and risks and the effectiveness of risk 

management practices should be completely 

evaluated through careful review 

of scientific literature and well-designed 

field experiments. 

The benefits of biotechnology in 

forestry go beyond economic advantages 

including increased production, lower 

costs to consumers and trees modified for 

easy wood processing or specific production 

objectives etc. to environmental surpluses 

viz. preservation of biodiversity, 

eco rehabilitation of degraded lands, eco 

restoration of contaminated sites and mitigation 

of global warming etc.. But biotechnological 

innovations raise immediate 

serious concerns about biosafety and the 

effects of transgenic plants on the resistance 

of pathogens and on the natural 

ecosystem too, particularly the question 


of genetic exchange between genetically 

modified trees and wild populations. 

Genetic engineering, by identifying 

and isolating specific genes to serve a 

particular purpose, can allow a novel trait 

to be transferred to any genotype in a single 

generation with little or no alteration 

to its other genetic properties. Thus, if 

there is to be a fertile crescent in forestry, 

it is to be found in the Biotechnology Laboratories 

and Field Experiments that operate 

at the interface of basic plant molecular 

genetics and forestry. 

Genetic engineering can provide 

exceptional particular genotypes that can 

be characterised by corresponding specific 

molecular markers and subsequently 

integrated in the clonal forestry and 

breeding programs. Further progress in 

this area will depend on reliable regeneration 

and automation for mass production 

of selected particular genotypes. It is expected 

that future research in biotechnology 

will provide insight into the control 

of maturation and rejuvenation, directed 

gene transfer, genome structure, gene sequence 

and basic mechanisms involved in 

growth and differentiation of trees. Biotechnology 

is going to play an important 

role in the 21 st century in boosting sustainability 

of Forests. 

References 

Bueno, A., Gomez, A. and Manzarena, 

J.A. (2003). Propagation and DNA 

markers characterization of Populus 

tremula L. and Populus alba L. In 

S.M. Jain & K. Ishii, eds. Micropropagation 

of woody trees and 

fruits, forestry sciences, pp. 37–74. 

Dordrecht, Netherlands, Kluwer. 

Cornu, D. (1994). Forêt, de la gélose à la 

terre. Biofutur, Février 1994, 25–31. 

FAO. (2001). Glossary of biotechnology 

for food and agriculture: a revised 

and augmented edition of the glossary 

of biotechnology and genetic 

engineering. FAO Research and 

Technology Paper No. 9. Rome. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 114



FAO. (2004). Preliminary review of biotechnology 

in forestry, including 

genetic modification. Rome. 

Fillatti, J.J., Sellmer, J., McCown, B., 

Haissig, B. and Comai, L. (1987). 

Agrobacterium mediated transformation 

and regeneration of populus. 

Mol. Gen. Genet. 206, 192-199. 

Frankenhuyzen,V. K.; Beardmore, T. 

(2004). Current status and environmental 

impact of transgenic trees. 

Canadian Journal of Forest Research 

34, 1163-1180. 

Galiana, A., Goh, D., Chevallier, M.H., 

Gidiman, J., Moo, H., Hattah, M. 

& Japarudin, Y. (2003). Micropropagation 

of Acacia mangium × 

A. auriculiformis hybrids in Sabah. 

Bois For. Trop., 275, 77–82. 

Goh, D. and Monteuuis, O. (2001). 

Production of tissue-cultured teak: 

the plant biotechnology laboratory 

experience. In Proceedings, 3rd Regional 

Seminar on Teak: „Potential 

and opportunities in marketing and 

trade of plantation teak: challenge 

for the new millennium‟, pp. 237- 

247. Yogyakarta, Indonesia, 31 July 

– 4 August, 2000. 

Jouanin, L., Goujon, T., de Nadaï,V., 

Martin, M.T., Mila, I., Vallet, C., 

Pollet, B., Yoshinaga, A., Chabbert, 

B., Petit-Conil, M. and 

Lapierre, C. (2000). Lignification 

in transgenic poplars with extremely 

reduced caffeic acid-Omethyltransferase 

activity. Plant 

Physiol., 123, 1363–1373. 

Kuusiene, S. (2002). Sexual and nonsexual 

plant reproduction in the laboratory 

(available at 

www.genfys.slu.se/staff/dagl/nova0 

2/Abstracts_Nova02.doc). 

Kjaer, E.D., Kaosa-Ard, A. and 

Suangtho, V. (2000). Domestication 

of teak through tree improvement: 

Options, potential gains and 

critical factors. In Site, technology 

and productivity of teak plantations, 

FORSPA Publication No. 24/2000, 


Teaknet Publication No. 3, pp. 161- 

189. 

Le Dantec, L., Chagné, D., Pot, D., Bedon, 

F., Géré-Garnier, P., De Daruvar, 

A. & Plomion, C. (2003). 

Data mining in pine ESTs: II. Development 

of SNP markers. In Treebiotechnologies, 

Umea, Sweden, 7– 

12 June 2003, s6.22. 

LeluWalter, M.A. and Harvengt, L. 

(2004). L‟emeryogenèse somatique 

des conifers, état et perspectives. 

Afocel Inf.-For., 694, 6. 

McCown, B.H., McCabe, D.E., Russell, 

D.R., Robinson, D.J., Barton, 

K.A. and Raffa, K.F. (1991). Stable 

transformation of Populus and 

incorporation of pest resistance by 

electric discharge particle acceleration. 

Plant Cell Rep. 9, 590-594. 

Monteuuis, O., Alloysius, D., Garcia, 

C., Goh, D. and Bacilieri, R. 

(2003). Field behavior of an in 

vitro-issued Acacia mangium mature 

selected clone compared to its 

seed-derived progeny. Aust. For., 

66, 87–89. 

Nehra, N. S. , Becwar, M. R., Rottmann, 

W. H. , Pearson, L. , 

Chowdhury, K. , Chang, S., Dayton 

Wilde, H., Kodrzycki, R. J. , 

Zhang, C., Gause, K. C. , Parks, 

D. W. and Hinchee, M. A. (2005). 

Forest biotechnology: innovative 

methods, emerging opportunities. In 


Biology–Plant 41(6),701-717. 

Nicodemus, A., Nagarajan, B., Mandal, 

A.K. and Suberbramanian, K. 

(2001). Genetic improvement of teak 

in India. In Proceedings, 3rd Regional 

Seminar on Teak: „Potential 

and opportunities in marketing and 

trade of plantation teak: challenge 

for the new millennium‟, pp. 277- 

294. Yogyakarta, Indonesia, 31 July 

– 4 August, 2000. 

Quoirin, M. (2003). Micropropagation of 

Acacia species. In S.M. Jain & K. 

Ishii, eds. Micropropagation of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 115



woody trees and fruits, pp. 245-268. 

Dordrecht, Netherlands, Kluwer. 

Rahman M.S., Messinamg, M.G. & 

Newton, R.J. (2003). Performance 

of loblolly pine (Pinus taeda L.) 

seedlings and micropropagated 

plantlets on an east Texas site. I. 

Above- and belowground growth. 

For. Ecol. Manage., 178, 245–255. 

Roy, S., Islam, M. & Hadiuzzaman, S. 

(1998). Micropropagation of Elaeocarpus 

robustus Roxb. Plant Cell 

Rep., 17(10), 810–813. 

Sarker, R.H., Rafiqul Islam, M. & 

Hoque, M.I. (1997). In vitro propagation 

of neem (Azadirachta indica 

A. Juss) plants from seedlings explants. 

Plant Tissue Cult., 7(2), 

125–133. 

Saxena, S. & Dhawan, V. (2001). Largescale 

production of Anogeissus pendula 

and A.latifolia by micropropagation. 

In Vitro Cell. Dev. Biol.- 

Plant 37, 586–591. 

Sedjo, R.A. (2001). Biotechnology in 

forestry: Considering the costs and 

benefits. Resources for the Future 

145, 10-12. 

Schuch. W. (1991). Advances in plant 

biotechnology and their implication 


for forestry research. In Vitro 

Cellular & Developmental Biology - 

Plant 27, 99-103. 

Scholes, R.J. and Noble, I.R. (2001). 

Storing carbon on land. Science, 

294, 1012. 

Sutton, B. (2002). Commercial delivery 

of genetic improvement to conifer 

plantations using somatic embryogenesis. 

Ann. For. Sci., 59, 657– 

661. 

Walter, C., Carson, Menzies, M.I. , 

Richardson T. and Carson, 

M..(1998). Review: Application of 

biotechnology to forestry – molecular 

biology of conifers. World Journal 

of Microbiology and Biotechnology 

14, 321-330. 

Watt, M.P., Blakeway, F.C., Mokotedi, 

Meo, Jain, S.M. (2003). Micropropagation 

of Eucalyptus. In S.M. 

Jain & K. Ishii, eds. Micropropagation 

of woody trees and fruits, pp. 

217–244. Dordrecht, Netherlands, 

Kluwer. 

Yanchuk, A.D. (2001). The role and implications 

of biotechnological tools 

in forestry. Unasylva 204, 53–61. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 116



Biotechnological Approaches for Conservation and Sustainable 

Supply of Medicinal Plants 

Sagar Satish Datir 1, * and Subhash Janardhan Bhore 2 

1 Department of Biotechnology, Savitribai Phule Pune University, Pune – 411007, MS, India; 

2 Department of Biotechnology, Faculty of Applied Sciences, AIMST University, 

Bedong-Semeling Road, 08100, Bedong, Kedah Darul Aman, Malaysia; 

subhashbhore@gmail.com / subhash@aimst.edu.my (SJB); 

*Correspondence: datirsagar2007@gmail.com; Tel.: +91 8412013810 

Abstract: Food and medicines are integral part of human life. Continuously increasing 

global population and food demand has created an alarm about sustainable use of natural 

resources. Due to adverse environmental conditions such as drought, salinity, temperature 

and pathogens, it is very challenging to achieve high yield with current agricultural practices. 

Due to deterioration of food quality and unpredictable environmental conditions, there is 

a major public health concern about various diseases. Plant-derived compounds are playing 

significant role in combating various human diseases since prehistoric times and therefore, 

there is an increasing demand for production of plant-derived secondary metabolites. However, 

due to mismanagement of natural resources and faulty agricultural practices, several 

medicinal plant species have become rare, vulnerable and endangered. Hence, alternative 

strategies are needed to protect medicinally important plant species. Biotechnology has become 

a center of attraction due to its innumerable advantages in agriculture, pharmaceuticals, 

forestry and food sectors. In recent years, plant-derived compounds (also called as 

natural compounds) are widely studied and biotechnological tools such as, in vitro propagation, 

transgenic for secondary metabolite production and cryopreservation not only provided 

alternative but also offer sustainable approaches towards conservation of medicinally 

important plant species. This brief review highlights various biotechnological approaches 

for conservation and sustainable supply of medicinal plants. Achievements, challenges and 

perspectives on in vitro propagation for the conservation of medicinal plants are also highlighted. 

Keywords: Biotechnology; conservation; medicinal plants; plant tissue culture; secondary 

metabolites; sustainable development 


Climate change, biotic and abiotic 

stress, depletion of natural resources, deforestation 

and loss of biodiversity are 

major challenges in the process of sustainable 

global development. In order to 

fulfill the basic requirements such as 

food, fuel, medicines and shelter, humans 

are completely dependent on natural resources. 

However, continuous increase in 

global population and associated food 

problems have magnified the aforementioned 

threats and necessitated the sustainable 

use of natural resources. Food 

and medicines are integral part of human 

life and to fulfil the growing demand, 

continuous global efforts are underway 

for increasing agricultural productivity. 

The United Nations Food and Agricultural 

Organization (FAO) assuming that 

global population will be about 9.1 billion 

in 2050 (Godfray et al., 2010). It is reported 

that 83% medicinal plants have 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 117


Biotechnological Approaches for Plants Conservation 

become endangered mainly due to the 

human activity (European Commission, 

2008; Ibrahim et al., 2013). Whereas, 

over-utilization of natural resources, pollution 

of the soil, water and the atmosphere, 

and introduction of invasive species 

have resulted into reduced biodiversity 

(Hunde, 2007). 

Medicinal plants are important for 

the wellbeing of human population and 

there is an increasing demand for the production 

of plant-derived secondary metabolites/ 

novel drug leads (Atanasov et 

al., 2015). Currently, there is constant 

demand for plants and plant parts in 

pharmaceutical industries as well as from 

Ayurveda professionals. Furthermore, due 

to major public concerns about dreadful 

diseases such as cancer, HIV etc., pharmaceutical 

industries are actively engaged 

in production of plant-derived drugs. Due 

to the toxicity and side effects of synthetic 

drugs, the plant-derived drugs are becoming 

more popular and as a result there 

is increase in the number of herbal drug 

manufacturers (Verma and Singh, 2008; 

Agrawal, 2005; Lahlou, 2013). 

The projected escalating demand 

for medicinal plants is increasing which 

leads to unscrupulous collection from the 

wild and adulteration of raw material 

supplied to the manufacturers. Ultimately, 

this practice has resulted into the overharvesting 

of many plants from wild and 

disturbed the population of various medicinal 

plant species and several species 

even became endangered (Kala et al., 

2006; Rao et al., 2004). It has been revealed 

that more than 50,000 plant species 

are used in phytotherapy and medicine 

of which 2/3 are harvested from nature 

leading to local extinction of many 

species or degradation of their habitats 

(Tasheva and Kosturkova, 2012). Due to 

the constant expansion of herbs trade, the 

insufficient cultivation fields, and the 

weak management of harvesting and 

overharvesting of medicinal plants have 

led to exhaustion of the natural resources 

and reduction in the biodiversity. Medicinal 

plants are always in demand even 

Datir and Bhore 

though they are facing the threat of becoming 

endangered and or extinct (Manohar, 

2012). 

One third of the global plant species 

are threatened at different level according 

to International Union of Conservation 

of Nature (IUCN, 2013). Furthermore, 

the habitat destruction and loss also 

leads to the fragmentation of the remaining 

habitat which eventually results in 

further isolation of the respective plant 

species population. The destructive harvest 

of underground parts of slow reproducing, 

slow growing and habitat-specific 

plant species are the crucial factors in 

making them vulnerable and rare 

(Ghimire et al., 2005; Kala, 2005). 

Providing high quality planting material 

for sustainable use and thereby saving the 

genetic diversity of plants in the wild is 

important (Krishnan et al., 2011). However, 

due to the human intervention there 

is rapid dwindling of plant resources for 

medicines; hence, alternative strategies 

and or innovative approaches are needed 

for their conservation. Bukuluki et al. 

(2014) had scrutinized the harvesting 

practices of medicinal plants in Uganda 

and identified harvesting methods for sustainable 

supply of medicinal plants. The 

good harvesting practices suggested include, 

careful harvesting of roots without 

affecting tap root, careful removal of stem 

bark to avoid damaging the innermost 

layer that contributes to drying of the 

plant, plucking of leaves without breaking 

the shoots, picking flowers those are fallen 

down or selecting only a few in order 

to allow the plant to bear fruits and reproduce. 

Recently, Hishe et al. (2016) reviewed 

the value chain of medicinal 

plants and the associated challenges. They 

have conducted detailed studies of modes 

of harvesting, storage, packaging, supply 

and distribution of medicinal plants. They 

highlighted that the medicinal plants supply 

chains have varying requirements for 

their cultivation, resource management in 

the wild, harvesting, processing and marketing. 

Considering these facts, they had 

concluded that in order to become com- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 118



petitive in the medicinal plants global 

market place, value chain must become 

more flexible, innovative and efficient, so 

it can bring to market new products in a 

timely fashion. 

As medicinal plants represent consistent 

part of biodiversity, their utilization 

and conservation strategies needs 

planned management for sustainability. 

Therefore, systematic efforts should not 

only be directed towards preservation of 

the plant populations but also elevating 

the level of knowledge for sustainable 

utilization of these plants in medicine 

(WHO 2010). Developing strategies for 

long-term sustainable supply of medicinal 

plants is challenging; therefore, it has 

been suggested that to meet future public 

food and healthcare demand, integration 

of conventional methods and biotechnology 

are essential. Biotechnological methods 

not only offer faster cloning and conservation 

of the genotype of the plants; 

but also enable genetic modification, gene 

regulation and expression for an efficient 

production of valuable natural substances 

in higher amounts or with better properties 

(Tasheva and Kosturkova, 2012). Because 

of innumerable advantages of biotechnology 

in agriculture, pharmaceuticals, 

forestry, food industry and other sectors, 

the field of biotechnology has become 

a center of attraction for conservation 

and sustainable supply of medicinal 

plants. 

2. Biotechnological approaches for conservation 

of medicinal plants 

It appears that biotechnology is 

emerging dramatically as a key enabling 

technology for environmental protection 

and stewardship in a sustainable manner 

(Cantor, 2000; Gavrilescu, 2004; Arai, 

2006). Biotechnological advances have 

encompassed almost every aspect of human 

life including food, fuel, cosmetics, 

medicines and beverages. Most importantly,, 

biotechnology based-methods 

are reliable and provides continuous supply 

of raw material and natural products 


for food, pharmaceutical and cosmetic 

industries (Nalawade et al., 2003). A systematic 

concept of sustainability was proposed 

by Prescott-Allen and Prescott- 

Allen (1996). According to them, both 

humans and ecosystem are interrelated 

and dependent on each other. Hence, in 

conceptual terms, the essence of sustainable 

development is expressed by the relationship 

between people and the ecosystem 

around them. They further stated that 

the society is thought to be sustainable 

when both the human condition and the 

condition of the ecosystem are satisfactory 

or improving. They concluded that the 

system improves only when both the condition 

of the ecosystem and the human 

condition improve (Prescott-Allen and 

Prescott-Allen, 1996). 

In order to supply medicinal 

plants or medicinal plant-based raw material 

in a sustainable manner, various in 

situ and ex situ strategies (which includes 

in vitro techniques, botanical gardens, 

plant banks, GenBank, gene sanctuaries 

and seed banks) have been suggested for 

the conservation of critically endangered 

plant species (Khan et al., 2012). Genetic 

diversity preservation is of prime importance 

while conserving plant genetic 

resources. For the conservation of plant 

and or their germplasm, ex situ and in situ 

strategies are used. The in situ approach 

includes the maintenance of plant species 

and or their populations in their habitats, 

where they can naturally occur, grow and 

reproduce. Whereas, ex situ approach of 

conservation focuses on the maintenance 

of plant species germplasm under controlled 

conditions (Pathak and Abido, 

2014; Rai et al., 2010). The multiplication 

of plants by classical methods such as 

cuttings, budding, layering, and or grafting 

in nurseries produces enormous number 

of plants. However, biotechnological 

methods such as micropropagation, metabolic 

engineering and genetic manipulations 

are especially appropriate for species 

which are difficult to propagate in 

vivo (Tasheva and Kosturkova, 2012). 

Hence, in situ approach of conservation 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 119



alone would not be efficient and effective 

strategy for conservation and multiplication 

of medicinal plants. Krishnan et al. 

(2011) suggested that prudent application 

of propagating biotechnology tools in 

plant conservation program is a prerequisite 

to succeed (in sustainable use of medicinal 

plants) and to complement the existing 

ex situ measures. The systematic 

study on genetic diversity of rare and endangered 

plant species is very important 

mainly because, it will be helpful in formulating 

plans for management and preserving 

their genetic diversity as well as 

ensuring their long term survival 


(Sreekumar and Renuka, 2006). Biotechnological 

approaches for sustainable supply 

and conservation of medicinal plants 

include micropropagation, mycorrhization, 

genetic transformation and development 

of the DNA banks (Sheikhpour et 

al., 2014; Rai et al., 2010). Figure 1 depicts 

the biotechnological approaches useful 

in sustainable supply of medicinal 

plants. Biotechnological approaches can 

be used to conserve plants from any 

group. Table 1 shows some examples of 

successfully conserved plant species using 

biotechnology approaches. 

Figure 1: Biotechnological approaches for the sustainable supply of medicinally important 

plants. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 120




Table 1: Names of some endangered medicinal plants those are propagated using plant tissue 

culture techniques 

Name Family Common Explant used Reference 

name 

Aerva lanata (L.) 

Juss. 

Amaranthaceae Chaya Node Shekhawat and 

Revathi, 2017 

ex Schult. 

Bacopa monnieri L. Scrophulariaceae Bramhi Leaves, node 

&shoot apex 

Rathore and 

Singh, 2013 

Commiphora wightii 

(Ar.) Bhandari 

Burseraceae Guggul node, shoot tip, 

& axillary bud 

Tejovathi et al., 

2011 

Gentiana kurroo L. Gentianaceae Indian 

gentian 

Node Verma et al., 

2012 

Paris polyphylla sm. Trilliaceae Bulb Verma et al., 

2012 

Picrorhiza kurroa Scrophulairaceae Kaur Nodal sector Jan et al., 2010 

Royle ex. Benth 

Picrorrhiza kurroa 

Royle ex Benth. 

Scrophulariaceae Satuva Leaf/ node Verma et al., 

2012 

Psoralea corylifolia 

Linn 

Fabaceae Indian 

bread root 

Apical meristem Pandey et al., 

2013 

Psoralea corylifolia 

Linn 

Fabaceae Kutki cotyledons, hypocotyls 

Sehrawat et al., 

2013 

Rheum emodii L. Polygonaceae Himalayan 

rhubarb 

Basal disc Verma et al., 

2012 

Salvia sclarea L. Labiateae Clary sage Node Verma et al., 

2012 

Saussurea esthonica 

Baer ex Rupr. 

Asteraceae -- Seeds Gailīte et al., 

2010 

Stevia rebaudiana 

(Bertoni) Bertoni 

Asteraceae Candy leaf Leaf/Node Verma et al., 

2012 

Table 2: Some examples of successfully conserved plant species using biotechnology approaches 

Plant species Approach Explant used Reference 

Calophyllum apetalum Micropropagation Lakshmi and Seeni, 

2003 

Cineraria maritima L. 

Cryopreservation- 

Encapsulation 

Shoot tips and 

nodal segments 

Srivastava et al., 2009 

Chlorophytum borivilianum 

Sant. Et Fernand 

Micropropagation Floral buds Sharma and Mohan, 

2006 

Decalepis arayalpathra Micropropagation Gangaprasad et al., 

2005 

Dioscorea floribunda Cryopreservation Shoot tip Ahuja et al., 2002 

Gomortega keule (Mol.) 

Baillon 

Micropropagation Zygotic embryos 

Psoralea corylifolia L. Micropropagation Apical meristem 

Muñoz-Concha and 

Dave, 2011 

Pandey et al., 2013 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 121




2.1. In vitro culture and micropropagation 

Plant tissue culture is the in vitro 

culture of plant cells, tissues, and or organs 

in sterile nutritionally and environmentally 

controlled conditions. In micropropagation, 

in vitro multiplication of 

large number of plants from explants such 

as leaves, seeds, nodes, anthers, ovary and 

tubers etc. is carried out. The plantlets 

thus propagated can be used for production 

and multiplication of plantlets which 

are genetically similar. In addition to the 

conservation of rare and endangered medicinal 

plants, micropropagation is considered 

as the oldest commercial biotechnology 

based clonal plant propagation 

method (Rai et al., 2010). Development 

of reliable in vitro culture protocols is of 

great importance for conservation of rare 

and endangered medicinal plants. So far 

number of in vitro protocols has been developed 

for rare/endangered medicinal 

plants to propagate them at large scale 

(Shahzad and Saeed, 2013). Table 2 

shows some examples of successfully 

propagated rare/endangered medicinally 

important plants using micropropagation 

technique. An efficient micropropagation 

system for endangered Chinese medicinal 

plant, Saussurea involucrata has been developed 

from leaf explants. Similarly, In 

vitro culture of Saussurea esthonica, an 

endangered wild plant species in Latvia 

was performed using seeds (Gailīte et al., 

2010). Tropical Botanic Garden and Research 

Institute, India has developed in 

vitro protocol for rapid regeneration and 

establishment of about 40 medicinally 

important rare and threatened plants of 

Western Ghats. Simila attempts of medicinal 

plants conservation were made by 

Verma et al. (2012). Their studies included 

in vitro conservation of 23 overexploited 

medicinal plants that belonging 

to the Indian Sub-Continent (Verma et al., 

2012). It is important to note that Synthetic 

seeds were also produced from highly 

proliferating shoot cultures of some 

plants. Out of 23 plants, 18 plants were 

successfully hardened under glasshouse 

conditions. 

Seed encapsulation techniques are 

considered as very promising for conservation 

purposes as the protection provided 

to the plant material by encapsulation 

could increase its resistance to dehydration 

and low temperature, thus opening 

new possibilities for medium-term storage 

(Ray and Bhattacharya, 2010). These 

studies clearly suggest that the efficient in 

vitro propagation system developed for 

rare, endangered and or economically important 

medicinal plants are very useful in 

their conservation and supply for a sustainable 

growth and development of the 

industry. 

2.2. Metabolite Engineering and Genetic 

Manipulations 

Plant-derived secondary metabolites 

are in great demand and researchers 

are actively engaged in secondary metabolite 

production using various in vitro 

techniques. For instance, transgenic for 

overexpression of gene/s in secondary 

metabolite pathway have not only provided 

alternative but also offer sustainable 

approaches towards conservation of medicinally 

important plants. Genetic transformation 

or transgenic technology (also 

referred as GM technology) have been 

successfully resulted in adjunct to classical 

plant breeding, in that it allows the 

targeted manipulation of specific characters 

using genes from a range of sources 

(Shewry et al., 2008). Agrobacterium 

mediated plant transformation approach 

was successful in a number of non-food 

and food crops mainly due to its simplicity 

and efficiency; but, it is still not used 

widely to improve the quality of medicinal 

plants (Tashdeva and Kosturkova, 

2012). One of the most appropriate methods 

for medicinal plants engineering is 

genetic transformation using Agrobacterium 

rhizogenes leading to increased synthesis 

of secondary metabolites in root 

cultures or in regenerated plantlets. For 

instance, studies on hairy root cultures 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 122



using Agrobacterium rhizogenes suggested 

that the intact plant synthesizes a 

large quantity of biologically active substances 

and therefore, their production 

could be increased significantly by transformed 

roots cultures as well as by transformed 

plants (Wang et al., 2015; 

Tashdeva and Kosturkova, 2012; 

Georgiev et al., 2007; Guillon et al., 

2006). For example, Psammosilene tunicoides 

is a medicinal herb endemic to 

China and due to excessive destructive 

exploration; natural resources of this plant 

species have dwindled and species become 

threatened (Wang et al., 2015). 

Considering its medicinal importance, 

successful hairy root culture has been established 

using genetic transformation of 

plant tissues by Agrobacterium rhizogenes 

aiming to enhance the secondary 

metabolites production (Wang et al., 

2015). As the continuous harvesting of 

medicinal plants lead to the exhaustion, 

use of Agrobacterium rhizogenes to transform 

medicinal plants for secondary metabolite 

production under laboratory conditions 

would not only provide the protection 

and conservation of rare or endangered 

medicinal plant species but also offers 

a sustainable approach. 

The sustainable plant production 

is also possible by using microbial inoculants 

as substitution for chemical fertilizers 

and pesticides (O'Gara, 1996). Inoculation 

of mycorrhizal fungi into the roots 

of plants is referred as mycorrhization 

(Williams et al., 1994). This can be 

achieved by delivering microbial inoculants 

via micropropagation (Dolcet- 

Sanjuan et al., 1996). Several reports 

suggest that inoculation of arbuscular 

mycorrhizal fungi (AMF) into the roots of 

micropropagated plantlets plays an advantageous 

role (Sylvia et al., 2003; Voets et 

al., 2005; Chandra et al., 2010). 

Tools such as in-vitro propagation, 

mycorrhization and genetic engineering 

not only hold tremendous potential 

to select, multiply and conserve the 

critical genotypes of medicinal plants but 

also offer the production of high-quality 


plant-based medicine (Sheikhpour et al., 

2014; Tripathi and Tripathi, 2003). As 

plant-derived drugs have lesser side effects 

in comparison to allopathic medicine, 

medicinal plant species have made 

an outstanding contribution in many traditional 

herbal therapies practiced in various 

parts of the world. Considering the 

importance of medicinal plants, efforts 

should be made at different levels for 

their sustainable supply (Kala et al., 

2006). Due to accelerated local, national 

and international interest, the demand for 

medicinal and aromatic plants has grown 

rapidly and therefore, public–private collaboration 

is essential (Van De Kp et al., 

2006). 

2.3. Cryopreservation 

One of the important biotechnological 

tools in conservation of plant species 

is cryopreservation which includes 

freeze- preservation or cryogenic storage 

of biological material at a very low temperature 

(Jain et al., 2012). Cryopreservation 

approach is used to conserve plant 

germplasm when other traditional approaches 

such as seed banking and vegetative 

propagation do not work efficiently 

for the respective plant species. Hence, 

for long-term conservation of in vitroderived 

plant germplasm is stored in liquid 

nitrogen (-196°C) (Engelmann, 2011). 

It has been reported that, as threatened 

and endangered species produce little or 

no viable seeds or are dormant; hence, the 

preservation of remaining individuals is 

considered of paramount importance 

(Bunn et al., 2007) and such problematic 

species needs to be maintained through 

cryo-collections (Kaczmarczyk et al., 

2012). Cryopreservation ensures safe and 

cost-efficient long-term conservation of 

species without the loss of viability, so 

when required, material can be readily 

retrieved and reinitiated, reestablishing 

desirable clonal lines (Shibli et al., 2004). 

Plant materials such as cells, tissues, 

gametes, oocytes, organs, DNA samples 

etc. are stored so that they can be used in 

future (Sharma and Sharma, 2013). Cryo- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 123



preservation is considered as one of the 

most important conservation techniques 

and has been a successfully approach for 

the conservation of number of medicinal 

plants. Conservation of Atropa belladonna, 

Digitalis lanata, Hyoscyamus sp. and 

Rauvolfia serpentine are some examples 

to state (Jain et al., 2012). Initiatives for 

conservation of medicinally important 

endangered plants have already been taken 

by several institutions. For instance, 

more than 110 accessions of rare or 

threatened plant species are stored using 

cryopreservation approach of conservation 

at the Kings Park and Botanic Garden 

in Perth, Australia (Touchell and 

Dixon, 1994). Likewise, The National 

Bureau for Plant Genetic Resources 

(NBPGR, New Delhi, India) has stored 

more than 1,200 accessions from 50 different 

plant species (Mandal, 2000). 

Shoot tips of endangered and medicinally 

important plant, Picrorhiza kurroa have 

been preserved using same approach 

(Sharma and Sharma, 2003). Cryopreserved 

embryogenic cultures of Dioscorea 

bulbifera was performed using an 

encapsulation-dehydration procedure. After 

cryopreservation, the sub-culturing 

showed 53.3% recovery of growth of embryogenic 

culture (Mandal et al., 2009). 

These studies clearly provide the insights 

about the usefulness of cryo-techniques in 

conservation of valuable medicinal plants 

(Tashdeva and Kosturkova, 2012). However, 

research on cryopreservation techniques 

is relatively limited; hence, further 

research and development is essential in 

this area to utilize this technique/approach 

efficiently for the conservation of medicinal 

plants. 


Understanding of the challenges 

and opportunities in conservation of medicinal 

plants and to develop the biotechnological 

approaches to deal with it is 

important for the sustainable supply of 

medicinal plants. Medicinally important 

plants are continuously harvested for their 


traditional applications and for the development 

of novel drugs and or supplementary 

products. However, plant-derived 

drugs as medicines have been based on 

the assumption that the plants will be 

available on a continuing basis. As much 

more attention is given for discovery of 

new drugs from medicinal plants, the demand 

for raw material is steadly growing. 

However, there are no enough meticulous 

efforts to ensure the availability of targeted 

medicinal plants for harvesting to fulfil 

the industry’s demand. Moreover, there 

are no sustainable approaches developed 

for harvesting as well as for conservation 

of in-demand medicinal plants. Due to 

this awful situation, many medicinal plant 

species are becoming rare, vulnerable, 

endangered and or extinct. Unfortunately, 

very meager efforts have been undertaken 

for their conservation and sustainable 

supply. Therefore, there is a need to intensify 

the efforts for not only for the 

conservation but also to ensure sustainable 

supply of medicinal plants. Biotechnological 

tools such as micropropagation, 

cryopreservation and transgenic approach 

should be used efficiently for the conservation 

of medicinal plants for the sustainable 

growth and development of the industry, 

people and the planet. 

References 

Agrawal, A. (2005). Current issues in 

quality control of natural products. 

Pharma Times 37, 9-11. 

Ahuja, S. Mandal, B.B. Dixit, S. and 

Srivastava, P. S. (2002). Molecular 

phenotypic and biosynthetic stability 

of plants recovered from cryopreserved 

shoot-tips of Dioscorea 

floribunda. Plant Science3, 971- 

977. 

Arai, K. (2006). Toward biotechnology: 

The mission of IUBMB in the 21st 

century. IUBMB Life 58, 267-268. 

Atanasov, A. G. Waltenberger, B. 

Pferschy-Wenzig, E. Linder, T. 

Wawrosch, C. Uhrin, P. Temml, 

V. Wang, L. Schwaiger, S. Heiss, 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 124




E. Rollinger, J. M. Schuster, D. 

Breuss, J. M. Bochkov, V. Mihovilovic, 

M. D. Kopp, B. Bauer, 

R. Dirsch, V. M. and Stuppner, 

H. (2015). Discovery and resupply 

of pharmacologically active plantderived 

natural products: A review. 

Biotechnology Advances 33, 1582- 

1614. 

Bunn, E. Turner S. Panaia M. and Dixon 

K. W. (2007). The contribution 

of invitro technology and cryogenic 

storage to conservation ofindigenous 

plants. Australian Journal of 

Botany 55, 345–355. 

Bukuluki, P. Luwangula, R. and Walakira 

E. J. (2014). Harvesting of 

Medicinal Plants in Uganda: Practices, 

Conservation and Implications 

for Sustainability of Supplies. 

Online International Journal of 

Medicinal Plant Research 3, 1-10. 

Cantor, C. R. (2000). Biotechnology in 

the 21st century. Trends in Biotechnology 

18, 6-7. 

Chandra, S. Bandopadhyay, R. Kumar, 

V.and Chandra, R. O. (2010). Acclimatization 

of tissue cultured 

plantlets: from laboratory to land. 

Biotechnology Letters 32, 1199- 

1205. 

Dolcet-Sanjuan, R. Claveria, E. 

Camprubi, A. Estaun, V. and 

Calvet, C. (1996). Micropropagation 

of walnut trees (Juglans regia 

L.) and response to arbuscular mycorrhizal 

inoculation. Agronomie 

16, 639-645. 

Engelmann, F. (2011). Use of biotechnologies 

for the conservation of 

plant Biodiversity. In Vitro Cellular 

& Developmental Biology 47, 5-16. 

Gailīte, A. Kļaviņa, D. and Ievinsh, G. 

(2010). In vitro propagation of an 

endangered plant Saussurea esthonica. 

Environmental and Experimental 

Biology 8, 43-48. 

Gangaprasad, A. Decruse, S. W. Seeni, 

S. and Nair, G. M. (2005). Micropropagation 

and restoration of (Joseph 

and Chandra) Venter- an endemic 

and endangered ethano medicinal 

plant of Western Ghats. Indian 

Journal of Biotechnology 4, 

265-270. 

Gavrilescu, M. (2004). Removal of 

heavy metals from the environment 

by bio-sorption. Engineering in 

Life Sciences 4, 219-232. 

Ghimire, S. K. McKey, D. and Aumeeruddy-Thomas, 

Y. (2005). Heterogeneity 

in ethno ecological 

knowledge and management of medicinal 

plants in the Himalayas of 

Nepal: Implication for conservation. 

Ecology and Society 9, 6. 

Georgiev, M.I. Pavlov, A.I. and Bley, T. 

(2007). Hairy root type plant in 

vitro systems as sources of bioactive 

substances. Applied Microbiology 

and Biotechnology 74, 1175- 

1185. 

Guillon, S. Tremouillaux-Guiller, J. 

Pati, P.K. Rideau, M. and Gantet, 

P. (2006). Harnessing the potential 

of hairy roots: dawn of a new era. 

Trends in Biotechnology 24, 403- 

409. 

Godfray, H. C. J. Beddington, J. R. 

Crute, I. R. Haddad, L. Lawrence, 

D. Muir, J. F. Pretty, J. 

Robinson, S. Thomas. S. M. and 

Toulmin, C. (2010). The Challenge 

of Feeding 9 Billion People. Science 

327, 812-818. 

Hishe, M. Asfaw, Z. and Giday, M. 

(2016). Review on value chain 

analysis of medicinal plants and the 

associated challenges. Journal of 

Medicinal Plants Studies 4, 45-55. 

Hunde, D. (2007). Human influence and 

threat to biodiversity and sustainable 

living. Ethiopian Journal of Education 

and Science 1, 85-94. 

Ibrahima, M. A. Nad, M. Oha J. Schinazie, 

R. F. McBrayerg, T. R. 

Whitakerg , T. Doerksenb, R. J. 

Newmani, D. J. Zachosj, L. J. and 

Hamanna, M. T. (2013). Significance 

of endangered and threatened 

plant natural products in the control 

of human disease. Proceeding of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 125



National Academy of Science USA 

110, 16832–16837. 

Jan, A. Thomas, J. Shawl, A. S. Jabeen, 

N. and Kozgar, M. I. (2010). Improved 

Micropropagation Protocol 

of an Endangered Medicinal Plant- 

Picrorhiza Kurroa Royleex Benth. 

Promptly Through Auxin Treatments. 

Chiang Mai Journal of Science 

37, 304-313. 

Jain, M. Johnson, T. S. and Krishnan, 

P. (2012). Biotechnological approaches 

to conserve the wealth of 

nature: endangered and rare medicinal 

plant species, a review. Journal 

of Natural Remedies 12, 93-102. 

Kaczmarczyk, A. Funnekotter, B. 

Menon, A. Phang, P. Y. Al- 

Hanbali, A. Bunn, E. and 

Mancera R. L. (2012). Current issues 

in plant cryopreservation. In: 

Current Frontiers in Cryobiology. 

Katkov I. (ed.). In Tech, Croatia, 

pp. 417-438. 

Kala, C. P. (2005). Ethnomedicinal botany 

of the Apatani in the Eastern 

Himalayan region of India. Journal 

of Ethnobiology and Ethnomedicine 

1, 111-118. 

Kala, C. P. Dhyani, P. P. and Sajwan, 

B. S. (2006). Developing the medicinal 

plants sector in northern India: 

challenges and opportunities. Journal 

of Ethnobiology and Ethnomedicine 

2, 1-15. 

Khan, S. Al-Qurainy, F. and Nadeem, 

M. (2012). Biotechnological approaches 

for conservation and improvement 

of rare and endangered 

plants of Saudi Arabia. Saudi Journal 

of Biological Sciences 19, 1-11. 

Krishnan, P. N. Decruse, S. W. Radha, 

R. K. (2011). Conservation of medicinal 

plants of Western Ghats, India 

and its sustainable utilization 

through in vitro technology. In 


Biology 47, 110-122. 

IUCN (2013). www.iucn.organization 

access on May 25, 2014. 


Lakshmi, G. N. and Seeni, S. (2003). In 

vitro multiplication of Calophyllum 

apetalum (Clusiaceae), an endemic 

medicinal tree of the Western 

Ghats. Plant Cell Tissue and Organ 

Culture 75, 169-174. 

Lahlou, M. (2013). The success of natural 

products in drug discovery. 

Pharmacology & Pharmacy 4, 17- 

31. 

Manohar, P. R. (2012). Sustainable harvesting 

of medicinal plants: Some 

thoughts in search for solutions. 

Ancient Science of Life 32, 1-2. 

Mandal, B. B. (2000). Cryopreservation 

research in India: current status and 

future perspectives. In: Cryopreservation 

of tropical plant germplasmcurrent 

research progress and applications. 

Engelmann F.; Takagi H. 

(eds). JIRCAS, Tsukuba, pp 282- 

286. 

Mandal, B. B. Sharma, D. S. Srivastava, 

P. S. (2009). Cryopreservation 

of embryogenic cultures of Dioscorea 

bulbifera L. by encapsulation- 

dehydration. Cryo Letters 30, 

440-448. 

Muñoz-Concha, D. and Davey, M. R. 

(2011). Micropropagation of the 

endangered Chilean tree, Gomortega 

keule. In Vitro Cellular & Developmental 

Biology 47, 170-175. 

Nalawade, S. M. Sagare, A. P. Lee, C. 

Y. Kao, C. L. and Tsay, H. S. 

(2003). Studies on tissue culture of 

Chinese medicinal plant resources 

in Taiwan and their sustainable utilization. 

Botanical Bulletin of Academia 

Sinica 44, 79-98. 

O'Gara, F. (1996). The biotechnology 

and ecology of rhizosphere microorganisms. 

In: Novel biotechnological 

approaches to plant production: 

from sterile root to mycorrhizosphere. 

Proceeding of the Joined 

Meeting between 8.21 and 8.22, Pisa, 

Italy, 14-15 July, 1996. 

Pandey, P. Mehta, R. and Upadhyay, 

R. (2013). In vitro propagation of 

an endangered medicinal plant Pso- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 126



ralea corylifolia L. Asian Journal of 

Pharmaceutical and Clinical Research, 

6, 115-118. 

Pathak, M. R. and Abido, M. S. (2014). 

The role of biotechnology in conservation 

of biodiversity. Journal of 

Experimental Biology and Agricultural 

Sciences 2, 352-363. 

Prescott-Allen, R. and Prescott-Allen, 

C. (1996). Assessing the sustainability 

of uses of wild species. Case 

studies and initial assessment procedure. 

– Gland & Cambridge, 

IUCN (Occasional Paper of the 

IUCN Species Survival Commission 

12). 

Rai, M. K. (2010). Review: Biotechnological 

strategies for conservation 

of rare and endangered medicinal 

plants. Biodiversitas 11, 157-166. 

Rao, M. R. Palada, M. C. and Becker, 

B. N. (2004). Medicinal and aromatic 

plants in agro-forestry systems. 

Agroforestry Systems 61, 107- 

122. 

Rathore, S. and Singh, N. (2013). In 

vitro conservation of bacopa monneri 

– An endangered medicinal 

plant. Global Journal of Bio Sciences 

and Biotechnology 2, 187- 

192. 

Ray, A. Bhattacharya, S. (2010). Storage 

and conversion of Eclipta alba 

synseeds and RAPD analysis of 

converted plantlets. Biologia 

Plantarum 54, 547-550. 

Report of the European Commission, 

(2008). Available online at 

http://ec.europa.eu/; accessed on 

April 18, 2017. 

Sehrawat, N. Yadav, M. and Jaiwal, P. 

K. (2013). Development of an efficient 

in vitro regeneration protocol 

for rapid multiplication and genetic 

improvement of an important endangered 

medicinal plant Psoralea 

corylifolia. Asian Journal of Plant 

Science and Research 3, 88-94. 

Shahzad. A. and Saeed, T. (2013). In 

Vitro Conservation Protocols for 

Some Rare Medicinal Plant Species. 


In: Recent Trends in Biotechnology 

and Therapeutic Applications of 

Medicinal Plants Mohd. S. A. 

Abida, M. and Aastha, S. (eds). pp 

263-292. 

Sharma, N. B. and Sharma B (2003). 

Cryopreservation of shoot tips of 

Picrorhiza kurroa Royle ex Benth, 

an indigenous endangered medicinal 

plant through vitrification. Cryo 

Letters 24, 181-90. 

Sharma, U. and Mohan, J. S. S. (2006). 

In vitro clonal propagation of Chlorophytum 

borivilianum Sant et Fernand., 

a rare medicinal herb from 

immature floral buds along with inflorescence 

axis. Indian Journal of 

Experimental Biology 44, 77-82. 

Sheikhpour, S. Yadollahi, P. Fakheri, 

B. A. and Amiri, A. (2014). Biotechnological 

strategies for conservation 

of rare medicinal plants. International 

Journal of Agriculture 

and Crop Sciences. International 

Journal of Agriculture and Crop 

Sciences 7, 79-82. 

Shekhawat, M. S. Manokari, M. and 

Revathi, J. (2017). In vitro propagation 

and ex vitro rooting of Aerva 

lanata (L.) Juss. ex Schult.: a rare 

medicinal plant. Indian Journal of 

Plant Physiology 22, 40-47. 

Shewry, P.R. Jones, H.D. and Halford, 

N.G. (2008). Plant biotechnology: 

transgenic crops. Advances in Biochemcial 

Engineering/ Biotechnology 

111, 149-86. 

Sylvia, D. M. Alagely, A. K. Kane, M. 

E. and Philman, N. L. (2003). 

Compatible host/mycorrhizal fungus 

combinations for micropropagated 

sea oats. I. Field sampling and 

greenhouse evaluations. Mycorrhiza 

13, 177-183. 

Shibli, R. A. Al-Ababneh, S. S. and 

Smith M. A. L. (2004). Cryopreservation 

of plant germplasm: a review 

(scientific review). Dirasat 

Agricultural Sciences 31, 60-73. 

Sreekumar, V. B. and Renuka, C. 

(2006). Assessment of genetic di- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 127



versity in Calamus thwaitesii BECC 

(Arecaceae) using RAPD markers. 

Biochemical Systematics and Ecology 

34, 397-405. 

Srivastava, V. Khan, S. A. and 

Banerjee, S. (2009). An evaluation 

of genetic fidelity of encapsulated 

microshoots of the medicinal plant: 

Cineraria maritima following six 

months of storage. Plant Cell Tissue 

and Organ Culture 99, 193-198. 

Tasheva, K. and Kosturkova, G. 

(2012). Towards Agrobacteriummediated 

transformation of the endangered 

medicinal plant golden 

root. AgroLife Scientific Journal 1, 

132-138. 

Tejovathi, G. Goswami, H. and 

Bhadauria, R. (2011). In vitro 

propagation of endangered medicinal 

plant–Commiphora wightii. Indian 

Journal of Science and Technology 

4, 1537-1540. 

Touchell, D. H. and Dixon, K. W. 

(1994). Cryopreservation for seed 

banking of Australian species. Annals 

of Botany 40, 541-546. 

Tripathi, L F. and Tripathi, J. N. 

(2003). Role of biotechnology in 

medicinal plants. Tropical Journal 

of Pharmaceutical Research 2, 243- 

253. 

Van De Kop, P. Alam, G. and De 

Steenhuijsen, B. P. (2006). Developing 

a sustainable medicinal-plant 

chain in India - Linking people, 

markets and values. In: Agro-food 

chains and networks for development. 

Ruben, R. Slingerland, M. 


and Nijhoff, H. (eds.). Springer, 

Netherlands pp 191-202. 

Verma, P. Mathur, A. K. Jain, S. P. 

and Mathur, A. (2012). In Vitro 

Conservation of Twenty-Three 

Overexploited Medicinal Plants Belonging 

to the Indian Sub Continent. 

The Scientific World Journal 

2012, 1-10. 

Verma, S. and Singh, S.P. (2008). Current 

and future status of herbal medicines. 

Veterinary World 11, 347- 

350. 

Voets, L. Dupré de Boulois, H. Renard, 

L. Strullu, D. G. and Declerck, S. 

(2005). Development of an autotrophic 

culture system for the in 

vitro mycorrhization of potato 

plantlets. FEMS Microbiology Letters 

248, 111-118. 

Wang, H. Gao, S. da Silva, J. A. T. and 

Guohua, M. (2015). Agrobacterium 

rhizogenes-mediated genetic 

transformation of Psammosilene tunicoides 

and identification of high 

saponin-yielding clones. Environmental 

and Experimental Biology 

13: 19-23. 

Williams, P. G. Roser, D. J. and Seppelt, 

R. D. (1994). Mycorrhizas of 

hepatics in continental Antarctica. 

Mycological Research 98, 34-36. 

WHO, (2010). URL, 

http://www.who.int/mediacentre/fac 

tsheets/fs-134/en/. Accessed on 

April 3, 2017. 

© 2017 by the authors. Licensee Editors and AIMST University, Malaysia. 




ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 128



Making Himalayas Sustainable: Opportunities and 

Challenges in Indian Himalayan Region 

Harsh Kumar Chauhan and Anil Kumar Bisht* 

Department of Botany, D.S.B. Campus, Kumaun University, Nainital, 263001, Uttarakhand, 

India; *Correspondence: bishtakb@rediffmail.com; Tel: +91 9412044500 

Abstract: Himalayas are among the most vulnerable ecosystems of the world. They harbor 

unique geology/geography, ecosystem, biodiversity, and several other life sustaining biotic 

and abiotic resources. Sustainability of Himalayas is being challenged by increased tourism 

activities, deforestation, pollution, unmanaged exploitation of bio-resources, climate change 

and unplanned developmental activities. Besides, the region has several resources of aesthetic 

and economic interest that can be harnessed for generation of income and employment 

to millions of the people residing in the region. Understanding the specific problems 

of Himalaya and carving out the prospects for its sustainable development is a difficult task. 

Looking at the present scenario, the chapter provides an overview of the opportunities and 

challenges for achieving sustainability in Indian Himalayan Region. The present trends 

suggest that the existing interventions in the region are unsustainable. Further, the unscientific 

exploitation of natural resources is increasing the environmental degradation in the region. 

Proper policies and their implementation for harnessing the potential of the natural 

resources are urgently needed for the sustainable development of the region. 

Keywords: Himalayas; hotspot; sustainability; unscientific exploitation; vulnerable 


The Himalayas are among the 

most vulnerable mountain ecosystem 

stretching between the Indus and Brahmaputra 

river valleys (Bawa et al., 2010). 

They spread across the eight countries; 

Afghanistan, Bangladesh, Bhutan, China, 

India, Myanmar, Nepal and Pakistan. The 

Himalayas harbor unique biodiversity, 

ecosystem composition and several other 

life sustaining resources. They are the 

source of 10 of the largest rivers in Asia 

which provides water to about 1.3 billion 

people (Xu et al., 2007; Bates et al., 

2008). In addition to provide water, the 

Himalayas provide huge inputs to agriculture 

through regulating micro-climates as 

well as wind and monsoon circulation 

(Rasul, 2010) supporting life of about 40 

million people (Zurik and Pacheco, 2006). 

They are known to facilitate vital ecological 

and economic security of the people 

living downstream. They are also considered 

as the repository of geological and 

agriculture assets and harvested wild 

goods (Badola et al., 2015). 

Unfortunately, the entire region is 

prone to several disasters due to fragile 

geophysical structures, high peaks, and 

high angle of slope and variable climatic 

conditions (Chhetri, 2001). This vulnerability 

increases several folds with the increasing 

human population, exploitation 

of the natural resources and the effects of 

the climate change (Liu and Chen, 2000; 

Dyurgerov and Meier, 2005). Poverty in 

the Himalayan region is high and persistent 

(Hunzai et al., 2011). Some of the 

areas in the region are under territory disputes 

between the nations which are associated 

with the military presence along 

the international border; for instant the 

degradation of the Hind Kush, Karako- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 129


Opportunities and Challenges in Indian Himalayan Region 

rum, Western Himalaya and the Kashmir. 

This situation is particularly damaging to 

fragile ecosystem whose recovery is particularly 

slow (Bawa et al., 2010). The 

political situation thus prevail prevent the 

proper policy implementation for developmental 

programs causing the unfair 

treatment of the people residing in these 

areas. These synergistic effects seem to 

make Himalayas the hotspot for the physical, 

economic and social vulnerability. 

The studies carried out on the 

mountain ecosystems throughout the 

world concluded that mountains are in 

dire need of relief from anthropogenic 

activities (Jodha, 2005). Sustainability of 

Himalayas is being challenged by increased 

tourism activities, deforestation, 

pollution, climate change and unplanned 

development. Besides all this, the region 

has several resources of symbolic and 

economic values whose harnessing can 

provide income and employment to millions 

of the people residing in the region. 

Understanding the specific Himalayan 

problems and prospects of the sustainable 

development is not an easy task. International 

failure to recognize the economic 

value of the issues of sustainability using 

policy tools in the Himalayas has been 

cited as the major cause of this continued 

Chauhan and Bisht 

degradation (Singh, 2002; Blaikie and 

Muldavin, 2004). Several scientific and 

political forums have emphasized the 

uniqueness, environmental challenges and 

political legacies of the Himalayan region 

so that sustainable planning and management 

in the region can be worked out. 

Global change and World’s Mountains 

conference held at Perth, Scotland in 2010 

also identified several research gaps in 

sustainable mountain development. Looking 

at the current scenario, the present 

chapter provides the overview of the opportunities 

and challenges of sustainability 

in the Himalayas with special reference 

to Indian Himalayan Region. 

2. Indian Himalayan region (IHR) 

IHR occupies a special place in 

the mountain ecosystem of the world 

(Singh, 2006). The region extends between 

latitude 26 o 20’ and 35 o 40’ North, 

and between longitudes 74 o 50’ and 95 o 40’ 

East covering 530, 795 sq. km of geographic 

area. It spreads across the states 

of Jammu and Kashmir, Himachal Pradesh, 

Uttarakhand, Sikkim, Arunachal 

Pradesh, Meghalaya, Nagaland, Manipur, 

Mizoram, Tripura and the hill regions of 

Assam and West Bengal (Figure 1). It co- 

Figure 1: A picture of the Himalayan landscape. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 130



nstitutes about 16.2% of India’s administered 

geographical area. Most of the area 

in the region is covered with the snow 

clad mountain peaks, glaciers in the higher 

Himalayas and dense forest in the mid- 

Himalayas thus harbouring a rich variety 

of flora, fauna and cultural diversity. 

3. Opportunities and challenges for 

sustainability in the IHR 


3.1. Water potential 

Himalayas has been rightly 

acknowledged as the ‘Water Tower of 

Asia’. Approximately 10-20 % of the area 

is covered by glaciers while 30-40% remains 

under seasonal snow cover (Bahadur, 

2004). Being the source of Asia’s 10 

largest rivers (Amu Darya, Indus, Ganges, 

Brahmaputra, Irrawaddy, Salween, Mekong, 

Yangtze, Yellow and Tarim) (Xu et 

al., 2007) they provide drinking water, 

irrigation, fisheries, hydropower, and 

supports several terrestrial and aquatic 

ecosystems. The Ganges River system is 

the main source of fresh water to more 

than half the population of India and 

Bangladesh and nearly entire population 

of Nepal (Rasul, 2014). About 60% of the 

India’s irrigated area of 546,820 Km 2 is in 

the Ganges basin (National Ganga River 

Basin Authority, 2011). Indus irrigation 

system irrigates about 14.3 million hectares 

of farmland constituting the world’s 

largest contiguous irrigation system; enabling 

the production of more than 80% 

food grains of Pakistan (GoP, 2010). Amu 

Darya irrigates 385,000 ha of farmland in 

Afghanistan contributing a reliable source 

of Afghanistan’s food and water security 

(NAS, 2012). The ground water flow 

through bedrocks is approximately six 

times the annual contribution from glacial 

ice melt and snow melt to central Himalayan 

Rivers (Andermann et al., 2012). 

The government of India had recognized 

the hydropower potential of the 

IHR. The country’s hydropower potential 

is 148,701 MW out of which more than 

75% (117,139 MW) resides in IHR; however 

only 22.37 % potential has been developed 

and 9.09% is under construction 

(CEA, 2009). Accordingly the Prime 

Minister of India launched a 50,000 MW 

hydroelectric initiative program, formulated 

by CEA for preparation of Preliminary 

Feasibility reports of 162 new hydropower 

schemes (47,930MW) and out 

of these 133 are in IHR (Agarwal et al., 

2010). 

The development of hydropower 

offers several advantages to the economy 

of the nation. It is the source of clean renewable 

source of energy offering the 

mitigation of climate change issues and 

achieving the sustainability goals. It provides 

the inexpensive power, especially 

when the project achieves financial 

breakeven. The development of the hydropower 

project is expected to improve 

the infrastructure of the remote areas as 

well as it helps in flood moderation, irrigation, 

navigation and providing drinking 

water all the year around. 

Unfortunately several challenges 

are associated with the development of 

the hydropower in IHR. It has been realized 

that the development of the hydropower 

projects has significant environmental 

and social impact (Goldsmith and 

Hildyard, 1984). These projects alter the 

vital ecological process such as flow of 

water, sediments, nutrients, energy and 

biota (Franklin et al., 1995). The IHR is 

among the most seismically active zone 

of the world; the construction of the hydropower 

project increases the chances of 

the earthquakes which may shatter the 

lives of millions of people. Proper Environment 

Impact Assessment appears to be 

the biggest challenge for the implementation 

of the hydropower project in IHR. In 

a nutshell it may cause multi-dimensional 

unpredictable ecological disturbances and 

loss of biodiversity, productive land, social 

and cultural heritage. Hence, the development 

interventions related to hydropower 

in the IHR should have different 

approach. As per the reports of Chopra 

committee, formulated by the order of 

Supreme Court of India in the year 2014 

to assess the role of hydroelectric projects 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 131



in Uttarakhand, the negative impacts of 

the small hydropower projects can be less 

intense and therefore mitigated more easily. 

On the other hand, large projects often 

lead to massive impacts that are hard to 

mitigate and may result in permanent 

scarring of nature and society. Further, 

the apex court also warned of negative 

impacts on geological environment, river 

ecosystem & forests and terrestrial biodiversity 

(Table 1). 

3.2. Biodiversity 

Climatic, topographic, geological 

and altitudinal variations have generated 

unique landscape (Figure 2), ecosystems 


and biodiversity in the Himalayas 

(Shrestha et al., 2012). The complex 

orogeny of the Himalayas, coupled with 

climatic and edaphic changes facilitate the 

colonization of floral and faunal diversity 

in the region (Pandit et al., 2000). Himalayas 

are the repository of the extremely 

rich and endemic biodiversity (Chatterjee, 

1939; Nayar, 1996; Pandit et al., 2000). 

The region hosts the parts of four global 

biodiversity hotspots (viz. the Himalayas 

hotspot, the Indo-Burma hotspot, South- 

West China Hotspot and the Mountains of 

Central Asia hotspot) (Mittermeier et al., 

2004). Himalayas are very important in 

terms of sustaining high levels of the 

Table 1: Negative impact of hydropower projects* 

Activity 

Impact 

I. Pre-project construction 

1. Construction of approach 

roads 

Land acquisition (displacement, loss of lands, 

homes and livelihoods) 

Deforestation (loss of tree cover, access to CPRs, 

soil erosion and landslides, loss of flora and fauna, 

changes in micro-climate) 

Disposal of debris and earth (loss of trees, river water 

pollution) 

2. Construction of housing Deforestation 

for staff and labour 

Pollution due to sewage release 

3.Quarrying Noise pollution, slop destabilization, disruption of 

underground seepage and damage to house. 

II. Project construction 

4.Tunneling 

5.Dam construction 

Air and soil pollution, destabilization of slopes, damage to 

houses, disturbing wildlife, drying of springs, disposal of 

muck into the river, psychological trauma to people and 

animals due to repeated blasts 

Disruption of river flows (biotic changes, disruption of natural 

functions, e.g. sediment disposal, land shaping, nutrient 

cycling), river pollution, loss of aesthetic, cultural, economic 

and recreational values. 

III. Project operation 

6. Testing of tunnels Slope destabilization (loss of tree cover, land, livelihoods, 

water sources and access to CPRs) 

7.Water storage and release 

 

 

 

Sedimentation (effect on river water quality) 

Disruption of river flow 

Secondary effects (release of greenhouse gases, 

warming of valleys, increased earthquake risks, 

floods, downstream urban and industrial development 

8.Laying of power lines Deforestation (loss of wild life habitat), soil erosion 

*Information source: http://iced.cag.gov.in 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 132




Figure 2: Indian Himalayan Region (IHR) (Source: 

www.http://gbpihedenvis.nic.in/indian_him_reg.htm) 

biodiversity. However, biodiversity loss 

from the region had become a matter of 

great concern. There are several factors 

such as anthropogenic pressure and the 

climate change which contributes to the 

loss of biodiversity in the region. 

Due to the collation of several geographic 

and climatic features IHR provides 

very suitable environment for flourishing 

the huge biodiversity. IHR supports 

nearly 50% of the total flowering 

plants in India of which 30% are endemic 

to the region (Singh et al., 2006). It harbours 

816 tree species, 675 edibles and 

1748 species of medicinal value (Samant 

et al., 1998). The forests of the region 

have phenomenal diversity that meets the 

diverse needs of the people (Singh and 

Singh, 1992). The forest of the region acts 

as the sink of the carbon dioxide and provides 

timber, wild edibles, gums, resins 

and other numerous products of immense 

value. 

Massive deforestation, extensive 

shifting cultivation and the demands for 

agricultural land are the primary drivers 

for the biodiversity loss in IHR. At the 

current rate of deforestation in IHR, the 

total forest cover (84.9 % in 2000) and 

coverage of dense forest (75.4% in 2000) 

is expected to be reduced to 52.8% and 

34% respectively by the year 2100 (Pandit 

et al. 2007). This could have serious 

implications on the diversity of the flora 

and fauna of the region. Looking at the 

ever-increasing threats to the biological 

diversity in the region; there is an urgent 

need of the proper actions or otherwise it 

may bring huge setback to the economic 

benefits of the local populations. 

3.3. Medicinal plants 

Among the biodiversity elements, 

the roles of medicinal plants is remarkable 

in the health care of the Himalayan 

people as most of the them resides in the 

remote locations where the allopathic system 

of medicines is not practiced to such 

an extent. Besides the health care, medicinal 

plants have high socio-cultural, symbolic 

and economic value, providing income 

and employment to millions of peo- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 133



ple living in the region (Ghimire, 2008). 

Himalayan medicinal plants meet most of 

the international demand. Overexploitation 

seems the biggest challenge 

for the survival of these plants though 

they also have slow growth rates, low 

population densities and narrow geographical 

ranges (Kala et al., 2006). 

IHR host 1748 plants species of 

medicinal value including 1685 angiosperms, 

12 gymnosperms and 51 pteridophytes 

(Samant et al., 1998). Ved et al. 


(2001) estimated 3200 medicinal plants 

from Indian Himalayas including 700 

from trans-Himalaya (Table 2). India is 

ranked 2 nd after China in terms of medicinal 

plants export and exports about 

32,600 tons of medicinal raw material 

worth about US $46 million annually 

(Lange, 1997). High nativity and endemism 

of medicinal plants is associated 

with the IHR. Out of 1748 medicinal 

plants, about 548 species are identified in 

HP, 707 in Sikkim and Darjeeling and 

Table 2: Medicinal plants, species diversity and representative species of different biogeographic 

zones of India # 

Biogeographic region 

Estimated no. of Examples of some typical medicinal species 

medicinal plants 

Trans-Himalayas 700 Ephedra geradiana Wall., Hippophae rhamnoides 

L., Arnebia euchroma (Royle) John 

Himalayan 2500 Aconitum heterophyllum Wall. ex Royle,.Ferula 

jaeshkeana Vatke and Saussurea costus (Balc). 

Lipsch., Nardostachys grandiflora D.C. Taxus 

wallichiana Zucc,. Rhododendron anthopogon 

D.Dun and Ponax pseudoginseng Wall. 

Desert 500 Convolvulus microphyllus Seib ex Spreng., 

Tecomella undulata (Sm.) Seem., Citrulus colocynthis 

(L.), Schraderand Cressa crertica L. 

Semi-Arid 1000 Commiphora wightii (Arn.) 

Bhandari, Caesalpinia 

bonduc (L.) 

Roxb, Balanites aegyptiaca (L.), Delilie 

and Tribulus rajasthanensis Bhandari & Sharma. 

Western Ghats 2000 Myristica malabarica Lam., Garcinia indica 

(Thou.) Choisy, Utleria salicifolia Bedd 

and Vateria indica L. 

Deccan Peninsula 3000 Pterocarpus santalinus L.f., Decalepis hamiltonii 

Wigh & Arn, Terminalia pallida Brandis 

and Shorea tumbuggaia Roxb 

Gangetic Plain 1000 Holarrhenaq pubescens (Buch-Ham.) Wall. ex 

DC., Mallotus philippensis (Lam.) Muell – 

Arg., Pluchea lanceolata C.B. Clarke 

and Peganum harmala L. 

North-East India 2000 Aquilaria malaccensis Lam., Smilax glabra 

Roxb., Ambroma augusts (L.) L.f. 

and Hydnocarpus hurzii (King) Warb. 

Islands 1000 Claophyllum inophyllum L. Adnanthera pavonina 

L., Barringtonia asiatica (L.), Kurz 

and Aisandra butyracea (Roxb.), Baehni. 

Coasts 500 Rhizophora mucronata Lam., Acanthus ilicifolius 

L., Avicennia marina Vierth and Sonneratia 

caseolaris (L.) engl. 

# Source: Ved et al., 2001; http://www.fao.org/docrep/007/ad871e/ad871e09.htm 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 134



701 in the Uttarakhand (Badola and Aitken, 

2003). The economic potential of the 

medicinal plants has been recognized in 

the IHR. It possesses various forest types 

with diverse habitats, slopes, aspects and 

altitudinal ranges which offers congenial 

environment for the natural and artificial 

propagation of the diverse medicinal 

plants. Such diversity of the medicinal 

plants would be helpful for further scientific 

research on exploring their medical 

efficacy (Kala et al., 2006). The aim set 

by India for establishing golden triangle 

between traditional medicine, modern 

medicine and modern science will be a 

boon for development of traditional herbal 

medicine and medicinal plant sector 

(Mashelkar et al., 2005). 

However, there is a paucity of research 

on the biology, habitat and adaptation 

mechanism of the majority of the 

threatened trade taxa of the medicinal 

plants (Dhar et al., 2000). Further, proper 

agro techniques for the cultivation of the 

medicinal plant are lacking in the Himalayas. 

It is noteworthy to mention here 

that < 90% of the plant raw material for 

herbal and industries in India and for export 

is drawn from natural habitats (Tandon, 

1996, Ved et al., 1998; Dhar et al., 

2000). Several challenges are associated 

with the sustainable development of the 

medicinal plant sector in IHR. Most important 

among these are low population 

size, habitat specificity, narrow distribution 

ranges, unscientific collection for 

commercial purposes, land use disturbances, 

introduction of non-native species, 

habitat loss and alteration, climate changes, 

heavy livestock grazing, unregulated 

tourism, construction of dams and roads, 

explosion of human population, population 

bottlenecks and genetic drift (Kala, 

2005). 

Recent advancement in the field of 

Plant Biotechnology especially Plant tissue 

culture has emerged as the promising 

technique for the conservation of these 

medicinal plants. In-vitro propagation of 

plants holds tremendous potential for the 

production of high-quality plant-based 


medicines (Fuentes et al., 1993). Using in 

vitro propagation methods several rare 

and endangered plant species can be 

quickly and successfully propagated with 

low impact on wild population (Cuenca et 

al., 1999). Plant cell cultures represent a 

potential source of valuable secondary 

metabolites which can be used as food 

additives, nutraceuticals, and pharmaceuticals. 

The major advantage of the synthesis 

of phytochemicals by the cell cultures 

is that they are independent of environmental 

conditions and quality fluctuations. 

Therefore, this sector undoubtedly 

offers several opportunities for the sustainable 

development of the region however 

there are lots of challenges associated 

with the sound development of the sector 

in the region. 

3.4. Agriculture assets and food security 

Agriculture assets and food security 

is a critical issue of Himalayas due to 

complete dependency on rain besides 

general characteristics of remoteness, low 

market integration and underdeveloped 

agrarian resources. However, agriculture 

and allied sector forms the pivotal part of 

the people living in the Himalayas. The 

huge diversity in the Himalayas has been 

maintained through a variety of crop 

composition, indigenous methods of 

maintaining soil fertility, socio-cultural 

and religious rituals (Negi and Maikhuri, 

2013). 

IHR is the storehouse for the diverse 

genetic stocks. For instance, farmers 

of the Central Himalaya grow about 100 

varieties of paddy, 170 varieties of kidney 

beans, 8 varieties of wheat,4 varieties of 

barley and about a dozen varieties of 

pulses and oil seeds each year and farmers 

of Uttarakhand Himalaya are known for 

cultivating 34 crop species comprising of 

6 types of cereals, 5 types of pseudo cereals, 

6 types of millets, 16 types of pulses, 

4 types of oilseeds, 5 types of condiments 

and 8 types of vegetables (Negi and 

Maikhuri, 2013). The wild fruits of IHR 

have significantly attracted the attention 

of the entire world from the Nutraceutical 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 135



point of view. However, at present the 

decline in the interest of farming has been 

observed as a result of climatic uncertainty 

and cultural transformation. The consequences 

of this may be disastrous because 

of genetic erosion of some unique 

and diverse gene bank. 


3.5. Tourism /aesthetic/ ecological services 

Tourism industry typically depends 

on the quality of the natural resources. 

Himalayas are blessed with natural 

beauty and pilgrim centers which attracts 

the millions of International tourists 

throughout the year. They have abundant 

of natural resources like spectacular landscapes, 

mountains, glaciers, rivers, lakes, 

fountains, snow clad peaks, forests having 

high floral and faunal diversity, which 

offers strong resonance with tourism. 

These bioresources make Himalayas suitable 

for establishing Sanatoria and rejuvenating 

centers. Thus, they have huge 

potential for promoting tourism in the 

form of natural and cultural heritage. 

Economic potential of tourism that 

could promote to sustainability in IHR is 

well recognized.IHR host several tourist 

destinations which provides livelihood to 

millions of people residing in the region. 

With the arrival of Britishers in 19 th century, 

several hill stations like Nainital, 

Darjelling, Mussoorie, Shimla etc. were 

established. They are the tourist hotspots 

for thousands of national and international 

tourist. In India, both the state and central 

government have declared tourism to be 

an industry and provides same concessions 

and incentivizes of the industrial 

sector (Cole and Sinclair, 2002). 

However, several challenges are 

associated with the sustainable development 

of the tourism industry in IHR. Hollen,(2010) 

had identified lack of confidence 

in the economic certainty of tourism 

as the major challenge in the sustainable 

development of the tourism in Himalayas. 

He also advocates the philosophy 

of sustainable development constructed 

upon conservation, community participation 

and social equality. In order to utilize 

the tourism industry market, uncontrolled 

number of tourist there should be proper 

infrastructural facilities. The ecological 

pressures are threatening land, water and 

wildlife resources through direct and indirect 

environmental impacts together with 

generation of solid and liquid wastes 

(Singh, 2002). So, ecotourism or green 

tourism should be promoted for the sustainable 

development of the region. 

3.6. Other challenges (disasters, climate 

change, population increase, poverty 

and waste management) 

People living in IHR, are facing the problems 

and damages due to some sudden 

events such as forest fire, avalanches, 

cloud bursting, land and mudslides, earthquakes, 

debris flows, flash floods, paralyses 

the life and property of the people 

(Nyaupane and Chhetri, 2009). There are 

several evidences of Climate change and 

its impact in Himalayas (Beniston, 2003; 

Cruz et al., 2007; Xu et al., 2009). 

Among these impacts, the most widely 

reported is the receding of glaciers which 

could have disastrous impact on the survival 

of the millions of people. The ongoing 

climate change over succeeding decades 

will likely to have additional negative 

impacts across these mountains, including 

significant cascading effects on 

the river flows, ground water recharge, 

natural hazards, and biodiversity; ecosystem 

composition, structure and human 

livelihoods (Xu et al., 2009). The population 

in Himalayas is rapidly increasing; 

however there are finite resources to enhance 

the production ultimately promoting 

the poverty. Recently proper waste 

management appears as the critical problem 

in the Himalayas. Reducing forest 

cover, accelerated soil erosion, drying 

springs, biodiversity loss etc. seems the 

ever increasing challenges for the sustainability 

in the Himalayas. 


ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 136



Himalayas have undoubtedly huge 

ecological, life sustaining, recreational, 

educational and scientific values. Several 

challenges are associated for the sustainability 

in the region. The present trends 

suggest that the existing interventions in 

the region are unsustainable; further unscientific 

exploitation of natural resources 

is increasing the environmental degradation 

in the region (Singh, 2006). Proper 

policies and their implementation for harnessing 

the potential of the natural resources 

is the need of the hour for the sustainable 

development in the region. 

References 

Agrawal, D. K., Lodhi, M. S. and 

Panwar, S. (2010). Are EIA studies 

sufficient for projected hydropower 

development in the Indian 

Himalayan region? Current Science 

98(2), 154-161. 

Andermann, C., Longuevergne, L., 

Bonnet, S., Crave, A., Davy, P., 

and Gloaguen, R. (2012). Impact 

of transient groundwater storage 

on the discharge of Himalayan 

rivers. Nature Geoscience 5(2), 

127-132. 

Badola, R., Hussain, S. A., Dobriyal, P. 

and Barthwal, S. (2015). Assessing 

the effectiveness of policies 

in sustaining and promoting 

ecosystem services in the Indian 

Himalayas. International Journal 

of Biodiversity Science, Ecosystem 

Services & Management 11(3), 

216-224. 

Bahadur, J. (2004). Himalayan Snow 

and Glaciers – Associated Environmental 

Problems, Progress and 

Prospects, Concept Publishing Co, 

New Delhi. 

Bates, B.C., Kundzewicz, Z. W., Wu, 

S., and Palutik J. P. 

(2008). Climate change and water. 

Technical paper of intergovernmental 

panel on climate change, 

IPCC Secretarist, Geneva. 


Bawa, K. S., Koh, L. P., Lee, T. M., 

Liu, J., Ramakrishnan, P., Yu, 

D. W., Zhang, Y.P. and Raven, 

P. H. (2010). China, India and the 

Environment. Science 327, 1457- 

1459. 

Beniston, M. (2003). Climatic change in 

mountain regions: a review of 

possible impacts. Climatic Change 

59, 5–31. 

Blaikie, P. M. and Muldavin, J. S. 

(2004). Upstream, downstream, 

China, India: the politics of environment 

in the Himalayan region. 

Annals of the Association of 

American Geographers 94(3), 

520-548. 

Central Electricity Authority (CEA). 

(2009). Status of hydroelectric potential 

development, 31 August 

2009; http://www.cea.nic.in/ 

Chatterjee, D. (1939). Studies on the endemic 

flora of India and Burma. J 

R Asiat Soc Bengal (Sci) 5, 19–67. 

Chhetri, M. B. P. (2001). A Practitioner's 

view of disaster management in 

Nepal: organisation, system, problems 

and prospects. Risk Management 

3(4), 63-72. 

Cole, V. and Sinclair, A. J. (2002). 

Measuring the ecological footprint 

of a Himalayan tourist center. 

Mountain Research and development 

22(2), 132-141. 

Cruz, R. V., Harasawa, H., Lal, M., 

Wu, S., Anokhin, Y., Punsalmaa, 

B., Honda, Y., Jafari, 

M., Li, C. and Huu Ninh, N. 

(2007). Asia. In: Parry, M. L., 

Canziani, O.F., Palutikof J.P., van 

der Linden, P. J. and Hanson, C.E. 

(eds). Climate change 2007: impacts, 

adaptation and vulnerability. 

Contribution of Working 

Group II to the Fourth Assessment 

Report of the Intergovernmental 

Panel on Climate Change. Cambridge 

University Press, Cambridge, 

UK. pp. 469-506. 

Cuenca, S., Amo-Marco J. B. and Parra, 

R. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 137



(1999). Micropropagation from inflorescence 

stems of Spanish endemic 

plant Centaurea pauri Loscos 

ex. Willk (Compositae). Plant 

Cell Reproduction 18, 674-679. 

Dhar, U., Rawal, R. S. and Upreti, J. 

(2000). Setting priorities for conservation 

of medicinal plants––a 

case study in the Indian Himalaya. 

Biological conservation 

95(1): 57-65. 

Dyurgerov, M. B. and Meier, M. F. 

(2005). Glaciers and the changing 

Earth system: a 2004 snapshot 

(Vol. 58). Boulder: Institute 

of Arctic and Alpine Research, 

University of Colorado. 

Franklin, J. (1995). Predictive vegetation 

mapping: geographic modelling of 

biospatial patterns in relation to 

environmental gradients. Progress 

in physical geography 19(4), 474- 

499. 

Fuentes, S. I., Suarez, R., Villegas, T., 

Acero, L. C. and Hernandez, G. 

(1993). Embryogenic response of 

Mexican alfalfa (Medicago sativa) 

varieties. Plant Cell Tissue Organ 

Culture 34, 299- 302. 

Ghimire, S.K. (2008). Sustainable harvesting 

and management of medicinal 

plants in the Nepal Himalaya: 

current issues, knowledge 

gaps and research priorities. 

Medicinal Plants in Nepal: 

an Anthology of Contemporary 

Research 25-44. 

Goldsmith, E. and Hildyard, N. 

(1984). The social and environmental 

effects of large dams. Volume 

1: overview. Wadebridge 

Ecological Centre. 

GoP (Government of Pakistan). (2010). 

Final Report of the Task Force on 

Climate Change. Islamabad: Planning 

Commission. 

Holden, A. (2010). Exploring stakeholders' 

perceptions of sustainable 

tourism development in the Annapurna 

Conservation Area: Issues 

and challenge. Tourism and Hos- 


pitality Planning & Development 

7(4), 337-351. 

Hunzai, K., Gerlitz, J. Y. and Hoermann, 

B.(2011). Understanding 

mountain poverty in the Hindu 

Kush-Himalayas: regional report 

for Afghanistan, Bangladesh, Bhutan, 

China, India, Myanmar, Nepal, 

and Pakistan. International 

Centre for Integrated Mountain 

Development (ICIMOD). 

Jodha, N. S. (2005). Economic globalisation 

and its repercussions for fragile 

mountains and communities in 

the Himalayas. In Global change 

and mountain regions, Springer 

Netherlands, 583-591. 

Kala, C. P. (2005). Indigenous uses, population 

density, and conservation 

of threatened medicinal plants in 

protected areas of the Indian Himalayas. 

Conservation Biology, 

19(2), 368-378. 

Kala, C. P., Dhyani, P. P. and Sajwan, 

B. S. (2006). Developing the medicinal 

plants sector in northern 

India: challenges and opportunities. 

Journal of Ethnobiology and 

Ethnomedicine 2(1), 32. 

Lange, D. (1997). Trade figures for botanical 

drugs worldwide. Medicinal 

Plant Conservation Newsletter 

3, 16-17. 

Liu, X. and Chen, B. (2000). Climatic 

warming in the Tibetan Plateau 

during recent decades. 

International journal of climatology 

20(14), 1729-1742. 

Mashelkar, R. A. (2005). India's R&D: 

Reaching for the 

Top. Science 307(5714), 1415- 

1417. 

Mittermeier, R. A. (2004). Hotspots revisited. 

Cemex. 

NAS (National Academy of Sciences). 

(2012). Himalayan Glaciers: Climate 

Change, Water Resources, 

and Water Security. Washington, 

DC. National Academies Press. 

National Ganga River Basin Authority. 

(2011). National Ganga River Ba- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 138



sin Authority. Environmental and 

Social Analysis, Vol. 1, Government 

of India, New Delhi, India. 

Nayar, M. P. (1996). Hot-spot of endemic 

plants of India, Nepal and Bhutan. 

Tropical Botanic Garden and 

Research Institute, Thiruvananthapuram, 

Kerala, India. 

Negi, V. S. and Maikhuri, R. K. (2013). 

Socio-ecological and religious 

perspective of agrobiodiversity 

conservation: issues, concern and 

priority for sustainable agriculture, 

Central Himalaya. Journal of agricultural 

and environmental ethics 

26(2), 491-512. 

Nyaupane, G. P. and Chhetri, N. 

(2009). Vulnerability to climate 

change of nature-based tourism in 

the Nepalese Himalayas. Tourism 

Geographies 11(1), 95-119. 

Pandit, M. K., Bhakar, A. and Kumar, 

V. (2000). Floral diversity of 

Goriganga Valley in the Central 

Himalayan highlands. J Bombay 

Nat Hist Soc 97,184–192. 

Pandit, M. K., Sodhi, N. S., Koh, L. P., 

Bhaskar, A. and Brook, B. W. 

(2007). Unreported yet massive 

deforestation driving loss of endemic 

biodiversity in Indian 

Himalaya. Biodiversity and Conservation 

16(1) 153-163. 

Rasul, G. (2010). The role of the Himalayan 

mountain systems in food 

security and agricultural sustainability 

in South Asia. International 

Journal of Rural Management 

6(1), 95-116. 

Rasul, G. (2014). Food, water, and energy 

security in South Asia: a nexus 

perspective from the Hindu Kush 

Himalayan region. Environmental 

Science & Policy 39, 35-48. 

Samant, S. S., Dhar, U. and Palni, L. 

M. S. (1998). Medicinal Plants of 

Indian Himalaya: Diversity, Distribution, 

Potential Values, 

Gyanodaya Prakashan, Nainital. 

Shrestha, U. B., Gautam, S. and Bawa, 

K. S. (2012). Widespread climate 


change in the Himalayas and associated 

changes in local ecosystems. 

PLoS One 7(5), e36741. 

Singh, J. S. (2002). The biodiversity crisis: 

a multifaceted review. Current 

Science 82(6), 638-647. 

Singh, J. S. (2006). Sustainable development 

of the Indian Himalayan 

region: Linking ecological and 

economic concerns. Current Science 

90(6), 784. 

Singh J. S. and Singh S. P. (1992). Forests 

of Himalaya: Structure, Functioning 

and Impact of Man, 

Gyanodaya Prakashan, Nainital. 

Singh, R. B. (2002). Tourism development 

and environmental implications 

for the Indian Frontier Region: 

A Study of Himachal Himalaya. 

In: S. Krakover and Y. Gradus. 

Tourism in Frontier Areas. 

Lanham: Lexington Books. 

Tandon, V. (1996). CAMP workshopplants 

under threat new list forged. 

Medicinal Plant Conservation 

Newsletter 2, 12-13. 

Ved, D. K., Mudappa, A. and Shankar, 

D. (1998). Regulating export of 

endangered medicinal plant species: 

Need for scientific rigour. 

Current Science 75(4), 341- 

344. 

Ved P. (2001). Indian Medicinal Plants; 

Current Status. In: Samant SS, 

Dhar U, Palni, LMS (eds), Himalayan 

Medicinal Plants: Potential 

and Prospects. Nainital: 

Gyanodaya Prakashan. pp. 45–65. 

Xu, J. C., Shrestha, A. B., Vaidya, R., 

Eriksson, M. and Hewitt, K. 

(2007). The melting Himalayas: 

regional challenges and local impacts 

of climate change on mountain 

ecosystems and livelihoods. 

Technical paper. International 

Center for Integrated Mountain 

Development, Kathmandu, Nepal. 

Xu, J., Grumbine, R. E., Shrestha, A., 

Eriksson, M., Yang, X., Wang, 

Y. U. N. and Wilkes, A. (2009). 

The melting Himalayas: cascading 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 139



effects of climate change on water, 

biodiversity, and livelihoods. 

Conservation Biology 

23(3), 520-530. 


Zurick, D., Pacheco, J., Shrestha, B. R., 

and Bajracharya, B. 

(2006). Illustrated atlas of the 

Himalaya. India Research Press. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 140



Natural Polyphenols and its Potential in Preventing Diseases 

Related to Oxidative Stress as an Alternative Green 

Nutraceutical Approach 

Sreenivasan Sasidharan 1,* , Shanmugapriy 1 , Subramanion Lachumy Jothy 1 , Mei Li 

Ng 2 , Nowroji Kavitha 1 , Chew Ai Lan 1 , Khoo Boon Yin 1 , Soundararajan Vijayarathna 

1 , Leow Chiuan Herng 1 and Chern Ein Oon 1 

1 Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 USM, Penang, 

Malaysia; 2 Integrative Medicine Cluster, Advanced Medical and Dental Institute 

(AMDI), Universiti Sains Malaysia, 13200, Kepala Batas Penang, Malaysia; 

*Correspondence: srisasidharan@yahoo.com; Tel: +60 46534820 

Abstract: Green bioactive polyphenol from Mother Nature especially from medicinal 

plants are a rich source of novel therapeutics. Therefore, the search for bioactive molecules 

from nature continues to play an important role in the invention of new medicinal agents. 

Most plants do provide an array of phytochemicals that may contribute to reduction of disease 

and slowing of aging. Oxidative stress which is continuously produced in vivo by oxygen-centred 

free radicals and other reactive oxygen species may leads to various diseases. 

Since the oxidative stress has a great impact on the human health, it is appropriate to examine 

the role of natural antioxidant as a defence system. In this line, medicinal plant based 

natural antioxidants such as polyphenols with free radical scavenging activity are emerging 

as the primary components of holistic approaches in impeding adverse effect of oxidative 

stress. This chapter focuses on biological effects of natural polyphenols on oxidative stress 

and related diseases as an aalternative green nutraceutical approach. 

Keywords: Free radicals; medicinal plant; natural antioxidants; oxidative stress; polyphenol 


Oxidative damage caused by free radicals 

to macromolecules outlines the 

foundation of what is arguably the most 

popular current explanation of ageing related 

diseases (Lawrence et al., 2005). 

Recent years have seen a surge of interest 

in the role of mitochondrial dysfunction, 

reactive oxygen species production and 

mitochondrial DNA mutation as driving 

factors in the ageing and various diseases 

(Balaban et al., 2005; Trifunovic et al., 

2005; Bender et al., 2006; Passos et al., 

2007). Hypothesized links between aging 

and oxidative stress are not new and were 

proposed over 50 years ago (Harman, 

1956). Mitochondria are found in nearly 

all eukaryotes. They vary in number and 

location according to cell type. Conversely, 

numerous mitochondria are found in 

human liver cells, with about 1000–2000 

mitochondria per cell, making up 1/5 of 

the cell volume (Alberts et al., 1994). 

Given the role of mitochondria as the 

cell's powerhouse, there may be some 

leakage of the high-energy electrons in 

the respiratory chain to form reactive oxygen 

species. This was thought to result 

in significant oxidative stress in the mitochondria 

with high mutation rates of mitochondrial 

DNA (mtDNA) (Richter et 

al., 1988). A vicious cycle was thought to 

occur, as oxidative stress leads to mitochondrial 

DNA mutations, which can lead 

to enzymatic abnormalities and further 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 141


Natural Polyphenols and Its Potential in … 

oxidative stress. The accumulation of these 

damaged macromolecules is proposed 

to contribute significantly to aging and 

various diseases (Ames et al., 1993). 

Hence, plants polyphenol with free radical 

scavenging activities are likely to 

make a considerable contribution for the 

protection against the free radical oxidation. 

Bioactive products from Mother Nature 

are a rich source of novel therapeutics. 

Therefore, the search for bioactive 

molecules from nature continues to play 

an important role in the invention of new 

medicinal agents. Most plants do provide 

an array of phytochemicals that may contribute 

to reduction of diseases and slowing 

of aging. This chapter focuses on biological 

effects of natural polyphenols on 

oxidative stress and related diseases as an 

alternative green nutraceutical approach. 

2. What are free radicals? 

The human body is, composed of different 

types of living cells which are organized 

into tissues, organs, and systems. 

Cells are made of different types of molecules 

which consist of atoms. Atoms 

comprise of a nucleus, neutrons, protons 

and electrons. Protons are positively 

charged particles in the nucleus that determine 

the number of negatively charged 

particles known as electrons in the atomic 

orbital. 

An atom‟s chemical behaviour is 

largely dictated by the number of electrons 

in its outermost shell. An atom is 

stable when its outermost shell is full. A 

free radical is an atom or group of atoms 

that has an unpaired electron due to splitting 

of weak bonds between electrons on 

the outermost shell and is therefore unstable 

and highly reactive. It will attempt to 

stabilize itself by reacting with another 

atom or molecule to donate its electron or 

to aggressively capture an electron to fill 

its outermost shell, thus stimulating a cascade 

of free radicals when the attacked 

atom or molecule receives an extra electron 

or loses its electron (Halliwell, 

1993). 

Sasidharan et al. 

Reactive nitrogen species (RNS) 

and reactive oxygen species (ROS) are 

free radicals that arise from normal cellular 

metabolism or as a consequence to 

pathological exposures. Nitric oxide, peroxynitrite 

and nitrogen dioxide are nitrogen-containing 

oxidants, often referred to 

as reactive nitrogen species. ROS are reactive 

molecules derived from oxygen 

molecules such as superoxide, hydroxyl, 

peroxyl, alkoxyl and singlet oxygen. They 

are usually generated as by-products in 

the mitochondria, peroxisomes, cytochrome 

P450 and other organelles. Free 

radicals include reactive oxygen and nitrogen 

species which are also collectively 

termed as reactive oxygen nitrogen species 

(RONS). RONS are known for being 

both beneficial and harmful. On the good 

side, they have been reported to have a 

crucial role in cell signalling (Adams et 

al., 2015; Reczek and Chandel, 2014), 

homeostasis (Kuster et al., 2010; Shadel 

and Horvath, 2015) and immune defence 

in response to inflammatory stimuli (Mizgerd 

and Brain, 1995; Reshi et al., 2014). 

However, these radicals are overgenerated 

in response to unfavourable environmental 

conditions such as poor nutrition, 

stress, smoking, alcohol, exercise, 

radiation, inflammation, drugs or exposure 

to air pollutants and chemicals (Figure 

1). At high concentrations, free radicals 

have the ability to alter proteins, carbohydrates, 

lipids and nucleic acids thus 

impairing cellular functions and leading 

to pathogenesis of cancer, aging, diabetes, 

atherosclerosis and other inflammatory 

diseases (Pham-Huy et al., 2008). 

Antioxidants are electron donors that 

can neutralize free radicals, thus preventing 

them from causing cellular damage 

(Figure 1). A balance between free radicals 

generated and antioxidant protective 

defence system is required for optimal 

physiological function (Bouayed and 

Bohn, 2010). Oxidative stress or nitrosative 

stress occurs when there is an imbalance 

between the production of free radicals 

and the ability of the body neutralize 

the deleterious effects through the role of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 142




Figure 1: Free radicals are generated due to factors such as stress, alcohol, tobacco, harmful 

chemicals including pesticides and drugs, fried food and radiation. Found abundantly in 

natural food, antioxidants are free radical scavengers that react with free radicals to neutralize 

them. Antioxidants function by donating an electron to the free radical before the latter 

oxidizes other components within the cell. The free radical is stabilized and becomes nondamaging 

to cells once it receives a free electron from the antioxidant. 

antioxidants. The human body manufactures 

endogenous antioxidant enzymes in 

order to control these destructive free radical 

chain reactions (Valko et al., 2007). 

However, the body also relies on dietary 

antioxidants to fulfil the needs of other 

antioxidants it cannot produce (Diplock et 

al., 1998), such as those found abundantly 

in bioactive food components including 

fruits, vegetables, grains and mushrooms. 

Examples of dietary antioxidants include 

lycopene, beta-carotene, lutein, zeaxanthin, 

alpha-tocopherol, vitamin A and vitamin 

C. 

3. Oxidative stress 

The origin of term „oxidative stress‟ 

from its very nature defined as the combination 

of electron transfer, free radicals, 

oxygen metabolites such as the superoxide 

anion radical, hydrogen peroxide, hydroxyl 

radical and singlet molecular oxygen 

with a biological concept of stress 

was first introduced by Selye (1955). A 

continuously activity of human body reacts 

with oxygen when breathing resulted 

in energy production by cells and generate 

highly reactive molecules within the 

cells known as free radicals. The effect 

exerted by free radicals in the body is 

called „oxidative stress‟ (Finaud et al., 

2006). 

Since overwhelming research has 

been developed, the contemporary concept 

of „oxidative stress‟ was updated and 

briefly redefined as disturbance in the 

balance between oxidants production and 

antioxidants defences (Sies and Jones, 

2007; Sies, 2015) which are associated 

with electron transfer influencing the redox 

state of cells and organism. Subsequently, 

the imbalance redox mechanism 

leads to the production of reactive oxygen 

species (ROS) that includes free radicals 

(superoxide, hydroxyl radical, peroxyl, 

alkoxyl, and hydroperoxyl) and non-free 

radicals (hydrogen peroxide, hypo- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 143



chlorous acid, ozone, singlet oxygen) are 

mainly involved in growth, differentiation, 

progression and death of the cells. 

(Rahman et al., 2012; Rajendran et al., 

2014). 

Free radicals known as small molecules 

and unpaired electron amid highly 

reactive proliferation characteristic which 

differ from most biological molecules that 

tend to involve in initiate chain reactions 

from single free radical until propagate to 

damage multiple molecules. Oxidative 

stress occurs when excessive free radical 

formation within a cell or organism as a 

form of ROS due to aerobic metabolism 

and immune activation (Delmastro- 

Greenwood and Piganelli, 2013), UV radiation 

(Halliwell and Gutteridge, 2015), 

heme-oxygenase accumulation (Vanella 

et al., 2013), and hypoxia (Yang et al., 

2011). Failure of the cell‟s defence mechanisms 

to neutralize or balance the accumulation 

of free radicals may leads to mitochondrial 

dysfunction, DNA damage 

and lipid peroxidation can trigger programmed 

cell death pathways (Dixon and 

Stockwell, 2014). The findings could explain 

that low concentration of ROS productions 

play role in intracellular signalling 

and defence against pathogens, while 

the higher concentration of ROS has been 

linked to clinically relevant diseases including 

cancer, cardiovascular disease, 

asthma, ischemia, diabetes and neurodegenerative 

diseases (Reynolds et al., 

2007; Halliwell and Gutteridge, 2015; 

Phaniendra et al., 2015). Mitochondria is 

a major part of cellular sources of ROS 

which consume oxygen with the process 

of oxidative phosphorylation. Excessive 

formation of free radicals is known to 

weaken defence mechanisms against oxidation 

and it results in more cell damages 

(Zorov, 2014). Consequently, the increasing 

level of oxidative stress involved in 

pathological redox reaction, process of 

ageing which can initiate tissue damage 

via apoptosis and necrosis (Cui et al., 

2012). 


4. Oxidative stress and immune response 

Immune system is important for our 

body to protect from the invasive pathogens. 

The human defence system has been 

well developed and can be divided into 

two immunity reactions, namely innate 

immunity and adaptive immunity. As its 

name suggests, the innate immunity is a 

non-specific first barrier of defence which 

is able to act fast towards microbial invasion. 

The adaptive immunity, on the other 

hand, is a highly specific barrier of defence 

due to possess immunological 

memory function that can rapidly and efficiently 

remove the pathogens that invaded 

into body (Poland et al., 2013). 

Components of the adaptive immune system 

are normally silent; however, when 

activated, these components „adapt‟ to the 

presence of infectious agents by activating, 

proliferating, and creating potent 

mechanisms for neutralizing or eliminating 

the microbes. In general, there are two 

types of adaptive immune responses 

where humoral immunity, mediated by 

antibodies produced by B lymphocytes 

(B-cells), and cell-mediated immunity, 

mediated by T lymphocytes (T-cells). Unlike 

B-cells, T-cells recognize circulating 

antigens of many chemical structures, the 

vast majority of T cells (> 95%) are only 

able to recognize peptide fragments that 

are displayed by specialized molecules, 

MHC molecules, on the surfaces of antigen 

presenting cells. Therefore, this system 

ensures that T-cells are able to recognize 

antigens that might be floating in the 

cytosol or contained within ingested vesicles 

of various cells (Thomas et al., 

2013). 

An imbalance between reactive 

oxygen species (ROS) and reducing 

agents (antioxidants) towards a prooxidant 

state can always result to oxidative 

stress (Sies, 1997). The damaging of 

macromolecular in the form of protein 

carbonylation, lipid peroxidation and 

DNA oxidation has historically has been 

proven as harmful to particular functional 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 144



cells and being part of the main factors to 

hypertension and aging problems (Annor 

et al., 2015; Zhao et al., 2017). Up to 

date, ROS is widely recognised as signalling 

molecular substances (Suzuki and 

Sevanian, 1997) that enable to trigger a 

wide range of biological effects. With regard 

to the immune system, high levels of 

ROS can also be beneficial. For instances, 

neutrophils generate ROS and release 

them intracellularly and extracellularly in 

the form of an “oxidative burst” to defend 

against and destroy pathogens, thus 

providing antimicrobial protection 

(Dahlgren and Karlsson, 1999). However, 

excessive ROS are generated in the presence 

of immune complexes with autoantigens 

where further macromolecular 

damage is induced. Prolonged exposure to 

high ROS concentrations can inhibit T- 

cell proliferation and lead to apoptosis 

(Thoren et al., 2007), and incubation of 

T-cells with the reactive nitrogen species 

(RNS) peroxynitrite can also inhibit their 

proliferation (Kasic et al., 2011). Previous 

study has revealed that different T-cell 

responses to ROS production may be due 

to the extent of change to the cellular redox 

environment (Griffiths, 2005). In 

some cases such as absence of antigen 

presenting cell, reactive carbonyls including 

4-hydroxy-2-nonenal and malondialdehyde 

(MDA), which are generated on 

proteins and lipids randomly in the presence 

of ROS, will promote differentiation 

towards a Th2 phenotype (Moghaddam et 

al., 2011). These data emphasize that the 

importance of ROS homeostasis and flux 

in governing cell maturation and that the 

balance between oxidising and reducing 

agents is a delicate process which must be 

tightly regulated and well managed, depending 

on whether the requirement is for 

protecting against bacteria, in an immune 

response, or requirements for T-cell signalling, 

activation and regulation of function. 

Owing to their pivotal role, the effect 

of the oxidative stress on T-cell biochemistry 

and its implications in autoimmune 

disease such as rheumatoid arthritis (RA) 


are greatly debated in healthcare management. 

5. Oxidative stress and cancer 

Oxidative stress is closely related to 

all aspects of cancer, from carcinogenesis 

to the tumor-bearing state, from treatment 

to prevention. Cancer cells generally display 

elevated ROS level compared to 

normal cells that give them a proliferative 

advantage and promote malignant progression. 

The excess level of ROS typically 

observed in cancer cells are the result 

of accumulation of intrinsic and environmental 

factors. In cancer cells, high 

levels of ROS can result from hypoxia, 

mitochondrial dysfunction, peroxisome 

activity, enhanced cellular metabolic activity, 

increased cellular receptor signaling, 

oncogene activity, increased activity 

of oxidases, cyclooxygenases, lipoxigenases 

and thymidine phosphorylase, and 

the crosstalk between cancer cells and 

immune cells recruited to the tumor site 

(Holmström and Finkel, 2014). Environmental 

sources of ROS that can significantly 

contribute to tumorigenesis include 

ionizing radiation, xenobiotics, tobacco 

components, chlorinated compounds, barbiturates, 

metal ions and phorbol esters 

etc. Figure 2 illustrates the potential outcomes 

when such ROS level exceeds the 

capacity of the oxidation-reduction system 

of the cell, may cause DNA, protein 

and lipid damage, leading to cellular responses 

such as chromosomal instability, 

genetic mutation, alterations in cellular 

metabolism and modulation of cell 

growth that may lead to malignant transformation 

by affecting crucial hallmarks 

of cancer. 

When present at high and sustained 

levels, ROS can cause severe deleterious 

modifications to DNA, protein, 

and lipids. During the initiation stage, 

ROS may produce DNA damage by introducing 

gene mutations and structural 

alterations into the DNA. ROS-induced 

DNA damage can result in single- or double-strand 

breakage, base modifications, 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 145




Inciting factors 

Intrinsic source of ROS 

Increased metabolic activity 

Mitochondrial dysfunction 

Peroxisome activity 

Oncogene activity 

Increased cellular receptor signalling 

Increased activity of enzymes 

Extrinsic source of ROS 

Ultraviolet rays 

Environmental agents 

Pharmaceuticals 

Industrial chemicals 

High oxidative stress 

in cancer cells 

ROS / Oxidative stress 

Cellular 

responses 

Oxidative damage 

to DNA, 

RNA 

Oxidative damage 

to proteins 

Increase 

lipid peroxidation 

Altered gene 

expression 

Altered signal 

transduction 

Loss of DNA repair 

activity (decreased 

efficiency of DNA polymerase 

and DNA repair 

enzymes) 

Chromosomal 

instability, 

gene mutations 

Altered cell growth, differentiation and apoptosis 

Carcinogenesis 

Initiation Promotion Progression Invasion Metastasis 

Figure 2: ROS and their role in the development of human cancer. 

deoxyribose modification and DNA 

cross-linking. Cell death, DNA mutation, 

replication errors and genomic instability 

can occur if the oxidative DNA damage is 

not repaired prior to DNA replication 

(Valko et al. 2006). Oxidative DNA damage/repair 

imbalance due to loss of DNA 

repair activity can lead to disease-causing 

mutations and may contribute to increased 

risk of carcinoma (Caramori et al., 2011). 

Proteins and lipids are also significant 

targets for oxidative attack and modification 

of these molecules can increase the 

risk of mutagenesis. Lipid peroxidation 

results in production of reactive metabolites 

which demonstrate high reactivity 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 146



with protein and DNA and have been implicated 

in the pathogenesis of cancer 

(Tuma, 2002). ROS also contribute to 

chromosomal instability and mutation 

through its role in increasing the rates of 

mutation, enhancing sensitivity to mutagenic 

agents and compromising the surveillance 

systems. In addition to inducing 

DNA, lipid and protein damage, oxidative 

damage to protein-coding or –non coding 

RNA may potentially cause errors in protein 

synthesis or dysregulation of gene 

expression. ROS-induced alteration of 

gene expression can occur through modulation 

of a host of signaling pathways including 

cAMP-mediated cascades, calcium-calmodulin 

pathways and intracellular 

signal transducers such as nitric oxide 

(Bertin and Averbeck, 2006). ROS stimulation 

on signal transduction pathways 

can lead to activation of key transcription 

factors such as Nrf2 and NF-κB. 

ROS interact with the multistage 

processes in carcinogenesis including initiation, 

promotion, proliferation, invasion, 

angiogenesis and metastasis. ROS act as 

essential signalling molecules and modulate 

a number of redox-sensitive signalling 

pathways in initiation of carcinogenesis. 

Well-characterized targets include 

ROS-mediated regulation of the mitogenactivated 

protein (MAP) kinase/Erk cascade, 

phosphoinositide-3-kinase 

(PI3K)/Akt-regulated signaling cascades, 

as well as the IκB kinase (IKK)/nuclear 

factor κ-B (NF-κB)-activating pathways 

(Ray et al., 2012). Oxidative stressmediated 

signaling pathways are persistently 

elevated in many types of cancers 

and affect all characters of cancer cell behavior, 

where they participate in cell 

growth/proliferation, differentiation, protein 

synthesis, glucose metabolism, cell 

survival and inflammation (Storz, 2005). 

In promotion stage, ROS can contribute 

to abnormal gene expression, inhibition 

of intercellular communication and 

modification of second-messenger systems, 

thus resulting in an increase in cell 

proliferation or a decrease in apoptosis of 

the initiated cell population (Klaunig and 


Kamendulis, 2007). Most initiating mutations 

affect proto-oncogenes or tumor 

suppressor genes. Proto-oncogenes code 

for a variety of growth factors, growth 

factor receptors, enzymes or transcription 

factors that promote cell growth and cell 

division while oncogenes are mutated 

versions of proto-oncogenes that promote 

abnormal cell proliferation. Activation of 

oncogenes and loss of tumor suppressors 

cause alterations to multiple intracellular 

signaling pathways that promote metabolic 

reprogramming in cancer, resulting in 

enhanced nutrient uptake to supply energetic 

and biosynthetic pathways for enhanced 

growth and survival (Zhang et al., 

2013). 

Oxidative stress may participate in 

the progression stage of the cancer process 

by adding further DNA alterations to 

the initiated cell population (Qanungo et 

al., 2005). It can also promote many aspects 

of tumor development and progression 

through various biological processes. 

It acts on cellular proliferation through 

extracellular-regulated kinase 1/2 

(ERK1/2) activation and ligand independent 

receptor tyrosine kinases (RTK) activation. 

ROS were shown as positive regulators 

of tumor cell proliferation by 

modulating key proteins in cell cycle progression 

such as cyclin, ATM (ataxia telangiectasia 

mutated) and antioxidant enzymes 

like MnSOD, catalase and glutathione 

peroxidase (Browne et al., 2004; 

Lewis et al., 2005). ROS are involved in 

Anoikis resistance, PTEN inactivation, 

activation of Src, NF-κB, CREB and 

phosphatidylinositol-3 kinase (PI3K)/Akt, 

enabling the tumor cells to escape from 

apoptosis (Giannoni et al., 2009; Zhu et 

al., 2011). In invasion and metastasis, 

ROS play a role in Met over-expression, 

matrix metalloproteinase secretion into 

the extracellular matrix (ECM), invadopodia 

formation, Rho-Rac interaction, 

plasticity in cell motility and epithelial– 

mesenchymal transition (EMT). These 

help cancer cells to escape the primary 

tumor, invade matrix of different organs, 

find a suitable metastatic niche and then 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 147



grow in the secondary site. In addition, 

oxidative stress is involved in continuous 

angiogenesis through its role in endothelial 

progenitor activation, release of VEGF 

and angiopoietin and recruitment of perivascular 

cells. 

6. oxidative stress (os) in diabetes pathology 


A growing body of evidence suggest 

that increased oxidative stress and deficit 

in antioxidant defense mechanism are 

central players in pathogenesis of diabetes 

complications, in particular β-cell dysfunction 

and failure (Folli et al., 2011). 

Under physiological condition, reactive 

oxygen species (ROS) serve as second 

messenger regulates signal transduction 

and gene expression. Oxidative stress develops 

from imbalance in redox homeostasis 

(overproduction of mitochondrial 

reactive oxygen species (ROS) that exceeds 

the level of antioxidants) leads to 

aberrant β-cell function and apoptosis. 

ROS are heterogenous molecules comprises 

of free radicals, such as nitric oxide 

(NO . ), superoxide (O . 2 - ), hydroxyl radical 

(OH . ), non-radicals such as hydrogen peroxide 

(H 2 O 2 ), anions such as superoxide 

(O 2 - ) and peroxynitrite (ONOOK) (Chang 

et al., 1993; Pieper et al., 1997; Lenzen, 

2008; Newsholme et al., 2012; Cao and 

Kaufman, 2014; Keane et al., 2015). 

Sources of free radicals production include 

the mitochondrial electron transport 

system, NADPH oxidases, xanthine oxidase 

(primary source in cardiomyocytes), 

uncoupled nitric oxide synthase (NOS) 

and arachidonic acid (primary source in 

vascular cells) pathway. Mitochondria are 

major source of free radicals production 

in cells. ROS was noted as a key upstream 

signaling event mediates downstream 

metabolic pathways, leading to loss of 

cellular biological function and ultimately 

cell death (Brownlee, 2005). Ample evidence 

indicate that ROS damage plays a 

major role in pathogenesis of micro- (diabetic 

retinopathy, nephropathy, and neuropathy) 

and cardiovascular complications 

(atherosclerotic diseases affecting 

arteries that supply heart, brain and lower 

extremities) (Greene et al., 1992; Rosen 

et al., 2001) and onset of diabetes (Kayama 

et al., 2015). Vincent and colleagues 

has demonstrated that ROS production 

and neuron injury are activated within 1-2 

hours of hyperglycaemic insult. Majority 

of the patients with impaired glucose tolerance 

have significant peripheral neuropathy, 

suggesting that ROS induced by 

hyperglycaemia is critical to cause major 

diabetes complications (Vincent et al., 

2002). 

Metabolic abnormalities such as 

hyperglycaemia, hyperlipidaemia, increased 

free fatty acids, insulin resistance 

and hyperinsulinaemia, each of which 

was noted to induce oxidative stress in 

endothelial cells of the blood vessels and 

myocardium. In addition, genetic susceptibility 

of an individual and presence of 

accelerating factors (e. g. hypertension 

and dyslipidaemia) also contribute to development 

of diabetes complications 

(general features of chronic hyperglycaemia-induced 

tissue damage are depicted 

in Figure 3). Several large scale perspective 

studies, such as the (Diabetes Control 

and Complication Trial DCCT/EDIC 

(The Diabetes Control and Complications 

Trial Research Group, 1993), UK prospective 

Diabetes Study (UKPDS) (UK 

Prospective Diabetes Study (UKPDS) 

Group, 1998), and Steno 2 Study have 

concluded that chronic hyperglycaemia as 

a key risk factor underlying diabetes pathology 

(Gaede et al., 2008). Hyperglycaemia 

is known to trigger oxidative 

stress through FIVE major molecular 

mechanisms (Figure 4 depicts the mechanism 

underlying oxidative stress and diabetes 

pathology): (1) Activation of Polyol 

pathway (2) Increased intracellular advanced 

glycation end products (AGEs) 

pathway activity and receptor expression 

for AGEs (RAGE) (3) Activation of Protein 

Kinase C isoforms (PKC) (4) Increased 

Hexosamine pathway flux (5) 

Decreased antioxidant defenses (Sima et 

al., 1990; Engerman et al., 1994; Brown- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 148




Figure 3: General features of chronic hyperglycaemia-induced diabetic tissue damage 

(Giacco and Brownlee, 2010). 

Figure 4: Mechanisms underlying hyperglycaemia-induced pathophysiology of diabetes 

via the generation of ROS and activation of stress-sensitive signaling pathways. Each 

mechanism is discussed in the text (Vincent et al., 2004). 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 149



lee, 1995; Lee et al., 1995; Ganz and 

Seftel, 2000; Brownlee, 2005). The effect 

of oxidative stress damage is aggravated 

by inactivation of anti-atherosclerotic enzymes 

(endothelial nitric oxide synthase 

(eNOS) and prostacyclin synthase. In addition, 

oxidative stress also activates 

stress-sensitive signaling pathways, such 

as nuclear redox sensitive transcription 

factor (NF-κB), p38 MAPK, and NH2- 

terminal Jun kinases/stress-activated protein 

kinases (JNK/SAPK) leads to both 

insulin resistance and impaired insulin 

secretion (Folli et al., 2011; Brownlee, 

2001). 

7. Molecular mechanisms of hyperglycaemia-induced 

oxidative stress in 

diabetes 

Hyperglycaemia-induced activation of 

polyol pathway was the first mechanism 

discovered (Gabbay et al., 1966). This 

pathway has been therapeutic target for 

diabetes neuropathy (Oates and Mylari, 

1999). Recent human genetic study has 

implicated polymorphisms of the aldose 

reductase gene associated with increased 

risk for diabetes complications (Oates and 

Mylari, 1999). Excess glucose activates 

polyol pathway. Aldose reductase (dependent 

upon NADPH as co-factor) increases 

conversion of glucose to polyalcohol 

sorbitol. Excessive activation of 

polyol pathway results in depletion of intracellular 

NADPH and GSH which is an 

important intracellular antioxidant (Lee 

and Chung, 1999). Accumulation of sorbitol 

forms cellular osmotic stress (Stevens 

et al., 1993). 

Excess glucose induces autooxidation 

through activation of the AGEs 

pathway. 

The AGE precursor damages cells by 

three mechanisms: modification of proteins 

involve in gene transcription 

(Giardino et al., 1994; Shinohara et al., 

1998), modification of extracellular matrix 

molecules (McLellan et al., 1994), 

and modification of circulating protein in 

the blood (e. g. albumin) (Vlassara et al., 


1988; Li et al., 1996). These results in 

auto-oxidation of glucose to glyoxals, decomposition 

of the Amadori product (glucose-derived 

1-amino 1-deoxyfructose 

lysine adducts, to 3-deoxyglucosone, and 

fragmentation of glyceraldehyde-3- 

phosphate and dihydroxyacetone phosphate 

to methylglyoxal (Brownlee, 2001). 

In addition, increased AGEs production 

promotes the binding of AGEs to its receptors 

(RAGE). Binding of AGEs to 

RAGE induces overproduction of ROS 

and activation of NF-kB signaling and 

upregulation of intracellular adhesion 

molecule-1(ICAM-1), vascular adhesion 

cell molecule-1 (VCAM-1), monocyte 

chemotactic protein-1 (MCP-1), PAI-1, 

tissue factor, and VEGF (Yamagishi et 

al., 1997; Bierhaus et al., 2001). 

Previous studies demonstrated that 

PCK activity was increased in the retina, 

kidney and microvasculature of diabetic 

rats (Craven, P.A. and F.R. DeRubertis, 

1989; Lee et al., 1989), suggested that the 

lipolytic pathway and production of diacylglycerol 

induces PKC activation 

(Ishii et al., 1998). Hyperglycaemia increases 

diacylglycerol synthesis, which is 

a critical activating co-factor for PKC 

isoforms (Derubertis and Craven, 1994; 

Xia et al., 1994; Koya et al., 1997; Koya 

and King, 1998). PKC activation has been 

shown to have diverse effects on gene expression 

in different cell types. PKC activation 

inhibits insulin-stimulated endothelial 

Nitric Oxide Synthase (eNOS) expression 

in the endothelial cells and decreases 

nitric oxide production in the 

smooth muscle cells (Vlassara et al., 

1995). In vascular smooth muscle cells, 

PKC activation induces over-expression 

of fibrinolytic inhibitor, plasminogen activator 

inhibitor (PAI-1) and activation of 

NF-kB (Abordo and Thornalley, 1997). 

PKC enhances accumulation of microvascular 

matrix protein by up-regulation of 

transforming growth factor (TGF-β), fibronectin 

and type 4 collagen in both culture 

mesangial cells and glomeruli of diabetic 

rats (Doi et al., 1992). PKC also enhances 

vascular permeability by increas- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 150



ing the expression of vascular endothelial 

growth factor (VEGF) (Skolnik et al., 

1991). 

Lastly, hyperglycaemia causes 

damage to the blood vessel through activation 

of hexosamine pathway. The end 

product of this pathway, uridine diphosphate 

(UDP)-N-acetyl glucosamine regulates 

gene expression implicated in vascular 

complications (such as PAI -, TGF-α, 

TGF-β1). In addition, activation of hexosamine 

pathway impairs Insulin Receptor 

Substrate (IRS)/phosphatidylinositol 

3-kinase (PI3-K)/Akt pathway, resulting 

in deregulation of eNOS activity (Bucala 

et al., 1991; Kolm-Litty et al., 1998). 

8. Oxidative stress and β-cell dysfunction 

in diabetes 


Diabetes mellitus (DM) is characterized 

by failure of the pancreatic β-cells to 

maintain glucose homeostasis. Physiologically, 

the pancreatic β-cells secrete hormone 

insulin and regulate glucose homeostasis. 

Insulin drives glucose uptake in 

the liver (reducing hepatic gluconeogenesis 

both directly and in conjunction with 

suppression of glucagon secretion), muscle 

and fat (Könner, 2007). Because of 

their high biosynthetic load and requirement 

for oxygen, pancreatic β-cells are 

very vulnerable to oxidative stress (Lenzen, 

2008; Newsholme et al., 2012; Cao 

and Kaufman, 2014; Kaneto and Matsuoka, 

2015). 

Oxidative stress and endoplasmic 

reticulum stress (ER) are key pathological 

features in particular type 2 diabetes 

mellitus (T2DM), contribute to pancreatic 

β-cell dysfunction, inducing inflammation 

(immune activation) and β-cell apoptosis. 

Previous studies have suggested the oxidative 

stress is able to suppress insulin 

transcription and associated with accumulation 

of β-amyloid in the human pancreatic 

islet (Kaneto and Matsuoka, 2015). In 

obesity and early stage of diabetes, nutrient 

overload leads to development of mild 

insulin resistance and hyperglycaemia. 

Increased insulin production by the pancreatic 

β-cells triggers oxidative stress 

and ER stress, exacerbated by high circulating 

glucose and lipids (non-esterified 

fatty acid). Oxidative stress and ER stress 

induce chemokine production and activates 

inflammatory cells in the pancreatic 

islet. In turn, the activated inflammatory 

cells produce cytokines that further exacerbate 

oxidative and ER stress and disrupt 

β-cell secretory pathway function. In addition, 

oxidative stress induces unfolded 

protein response (UPR) and NF-κB activation. 

In early diabetes (manifested by 

chronic ER stress and inflammation), increased 

proinsulin:insulin ratio impairs 

insulin signaling further aggravates hyperglycaemia. 

Overall, this vicious cycle 

leads to β-cell apoptosis and progression 

to diabetes (summarized schematically in 

Figure 5). Several mechanisms have been 

implicated in β-cell apoptosis (Nakagawa 

et al., 2000; Oyadomari et al., 2002; 

Puthalakath et al., 2007; Song et al., 

2008; Mahdi et al., 2012; Supale et al., 

2012; Uruno et al., 2015). The 

PERK/ATF4-mediated activation of 

CHOP and IRE1a/TRAF2/ASK1- 

mediated activation of JNK are important 

molecular mechanisms (reviewed in Papa 

FR 2012) (Papa, 2012). A growing body 

of evidence suggests that ER stress induces 

autophagy (an important mechanism 

for removal of terminally misfolded protein 

from the endoplasmic reticulum (ER) 

leads to induction of apoptosis (Wang et 

al., 2014; Li et al., 2012; Quan et al., 

2012). Another mechanism involves NFkB 

and interleukin 1 beta (IL1b) activation 

(reviewed in Hasnain SZ et al., 2012; 

Hasnain et al., 2014). 

9. Current therapeutics in diabetes 

Good glycaemic control is the most effective 

mean of mitigating diabetes complications 

in particular type 1 diabetes 

(Greene et al., 1992; Molitch et al., 

1993). In general, drug available for diabetes 

work by reducing stress on β-cells 

biosynthesis pathway. In diabetes patients, 

the use of drug that promotes insul- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 151




Figure 5: Schematic representation of the cycle of oxidative and ER stress and its effects 

on glucose homeostasis in diabetes (Hasnain et al., 2015). 

-in secretion (such as sulfonylureas) is 

known to causes loss of β-cell function. 

Another class of GLP-1 receptor agonist, 

which promotes insulin secretion in a glucose 

dependent-manner, may also have 

long-term damaging effect on β-cell 

(Hasnain et al., 2014). Drug that suppresses 

gluconeogenesis (metformin), increase 

glucose excretion (SGLT-2 inhibitor) 

or reduces peripheral insulin resistance 

(thiazolidinediones) or exogenous 

insulin. 

Given that pronounced oxidative 

stress mediates major diabetes complications, 

antioxidant therapy remains a novel 

therapeutic approach for diabetes patients. 

Antioxidant drugs target NADPH oxidases 

are unable to combat high level of oxidative 

stress (Li et al., 2012). In view that 

the IL22 receptor is the most highly expressed 

in human pancreatic islet cells, 

studies have been identifying IL22 as 

novel antioxidant target in diabetes (Cobleigh 

and Robek, 2013; Kumar et al., 

2013; Rutz et al., 2013; Hasnain et al., 

2014; Sabat et al., 2014). 

10. Biological effects of natural polyphenols 

on oxidative stress 

Oxygen is an essential element of life 

used by cells to generate energy in the 

form of ATP whereby this process occurs 

within the mitochondria (Turrens, 2003). 

The process, however, causes the production 

of free radicals, such as reactive oxygen 

species (ROS) and reactive nitrogen 

species (RNS) due to the cellular redox 

process in the cells (Pham-Huy et al., 

2008). At low or moderate concentration, 

these species exert beneficial effects on 

cellular responses and immune function, 

but when the species exist at higher levels, 

oxidative stress is generated (Young 

and Woodside, 2001; Halliwell, 2007; 

Pham-Huy et al., 2008). Oxidative stress 

refers to the balance between the production 

of free radicals and antioxidant defences 

in a cell (Betteridge, 2000). The 

mechanism arises when there is an unfavourable 

balance between the free radical 

production and antioxidant defences, 

which result in the damage of a broad 

range of molecular species, including li- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 152



pids, proteins and nucleic acid in the cells 

(Rock et al., 1996; McCord, 2000). 

The concept of oxidative stress 

was first hypothesised in the 1950s by 

researchers that investigated the toxic effects 

of ionizing radiation, free radicals, 

and the similar toxic effects of molecular 

oxygen (Gerschman et al., 1954), as well 

as its possible contribution to the aging 

process (Harman, 1956). Interest in this 

field of research grew (Hybertson et al., 

2011) when studies reported that the biological 

systems are capable of producing 

substantial amounts of superoxide free 

radical, O 2 - through the natural metabolic 

pathways (McCord & Fridovich, 1968) 

and the activity of antioxidant enzymes, 

superoxide dismutases (SODs) is necessary 

for the survival of aerobic organisms 

(McCord and Fridovich, 1969; McCord et 

al., 1971). Besides the aging process, oxidative 

stress has been postulated to play a 

role in many other conditions, as well. 

These conditions includes inflammatory 

diseases (arthritis, vasculitis, glomerulonephritis, 

lupus erythematous, adult respiratory 

diseases syndrome), ischemic 

diseases (heart diseases, stroke, intestinal 

ischemia), hemochromatosis, acquired 

immunodeficiency syndrome, emphysema, 

carcinogenesis, gastric ulcers, hypertension 

and preeclampsia, neurological 

disorders (Alzheimer's disease, Parkinson's 

disease, muscular dystrophy), alcoholism 

and smoking-related diseases 

(Pham-Huy et al., 2008; Lobo et al., 

2010). 

To protect cellular components 

from the free radical-induced damage, the 

body possess several mechanisms to 

counteract oxidative stress by producing 

an extensive range of antioxidant, both 

endogenous (generated in situ) and exogenous 

(supplied through food, e.g. polyphenols) 

in the cells. The antioxidants can 

be divided into three main categories 

which are antioxidant enzymes, chain 

breaking antioxidants and transition metal 

binding proteins (Young and Woodside, 

2001; Pham-Huy et al., 2008). 


Polyphenols are a large group of 

natural antioxidants found mostly in 

fruits, vegetables, cereals and beverages 

(Arts and Hollman, 2005; Pandey and 

Rizvi, 2009). There are more than 8,000 

polyphenolic compounds that have been 

identified in various plant species, which 

arise from a common intermediate, phenylalanine 

or an immediate precursor, and 

shikimic acid. Polyphenols contain phenol 

rings in the basic structure. Based on the 

number of phenol rings and the basis of 

the structural elements that binds to these 

rings, polyphenols can be classified as 

phenolic acids, flavonoids, stilbenes and 

lignans (Spencer et al., 2008; Pandey and 

Rizvi, 2009). Epidemiological studies 

have shown that high consumption of 

polyphenols lowered the risk of chronic 

human diseases (Scalbert et al., 2005; 

Arts and Hollman, 2005). Besides that, 

the consumption of polyphenols have also 

been linked with cardio-protective effect, 

anti-cancer effect, anti-diabetic effect, 

anti-aging effect and neuroprotective effect 

(Pandey and Rizvi, 2009). Several 

types of research have also concluded that 

high phenolic content correlates with a 

good antioxidant capability and lower oxidative 

stress-related chronic diseases 

(Aruoma, 1998; Kohen and Nyska, 2002). 

Clinacanthus nutans is an example of a 

traditional herb that is rich in polyphenols 

and has potent antioxidant capabilities 

(Aromdee et al., 2007; Yong et al., 2013). 

It is traditionally used to treat oxidative 

stress-related diseases, such as diabetes 

and various kinds of cancers (Ching et al., 

2013; P‟ng et al., 2013). 

The antioxidants protect cellular 

components by acting as radical scavengers, 

hydrogen and electron donors, peroxide 

decomposer, singlet oxygen 

quencher, enzyme inhibitors, synergist, 

and metal-chelating agents or by gene expression 

regulation (Frei et al., 1988; Lobo 

et al., 2010). The antioxidant process 

has been proposed to have two principle 

mechanisms of action, a chain-breaking 

mechanism and a prevention mechanism. 

The first mechanism donates an electron 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 153



from the primary antioxidant to the free 

radical. The free radical is unstable and 

highly reactive. Therefore, it either 

releases or steals an electron from another 

molecule in order to stabilise itself. This 

activity causes the molecule to become 

unstable, thus forming a second radical. 

The second radical then releases or steals 

an electron from another molecule, thus 

forming the following radical.This 

process is continued until a chainbreaking 

antioxidant helps to stabilise the 

free radical. On the other hand, the second 

mechanism involves the removal of 

ROS/RNS initiators by inhibiting chaininitiation 

catalyst in the cells (Pham-Huy 

et al., 2008; Lobo et al., 2010). Both 

mechanisms play a significant role in preventing 

oxidative stress-related diseases. 

11. The defences against free radical 

attack and oxidative stress 

Human body is naturally adapted with 

several defence mechanisms to counterbalance 

the excessive free radicals produced 

through the oxidative phosphorylation. 

Enzymatic antioxidant mechanism is 

the most important defence system 

against free radical. One such major free 

radical scavenging enzyme is the superoxide 

dismutase (SOD). Manganese SOD 

(MnSOD) was reported to be directly involved 

in the suspension of superoxide 

anions which was evident by the death of 

sod2 gene-knocked out mice within 10 

days after birth due to dilated cardiomyopathy, 

lipid amassing in liver and skeletal 

muscle and metabolic acidosis caused by 

the deficiency of MnSOD (Li et al., 

1995). Furthermore, tissues of those mice 

lacking MnSOD exhibited an elevated 

degree of oxidative damage DNA with 

higher rate cancer prevalence (Remmen et 

al., 2003). Another type of SOD is the 

Copper, Zinc SOD (CuZnSOD) which is 

mainly localized in cytoplasm, nucleus, 

lysosome and intermembrane space of 

mitochondria (Chang et al., 1988; Keller 

et al., 1991; Crapo et al., 1992; Sturtz et 

al., 2001). Based on the study conducted 


by Yim et al. (1990), CuZnSOD catalyzes 

hydroxyl radical (OH●) from H 2 O 2 . Deletion 

of sod1 gene in knock-out mice was 

reported to show high level of oxidative 

damage to proteins, lipids, and DNA in 

skeletal muscle tissue due to lack of 

CiZnSOD (Muller et al., 2006). Comprehensive 

oxidative damage was also observed 

in liver tissues of sod1 -/- mice 

(Elchuri et al., 2005). Hence, CuZnSOD 

is a potential antioxidant enzyme. 

Another enzymatic antioxidant defence 

mechanism is glutathione peroxidise 

(GSHPx) activity where free radicals 

are reduced by glutathione into water and 

its analogous alcohols (Wendel, 1980). 

The selenieum-containing GSHPx is 

mainly localized in cytoplasm and mitochondria 

(Zarowski and Tappel, 1978; 

Epp et al., 1983; Tappel, 1984; Timcenko-Youssef, 

1985). The free radical defence 

mechanism through GSHPx is evident 

through the study conducted by 

Hawker et al. (1993) who demonstrated 

an excessive ROS generation in seleniumdeficient 

mice. Although GSHPx-1 was 

reported to be insignificantly contribute 

against oxidative damage by evaluating 

carbonyl content, lipid peroxidation activity 

and rate of extracellular H 2 O 2 consumption 

in several tissues of GPSH-1 -/- 

knock-out mice samples (Ho et al., 1997), 

studies conducted by Zhang et al. (2009) 

revealed an elevation of free radicals in 

Sod2 +/− Gpx1 −/− mice through evaluation 

of DNA and protein oxidation in several 

tissues of the transgenic mice. In addition, 

Gpx4 +/− mice was reported to be highly 

sensitive to oxidative stress (Yant et al., 

2003; Ran et al., 2003). 

Furthermore, catalase is also a potential 

free radical defence enzyme which 

eliminates hydrogen peroxide by catalysing 

it into water and oxygen in which one 

molecule of catalase decomposes approximately 

six million of H 2 O 2 molecules per 

minute (Kellin and Hartree, 1938; Mates 

et al., 1999). Several studies have demonstrated 

the defence mechanism of free 

radicals by catalyse. Transgenic mice 

over expressing catalase (hCatTg +/0 ) was 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 154



shown to decrease the generation of H 2 O 2 

which eventually resulted in reduced 

blood pressure in contrast to the wild-type 

mice (Yang et al., 2002). In addition, 

knockout mice with under expression of 

catalase by disruption of cat or cas1 gene 

was reported to show decreased rate of 

decomposition of hydrogen peroxide with 

an increased tendency to oxidative stress 

(Ho et al., 2004). 

On the other hand, there are several 

other non-enzymatic defence mechanisms 

against free radicals such as vitamin 

E, vitamin C and Thiol antioxidants. 

Vitamin E is a fat soluble vitamin which 

was reported to act against lipid peroxidation 

(Pryor, 2000). Vitamin C is a water 

soluble vitamin which acts in correspondence 

with vitamin E. α-tocopherol is an 

active form of vitamin E which eliminates 

lipid peroxyl radical by donating a hydrogen 

atom and itself becoming an α- 

tocopherol radical while vitamin C reduces 

α-tocopherol radical into its original 

form (Carr and Frei, 1999; Kojo, 2004). 

Thiol antioxidants include the tripeptide 

glutathione (GSH), thioredoxin (TRX), 

and α-lipoic acid (ALA) also acts against 

free radical through redox reactions 

(Rahman, 2007). 

12. Medicinal plants as green approach 

defences against free radical attack 

In recent times, substantial evidence 

emerged indicating a mutual relationship 

between the consumption of antioxidantrich 

foods and the episodes of human 

health deterioration (Sies, 1997). Conversely, 

when synthetic antioxidants 

namely butylated hydroxytoluene (BHT) 

and butylated hydroxyanisole (BHA) 

were extensively employed as additives in 

food manufacturing business, a multitude 

of cases were reported on liver injuries 

and carcinogenesis (Grice, 1988; Wichi, 

1986). Considering this fact, attentions 

were shifted into utilizing natural antioxidants. 

Plants, given the fact that can grow 

successfully adapting to the exposure of 

ultraviolet, are also reservoir for potent 


antioxidants that quench free radicals, a 

physiological response usually not present 

in lower elevation plants (Alonso-Amelot, 

2008). 

12.1. Verbascum sinaiticum Benth. 

(Scrophulariaceae) 

The plant, Verbascum sinaiticum 

is a biennial rosette plant (a taproot with a 

cluster of leaves on the soil surface), upon 

where this species is listed as rare in 

Egypt and wide-reaching to Sinai Peninsula 

(Hegazy, 2000). The leaves and 

flowers from Verbascum species had been 

utilized in the treatment of bronchitis, dry 

coughs, tuberculosis and asthma while 

studies have confirmed expectorant, mucolytic 

and demulcent properties (Kozan 

et al., 2011). Extraordinary results were 

obtained from V. sinaiticum when it was 

observed for selectivity in antiproliferation 

response against carcinoma 

and normal cells. The effect of antiproliferate 

was evaluated in hepatocellular 

carcinoma (Hep-G2) and normal 

(MRC-5) cells after exposing the cells to 

the extract for 48 hours (Tauchen et al., 

2015). The extract comprises compositions 

of luteolin, chrysoeriol, hydrocarpin 

and sinaiticin which were earlier tested 

for cytotoxicity reaction towards leukaemia 

P-388 cells, providing a positive 

feedback (Afifi et al., 1993). These compounds 

falls under the classification of 

flavonoids and flavonolignans (Afifi et 

al., 1993 and Mahmoud et al., 2007) to 

which they will not cause possible hazards 

to consumer and consequently an 

attribution to the selective antiproliferative 

activity of V. sinaiticum 

against Hep-G2 and MRC-5 cell line 

(Tauchen et al., 2015). 

12.2. Ugni myricoides (Kunth) O.Berg 

Ugni myricoides are inhabitants of 

western Latin America (WCSP, 2016), 

possessing the properties for antihypernociceptive 

effect (Quintão et al., 

2010) and anti-nociceptive effects in mice 

(Campêlo, 2011). Brovo et al., had carried 

out an investigation between U. my- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 155



ricoides fruits antioxidant properties and 

their inhibitory interaction against skin 

aging-related enzymes. U. myricoides has 

been measured for high values in mitigating 

free radical damages of UV light in 

skin, by both the ORAC (Oxygen radical 

absorbance capacity) and TEAC assays 

(The Trolox Equivalent Antioxidant Capacity). 

As a result, U. myricoides were 

determine to retain high total phenolic 

content, with high correlation coefficient 

above 0.80 and its aptitude in hindering 

collagenase, elastase, hyaluronidase and 

tyrosinase enzymes propose that the presence 

of high values of polyphenols are 

potential conducive for these responses 

(Bravo et al., 2016). 

12.3. Black highland barley (BHLPE) 

The black highland barley known 

in Tibet for a crop of high importance is 

referred as Qing Ke in Chinese. The potential 

of BHLPE was measured and the 

findings exhibited potent superoxide radical, 

hydroxyl radical and 2,2-diphenyl-1- 

picrylhydrazyl radical-scavenging activity, 

ferric reducing antioxidant power and 

moderate metal ion-chelating activity 

(Shen et al., 2016). Two groups of mice 

were prepared; a high fat diet (HFD) 

group and a group that has been administrated 

with 600 mg BHLPE/kg body 

weight. The treated group of mice displayed 

remarkable reduction in total cholesterol, 

low-density lipoprotein cholesterol 

and the atherosclerosis index. The in 

vivo test further provides information on 

antioxidant defence system and antioxidant 

gene expression where significant 

amelioration had occurred within BHLPE 

treated group as compared to HFD mice 

group. Conclusively, the study highlighted 

the tendency of natural antioxidant of 

BHLPE in generating good health by reducing 

the incident of disease (Shen et al., 

2016). 

12.4. Curcuma longa L. 

Curcuma longa L is a spice that 

has considerable usage in the practise of 

Ayurveda, Unani, Siddha, and Chinese 


indigenous medicine for the administration 

of many ailments. It also has measurably 

multitude biological activities associated 

to antioxidant, anti-inflammatory 

and cancer preventive properties (Teiten 

et al., 2010; Goel et al., 2008; Hatcher et 

al., 2008). 

In a paper reported by Dall'Acqua 

et al., a pattern of significant changes 

were observed in the urinary metabolome 

of healthy rats, orally treated with curcumin, 

compared to controls in data sets obtained 

both by NMR and HPLC–MS 

(Dall'Acqua et al., 2016). The effect of 

Curcuma extract on urinary composition 

in healthy rats disclosed evidence for in 

vivo reduction of the amount of urinary 

biomarkers of oxidative stress namely allantoin, 

3-nitrotyrosine, m-tyrosine, 8- 

OHdG. The m-Tyrosine is examined as a 

biomarker for oxidative damage to proteins 

while urinary 8-OHdG (Orhan et al., 

2004) is measured as a biomarker of 

comprehensive cellular oxidative stress 

due to its prevalent generation of oxidized 

DNA repair (Tsikas et al., 2005). 

12.5. Beta vulgaris L. 

Beetroot (Beta vulgaris) is well 

known for its high total phenolic content 

(50–60 μmol/g dry weight) (Kähkönen et 

al., 1999; Vinson et al., 1998), and the 

presence of considerable amount of phenolic 

acids: ferulic, protocatechuic, vanillic, 

p-coumaric, p-hydroxybenzoic and 

syringic acids (Kujala, et al., 2000). It also 

a source of betalains, water-soluble 

nitrogenous pigments that feed on reactive 

oxygen species (ROS) to protect its 

plant from wound injuries and pathogenic 

penetration as studied in red beet 

(Sepúlveda-Jiménez et al., 2004). Five 

beetroot pomace extracts from different 

cultivar were studied upon where Detroit 

beetroot pomace extract (DBPE) exhibited 

the highest antiradical properties in 

vitro, by effectively scavenging DPPH 

radicals (EC 50 =2.06±0.10 μg/ml) and high 

reducing power (EC 50 =123.39±06.05 

μg/ml). The effect of in vivo antioxidant 

and hepatoprotective activities indicated 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 156



reduction in the measurement levels of 

(xanthine oxidase, catalase-CAT, peroxidase, 

glutathione peroxidase-GSHPx, glutathione 

reductase, glutathione-GSH and 

thiobarbituric acid reactive substance with 

the administration of DBPE in doses of 2 

and 3 ml/kg body weight (Vulic et al., 

2012; Vulic et al., 2014). 

12.6. Syzygium aromaticum L. 

Clove (Syzygium aromaticum) is a 

spice regularly used in Asia and many 

parts of the world for cooking. The composition 

of S. aromaticum widens its 

properties ranging from antioxidant, antifungal, 

anti-viral, anti-microbial to antidiabetic, 

anti-inflammatory, antithrombotic, 

anaesthetic, pain reliving and insect 

repellent properties (Parle and Khanna, 

2011). Clove buds creates insulin-like interaction 

with hepatocytes and hepatoma 

cells by reducing phosphoenolpyruvate 

carboxykinase and glucose 6-phosphatase 

gene expression (Prasad et al., 2005). It is 

a vital step to reduce postprandial hyperglycemia 

peak for the treatment of diabetes 

(Aguilar-Santamaría et al., 2009).The 

presence of phenolic (free and bound) appears 

to hinder alpha-amylase and alphaglucosidase 

in a dose-dependent manner. 

The extracts displayed high antioxidant 

activities as exemplified by their high 

reducing power, 1,1 diphenyl-2- picrylhydrazyl 

(DPPH) and 2, 2-azinobis-3- 

ethylbenzo-thiazoline-6-sulfonate (ABTS) 

radical scavenging abilities, as well as 

inhibition of Fe 2+ -induced lipid peroxidation 

in rat pancreas in vitro (Adefegha and 

Oboh, 2012). 

References: 

Abordo, E. A. and Thornalley, P. J. 

(1997). Synthesis and secretion of tumour 

necrosis factor-α by human 

monocytic THP-1 cells and chemotaxis 

induced by human serum albumin 

derivatives modified with methylglyoxal 

and glucose-derived advanced 

glycation endproducts. Immunology 

Letters 58, 139-147. 


Adams, L., Franco, M. C. and Estevez, 

A. G. (2015). Reactive nitrogen species 

in cellular signaling. Experimental 

Biology and Medicine (Maywood) 

240, 711-7. 

Adefegha, S.A. and Oboh, G. (2012). In 

vitro inhibition activity of polyphenolrich 

extracts from Syzygium aromaticum 

(L.) Merr. & Perry (Clove) buds 

against carbohydrate hydrolyzing enzymes 

linked to type 2 diabetes and 

Fe2+-induced lipid peroxidation in rat 

pancreas. Asian Pacific Journal of 

Tropical Biomedicine 2, 774–781. 

Afifi, M. S. A., Ahmed, M. M., Pezzuto, 

J. M. and Kinghornt, D. A. 

(1993). Cytotoxic flavonolignans and 

flavones from verbascum sinaiticum 

leaves. Phytochemistry 34, 839–841. 

Aguilar-Santamaría, L., Ramírez, G., 

Nicasio, P., Alegría-Reyes, C. and 

Herrera-Arellano, A. (2009). Antidiabetic 

activities of Tecoma stans (L.) 

Juss. ex Kunth. Journal of Ethnopharmacology 

124, 284–8. 

Alberts, B., Alexander, J., Julian, L., 

Martin, R., Keith, R. and Peter, W. 

(1994). Molecular Biology of the Cell, 

Garland Publishing Inc., New York, 

USA. 

Alonso-Amelot, M. E. (2008). High altitude 

plants, chemistry of acclimation 

and adaptation. Studies in Natural 

Products Chemistry 34, 883–982. 

Ames, B.N., Shigenaga, M.K. and Hagen, 

T.M. (1993). Oxidants, Antioxidants, 

and the Degenerative Diseases 

of Aging. Proceedings of the National 

Academy of Sciences USA 90, 7915- 

7922. 

Annor, F. B., Goodman, M., Okosun, I. 

S., Wilmot, D. W., Il'yasova, D., 

Ndirangu, M. and Lakkur, S. 

(2015). Oxidative stress, oxidative 

balance score, and hypertension 

among a racially diverse population. 

Journal of the American Sociecty of 

Hypertension 9, 592-599. 

Aromdee, C., Kongyingyoes, B., Laupattarakasem, 

P., Kukongviriyapan, 

U., Kukongviriyapan, V. and 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 157



Pannangpetch, P. (2007). Antioxidant 

activity and protective effect 

against oxidative hemolysis of Clinacanthus 

nutans (Burm.f) Lindau. 

Songklanakarin Journal of Science 

and Technology 29, 1–9. 

Arts, I.C.W. and Hollman, P.C.H. 

(2005). Polyphenols and disease risk 

in epidemiologic studies. The American 

Journal of Clinical Nutrition 81, 

317S–325S. 

Aruoma, D.O.I. (1998). Free radicals, 

oxidative stress, and antioxidants in 

human health and disease. Journal of 

the American Oil Chemists' Society 

75, 199–212. 

Balaban, R.S., Nemoto, S. and Finkel, 

(2005). Mitochondria, oxidants, and 

aging. Cell 120, 483–495. 

Bertin G., Averbeck D. (2006). Cadmium: 

cellular effects, modifications of 

biomolecules, modulation of DNA repair 

and genotoxic consequences. Biochimie 

88, 1549–59. 

Betteridge, D. J. (2000). What is oxidative 

stress? Metabolism, Clinical and 

Experimental 49, 3–8. 

Bender, A., Krishnan, K.J., Morris, 

C.M., Taylor, G.A., Reeve, A.K., 

Perry, R.H., Jaros, E., Hersheson, 

J.S., Betts, J., Klopstock, T., Taylor, 

R.W. and Turnbull, D.M. (2006). 

High levels of mitochondrial DNA deletions 

in substantia nigra neurons in 

aging and Parkinson disease. Nature 

Genetics 38, 515–517. 

Bierhaus, A., Schiekofer, S., Schwaninger, 

M., Andrassy, M., Humpert, 

P. M., Chen, J., Hong, M., Luther, 

T., Henle, T., Klöting, I., Morcos, 

M., Hofmann, M., Tritschler, H., 

Weigle, B., Kasper, M., Smith, M., 

Perry, G., Schmidt, A. M., Stern, D. 

M., Häring, H. U., Schleicher, E. 

and Nawroth, P. P. (2001). Diabetesassociated 

sustained activation of the 

transcription factor nuclear factorkappaB. 

Diabetes 50, 2792-808. 

Bouayed, J. and Bohn, T. (2010). Exogenous 

antioxidants-Double-edged 


swords in cellular redox state: Health 

beneficial effects at physiologic doses 

versus deleterious effects at high doses. 

Oxidative Medicine and Cellular 

Longevity 3, 228-237. 

Bravo,K., Alzate, F. and Osorio, E. 

(2016). Fruits of selected wild and 

cultivated Andean plants as sources of 

potential compounds with antioxidant 

and anti-aging activity. Industrial 

Crops and Products, In Press, Corrected 

Proof. 

Browne, S.E., Roberts, L. J., Dennery, 

P. A., Doctrow, S. R., Beal, M. F., 

Barlow, C. and Levine, R. L. (2004). 

Treatment with a catalytic antioxidant 

corrects the neurobehavioral defect in 

ataxia-telangiectasiamice. Free Radical 

Biology and Medicine 36, 938– 

942. 

Brownlee, M. (1995). Advanced protein 

glycosylation in diabetes and aging. 

Annual Review of Medicine 46, 223- 

234. 

Brownlee, M. (2001). Biochemistry and 

molecular cell biology of diabetic 

complications. Nature 414, 813-820. 

Brownlee, M. (2005). The pathobiology 

of diabetic complications: a unifying 

mechanism. Diabetes 54, 1615-1625. 

Bucala, R., Tracey, K. J. and Cerami, 

A. (1991). Advanced glycosylation 

products quench nitric oxide and mediate 

defective endotheliumdependent 

vasodilatation in experimental 

diabetes. Journal of Clinical 

Investigation 87, 432-438. 

Campêlo, L. M. L., Almeida, A. A. C., 

Freitas, R. L. M., Cerqueira, G. S., 

Sousa, G. F., Saldanha, G. B., 

Feitosa, C. M. and Freitas, R. M. 

(2011). Antioxidant and Antinociceptive 

Effects of Citrus limon Essential 

Oil in Mice. Journal of Biomedicine 

and Biotechnology 2011, 678673. 

Cao, S. S. and Kaufman, R. J. (2014). 

Endoplasmic reticulum stress and oxidative 

stress in cell fate decision and 

human disease. Antioxidants and Redox 

Signaling 21, 396-413. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 158




Caramori, G., Adcock, I. M., Casolari, 

P., Ito, K., Jazrawi, E., Tsaprouni, 

L., Villetti, G., Civelli, M., Carnini, 

C., Chung, K. F., Barnes, P. J. and 

Papi A. (2011). Unbalanced oxidantinduced 

DNA damage and repair in 

COPD: a link towards lung cancer. 

Thorax 66, 521-527. 

Carr, A., and Frei, B. (1999). Does vitamin 

C act as a pro-oxidant under 

physiological conditions? FASEB 

Journal 13, 1007-1024. 

Chang, K.C., Chung, S. Y., Chong, W. 

S., Suh, J. S., Kim, S. H., Noh, H. 

K., Seong, B. W., Ko, H. J. and 

Chun, K. W. (1993). Possible superoxide 

radical-induced alteration of 

vascular reactivity in aortas from 

streptozotocin-treated rats. Journal of 

Pharmacology and Experimental 

Therapeutics 266, 992-1000. 

Chang, L. Y., Slot, J. W., Geuze, H. J., 

and Crapo, J. D. (1988). Molecular 

immunocytochemistry of the CuZn 

superoxide dismutase in rat hepatocytes. 

The Journal of Cell Biology 

107, 2169-2179. 

Ching, S. M., Zakaria, Z. A., Paimin, F. 

and Jalalian, M. (2013). Complementary 

alternative medicine use 

among patients with type 2 diabetes 

mellitus in the primary care setting: a 

cross-sectional study in Malaysia. 

BMC Complementary and Alternative 

Medicine 13, 148. 

Cobleigh, M. A. and Robek, M. D. 

(2013). Protective and pathological 

properties of IL-22 in liver disease: 

implications for viral hepatitis. American 

Journal of Pathology 182, 21-28. 

Crapo, J. D., Oury, T., Rabouille, C., 

Slot, J. W. and Chang, L. Y. (1992). 

Copper,zinc superoxide dismutase is 

primarily a cytosolic protein in human 

cells. Proceedings of The National 

Academy of Sciences of the United 

States of America 89(21), 10405- 

10409. 

Craven, P. A. and DeRubertis, F. R. 

(1989). Protein kinase C is activated 

in glomeruli from streptozotocin diabetic 

rats. Possible mediation by glucose. 

Journal of Clinical Investigation, 

83, 1667-1675. 

Cui, H., Kong, Y. and Zhang, H. 

(2012). Oxidative stress, mitochondrial 

dysfunction, and aging. Journal of 

signal transduction 2012, 646354. 

Dahlgren, C. and Karlsson, A. (1999). 

Respiratory burst in human neutrophils. 

Journal of Immunological 

Methods 232, 3-14. 

Dall'Acqua, S., Stocchero, M., 

Boschiero, I., Schiavon, M., Golob,S., 

Uddin,J., Voinovich, D., 

Mammi,S. and Schievano E. (2016). 

New findings on the in vivo antioxidant 

activity of Curcuma longa extract 

by an integrated 1H NMR and HPLC– 

MS metabolomic approach. Fitoterapia 

109, 125-131. 

Delmastro-Greenwood, M. M. and Piganelli, 

J. D. (2013). Changing the 

energy of an immune response. American 

Journal of Clinical and Experimental 

Immunology 2, 30-54. 

Derubertis, F. R. and Craven, P. A. 

(1994). Activation of protein kinase C 

in glomerular cells in diabetes. Mechanisms 

and potential links to the pathogenesis 

of diabetic glomerulopathy. 

Diabetes 43, 1-8. 

Diplock, A. T., Charleux, J. L., Crozier-Willi, 

G., Kok, F. J., Rice- 

Evans, C., Roberfroid, M., Stahl, 

W. and Viña-Ribes, J. (1998). Functional 

food science and defence 

against reactive oxidative species. The 

British Journal of Nutrition 80, S77- 

112. 

Dixon, S. J. and Stockwell, B. R. (2014). 

The role of iron and reactive oxygen 

species in cell death. Nature chemical 

biology 10, 9-17. 

Doi, T., Vlassara, H., Kirstein, M., 

Yamada, Y., Striker, G. E. and 

Striker, L. J. (1992). Receptorspecific 

increase in extracellular matrix 

production in mouse mesangial 

cells by advanced glycosylation end 

products is mediated via plateletderived 

growth factor. Proceedings of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 159



the National Academy of Sciences of 

the United States of America 89, 

2873-2877. 

Elchuri, S., Oberley, T. D., Qi, W., Eisenstein, 

R. S., Roberts, L. J., 

Remmen, V. H., Epstein, C. J., and 

Huang, T. (2005). CuZnSOD deficiency 

leads to persistent and widespread 

oxidative damage and hepatocarcinogenesis 

later in life. Oncogene 

24 367–380. 

Engerman, R. L., Kern, T. S. and Larson, 

M. E. (1994). Nerve conduction 

and aldose reductase inhibition during 

5 years of diabetes or galactosaemia 

in dogs. Diabetologia 37, 141-144. 

Epp, D., Landenstein, R., and Wendel, 

A. (1983). The refined structure of the 

selenoenzyme glutathione peroxidase 

at 0.2-nm resolution. European Journal 

of Biochemostry 133(1): 51–69 

Finaud, J., Lac, G., and Filaire, E. 

(2006). Oxidative stress. Sports medicine 

36, 327-358. 

Folli, F., Corradi, D., Fanti, P., Davalli, 

A., Paez, A., Giaccari, A., Perego, 

C. and Muscogiuri, G. (2011). The 

role of oxidative stress in the pathogenesis 

of type 2 diabetes mellitus 

micro- and macrovascular complications: 

avenues for a mechanistic-based 

therapeutic approach. Current Diabetes 

Reviews 7, 313-24. 

Frei, B., Stocker, R., Ames, B.N. (1988). 

Antioxidant defenses and lipid peroxidation 

in human blood plasma. Proceedings 

of the National Academy of 

Sciences of the United States of America 

85, 9748–9752. 

Gabbay, K. H., Merola, L. O. and 

Field, R. A. (1966). Sorbitol Pathway: 

Presence in Nerve and Cord with 

Substrate Accumulation in Diabetes. 

Science 151, 209-210. 

Gaede, P., Lund-Andersen, H., 

Parving, H. and Pedersen, O. 

(2008). Effect of a multifactorial intervention 

on mortality in type 2 diabetes. 

The New England Journal of 

Medicine 358, 580-591. 


Ganz, M. B. and Seftel, A. (2000). Glucose-induced 

changes in protein kinase 

C and nitric oxide are prevented 

by vitamin E. American Journal of 

Physiology. Endocrinology and Metabolism 

278, E146-152. 

Gerschman, R., Gilbert, D. L., Nye, S. 

W., Dwyer, P., Fenn, W. O. (1954). 

Oxygen Poisoning and X-irradiation: 

A Mechanism in Common. Science 

119, 623–626. 

DOI:10.1126/science.119.3097.623. 

Giacco, F. and Brownlee, M. (2010). 

Oxidative stress and diabetic complications. 

Circulation Research 107, 

1058-1070. 

Giannoni, E., Fiaschi, T., Ramponi, G., 

Chiarugi, P. (2009). Redox regulation 

of anoikis resistance of metastatic 

prostate cancer cells: key role for Src 

and EGFR-mediated pro-survival signals. 

Oncogene 28, 2074–2086. 

Giardino, I., Edelstein, D. and Brownlee, 

M. (1994). Nonenzymatic glycosylation 

in vitro and in bovine endothelial 

cells alters basic fibroblast 

growth factor activity. A model for intracellular 

glycosylation in diabetes. 

Journal of Clinical Investigation 94, 

110-117. 

Goel, A., Kunnumakkara, A. B. and 

Aggarwal, B. B. (2008). Curcumin as 

"Curecumin": from kitchen to clinic. 

Biochemical Pharmacology 75, 787– 

809. 

Greene, D. A., Sima, A. A. F., Stevens, 

M. J., Feldman, E. L. and Lattimer, 

S. A. (1992). Complications: neuropathy, 

pathogenetic considerations. Diabetes 

Care 15, 1902-1925. 

Grice, H. P. (1988). Enhanced tumour 

development by butylated hydroxyanisole 

(BHA) from the prospective of 

effect on fore-stomach and oesophageal 

squamous epithelium. Food 

Chemical Toxicology 26, 717-723. 

Griffiths, H. R. (2005). ROS as signalling 

molecules in T cells--evidence for 

abnormal redox signalling in the autoimmune 

disease, rheumatoid arthritis. 

Redox Report 10, 273-280. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 160



Halliwell, B. (1993). The chemistry of 

free radicals. Toxicology and Industrial 

Health 9, 1-21. 

Halliwell, B. (2007). Biochemistry of oxidative 

stress. Biochemical Society 

Transactions. 35, 1147–1150. 

DOI:10.1042/BST0351147 

Halliwell, B. and Gutteridge, J. M. 

(2015). Free radicals in biology and 

medicine. Oxford University Press, 

USA. 

Harman, D. (1956). Aging: a theory 

based on free radical and radiation 

chemistry. The Journals of Gerontology 

11, 298–300. 

Hasnain, S. Z., Lourie, R., Das, I., 

Chen, A. C. and McGuckin, M. A. 

(2012). The interplay between endoplasmic 

reticulum stress and inflammation. 

Immunology and Cell Biology 

90, 260-270. 

Hasnain, S. Z., Prins, J. B. and 

McGuckin, M. (2015). Oxidative and 

endoplasmic reticulum stress in β-cell 

dysfunction in diabetes. Journal of 

Molecular Endocrinology. DOI: 

10.1530/JME-15-0232. 

Hasnain, S.Z., Borg, D. J., Harcourt, 

B. E., Tong, H., Sheng, Y. 

H., Ng, C. P, Das, I., Wang, R., 

Chen, A. C., Loudovaris, T., 

Kay, T. W., Thomas, H. E., Whitehead, 

J. P., Forbes, J. M., 

Prins, J. B. and McGuckin, M. 

A. (2014). Glycemic control in diabetes 

is restored by therapeutic manipulation 

of cytokines that regulate beta 

cell stress. Nature Medicine 20, 1417- 

1426. 

Hatcher, H., Planalp, R., Cho, J., Torti, 

F. M. and Torti, S. V. (2008). Curcumin: 

from ancient medicine to current 

clinical trials. Cellular and Molecular 

Life Science 65, 1631–1652. 

Hawker, F. H., Ward, H. E., Stewart, P. 

M., Wynne, L. A., and Snitch, P. J. 

(1993). Selenium deficiency augments 

the pulmonary toxic effects of oxygen 

exposure in the rat. European Respiratory 

Journal 6: 1317–1323. 


Hegazy, A. K. (2000). Intra-population 

variation in reproductive ecology and 

resource allocation of the rare biennial 

species Verbascum sinaiticum Benth., 

in Egypt. Journal of Arid Environments 

44, 185–196. 

Ho, Y. S., Xiong, Y., Ma, W., Spector, 

A., and Ho, D. S. (2004). Mice lacking 

catalase develop normally but 

show differential sensitivity to oxidant 

tissue injury. The Journal of Biological 

Chemistry 279(31), 32804-32812. 

Ho, Y., Magnenat, J., Bronsoni, R. T., 

Cao, J., Gargano, M., Sugawara, 

M., and Funk, C. D. (1997). Mice 

Deficient in Cellular Glutathione Peroxidase 

Develop Normally and Show 

No Increased Sensitivity to Hyperoxia. 

The Journal of Biological Chemistry 

272(26), 16644–16651. 

Holmström, K. M. and Finkel T. 

(2014). Cellular mechanisms and 

physiological consequences of redoxdependent 

signalling. Nature Reviews 

Molecular Cell Biology 15, 411–421. 

Hybertson, B. M., Gao, B., Bose, S. K., 

McCord, J. M. (2011). Oxidative 

stress in health and disease: The therapeutic 

potential of Nrf2 activation. 

Molecular Aspects of Medicine, Oxidative 

Damage and Disease 32, 234– 

246. 

Ishii, H., Koya, D. and King, G. L. 

(1998). Protein kinase C activation 

and its role in the development of vascular 

complications in diabetes mellitus. 

Journal of Molecular Medicine 

(Berlin, Germany) 76, 21-31. 

Kähkönen, M. P., Hopia, A. I., Vuorela, 

H. J., Rauha, J. P., Pihlaja, K., 

Kujala, T. S. and Heinonen, M. 

(1999). Antioxidant activity of plant 

extracts containing phenolic compounds. 


Food Chemistry 47, 3954–3962. 

Kaneto, H. and Matsuoka, T. A. (2015). 

Role of Pancreatic Transcription Factors 

in Maintenance of Mature β-Cell 

Function. International Journal of 

Molecular Sciences 16, 6281. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 161



Kasic, T., Colombo, P., Soldani, C., 

Wang, C. M., Miranda, E., Roncalli, 

M., Bronte, V. and Viola, A. 

(2011). Modulation of human T-cell 

functions by reactive nitrogen species. 

European Journal of Immunology 41, 

1843-1849. 

Kayama, Y., Raaz, U., Jagger, 

A., Adam, M., Schellinger, I. 

N., Sakamoto, M., Suzuki, 

H., Toyama, K., Spin, J. M. 

and Tsao, P. S. (2015). Diabetic Cardiovascular 

Disease Induced by Oxidative 

Stress. International Journal of 

Molecular Sciences 16, 25234-25263. 

Keane, K.N., Cruzat, V. F., Carlessi, 

R., Bittencourt, P. I. 

H. and Newsholme, P. (2015). Molecular 

Events Linking Oxidative 

Stress and Inflammation to Insulin 

Resistance and beta-Cell Dysfunction. 


Longevity 2015, 181643. 

Keller, G. A., Warner, T. G., Steimer, 

K. S., and Hallewell, R. A. (1991). 

Cu,Zn superoxide dismutase is a peroxisomal 

enzyme in human fibroblasts 

and hepatoma cells. Proceedings 

of The National Academy of Sciences 

of the United States of America 

88(16), 7381-7385. 

Kellin, D., and Hartree, E. F. (1938). 

On the Mechanism of the Decomposition 

of Hydrogen Peroxide by Catalase. 

Proceedings of the Royal Society 

of London. Series B, Biological Sciences 

124, 397-405. 

Klaunig, J. E. and Kamendulis, L. M. 

(2007). Chapter 8: chemical carcinogenesis. 

In Casarett & Doull‟s Toxicology: 

The Basic Science of Poisons 

(Klaassen C. D., ed.), 7th ed. 

McGraw-Hill, New York. pp. 329– 

380. 

Kohen, R. and Nyska, A. (2002). Invited 

Review: Oxidation of Biological Systems: 

Oxidative Stress Phenomena, 

Antioxidants, Redox Reactions, and 

Methods for Their Quantification. 

Toxicologic Pathology 30, 620–650. 


Kojo, S. (2004). Vitamin C: basic metabolism 

and its function as an index of 

oxidative stress. Current Medical 

Chemistry 11, 1041-1064. 

Kolm-Litty, V., Sauer, U., Nerlich, A., 

Lehmann, R. and Schleicher, E. D. 

(1998). High glucose-induced transforming 

growth factor beta1 production 

is mediated by the hexosamine 

pathway in porcine glomerular 

mesangial cells. Journal of Clinical 

Investigation 101, 160-169. 

Könner, A.C., Janoschek, R., Plum, L., 

Jordan, S. D., Rother, E., Ma, X., 

Xu, C., Enriori, P., Hampel, B., 

Barsh, G. S., Kahn, C. R., Cowley, 

M. A., Ashcroft, F. M. and Brüning, 

J. C. (2007). Insulin Action in AgRP- 

Expressing Neurons Is Required for 

Suppression of Hepatic Glucose Production. 

Cell Metabolism 5, 438-449. 

Koya, D. and King, G. L. (1998). Protein 

kinase C activation and the development 

of diabetic complications. 

Diabetes 47, 859-66. 

Koya, D., Jirousek, M. R., Lin, Y. W., 

Ishii, H., Kuboki, K. and King, G. 

L. (1997). Characterization of protein 

kinase C beta isoform activation on 

the gene expression of transforming 

growth factor-beta, extracellular matrix 

components, and prostanoids in 

the glomeruli of diabetic rats. Journal 

of Clinical Investigation 100, 115- 

126. 

Kozan, E., Çankaya, I. T., Kahraman, 

C., Akkol, E. K. and Akdemir, Z. 

(2011). The in vivo anthelmintic efficacy 

of some Verbascum species 

growing in Turkey. Experimental 

Parasitology 129, 211-214. 

Kujala, T. S., Loponen, J. M., Klika, K. 

D. and Pihlaja, K. (2000). Phenolics 

and betacyanins in red beetroot (Beta 

vulgaris) root: distribution and effect 

of cold storage on the content of total 

phenolics and three individual compounds. 



Kumar, P., Rajasekaran, K., Palmer, J. 

M., Thakar, M. S. and Malarkan- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 162



nan, S. (2013). IL-22: An Evolutionary 

Missing-Link Authenticating the 

Role of the Immune System in Tissue 

Regeneration. Journal of Cancer 4, 

57-65. 

Kuster, G. M., Hauselmann, S. P., 

Rosc-Schluter, B. I., Lorenz, V. and 

Pfister, O. (2010). Reactive oxygen/nitrogen 

species and the myocardial 

cell homeostasis: an ambiguous 

relationship. Antioxidants and Redox 

Signaling 13: 1899-910. 

Lawrence, A.L., Douglas, C.W. and 

George, M.M. (2005). The mitochondrial 

theory of aging and its relationship 

to reactive oxygen species 

damage and somatic mtDNA mutations”, 

Proceedings of the National 

Academy of Sciences USA 102(52), 

18769–18770. 

Lee, A. Y. and Chung, S. S. (1999). 

Contributions of polyol pathway to 

oxidative stress in diabetic cataract. 

The FASEB Journal 13, 23-30. 

Lee, A. Y., Chung, S. K., & Chung, S. 

S. (1995). Demonstration that polyol 

accumulation is responsible for diabetic 

cataract by the use of transgenic 

mice expressing the aldose reductase 

gene in the lens. Proceedings of the 

National Academy of Sciences of the 

United States of America, 92(7), pp. 

2780–2784. 

Lee, T. S., Saltsman, K. A., Ohashi, H. 

and King, G. L. (1989). Activation of 

protein kinase C by elevation of glucose 

concentration: proposal for a 

mechanism in the development of diabetic 

vascular complications. Proceedings 

of the National Academy of 

Sciences U S A. 86, pp. 5141-5145. 

Lenzen, S. (2008). Oxidative stress: the 

vulnerable β-cell. Biochemical Society 

Transactions 36, 343-347. 

Lewis, A., Du, J., Liu, J., Ritchie, J. M., 

Oberley, L. W. and Cullen, J. J. 

(2005). Metastatic progression of pancreatic 

cancer: changes in antioxidant 

enzymes and cell growth. Clinical and 

Experimental Metastasis 22, 523–532. 


Li, N., Li, B., Brun, T., Deffert- 

Delbouille, C., Mahiout, Z., Daali, 

Y., Ma, X. J., Krause, K. H. and 

Maechler, P. (2012). NADPH Oxidase 

NOX2 Defines a New Antagonistic 

Role for Reactive Oxygen Species 

and cAMP/PKA in the Regulation 

of Insulin Secretion. Diabetes 61, 

2842-2850. 

Li, Y. M., Mitsuhashi, T., Wojciechowicz, 

D., Shimizu, N., Li, J., Stitt, A., 

He, C., Banerjee, D. and Vlassara, 

H. (1996). Molecular identity and cellular 

distribution of advanced glycation 

endproduct receptors: relationship 

of p60 to OST-48 and p90 to 

80K-H membrane proteins. Proceedings 

of the National Academy of Sciences 

USA. 93, 11047-11052. 

Li, Y., Huang, T. T., Carlson, E. J., Melov, 

S., Ursell, P. C., Olson, J. L., 

Noble, L. J., Yoshimura, M. P., 

Berger, C., Chan, P. H., Wallace, D. 

C., and Epstein, C. J. (1995). Dilated 

cardiomyopathy and neonatal lethality 

in mutant mice lacking manganese 

superoxide dismutase. Nature Genetics 

11, 376-381. 

Lobo, V., Patil, A., Phatak, A. and 

Chandra, N. (2010). Free radicals, 

antioxidants and functional foods: 

Impact on human health. Pharmacognosy 

Reviews 4, 118–126. 

DOI:10.4103/0973-7847.70902 

Mahdi, T., Hänzelmann, S., Salehi, A., 

Muhammed, S. J., Reinbothe, T. 

M., Tang, Y., Axelsson, A. S., Zhou, 

Y., Jing, X., Almgren, P., Krus, U., 

Taneera, J., Blom, A. M., Lyssenko, 

V., Esguerra, J. L., Hansson, O., 

Eliasson, L., Derry, J., Zhang, E., 

Wollheim, C. B., Groop, L., 

Renström, E. and Rosengren, A. H. 

(2012). Secreted Frizzled-Related 

Protein 4 Reduces Insulin Secretion 

and Is Overexpressed in Type 2 Diabetes. 

Cell Metabolism. 16, 625-633. 

Mahmoud, S. M., Abdel-Azim, N. S., 

Shahat, A. A., Ismail, S. I. and 

Hammouda, F. M. (2007). Phytochemical 

and biological studies on 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 163




verbascum sinaiticum growing in 

Egypt. Natural Product Sciences 13, 

186–189. 

Mates, J. M., Perez-Gomez, C., and 

Nunez de Castro, I. (1999). Antioxidant 

enzymes and human diseases. 

Clinical Biochemistry 32, 595-603. 

McCord, J. M. (2000). The evolution of 

free radicals and oxidative stress. 

American Journal of Medicine 108, 

652–659. 

McCord, J. M. and Fridovich, I. (1969). 

Superoxide dismutase. An enzymic 

function for erythrocuprein (hemocuprein). 

The Journal of Biological 

Chemistry 244, 6049–6055. 

McCord, J. M., Fridovich, I. (1968). 

The Reduction of Cytochrome c by 

Milk Xanthine Oxidase. The Journal 

of Biological Chemistry 243, 5753– 

5760. 

McCord, J. M., Keele, B. B. and Fridovich, 

I. (1971). An Enzyme-Based 

Theory of Obligate Anaerobiosis: The 

Physiological Function of Superoxide 

Dismutase. Proceedings of the National 

Academy of Sciences of the 

United States of America 68, 1024– 

1027. 

McLellan, A. C., Thornalley, P. J., 

Benn, J. and Sonksen, P. H. (1994). 

Glyoxalase system in clinical diabetes 

mellitus and correlation with diabetic 

complications. Clinical Science 

(Lond) 87, 21-29. 

Mizgerd, J. P. and Brain, J. D. (1995). 

Reactive oxygen species in the killing 

of Pseudomonas aeruginosa by human 

leukocytes. Current Microbiology 31, 

124-128. 

Moghaddam, A. E., Gartlan, K. H., 

Kong, L. and Sattentau, Q. J. 

(2011). Reactive carbonyls are a major 

Th2-inducing damage-associated 

molecular pattern generated by oxidative 

stress. The Journal of Immunology 

187, 1626-1633. 

Molitch, M. E., Steffes, M. W., Cleary, 

P. A. and Nathan, D. M. (1993). 

Baseline analysis of renal function in 

the Diabetes Control and Complications 

Trial. Kidney International 43, 

668-674. 

Muller, F. L., Song, W., Liu, Y., 

Chaudhuri, A., Pieke-Dahl, S., 

Strong, R., Huang, T., Epstein, C. 

J., Roberts II, L. J, Csete, M., 

Faulkner, J. A., and Remmen, V. H. 

(2006). Absence of CuZn superoxide 

dismutase leads to elevated oxidative 

stress and acceleration of agedependent 

skeletal muscle atrophy. 

Free Radical Biology and Medicine 

40, 1993–2004. 

Nakagawa, T., Zhu, H., Morishima, N., 

Li, E., Xu, J., Yankner, B. A. and 

Yuan, J. (2000). Caspase-12 mediates 

endoplasmic-reticulum-specific apoptosis 

and cytotoxicity by amyloid- 

[beta]. Nature 403, 98-103. 

Newsholme, P., Rebelato, E., Abdulkader, 

F., Krause, M., Carpinelli, A. 

and Curi, R. (2012). Reactive oxygen 

and nitrogen species generation, antioxidant 

defenses, and β-cell function: 

a critical role for amino acids. Journal 

of Endocrinology 214, 11-20. 

The Diabetes Control and Complications 

Trial Research Group. (1993). 

The effect of intensive treatment of 

diabetes on the development and progression 

of long-term complications 

in insulin-dependent diabetes mellitus. 

The Diabetes Control and Complications 

Trial Research Group. The New 

England Journal of Medicine 329, 

977-986. 

UK Prospective Diabetes Study 

(UKPDS) Group. (1998). Intensive 

blood-glucose control with sulphonylureas 

or insulin compared with 

conventional treatment and risk of 

complications in patients with type 2 

diabetes (UKPDS 33). UK Prospective 

Diabetes Study (UKPDS) Group. 

Lancet 352, 837-853. 

Oates, P. J. and Mylari, B. L. (1999). 

Aldose reductase inhibitors: therapeutic 

implications for diabetic complications. 

Expert Opinion on Investigational 

Drugs 8, 2095-2119. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 164




Orhan, H., Vermeulen, N. P. E., Tump, 

C., Zappey, H. and Meerman J. H. 

N. (2004). Simultaneous determination 

of tyrosine, phenylalanine and 

deoxyguanosine oxidation products by 

liquid chromatography-tandem mass 

spectrometry as non-invasive biomarkers 

for oxidative damage. Journal 

of Chromatography, B: Analytical 

Technologies in the Biomedical and 

Life Sciences 799, 245–254. 

Oyadomari, S., Koizumi, A., Takeda, 

K., Gotoh, T., Akira, S., Araki, E. 

and Mori, M. (2002). Targeted disruption 

of the Chop gene delays endoplasmic 

reticulum stress-mediated 

diabetes. Journal of Clinical Investigation 

109, 525-532. 

P’ng, X. W., Akowuah, G. A. and Chin, 

J. H. (2013). Evaluation of the Sub– 

acute Oral Toxic Effect of Methanol 

Extract of Clinacanthus nutans Leaves 

in Rats. Journal of Acute Disease 2, 

29–32. 

Pandey, K. B. and Rizvi, S. I. (2009). 

Plant polyphenols as dietary antioxidants 

in human health and disease. 


Longevity 2, 270–278. 

Papa, F. R. (2012). Endoplasmic Reticulum 

Stress, Pancreatic β-Cell Degeneration, 

and Diabetes. Cold Spring 

Harbor Perspectives in Medicine 2(9), 

a007666. 

Parle, M. and Khanna D. (2011). Clove: 

a champion spice. International Journal 

of Research in Ayurveda and 

Pharmacy 2, 47–54. 

Passos, J.F. Saretzki, G. and Von 

Zglinicki, T. (2007). DNA damage in 

telomeres and mitochondria during 

cellular senescence: is there a connection? 

Nucleic Acids Research 35, 

7505–7513. 

Pham-Huy, L. A., He, H. and Pham- 

Huy, C. (2008). Free radicals, antioxidants 

in disease and health. International 

Journal of Biomedical Sciences 

4, 89-96. 

Pham-Huy, L. A., He, H. and Pham- 

Huy, C. (2008). Free Radicals, Antioxidants 

in Disease and Health. International 

Journal of Biomedical Science 

4, 89–96. 

Phaniendra, A., Jestadi, D. B. and Periyasamy, 

L. (2015). Free radicals: 

properties, sources, targets, and their 

implication in various diseases. Indian 

Journal of Clinical Biochemistry 30, 

11-26. 

Pieper, G. M., Langenstroer, P. and 

Siebeneich, W. (1997). Diabeticinduced 

endothelial dysfunction in rat 

aorta: role of hydroxyl radicals. Cardiovascular 

Research 34, 145-156. 

Poland, G. A., Quill, H. and Togias, A. 

(2013). Understanding the human 

immune system in the 21st century: 

the Human Immunology Project Consortium. 

Vaccine 31, 2911-2912. 

Prasad, R. C., Herzog, B., Boone, B., 

Sims, L. and Waltner-Law, M. 

(2005). An extract of Syzygium aromaticum 

represses genes encoding 

hepatic gluconeogenic enzymes. 

Journal of Ethnopharmacology 96, 

295–301. 

Pryor, W. A. (2000). Vitamin E and heart 

disease: basic science to clinical intervention 

trials. Free Radical Biology 

and Medicine 28, 141-164. 

Puthalakath, H., O'Reilly, L. A., Gunn, 

P., Lee, L., Kelly, P. N., Huntington, 

N. D., Hughes, P. D., Michalak, E. 

M., McKimm-Breschkin, J., Motoyama, 

N., Gotoh, T., Akira, S., 

Bouillet, P. and Strasser, A. (2007). 

ER Stress Triggers Apoptosis by Activating 

BH3-Only Protein Bim. Cell 

129, 1337-1349. 

Qanungo, S., Das, M., Haldar, S. and 

Basu, A. (2005). Epigallocatechin-3- 

gallate induces mitochondrial membrane 

depolarization and caspasedependent 

apoptosis in pancreatic 

cancer cells. Carcinogenesis 26, 958– 

67. 

Quan, W., Hur, K. Y., Lim, Y., Oh, S. 

H., Lee, J. C., Kim, K. H., Kim, G. 

H., Kim, S. W., Kim, H. L., Lee, M. 

K., Kim, K. W., Kim, J., Komatsu, 

M. and Lee, M. S. (2012). Autophagy 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 165



deficiency in beta cells leads to compromised 

unfolded protein response 

and progression from obesity to diabetes 

in mice. Diabetologia 55, 392-403. 

Quintão, N. L., da Silva, G. F., Antonialli, 

C. S., Rocha, L. W., Cechinel, 

A., Filho, V. and Cicció, J. F. 

(2010). Chemical composition and 

evaluation of the antihypernociceptive 

effect of the essential 

oil extracted from the leaves of 

Ugni myricoides on inflammatory and 

neuropathic models of pain in mice. 

Planta medica 76, 1411-1418. 

Rahman, K. (2007). Studies on free radicals, 

antioxidants, and co-factors. 

Clinical Interventions in Aging 2, 

219-236. 

Rahman, T., Hosen, I., Islam, M. T. 

and Shekhar, H. U. (2012). Oxidative 

stress and human health. Advances 

in Bioscience and Biotechnology 3, 

997. 

Rajendran, P., Nandakumar, N., 

Rengarajan, T., Palaniswami, R., 

Gnanadhas, E. N., Lakshminarasaiah, 

U., Gopas, J. and Nishigaki, 

I. (2014). Antioxidants and human 

diseases. Clinica Chimica Acta 436, 

332-347. 

Ran, Q., Remmen, H. V., Gu, M., Qi, 

W., Roberts II, L. J., Prolla, T., and 

Richardson, A. (2003). Embryonic 

fibroblasts from Gpx4+/− mice: a 

novel model for studying the role of 

membrane peroxidation in biological 

processes. Free Radical Biology and 

Medicine 35, 1101-1109. 

Ray P.D., Huang B.W., Tsuji Y. (2012). 

Reactive oxygen species (ROS) homeostasis 

and redox regulation in cellular 

signaling. Cell Signal 24, 981– 

90. 

Reczek, C. R. and Chandel, N. S. 

(2014). ROS-dependent signal transduction. 

Current Opinion in Cell Biology 

33, 8-13. 

Remmen, V. H., Ikeno, Y., Hamilton, 

M., Pahlavani, M., Wolf, N., 

Thorpe, S. R., Alderson, N. L., 

Baynes, J. W., Epstein, C. J., 


Huang, T. T., Nelson, J., Strong, R., 

and Richardson, A. (2003). Lifelong 

reduction in MnSOD activity results 

in increased DNA damage and 

higher incidence of cancer but does 

not accelerate aging. Physiological 

Genomic 16, 29-37. 

Reshi, M. L., Su, Y. C. and Hong, J. R. 

(2014). RNA viruses: ROS-mediated 

cell death. International Journal of 

Cell Biology 2014, 467452. 

Reynolds, A., Laurie, C., Mosley, R. L. 

and Gendelman, H. E. (2007). Oxidative 

stress and the pathogenesis of 

neurodegenerative disorders. International 

review of neurobiology 82, 297- 

325. 

Richter, C., Park, J.W. and Ames, B.N. 

(1988). Normal oxidative damage to 

mitochondrial and nuclear DNA is extensive. 


Academy of Sciences USA 85, 6465– 

6467. 

Rock, C. L., Jacob, R. A. and Bowen, P. 

E. (1996). Update on the biological 

characteristics of the antioxidant micronutrients: 

vitamin C, vitamin E, 

and the carotenoids. Journal of the 

American Dietetic Association 96, 

693–702. 

Rosen, P., Nawroth, P. P., King, G., 

Möller, W., Tritschler, H. J. and 

Packer, L. (2001). The role of oxidative 

stress in the onset and progression 

of diabetes and its complications: a 

summary of a Congress Series sponsored 

by UNESCO-MCBN, the 

American Diabetes Association and 

the German Diabetes Society. Diabetes/Metabolism 

Research and Reviews 

17, 189-212. 

Rutz, S., Eidenschenk, C. and Ouyang, 

W. (2013). IL -22, not simply a Th17 

cytokine. Immunological Reviews 

252, 116-132. 

Sabat, R., Ouyang, W. and Wolk, K. 

(2014). Therapeutic opportunities of 

the IL-22-IL-22R1 system. Nature 

Reviews Drug Discovery 13, 21-38. 

Scalbert, A., Manach, C., Morand, C., 

Rémésy, C. and Jiménez, L. (2005). 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 166




Dietary polyphenols and the prevention 

of diseases. Critical Reviews in 

Food Science and Nutrition 45, 287– 

306. doi:10.1080/1040869059096 

Selye, H. (1955). Stress and disease. The 

Laryngoscope 65, 500-514. 

Sepúlveda-Jiménez, G., Rueda-Benítez, 

P., Porta, H. and Rocha-Sosa, M. 

(2004). Betacyanin synthesis in red 

beet (Beta vulgaris) leaves induced by 

wounding and bacterial infiltration is 

preceded by an oxidative burst. Physiological 

and Molecular Plant Pathology 

64, 125–133. 

Shadel, G. S. and Horvath, T. L. (2015). 

Mitochondrial ROS signaling in organismal 

homeostasis. Cell 163, 560- 

9. 

Shen, Y., Zhang, H., Cheng, L., Wang, 

L., Qian, H. and Qi, X. (2016). In 

vitro and in vivo antioxidant activity 

of polyphenols extracted from black 

highland barley. Food Chemistry 194, 

1003-1012. 

Shinohara, M., Thornalley, P. J., 

Giardino, I., Beisswenger, P., 

Thorpe, S. R., Onorato, J. and 

Brownlee, M. (1998). Overexpression 

of glyoxalase-I in bovine endothelial 

cells inhibits intracellular advanced 

glycation endproduct formation and 

prevents hyperglycemia-induced increases 

in macromolecular endocytosis. 

Journal of Clinical Investigation 

101, 1142-1147. 

Sies, H. (1997). Oxidative stress: oxidants 

and antioxidants. Experimental Physiology 

82, 291-295. 

Sies, H. (2015). Oxidative stress: a concept 

in redox biology and medicine. 

Redox biology 4, 180-183. 

Sies, H. and Jones, D. (2007). Oxidative 

stress, 2nd ed.,in: G. Fink (Ed.), Encyclopedia 

of Stress, 3, Elsevier, Amsterdam. 

pp. 45–48. 

Sima, A. A., Prashar, A., Zhang, W. 

X., Chakrabarti, S. and Greene, D. 

A. (1990). Preventive effect of longterm 

aldose reductase inhibition 

(ponalrestat) on nerve conduction and 

sural nerve structure in the spontaneously 

diabetic Bio-Breeding rat. The 


1410-1420. 

Skolnik, E.Y., Yang, Z., Makita, Z., 

Radoff, S., Kirstein, M. and Vlassara, 

H. (1991). Human and rat mesangial 

cell receptors for glucosemodified 

proteins: potential role in 

kidney tissue remodelling and diabetic 

nephropathy. The Journal of Experimental 

Medicine 174, 931-939. 

Song, B., Scheuner, D., Ron, D., Pennathur, 

S. and Kaufman, R. J. 

(2008). Chop deletion reduces oxidative 

stress, improves beta cell function, 

and promotes cell survival in 

multiple mouse models of diabetes. 


3378-3389. 

Spencer, J. P. E., Abd El Mohsen, M. 

M., Minihane, A.-M. and Mathers, 

J. C. (2008). Biomarkers of the intake 

of dietary polyphenols: strengths, limitations 

and application in nutrition research. 

British Journal of Nutrition 

99, 12–22. 

Stevens, M. J., Lattimer, S. A., Kamijo, 

M., Van Huysen, C., Sima, A. A. 

and Greene, D. A. (1993). Osmotically-induced 

nerve taurine depletion 

and the compatible osmolyte hypothesis 

in experimental diabetic neuropathy 

in the rat. Diabetologia 36, 608- 

614. 

Storz, P. (2005). Reactive oxygen species 

in tumor progression. Frontiers in Bioscience 

10, 1881–1896. 

Sturtz, L. A., Diekert, K., Jensen, L. T., 

Lill, R., and Culotta, V. C. (2001). A 

fraction of yeast Cu,Zn-superoxide 

dismutase and its metallochaperone, 

CCS, localize to the intermembrane 

space of mitochondria. A physiological 

role for SOD1 in guarding against 

mitochondrial oxidative damage. The 

Journal of Biological Chemistry 276, 

38084-38089. 

Supale, S., Li, N., Brun, T. and Maechler, 

P. (2012). Mitochondrial dysfunction 

in pancreatic β cells. Trends in 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 167



Endocrinology & Metabolism 23, 

477-487. 

Suzuki, Y. J., Forman, H. J. and 

Sevanian A. (1997). Oxidants as 

stimulators of signal transduction. 

Free Radical Biology and Medicine 

22, 269-285. 

Tappel, A. L. (1984). Selenium-- 

glutathione peroxidase: properties and 

synthesis. Current Topics in Cellular 

Regulations 24, 87–97 

Tauchen, J., Doskocil, I., Caffi, C., 

Lulekal, E., Marsik, P., Havlik, J., 

Van Damme, P. and Kokoska L. 

(2015). In vitro antioxidant and antiproliferative 

activity of Ethiopian medicinal 

plant extracts. Industrial Crops 

and Products 74, 671-679. 

Teiten, M., Eifes, S., Dicato, M. and. 

Diederich M. (2010). Curcumin-the 

paradigm of a multi-target natural 

compound with applications in cancer 

prevention and treatment. Toxins 2, 

128–162. 

Thomas, N., Heather, J., Pollara, G., 

Simpson, N., Matjeka, T., Shawe- 

Taylor, J., Noursadeghi, M. and 

Chain B. (2013). The immune system 

as a biomonitor: explorations in innate 

and adaptive immunity. Interface Focus 

3(2), 20120099. 

Thoren, F. B., Betten, A., Romero, A. I. 

and Hellstrand, K. (2007). Cutting 

edge: Antioxidative properties of myeloid 

dendritic cells: protection of T 

cells and NK cells from oxygen radical-induced 

inactivation and apoptosis. 

The Journal of Immunology 179, 

21-25. 

Timcenko-Youssef, L., Yamazaki, R. 

K., and Kimura, T. (1985). Subcellular 

localization of adrenal cortical glutathione 

peroxidase and protective 

role of the mitochondrial enzyme 

against lipid peroxidative damage. 


260, 13355–13359. 

Trifunovic, A., Hansson, A., Wredenberg, 

A., Rovio, A.T., Dufour, E., 

Khvorostov, I., Spelbrink, J.N., Wibom, 

R., Jacobs, H.T. and Larsson, 


N.G. (2005). Somatic mtDNA mutations 

cause aging phenotypes without 

affecting reactive oxygen species production. 


Academy of Sciences USA, 102, 

17993–17998. 

Tsikas, D., Mitschke, A., Suchy, M., 

Gutzki, F. and Stichtenoth, D. O. 

(2005). Determination of 3- 

nitrotyrosine in human urine at the basal 

state by gas chromatographytandem 

mass spectrometry and evaluation 

of the excretion after oral intake. 

Journal of Chromatography, B: Analytical 

Technologies in the Biomedical 

and Life Sciences 827, 146–156. 

Tuma, D. J. (2002). Role of malondialdehyde-acetaldehyde 

adducts in liver 

injury. Free Radical Biology and 

Medicine 32, 303–308. 

Turrens, J. F. (2003). Mitochondrial 

formation of reactive oxygen species. 

The Journal of Physiology 552, 335– 

344. 

DOI:10.1113/jphysiol.2003.049478 

Uruno, A., Yagishita, Y. and Yamamoto, 

M. (2015). The Keap1–Nrf2 system 

and diabetes mellitus. Archives of 

Biochemistry and Biophysics 566, 76- 

84. 

Valko, M., Leibfritz, D., Moncol, J., 

Cronin, M. T., Mazur, M. and 

Telser, J. (2007). Free radicals and 

antioxidants in normal physiological 

functions and human disease. The International 

Journal of Biochemistry & 

Cell Biology 39, 44-84. 

Valko, M., Rhodes, C. J., Moncol, J., 

Izakovic, M. and Mazur M. (2006). 

Free radicals, metals and antioxidants 

in oxidative stress-induced cancer. 

Chemico-Biological Interactions 160, 

1–40. 

Vanella, L., Sodhi, K., Kim, D. H., Puri, 

N., Maheshwari, M., Hinds, T. D. 

and Abraham, N. G. (2013). Increased 

heme-oxygenase 1 expression 

in mesenchymal stem cell-derived adipocytes 

decreases differentiation and 

lipid accumulation via upregulation of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 168



the canonical Wnt signaling cascade. 

Stem Cell Research & Therapy 4, 28. 

Vincent, A. M., Brownlee, M. and Russell, 

J. W. (2002). Oxidative stress 

and programmed cell death in diabetic 

neuropathy. Annals of the New York 

Academy of Sciences 959, 368-383. 

Vincent, A. M., Russell, J. W., Low, P. 

and Feldman, E. L. (2004). Oxidative 

stress in the pathogenesis of diabetic 

neuropathy. Endocrine Reviews 

25, 612-628. 

Vinson, J. A., Hao, Y., Su, X. and Zubik, 

L. (1998). Phenol antioxidant 

quantity and quality in foods: vegetables. 



Vlassara, H., Brownlee, M., Manogue, 

K. R., Dinarello, C. A. and Pasagian 

A. (1988). Cachectin/TNF and IL-1 

induced by glucose-modified proteins: 

role in normal tissue remodeling. Science 

240, 1546-1548. 

Vlassara, H., Li, Y. M., Imani, F., 

Wojciechowicz, D., Yang, Z., Liu, F. 

T. and Cerami, A. (1995). Identification 

of galectin-3 as a high-affinity 

binding protein for advanced glycation 

end products (AGE): a new 

member of the AGE-receptor complex. 

Molecular Medicine 1, 634-646. 

Vulić, J., Ĉanadanović-Brunet, J., 

Ćetković, G., Tumbas, V., Djilas, S., 

Ĉetojević-Simin, D. and Ĉanadanović, 

V. (2012). Antioxidant and 

cell growth activities of beet root 

pomace extract. Journal of Functional 

Foods 4, 670-678. 

Vulić, J., Ćebović, X., Ĉanadanović- 

Brunet, T.N., Ćetković, J.M., Ĉanadanović, 

G.S., Djilas, V.M. and 

Tumbas Šaponjac, S.M. (2014). In 

vivo and in vitro antioxidant effects of 

beetroot pomace extracts. Journal of 

Functional Foods 6, 168-175. 

Wang, X., Ota, N., Manzanillo, P., 

Kates, L., Zavala-Solorio, J., Eidenschenk, 

C., Zhang, J., Lesch, J., 

Lee, W. P., Ross, J., Diehl, L., van 

Bruggen, N., Kolumam, G. and 

Ouyang, W. (2014). Interleukin-22 


alleviates metabolic disorders and restores 

mucosal immunity in diabetes. 

Nature 514, 237-241. 

WCSP. (2016). 'World Checklist of Selected 

Plant Families. Facilitated by 

the Royal Botanic Gardens, Kew. 

Published on the Internet; 

http://apps.kew.org/wcsp/. Retrieved 

on 1 Feb 2016. 

Wendel, A. (1980). in Enzymatic Basis of 

Detoxification (Jakoby, W., ed) Vol. 

1. Academic Press, New York. pp. 

333–353. 

Wichi, H. C. (1986). Safety evaluation of 

butylated hydroxytoluene (BHT) in 

the liver, lung and gastrointestinal 

tract. Food Chemical Toxicology 24, 

1127-1130. 

Xia, P., Inoguchi, T., Kern, T. S., 

Engerman, R. L., Oates, P. J. and 

King, G. L. (1994). Characterization 

of the mechanism for the chronic activation 

of diacylglycerol-protein kinase 

C pathway in diabetes and hypergalactosemia. 

Diabetes 43, 1122- 

1129. 

Yamagishi, S., Yonekura, H., Yamamoto, 

Y., Katsuno, K., Sato, F., Mita, 

I., Ooka, H., Satozawa, N., Kawakami, 

T., Nomura, M. and Yamamoto, 

H. (1997). Advanced glycation 

end products-driven angiogenesis in 

vitro. Induction of the growth and 

tube formation of human microvascular 

endothelial cells through autocrine 

vascular endothelial growth factor. 


272, 8723-8730. 

Yang, C., Ling, H., Zhang, M., Yang, 

Z., Wang, X., Zeng, F. and Feng, J. 

(2011). Oxidative stress mediates 

chemical hypoxia-induced injury and 

inflammation by activating NF-κB- 

COX-2 pathway in HaCaT cells. Molecules 

and cells 31, 531-538. 

Yang, H., Shi, M., VanRemmen, H., 

Chen, X, L., Vijg, J., Richardson, 

A., and Guo, Z. M. (2002). Reduction 

of pressor response to vasoconstrictor 

agents by overexpression of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 169



catalase in mice. American Journal of 

Hypertension 16, 1-5. 

Yant, L. J., Ran, Q., Rao, L., Remmen, 

H. V., Shibatani, T., Belter, J. G., 

Motta, L.,Richardson, A., and 

Prolla, T. A. (2003). The selenoprotein 

GPX4 is essential for mouse development 

and protects from radiation 

and oxidative damage insults. Free 

Radical Biology and Medicine 34, 

496-502. 

Yim, M. B., Chock, P. B., Stadtman, E. 

R. (1990). Copper, zinc superoxide 

dismutase catalyzes hydroxyl radical 

production from hydrogen peroxide. 

Proceedings of the National Academy 

of Sciences USA 87, 5006-5010. 

Yong, Y. K., Tan, J. J., Teh, S. S., Mah, 

S. H., Ee, G. C. L., Chiong, H. S. 

and Ahmad, Z. (2013). Clinacanthus 

nutans Extracts Are Antioxidant with 

Antiproliferative Effect on Cultured 

Human Cancer Cell Lines. Evidencebased 

Complementary and Alternative 

Medicine, 2013, 462751. 

Young, I. and Woodside, J. (2001). Antioxidants 

in health and disease. Journal 

of Clinical Pathology 54, 176– 

186. 

Zarowski, J., and Tappel, A. L. (1978). 

Purification and properties of rat liver 

mitochondrial glutathione peroxidise. 

Biochimica et Biophysica Acta 526, 

65–76. 

Zhang, R., Kang, K. A., Kim, K.C., Na, 

S-Y, Chang, W.Y., Kim, G.Y., Kim, 

H.S., Hyun, J.W. (2013). Oxidative 


stress causes epigenetic alteration of 

CDX1 expression in colorectal cancer 

cells. Gene 524, 214–219. 

Zhang, Y., Ikeno, Y.,Qi, W., 

Chaudhuri, A., Li, Y., Bokov, A., 

Thorpe, S. R., Baynes, J. W., Epstein, 

C., Richardson, A., and 

Remmen, H. V. (2009). Mice Deficient 

in Both Mn Superoxide Dismutase 

and Glutathione Peroxidase-1 

Have Increased Oxidative Damage 

and a Greater Incidence of Pathology 

but No Reduction in Longevity. The 

Journals of Gerontology Series A: Biological 

sciences and Medical Sciences 

64, 1212-1220. 

Zhao, J., Yu, S., Zheng, Y., Yang, H. 

and Zhang J. (2017). Oxidative 

Modification and Its Implications for 

the Neurodegeneration of Parkinson's 

disease. Molecular Neurobiology 54, 

1404-1418. 

Zhu, P., Tan, M.J., Huang, R.L., Tan, 

C. K., Chong, H. C., Pal, M., Lam, 

C. R., Boukamp, P., Pan, J. Y., Tan, 

S. H., Kersten, S., Li, H. Y., Ding, J. 

L. and Tan, N. S. (2011). Angiopoietin-like 

4 protein elevates the prosurvival 

intracellular O2-:H2O2 ratio and 

confers anoikis resistance to tumors. 

Cancer Cell 19, 401–415. 

Zorov, D. B., Juhaszova, M. and Sollott, 

S. J. (2014). Mitochondrial reactive 

oxygen species (ROS) and ROSinduced 

ROS release. Physiological 

reviews 94, 909-950. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 170



A Review on Green Synthesis of Nanoparticles and its 

Antimicrobial Properties 

Karthika Arumugam 1, * and Naresh Kumar Sharma 2 

1 Department of Biotechnology, Kalasalingam University, Krishnankoil, Srivilliputtur, 

Tamil Nadu, India; 2 Department of Biotechnology, Kalasalingam University, Krishnankoil, 

Srivilliputtur, Tamil Nadu, India; *Correspondence: karthika1386@gmail.com; Tel: +91- 

9489142440 

Abstract: The most important aspect of nanotechnology is the synthesising and characterizing 

the nanoparticles (NPs). Nano particles are manufactured in large quantities because of 

their wide range of applications. All Physical, Chemical and Biological methods have been 

used in the metal nanoparticles production. The major problems in the production are the 

stability, aggregation, morphology, crystal growth, size and size distribution. In recent 

years, the green synthesis of metal NPs has become more attractive because of cost effectiveness 

and its various applications in developing new technologies. It is considered as an 

eco-friendly technology for the production of well characterized NPs. The metal nanoparticles 

produced by the plant are more stable and the rate of synthesis is faster as compared to 

other methods. Currently, researchers are mainly focused on searching new antimicrobial 

agents against various pathogenic bacteria which cause infectious diseases. Moreover, the 

NPs have effective antimicrobial properties against infectious pathogens. In this review, we 

have discussed different kind of plants which are used in synthesising NPs and highlighted 

their antimicrobial applications. We also discussed the basic mechanism by which nanoparticles 

interact with microbes. 

Keywords: Antimicrobial activities; bacteria; green synthesis; nanoparticles; plants 


In modern science, nanotechnology 

is one of the most interesting areas of 

research. The synthesis of nano particle 

and nano materials is an important area of 

the nanotechnology. The size of synthesized 

nano particle could be in the range 

of 1 to 100 nm. Nowdays it has gaining a 

great importance in area such as cosmetics, 

health care, food and feed, biomedical, 

environment, mechanics, drug-gene 

delivery, health, optics, electronics, energy 

science, space industries, chemical industries, 

catalysis, light emitters, single 

electron transistors, nonlinear optical devices 

(Hoffman et al., 1992) and photoelectrochemicals 

(Colvin et al., 1994; 

Wang and Herron et al., 1991). 

Additionally, tremendous application 

are increasing in the field of antimicrobials 

catalysis, microelectronics, bimolecular 

detection, diagnostics and therapeutics 

(Veera et al., 2013) This is because 

of newly enhance physical, chemical 

and biological properties based on 

their size and morphology distribution. It 

was found that metallic nano particles are 

considered to have high antibacterial 

properties because of their large surface 

area. These nanoparticles find their application 

in the field of nanocomposites, 

medical imaging, filters, hyperthermia of 

tumors and drug delivery (Tan et al., 

2006; Panigrahi et al., 2004). The antibac- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 171


A Review on Green Synthesis of Nanoparticles… 

Arumugam and Sharma 

terial efficiency of nanoparticles made 

researcher to find the resistant strains 

against metal ions, antibiotics (Khalil et 

al., 2013). Since there are the many approaches 

are available for the synthesis of 

nano particle like photochemical, thermal 

decomposition, microwave decomposition 

(Akl et al., 2012). 

Comparing all this, the best one is 

the eco-friendly way of green synthesis 

approach for the production of nanoparticles. 

This can be done by using plant extract, 

fungi, bacteria and enzyme. As there 

is the lack of chemicals this green 

synthezied nanoparticles have numerous 

benefits in the field of pharmaceutical genomics, 

immune response enhancement, 

biosensors, clinical chemistry and other 

biomedical applications (Diva et al., 

2012). These biosynthesized nanoparticles 

were found to be highly toxic against 

human pathogen which varies from simple 

prokaryotes to complex eukaryotes. 

The development of natural nano factories 

will depend on the ability of organism in 

the production of metal nano particles 

(Korbekandi et al., 2009). The main aspects 

of producing highly stable and well 

characterized nano particle is achieved by 

selecting best organism and providing 

best optimal condition for the growth and 

enzyme activity. 

Instead of using organism for synthesis 

of nanoparticles, plant extracts is 

consider to be cost effective and therefore 

can be used as an economic and best alternative 

for the large-scale production of 

metal nanoparticles. The biomolecules 

found in the plant extract will help in the 

bioreduction of metal nano particles in an 

eco-friendly way. Several plants are act as 

a source for green synthesizing nano particles. 

In present study the microbial 

routes and the plant extract that are used 

for synthesising nanoparticles were reviewed. 

And also the antibacterial mechanism 

of the NPs was discussed. 

2. Green synthesis 

Green synthesis is a green biologically 

based method of synthesising nano 

particles using microorganisms and plants 

in a cost effective, and an environmentfriendly 

manner (Gowramma et al., 2015; 

Makarov et al., 2014). In Green synthesis 

method nano particle can be synthesized 

by utilizing (a) microorganisms like fungi, 

yeasts (eukaryotes), bacteria and actinomycetes 

(prokaryotes), (b) plants and 

plant extracts (c) templates like membranes, 

viruses DNA and diatoms. Microorganism 

and plants are very stable to absorb 

and accumulate inorganic metallic 

ions and heavy metals from their surrounding 

environment (Beveridge and 

Murray, 1980; Singh et al., 2011). 

During synthesising nano particles 

using microorganism, optimisation of culturing 

medium, light, pH and temperature 

are very important. Thereby significantly 

increase enzyme activity (Iravani, 2011; 

Mukherjee et al., 2001). Accoring to Mittal 

et al. (2013) biosynthesis of nanoparticles 

using plants or plant based extracts 

are biologically safe and cost effective. 

Moreover nano particle synthesis using 

plant extract perform both reducing and 

stabilizing (capping) agents (Singh et al., 

2010; Sathishkumar et al., 2009a). 

3. Nano particle synthesized using 

plant 

3.1. Gold nanoparticle 

Gold nano particle act as a good 

source of green chemistry based techniques. 

The flower extract of Nyctanthes 

arbortristis (night jasmine) are used to 

synthesis spherical shaped Gold nanoparticle 

(Das et al., 2011). Coriandrum sativum 

(coriander) leaf extracts are used to 

produce Au nanoparticles at different 

shape ranging in size from 7 to 58 nm 

(Narayanan and Sakthivel, 2008) According 

to Poinern et al. (2013) Eucalyptus 

macrocarpa leaf extract could be utilised 

to synthesize Au nanoparticles as well as 

silver nanoparticle with in a size from 50 

to 200 nm. Moreover variety of plants 

sources such as the leaves and bark of Fi- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 172




cus carica (Singh and Bhakat, 2012), 

Sphaeranthus amaranthoides (Nellore et 

al., 2012) and Putranjiva roxburghii 

(Badole and Dighe, 2012), plant extract of 

Mango (Yang et al., 2014); Gymnocladus 

assamicus (Tamuly et al., 2013); Cacumen 

platycladi (Wu et al., 2013); Pogestemon 

benghalensis (Paul et al., 

2015); Nerium oleander (Tahir et al., 

2015); Butea monosperma (Patra et al., 

2015); Pea nut (Raju et al., 2014); Hibiscus 

cannabinus (Bindhu et al., 2014); 

Sesbania grandiflora (Das and Velusamy, 

2014). 

3.2. Silver nanoparticle 

Normaly silver (Ag) nanoparticles 

ranged in size from 15 to 65 nm with an 

average size of 34 nm and cuboidal, rectangular 

in shape. The medicinally important 

plants like Boerhaavia diffusa 

(Vijaykumar et al., 2014), Aloe vera 

(Chandran et al., 2006), Terminalia 

chebula (Edison and Sethuraman 2012), 

Catharanthus roseus (Mukunthan et al., 

2011), Ocimum tenuiflorum (Patil et al., 

2012) Azadirachta indica (Tripathi et al., 

2009), Emblica officinalis (Ankamwar et 

al., 2005) Cocos nucifera (Roopan et al., 

2013), common spices Piper nigrum 

(Shukla et al., 2010), Cinnamon zeylanicum 

(Satishkumar et al., 2009a) have also 

been used for Ag NP’s synthesis. 

Au-Ag bimetallic nanoparticles also synthesize 

successfully by plants include 

Azadirachta indica (neem) (Shankar et 

al., 2004), Anacardium occidentale (cashew 

nut) (Sheny et al., 2011), Swietenia 

mahagony (West Indies mahogany) 

(Mondal et al., 2011) and cruciferous 

vegetable extracts (Jacob et al., 2012). 

3.3. Copper nanoparticles 

Many variety of plant extracts 

have been used for the synthesize of Copper 

(Cu) and copper oxide (CuO) nanoparticles. 

Magnolia leaf extract and 

Syzygium aromaticum (Clove) extracts is 

used to Cu nanoparticles with the size 

ranging from 40 to 100 nm (Subhankari 

and Nayak, 2013). Cu nanoparticles can 

be synthesised using stem latex of Euphorbia 

nivulia (Common milk hedge). 

Some green methods for synthesis of 

copper nanoparticles are reported using 

plant leaf extracts such as Capparis 

zeylanica Linn (Saranyaadevi et al., 

2014), tamarind, lemon juice (Sastry et 

al., 2013); Ocimum sanctum as capping 

agents (Kulkarni and Kulkarni 2013); 

Magnolia kobus leaf extract (Lee et al., 

2013); Syzygium aromaticum (cloves) 

aqueous extract (Subhankari et al., 2013) 

and Nerium oleander (Gopinath et al., 

2014). Brassica juncea, Medicago sativa 

and Helianthus annus and Tridax procumbens 

(Abdul and Samarrai, 2012; 

Asim et al., 2012), Lantana camara (Majumder, 

2012), Zingiber officinale (Ipsa 

and Nayak, 2013) and Ocimum sanctum 

(Vasudev and Pramod, 2013). Copper nanoparticles 

could be directly used for administration/in 

vivo delivery of nanoparticles 

for cancer therapy (Valodkar et al., 

2011). 

3.4. Copper oxide nanoparticles 

According to Padile et al. (2013) 

Sterculia urens (Karaya gum) synthesizes 

Cuprous Oxide (CuO at a range of 4.8 nm 

size). Jayalakshmi and Yogamoorthi, 

(2014) tried out the copper oxide nanoparticles 

synthesis using flower extract of 

Cassia alata. Rinkesh et al. (2016) use 

Floral extract of Caesalpinia pulcherrima 

for the synthesis of CuO in an ecofriendly 

method. 

3.5. Palladium nanoparticles 

Palladium nanoparticles were synthesised 

using an extract of C. Zeylanicum 

taken from (cinnamon) bark Satishkumar 

et al. (2009b) and also using Annona 

squamosa (Custard apple) peel extract 

with the size ranging from 75 to 85 nm 

(Roopan et al., 2011). The leaf extract of 

soybean (Glycine max) have been able to 

synthesise nanoparticles with a mean size 

of 15 nm (Kumar et al., 2012). According 

to Nadagouda et al. (2008) Coffea arabica 

(Coffee) and Camellia sinensis (Tea) 

extracts have been utilised to synthesise 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 173




palladium nanoparticles at 20 to 60 nm in 

size. Gardenia jasminoides (Cape jasmine) 

act as a good source for synthesis 

of NPs. Many plant leaf extract are used 

for the synthesis of palladium nano particle 

such as Pulicaria glutinosa (Mujeeb et 

al., 2014); Anogeissus latifolia (Kora et 

al., 2015); Cinnamomum camphora 

(Yang et al., 2010); Curcuma longa 

(Sathishkumar et al., 2009c); Gardenia 

jasminoides (Jia et al., 2009); Glycine 

max (Petla et al., 2012); Musa paradisica 

(Bankar et al., 2010); Pinus resinosa 

(Coccia et al., 2012); Pulicaria glutinosa 

(Khan et al., 2014) and Moringa oleifera 

(Anand et al., 2016) have been reported. 

3.6. Platinium nanoparticles 

Song et al. (2010) was first reported 

the leaf extract of Diospyros kaki (Persimmon) 

were utilized for the synthesis of 

platinium nanoparticles n this 

tinu i ns we e nve te int 

e i ss t within the range of 2 

to 12 nm. Platinum nano particle can also 

synthesized by using Ocimum sanctum 

(Holy basil) leaf extract within a range of 

23 n t (Soundarrajan et al., 

2012). Recently, very few reports available 

for the synthesis of Pt NPs using several 

plant extracts including Cacumen 

platycladi, Prunus yedoensis, Azadirachta 

indica, Cochlospermum gossypium, honey 

(Zheng et al., 2013; Velmurugan et al., 

2016; Thirumurugan et al., 2016; Vinod 

et al., 2011; Venu et al., 2011). Similarly 

Diopyros kaki plant (Jae et al., 2010); 

Lantana camara (Musthafa et al., 2016); 

Quercus glauca (Qg); Azadirachta indica 

(Thirumurugan et al., 2016); Ocimun 

sanctum (Soundarrajan et al., 2012); Pinus 

resinosa (Manikandan et al., 2016). 

3.7. Titanium dioxide nanoparticles 

According to Roopan et al. (2012) 

TiO 2 nanoparticles was effectively synthesize 

by Annona squamosa peel. Sundrarajan 

and Gowri (2011) found that 

spherical sized titanium oxide with a 

range of 100 to 150 nm was synthesized 

by utilizing Nyctanthes arbor-tristis leaf. 

Eclipta prostrata leaf extracts can able to 

produce titanium particles ranging in size 

from 36 to 68 nm (Rajakumar et al., 

2012; Zahir et al., 2015). TiO 2 nanoparticles 

was found to be biologically synthesized 

by using Catharanthus roseus leaf 

extract ranged in size from 25 up to 110 

nm Velayutham et al. (2011). Various 

plants are used for the synthesis of TiO 2 

such as Psidium guajava 

(Thirunavukkarasu et al., 2014); Aloe 

Vera plant extract (Ganapathi et al., 

2015); Nyctanthes, Annona squamosapeel 

extract, (Roopan, 2012) and Ecliptaprostrata 

(Gong et al., 2007), Azadirachta 

indica (Siegel et al., 1999); Azadirachta 

indica (Anbalagan et al., 2015). 

3.8. Zinc oxide nanoparticles 

ZnO nanoparticles have been synthesized 

using Aloe vera extract (Sangeetha 

et al., 2011) in spherical shape. In 

addition, Physalis alkekengi extract was 

used to synthesis crystalline polydispersed. 

ZnO nanoparticles with size 

range of 72.5 nm (Qu et al., 2011a) and 

were pseudo-spherical shape and with a 

size of 53.7 nm from Sedum alfredii (Qu 

et al., 2011b). Pragati et al. (2016) was 

first to report the synthesis of zinc oxide 

nanoparticles using flower extract of Nyctanthes 

arbor-tristis. Mimosa pudica 

leaves extract and coffee powder extract 

were utilized for the synthesis of ZnO 

nano particle. Various plants are used for 

the synthesis of ZnO nanoparticles such 

as Solanum nigrum (Ramesh et al., 2015); 

Ocimum Tenuiflorum (Sagar et al., 2015); 

Hibiscus subdariffa (Niranjan et al., 2015); 

Cassia fistula (Vidya et al., 2013); 

Agathosma betulina (Thema et al., 2015). 

3.9. Indium oxide nanoparticles 

Indium Oxide (In 2 O 3 ) nanoparticles 

were synthesized by utilizing the leaf 

extracts from Aloe vera (Aloe barbadensis 

Miller) in spherical shaped with the size 

range from 5 to 50 nm (Maensiri et al., 

2008). Astragalus gummifer (Katira Gum) 

is used for the synthesis of Indium oxide 

nano particle (Kanchana et al., 2016). 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 174




3.10. Iron nanoparticles 

Fe nanoparticles was biologically 

synthesiszed using aqueous extract of 

sorghum bran at room temperature (Njagi 

et al., 2011). According to Pattanayak et 

al. (2013), Azadirachta indica (Neem) 

were utilized to synthesis Fe nanoparticles 

with the size range from 100 nm. Shah et 

al. (2014) able to utilize stem extract of 

Euphorbia milii and leaf extracts of 

Cymbopogon citrates to synthesise Fe 

nano particle with the range of 43-42 nm. 

Additionally Fe nanoparticle can also synthesized 

using Euphorbia milii, Tridax 

procumbens, Tinospora cordifolia, Datura 

innoxia, Calotropis procera and 

Cymbopogon citratus (lemon grass tea). 

Plant parts like Mango leaves, Clove 

buds, Black Tea, Green tea leaves, Coffee 

seeds, Rose leaves, Cumin seeds, Origano 

leaves, Thymol seeds and Curry leaves 

for synthesising Fe nano particle (Monalisa 

and Nayak, 2013). Iron nano particle in 

association with silver can be able to synthesis 

by utilizing aqueous sorghum extract 

(Eric et al., 2011). 

3.11. Iron oxide nanoparticles 

Iron oxide was successfully synthesized 

by (Yen et al., 2016) using Seaweed 

Kappaphycus alvarezii. Latha and 

Gowri, (2014) found that caricaya papaya 

leaves extract were able to synthesis 

Fe 3 O 4 nanoparticles at room temperature. 

Eucalyptus globulus leaf extract was utilized 

for the synthesis of Iron oxide by 

adding the extract into the aqueous solution 

of Ferric chloride (Matheswaran, et 

al., 2014). Makarov et al. (2014) reported 

that aquous extract of monocotyledonous 

(Hordeum vulgare) and dicotyledonous 

(Rumex acetosa) were utilized for the 

synthesis of iron oxide with the size ranging 

from 10 to 40 nm. Iron oxide magnetic 

nanoparticles (Fe 3 O 4 -MNPs) were synthesized 

using the aqueous extract of 

White tea (Camelia sinensis) (Sara and 

Mahnaz, 2016). Mahnaz et al. (2013) 

work focused on the development of a 

biosynthetic method for the production 

of Fe 3 O 4 -NPs using brown seaweed 

(Sargassum muticum) extract. 

3.12. Lead nanoparticles 

Spherical shape Pb nanoparticles 

with the size of 10 to 12 nm were able by 

utilizing the latex from Jatropha curcas 

by Joglekar et al. (2011). Delma et al. 

2016 tried out the green synthesis of Lead 

in association with copper nanoparticles 

by utilizing Zingiber officinale stem extract. 

3.13. Selenium nanoparticles 

Recently, Sasidharan et al. (2014) 

were able to synthesise spherically shaped 

particles Selenium (Se) nanoparticles using 

the extracts taken from the peel of citrus 

reticulata to produce with a mean particle 

size of 70 nm. Similarly Garima et 

al. (2014) approach is to utilize dried Vitis 

vinifera (raisin) extracts for biosynthesize 

selenium nanoparticles (Se-Nps) using. 

Fenugreek seed is used to synthesis selenium 

nanoparticle (Ramamurthy et al., 

2013). Various plants are used for synthesis 

selenium nano particle such as Vitis 

vinifera (raisin) extracts (Sharma et al., 

2014); Clausena dentata (Sowndarya et 

al., 2016); Leucas lavandulifolia 

(Kirupagaran et al., 2016) and Capsicum 

annuum (Shikuo et al., 2007). 

4. Antimicrobial properties 

In the field of biotechnology, biominearlization, 

bioremediation and microbial 

corrosion, Metal microbes interaction 

plays an important role (Prabhu et al., 

2012). Researchers found that metal oxide 

nanoparticles have good antimicrobial 

activity against fungi, virus and bacteria. 

Antimicrobial NPs have nanosized carrier 

for efficient delivery of antibiotics. This 

can prove the effectiveness for treating 

infectious diseases (Huh and Kwon, 

2011). The susceptibility or tolerance of 

bacteria against Np differs among gram + 

ve and gram –ve. The mechanisms of NP 

toxicity depend on composition, surface 

modification and intrinsic properties. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 175




The ionic silver released by the silver nanoparticles 

inactivates bacterial enzymes 

by interacting with thiol group. It inhibits 

bacterial DNA replication and damage the 

bacterial cytoplasm membranes thereby 

depleting the level of ATP and inhibit 

DNA unwinding, which leads to cell 

death. (Parveen et al., 2012). In cause of 

copper NPs Metallic and ionic forms of 

copper produce hydroxyl radicals that 

damage essential proteins and DNA 

thereby inhibit the growth (Wang et al., 

2011). In case of Au, oxidation of Au 3 

+ 

and decarboxylation of citrate generate 

free radicals in the presence of light. This 

will damage the DNA and essential protein 

of bacterial growth (Santo et al., 

2008). The ZnO NPs induce frame shift 

mutation in the bacteria and cause cell 

death. TiO 2 NPs peroxidise the phospholipid 

component of the lipid membrane 

thereby disturb the cell respiration and 

cause cell death. The exact mechanism of 

action of CdS Nps is not known. But it 

has antibacterial activity against E. Coli 

(Sarita, 2010) Fe 2 O 3 nanoparticles to interact 

closely with microbial membranes, 

damaging their structure and inactivate 

bacteria. 


This review has summarized the 

recent research work in the field of green 

synthesis of nanoparticles using plants. 

Plants are able to reduce metal ions faster 

than fungi or bacteria. Hence it is evident 

that the metal nanoparticles produced by 

plants are more stable while compared 

with nanoparticles produced by microbes. 

The capacity of plants in reducing ions 

depends on the presence of polyphenols, 

enzymes, and other chelating agents (in 

the plants). This could have the critical 

effects on the amounts of nanoparticle 

production. Nanoparticles are also 

demonstrated to have interesting antimicrobial 

activity against toxic pathogens. 

Usually, antibacterial activities of NPs 

depend on its physicochemical properties 

and type of targeted bacteria. The synthesised 

green NPs have a good potential as 

an antimicrobial agent against different 

microorganism. However, further research 

is required to tap the potential. 

References 

Abdul, H.M. and Al-Samarrai (2012). 

Nanoparticles as Alternative to Pesticides 

in Management Plant Diseases-A 

Review, International 

Journal of Scientific and Research 

Publications, 2(4), 1- 4. 

Akl, M. Awwad and Nida, S.M. (2012). 

Green synthesis of silver nanoparticles 

by mulberry leaf extract. Nanoscience 

and Nanotechnology. 

2(4), 125-128. 

Anand, K. Tiloke, C. Phulukdaree, A. 

Ranjan, B. Chuturgoon, A. Singh, 

S. and Gengan, R.M. (2016). Biosynthesis 

of palladium nanoparticles 

by using Moringa oleifera flower 

extract and their catalytic and biological 

properties. Photochem Photobiol. 

doi:10.1016/j.jphotobiol.2016.09.0 

39 

Anbalagan, K, Mohanraj, S. and Pugalenthi 

,V. (2015). Rapid phytosynthesis 

of nano-sized titanium using 

leaf extract of Azadirachta indica. 

International Journal of ChemTech 

Research. 8 (4), 2047-2052. 

Ankamwar, B. Damle, C. Ahmad, A. 

and Sastry, M. (2005). Biosynthesis 

of Gold and Silver Nanoparticles 

Using Emblica officinalis Fruit Extract, 

Their Phase Transfer and 

Transmetallation in an Organic Solution. 

Journal of Nanoscience and 

Nanotechnology. 5, 1665-1671. 

Anuj, S.A. and Ishnava, K.B. (2013). 

Plant Mediated Synthesis of Silver 

Nanoparticles Using Dried Stem 

Powder of Tinospora cordifolia, Its 

Antibacterial Activity and Its Comparison 

with Antibiotics. International 

Journal of Pharmacy and Biological 

Sciences. 4, 849-863. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 176




Armendariz, V. Herrera, I. Peralta- 

Videa, J.R. Jose-Yacaman, M. 

Troiani, H. Santiago, P. and 

Gardea-Torresdey, J.L. (2004). 

Size controlled gold nanopartilce 

formation by Avena sativa biomass: 

Use of plants in nanobiotechnology. 

Journal of Nanoparticle. Research. 

6, 377–382. 

Asim, U. Shahid, N. and Naveed, R. 

(2012). Selection of a Suitable 

Method for the Synthesis of Copper 

Nanoparticles. NANO: Brief Reports 

and Reviews. 7(5), 1- 18. 

Badole, M.R. and Dighe, V.V. (2012). 

Synthesis of Gold Nano particles using 

Putranjiva roxburghiiWall. 

leaves Extract. Int. J. Drug Discov. 

Herb. Res. 2, 275–278. 

Badole, M.R. and Dighe, V.V. (2012). 

Synthesis of Gold Nano particles using 

Putranjiva roxburghii Wall 

leaves Extract. Int. J. Drug Discov. 

Herb. Res. 2, 275–278. 

Bankar, A. Joshi, B. Kumar, A.R. and 

Zinjarde, S. (2010). Banana peeled 

extract mediated noval route for the 

synthesis of palladium nanoparticles. 

Mater. Lett .64, 1951–1953. 

Beveridge, T.J. and Murray, R.G.E. 

(1980). Sites of metal deposition in 

the cell wall of Bacillus subtilis. J. 

Bacteriol. 141, 876–887. 

Bindhu, M.R. VijayaRekha, P. 

Umamaheswari, T. and Umadevi, 

M. (2014). Antibacterial activities 

of Hibiscus cannabinus stemassisted 

silver and gold nanoparticles. 

Mater. Lett. 131, 194-197. 

Brayner, R. Ferrari-Iliou, R. Brivois, 

N. Djediat, S. Benedetti, M. and 

Fiévet, F. (2006). Toxicological 

impact studies based on Escherichia 

coli bacteria in ultrafine ZnO nanoparticles 

colloidal medium. Nano. 

Lett. 6, 866–870. 

Cai, W. Gao, T. Hong, H. and Sun, J. 

(2008). Applications of gold nanoparticles 

in cancer nanotechnology. 

Nanotechnol. Sci. Appl. 1, 17–32. 

Chandran, S.P. Chaudhary, M. 

Pasricha, R. Ahmad, A. and 

Sastry, M. (2006). Synthesis of 

Gold Nanotriangles and Silver Nanoparticles 

Using Aloe vera Plant 

Extract. Biotechnology Progress. 

22, 577-583. 

Chen, X. and Schluesener, H.J. (2008). 

Nanosilver: A nanoproduct in medical 

applications. Toxicol. Lett. 176, 

1–12. 

Coccia, F. Tonucci, L. Bosco, D. Bressan, 

M. and d’Alessandro, N. 

(2012). One pot synthesis of ligninstabilized 

platinum and palladium 

nanoparticles and their catalytic behaviours 

in oxidation and reduction 

reactions. Green Chem. 14, 1073– 

1078. 

Colvin, V. L. Schlamp, M. C. and Alivisatos, 

A.P. (1994). Light-emitting 

diodes made from cadmium selenide 

nanocrystals and a semiconducting 

polymer. Nature. 370, 354–357. 

Das, J. and Velusamy, P. (2014). Catalytic 

reduction of methylene blue 

using biogenic gold nanoparticles 

from Sesbaniagrandiflora L. J. Taiwan 

Inst. Chem. Eng. 45, 2280- 

2285. 

Das, R.K. Gogoi, N. and Bora, U. 

(2011). Green synthesis of gold nanoparticles 

using Nyctanthes arbortristis 

flower extract. Bioprocess. 

Biosyst. Eng. 34, 615–619. 

Delma, B.T. Vijila, B. and Jaya Rajan, 

M. (2016). Green Synthesis of Copper 

and Lead Nanoparticles using 

Zingiber Officinale stem extract. International 

Journal of Scientific and 

Research Publications. 6, 11. 

Diva, B. Lingappa, K. and Dayanand. 

A. (2012). Antibacterial activity of 

nano gold particles synthesezd by 

Bacillus sps. Journalecobiotechnology. 

4 (1), 43-45. 

Edison, T.J.I. and Sethuraman, M.G. 

(2012). Instant Green Synthesis of 

Silver Nanoparticles Using Terminalia 

chebula Fruit Extract and 

Evaluation of Their Catalytic Ac- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 177




tivity on Reduction of Methylene 

Blue. Process Biochemistry. 47, 

1351-1357. 

Eric, C.N. Hui, H.Lisa, S.Homer, 

G.Hugo, M.G.John, B.C.George, 

E. H. andSteven, L. S. (2011). 

Biosynthesis of Iron and Silver 

Nanoparticles at Room Temperature 

Using Aqueous sorghum Bran 

Extracts. Langmuir. 27 (1). 264– 

271. 

Ganapathi, R.K, Ashok, C.H. and Venkateswara 

R.K. Shilpa C.C.H. and 

Pavani, T. (2015). Green Synthesis 

of TiO 2 Nanoparticles Using Aloe 

Vera Extract. International Journal 

of Advanced Research in Physical 

Science. 2, 1A, 28-34. 

Garima, S. Ashish, R.S. Riju, B. Jongbong, 

P. Bilguun, G. Ju-Suk, N. 

and Sang-Soo, L. (2014). Biomolecule-Mediated 

Synthesis of Selenium 

Nanoparticles using Dried Vitis 

vinifera (Raisin) extract. Molecules. 

19, 2761-2770. 

Geetha, N. Geetha, T.S. Manonmani, P. 

and Thiyagarajan, M. (2014). 

Green Synthesis of silver nanoparticles 

using Cymbopogan Citratus 

(Dc) Stapf. Extract and its antibacterial 

activity. Aust. J. Basic Appl. Sci. 

8, 324–331. 

Ghosh, S.K. and Pal, T. (2007). Interparticle 

coupling effect on the surface 

plasmon resonance of gold nanoparticles: 

From theory to application. 

Chem. Rev. 107, 4797–4862. 

Gong, P. Li, H. He, X. Wang, K. Hu, J. 

and Zhang, S. (2007). Preparation 

and antibacterial activity of Fe 3 O 4 , 

Ag nanoparticles. Nanotechnology, 

18(28), 604-611. 

Gopinath, M. Subbaiya, R. Selvam, 

M.M. and Suresh, D. (2014). Synthesis 

of copper nanoparticles from 

Nerium oleander leaf aqueous extract 

and its antibacterial activity. 

International Journal of Current 

Microbiology and Applied Science. 

3(9), 814–818. 

Gowramma, B. Keerthi, U. Mokula, R. 

and Rao, D.M. (2015). Biogenic 

silver nanoparticles production and 

characterization from native stain of 

Corynebacterium species and its antimicrobial 

activity. Biotech. 5, 195– 

201. 

Hoffman, J. Mills, G. Yee, H. and 

Hoffmann, M. (1992). Q-Sized 

CdS: Synthesis, Characterization, 

and Efficiency of Photoinitiation of 

Polymerization of Several Vinylic 

Monomers. Journal of Physical 

Chemistry. 96(13), 5546–5552. 

Huang, X. Jian, P.K. El-Sayed, I.H. and 

El-Sayed, M.A. (2006). Determination 

of the minimum temperature 

required for selective photothermal 

destruction of cancer cells with the 

use of immune-targeted gold nanoparticles. 

Photochem. Photobiol. 82, 

412–417. 

Huh, A. J. and Kwon, Y.J. (2011). 

Nanoantibiotic: a new paradigm for 

treating infectious diseases using 

nanomaterials in the antibiotics resistant 

era. Journal of Controlled 

Release. 156(2), 128–145. 

Ipsa, S. and Nayak, P.L. (2013). Antimicrobial 

Activity of Copper Nanoparticles 

Synthesised by Ginger 

(Zingiber officinale) extract. World 

Journal of Nano Science & Technology. 

2(1), 10-13. 

Iravani, S. (2011). Green Synthesis of 

Metal Nanoparticles Using Plants. 

Green Chemistry, 13, 2638-2650. 

Jacob, J. Mukherjee, T. and Kapoor, S. 

(2012). A simple approach for facile 

synthesis of Ag, anisotropic Au and 

bimetallic (Ag/Au) nanoparticles using 

cruciferous vegetable extracts. 

Mater. Sci. Eng. C. 32, 1827–1834. 

Jae, Y.S. Eun-Yeong, K. and Beom, 

S.K. (2010). Biological synthesis of 

platinum nanoparticles using Diopyros 

kaki leaf extract. Bioprocess Biosyst. 

Eng. 33, 159–164. 

Jayalakshmi, and Yogamoorthi, A. 

(2014). Green synthesis of copper 

oxide nanoparticles using aqueous 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 178




extract of flowers of Cassia alata 

and particles characterisation. International 

Journal of Nanomaterials 

and Biostructures. 4(4), 66-71. 

Jia, L. Zhang, Q. Li, Q. and Song, H. 

(2009). The biosynthesis of palladium 

nanoparticles by antioxidants 

in Gardenia jasminoides Ellis: Long 

lifetime nanocatalysts for p- 

nitrotoluene hydrogenation. Nanotechnology. 

20,385601. 

Joglekar, S. Kodam, K. Dhaygude, M. 

and Hudlikar, M. (2011). Novel 

route for rapid biosynthesis of lead 

nanoparticles using aqueous extract 

of Jatropha curcas L. latex. Mater. 

Lett. 65, 3170–3172. 

Kanchana, L.C. Aparna, Y. Ramchander, 

M. Ravinder, D. and Jaipal, 

K. (2016). Synthesis and Characterisation 

of In 2 O 3 Nanoparticles from 

Astragalus gummifer. Advances in 

Nanoparticles. 5, 114-122. 

Karthik, R. Sasikumar, R. Shen-Ming, 

C. Govindasamy, M. Vinoth Kumar, 

J. and Muthuraj, V. (2016). 

Green Synthesis of Platinum Nanoparticles 

Using Quercus Glauca extract 

and Its Electrochemical Oxidation 

of Hydrazine in Water Samples. 

Int. J. Electrochem. Sci. 11, 8245 – 

8255. 

Khalil, K.A. Fouad, H. Elsarnagawy, T. 

and Almajhdi, F.N. (2013). Preparation 

and characterization of electrospun 

PLGA/silvercomposite nanofibers 

for biomedical applications. 

Int J Electrochem Sci .8, 3483–93. 

Khalil, M.M.H. Ismail, E.H. El- 

Baghdady, K.Z. and Mohamed, D. 

(2013). Green synthesis of silver 

nanoparticles using olive leaf extract 

and its antibacterial activity. Arabian 

Journal of Chemistry. 7, 1131– 

1139. 

Khan, M. Khan, M. Kuniyil, M. Adil, 

S.F. Al-Warthan, A. Alkhathlan, 

H.Z. Tremel, W. Tahir, M.N. and 

Siddiqui, M.R.H. (2014). Biogenic 

synthesis of palladium nanoparticles 

using Pulicaria glutinosa extract 

and their catalytic activity towards 

the Suzuki coupling reaction. Dalton 

Trans. 43, 9026. 

Kirupagaran, R. Saritha, A. and Bhuvaneswari, 

S. (2016). Green Synthesis 

of Selenium Nanoparticles 

from Leaf and Stem Extract of Leucas 

lavandulifolia Sm. and their 

Application. Journal of Nanoscience 

and Technology. 2(5), 224–226. 

Kora, A.J. and Rastog,i L. (2015). 

Green synthesis of palladium nanoparticles 

using gum ghatti (Anogeissus 

latifolia) and its application as 

an antioxidant and catalyst. Arab. J. 

Chem. doi; 2015.06.024 

Korbekandi, H. Iravani S. and Abbasi, 

S. (2009). Production of nanoparticles 

using organisms. Critical Reviews 

in Biotechnology. 29, 279– 

306. 

Kulkarni, V.D. and Kulkarni, P.S. 

(2013). Green Synthesis of Copper 

Nanoparticles Using Ocimum Sanctum 

Leaf Extract. International 

Journal of Chemical Studies. 1(3), 

1–4. 

Kumar, P.R. Vivekanandhan, S. Misra, 

M. Mohanty, A. and Satyanarayana, 

N. (2012). Soybean (Glycine 

max) leaf extract based green 


J. Biomater. Nanobiotechnol. 

3, 14–19. 

Latha, N. and Gowri. M. (2014). Bio 

Synthesis and Characterisation of 

Fe 3 O 4 Nanoparticles Using Caricaya 

Papaya Leaves Extract. International 

Journal of Science and Research. 

3, 11. 

Lee, H.J. Song, J.Y. and Kim, B.S. 

(2013). Biological synthesis of copper 

nanoparticles using Magnolia 

kobus leaf extract and their antibacterial 

activity. Journal of Chemical 

Technology and Biotechnology. 

88(11), 1971–1977. 

Maensiri, S. Laokul, P. Klinkaewnarong, 

J. Phokha, S. Promarak, V. 

and Seraphin, S. (2008). Indium 

oxide (In 2 O 3 ) nanoparticles using 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 179




Aloe vera plant extract: Synthesis 

and optical properties. J. Optoelectron. 

Adv. Mater. 10, 161–165. 

Mahnaz, M. Farideh, N. Mansor, B.A. 

and Rosfarizan, M. (2013). Green 

Biosynthesis and Characterization 

of Magnetic Iron Oxide (Fe 3 O 4 ) 

Nanoparticles Using Seaweed 

(Sargassum muticum) Aqueous Extract. 

Molecules. 18(5), 5954-5964. 

Majumder, B.R. (2012). Bioremediation: 

Copper Nanoparticles from Electronic-waste, 

International Journal 

of Engineering Science and Technology, 

4. 

Makarov, V.V. Love, A.J. Sinitsyna, 

O.V. Makarova, S.S. Yaminsky, 

I.V. Taliansky, M.E. and Kalinina, 

N.O. (2014). Green nanotechnologies: 

Synthesis of metal nanoparticles 

using plants. Acta Naturae. 

6, 35–44. 

Makarov, V.V. Makarova, S.S. Love, 

A.J. Sinitsyna, O.V. Dudnik, 

A.O.Yaminsky, I.V. Taliansky, 

M.E. Kalinina, N.O. (2014). Biosynthesis 

of Stable Iron Oxide Nanoparticles 

in Aqueous Extracts of 

Hordeum vulgare and Rumex acetosa 

plants. J. Langmuir. 30 (20), 

5982-5988. 

Manikandan, V. Velmurugan, P. Park, 

J.H. Lovanh, N. Seo, S.K. Jayanthi, 

P. Park, Y.J. Cho, M. and Oh, 

B.T. (2016). Synthesis and antimicrobial 

activity of palladium nanoparticles 

from Prunus x yedoensis 

leaf extract. Materials Letter. 185, 

335–338. 

Matheswaran, B. (2014). Synthesis of 

Iron Oxide Nanoparticles by Using 

Eucalyptus globulus Plant Extract. 

J. Surf. Sci. Nanotech. 12, 363-367. 

Mittal, A.K. Chisti, Y. and Banerjee, 

U.C. (2013). Synthesis of metallic 

nanoparticles using plants. Biotechnol. 

Adv. 31, 346–356. 

Monalisa, P. and Nayak P.L. (2013). 

Eco-friendly green synthesis of iron 

nanoparticles from various plants 

and spices extract. International 

Journal of Plant, Animal and Environmental 

Sciences. 3, 1. 

Mondal, S. Roy, N. Laskar, R.A. Sk, I. 

Basu, S. Mandal, D. and Begum, 

N.A. (2011). Biogenic synthesis of 

Ag, Au and bimetallic Au/Ag alloy 

nanoparticles using aqueous extract 

of mahogany (Swietenia mahogani 

JACQ) leaves. Colloids Surf. B. 82, 

497–504. 

Mujeeb, K. Merajuddin, K. Mufsir, K. 

Syed, F.A. Abdulrahman, A.W. 

Hamad, Z. A. Wolfgang, T. Muhammad, 

N.T. and Mohammed, 

R.H.S. (2014). Biogenic synthesis of 

palladium nanoparticles using Pulicaria 

glutinosa extract and their 

catalytic activity towards the Suzuki 

coupling reaction. Dalton Transactions. 

24. 

Mukherjee, P. Ahmad, A. Mandal, D. 

Senapati, S. Sainkar, S.R. Khan, 

M.I. Ramani, R. Parischa, R. 

Ajayakumar, P.V. and Alam, M. 

(2001). Bioreduction of AuCl 4 ions 

by the fungus, Verticillium sp. and 

surface trapping of the gold nanoparticles 

formed. Angew. Chem. Int. 

Ed. 40, 3585–3588. 

Mukunthan, K.S. Elumalai, E.K. Patel, 

E.N. and Murty, V.R. (2011). 

Catharanthus roseus: A Natural 

Source for Synthesis of Silver Nanoparticles. 

Asian Pacific Journal of 

Tropical Biomedicine. 1, 270-274. 

Musthafa, O.M. Sudip, C. Tasneem, A. 

Shahid, A. and Abbasi, A. (2016). 

Clean-Green Synthesis of Platinum 

Nanoparticles Utilizing a Pernicious 

Weed Lantana (Lantana Camara). 

American Journal of Engineering 

and Applied Sciences. 9 (1), 84-90. 

Nadagouda, M.N. and Varma, R.S. 


and palladium nanoparticles at room 

temperature using coffee and tea extract. 

Green Chem. 10, 859–862. 

Narayanan, K.B. and Sakthivel, N. 

(2008). Coriander leaf mediated bi- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 180




osynthesis of gold nanoparticles. 

Mater. Lett. 62, 4588–4590. 

Nellore, J. Pauline, P.C. and Amarnath, 

K. (2012). Biogenic synthesis 

of Sphearanthus amaranthoids 

towards the efficient production of 

the biocompatible gold nanoparticles. 

Dig. J. Nanomater. Biostruct. 

7, 123–133. 

Niranjan, B. Saha, S. Chakraborty, M. 

Maiti, M. Das, S. Basu, R. and 

Nandy, P. (2015). Green synthesis of 

zinc oxide nanoparticles using Hibiscus 

subdariffa leaf extract: Effect of 

temperature on synthesis, antibacterial 

activity and anti-diabetic activity. 

RSC Advances. 5, 4993-5003. 

Njagi, E.C. Huang, H. Stafford, L. 

Genuino, H. Galindo, H.M. and 

Collins, J.B. (2011). Biosynthesis 

of iron and silver nanoparticles at 

room temperature using aqueous 

sorghum bran extracts. Langmuir. 

27, 264–271. 

Paciotti, G.F. Mayer, L. Weinreich, D. 

Goia, D. Pavel, N. McLaughlin, 

R.E. and Tamarkin, L. (2006). 

Colloidal gold: A novel nanoparticle 

vector for tumour directed drug delivery. 

Drug Deliv.11, 169–183. 

Padil, V.V.T. and Cerník, M. (2013). 

Green synthesis of copper oxide nanoparticles 

using Gum karaya as a 

biotemplate and their antibacterial 

application. Int. J. Nanomed. 8, 

889–898. 

Panigrahi, S. Kundu, S. Ghosh, S. 

Nath, S. and Pal, T. (2004). General 

method of synthesis for metal 

nanoparticles. Journal of Nanoparticle 

Research. 6(4), 411–414. 

Parak,W.J. Gerion, D. Pellegrino, T. 

Zanchet, D. Micheel, C. Williams, 

S.C. Boudreau, R. Le Gros, M.A. 

Larabell, G.A. and Alivisatos, 

A.P. (2003). Biological applications 

of colloidal nanocrystals. Nanotechnology. 

14, 15–27. 

Parveen, A. Ashish, S.R. and R. 

Srinath. (2012). Biosynthesis and 

characterization of silver nanoparticles 

from Cassia auriculata leaf extract 

and in-vitro evaluation of antimicrobial 

activity. International 

Journal of Applied Biology and 

Pharmaceutical Technology. 3(2), 

222-228. 

Patil, R.S. Kokate, M.R. and Kolekar, 

S.S. (2012). Bioinspired Synthesis 

of Highly Stabilized Silver Nanoparticles 

using Ocimum tenuiflorum 

Leaf Extract and Their Antibacterial 

Activity. Spectrochimica Acta Part 

A: Molecular and Biomolecular 

Spectroscopy. 91, 234-238. 

Patra, S. Mukherjee, S. Barui, A.K. 

Ganguly, A. and Sreedhar, B. 

(2015). Green synthesis, characterization 

of gold and silver nanoparticles 

and their potential application 

for cancer therapeutics. Mater. Sci. 

Eng. C. 53, 298-309. 

Pattanayak, M. and Nayak, P.L. (2013). 

Green synthesis and characterization 

of zero valent iron nanoparticles 

from the leaf extract of Azadirachta 

indica (Neem). World J. Nano 

Sci.Technol. 2, 06–09. 

Paul, B. Bhuyan, B. DharPurkayastha, 

D. Dey, M. and Dhar, S.S. (2015). 

Green synthesis of gold nanoparticles 

using Pogestemonbenghalensis 

(B) O. Ktz. leaf extract and studies 

of their photocatalytic activity in 

degradation of methylene blue. Mater 

Lett .148, 37-40. 

Petla, R.K. Vivekanandhan, S. Misra, 

M. Mohanty, A.K. and Satyanarayana, 

N. (2012). Soybean (Glycine 

max) leaf extract based green 


J. Biomater Nanobiotechnol. 3, 

14–19. 

Poinern, G.E.J. Shah, M. Chapman, P. 

and Fawcett, D. (2013). Green biosynthesis 

of silver nanocubes using 

the leaf extracts from Eucalyptus 

macrocarpa. Nano Bull. 2, 1–7. 

Prabhu S. and Paulose E. (2012). Silver 

nanoparticles mechanism of antimicrobial 

action, synthesis medical 

applications and toxicity effects. In- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 181




ternational Journal of Nano Letters. 

2. 

Pragati, J. Poonam, K. and Rana J.S. 

(2016). Green synthesis of zinc oxide 

nanoparticles using flower extract 

of Nyctanthes arbor-tristis and 

their antifungal activity Journal of 

King Saud University – Science. 

doi.2016.10.002 

Qu, J. Luo, and C. Hou, J. (2011b). 

Synthesis of ZnO nanoparticles 

from Zn hyper-accumulator (Sedum 

alfredii Hance) plants. Micro Nano 

Lett. 6, 174–176. 

Qu, J. Yuan, X. Wang, X. and Shao, P. 

(2011a). Zinc accumulation and 

synthesis of ZnO nanoparticles using 

Physalis alkekengi L. Environ. 

Pollut. 159, 1783–1788. 

Rajakumar, G. Abdul-Rahuman, A. 

Priyamvada, B. Gopiesh-Khanna, 

V. Kishore-Kumar, and D. Sujin, 

P.J. (2012). Eclipta prostrata leaf 

aqueous extract mediated synthesis 

of titanium dioxide nanoparticles. 

Mater. Lett. 68, 115–117. 

Raju, D. Vishwakarma, R.K. Khan, 

B.M. Mehta, U.J. and Ahmad, A. 

(2014). Biological synthesis of cationic 

gold nanoparticles and binding 

of plasmid DNA. Mater Lett. 129, 

159-161. 

Ramamurthy, C.H. Sampath, K.S. 

Arunkumar, P. Suresh, K.M. 

Sujatha, V. Premkumar, K. and 

Thirunavukkarasu, C. (2013). 


of selenium nanoparticles and its 

augmented cytotoxicity with doxorubicin 

on cancer cells. Bioprocess 

Biosyst. Eng. DOI 10.1007/s00449- 

012-0867-1 

Ramesh, M. Anbuvannan, M. and 

Viruthagiri, G. (2015). Green 

synthesis of ZnO nanoparticles 

using Solanum nigrum leaf extract 

and their antibacterial activity. 

Spectrochim Acta A Mol Biomol 

Spectrosc. 136, 864-70. 

Rinkesh, V.K. Sandesh, J. and Abhijeet, 

S. (2016). Synthesis of Copper 

/ Copper Oxide Nanoparticles in 

Eco-Friendly and NonToxic Manner 

from Floral Extract of Caesalpiniapulcherrima. 

International Journal 

on Recent and Innovation Trends in 

Computing and Communication. 4, 

4. 

Roopan, S.M. Bharathi, A. Kumar, R. 

Khanna, V.G. and Prabhakarn, 

A. (2011). Acaricidal, insecticidal, 

and larvicidal efficacy of aqueous 

extract of Annona squamosa L peel 

as biomaterial for the reduction of 

palladium salts into nanoparticles. 

Colloids Surf. B Biointerfaces. 92, 

209–212. 

Roopan, S.M. Bharathi, A. Prabhakarn, 

A. Rahuman, A.A. Velayutham, 

K. and Rajakumar, G. 

(2012). Efficient phyto-synthesis 

and structural characterization of rutile 

TiO 2 nanoparticles using Annona 

squamosa peel extract. Spectrochim 

Acta. A Mol. Biomol. Spectrosc. 

98, 86-90. 

Roopan, S.M. Rohit, M.G. Rahuman, 

A.A. Kamraj, C. Bharathi, A. and 

Surendra, T.V. (2013). Low-Cost 

and Eco-Friendly Phyto-Synthesis 

of Silver Nanoparticles Using Coos 

nucifera Coir Extract and Its Larvicidal 

Activity. Industrial Crops and 

Products. 43, 631-635. 

Sagar, R. Thorat, P.V. and Rohini, T. 

(2015). Green Synthesis of Zinc Oxide 

(ZnO) Nanoparticles Using 

Ocimum Tenuiflorum Leaves. International 

Journal of Science and 

Research (IJSR). 4, 5. 

Sangeetha, G. Rajeshwari, S. and 

Venckatesh, R. (2011). Green synthesis 

of zinc oxide nanoparticles by 

aloe barbadensis miller leaf extract: 

Structure and optical properties. 

Mater. Res. Bull. 46, 2560–2566. 

Santo, C.E. Taudte, N. Nies D. H. and 

Grass G. (2008). Contribution of 

copper ion resistance for survival of 

Escherichia coli on metallic copper 

surfaces. Applied and Environmental 

Microbiology. 74(4), 977-986. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 182




Sara, S. and Mahnaz, M.S. (2016). 


of iron oxide magnetic nanoparticles 

using Shanghai White tea (Camelia 

sinensis) aqueous extract. Journal of 

Chemical and Pharmaceutical Research. 

8(5), 138-143. 

Saranyaadevi, K. Subha, V. Ravindran, 

R.E. and Renganathan, S. (2014). 

Synthesis and characterization of 

copper nanoparticle using Capparis 

zeylanica leaf extract. International 

Journal of Chemistry, Technology 

and Research. 6(10), 4533–4541. 

Sarita. (2010). Synthesis, Characterization 

and applications of CdO, CdS 

nanoparticles and nanocomposites , 

M.Phil.Thesis, Shoolini University 

of Biotechnology, 57-58. 

Sasidharan, S. Sowmiya, R. and Balakrishnaraja, 

R. (2014). Biosynthesis 

of selenium nanoparticles using 

citrus reticulata peel extract. World 

J. Pharm. Res. 4, 1322–1330. 

Sastry, A.B.S. Karthik, A.R. Linga, 

P.R. and Murthy, B.S. (2013). 

Large-scale green synthesis of Cu 

nanoparticles. Environmental chemistry 

letters. 11(2), 183–187. 

Satishkumar, M., Sneha, K., Won, 

S.W., Cho, C.W., Kim, S. and 

Yun, Y.S. (2009a). Cinnamon 

zeylancium Bark Extract and Powder 

Mediated Green Synthesis of 

Nano-Crystalline Silver Particles 

and Its Antibacterial Activity. Colloids 

and Surfaces B: Biointerfaces. 

73, 332-338. 

Sathishkumar, M. Sneha, K. Kwak, I.S. 

Mao, J. Tripathy, S.J. and Yun, 

Y.S. (2009b). Phyto-crystallization 

of palladium through reduction process 

using Cinnamon zeylanicum 

bark extract. J. Hazard Mater. doi: 

171:404–404. 

Sathishkumar, M. Sneha, K. Yun, and 

Y.S. (2009c). Palladium nanocrystals 

synthesis using Curcuma longa 

tuber extract. Int. J. Mater. Sci .4, 

11–17. 

Schmid, G. (1992). Large clusters and 

colloids. Metals in the embryonic 

state. Chemical Reviews. 92(8), 

1709–1727. 

Shah, S. Dasgupta, S. Chakraborty, M. 

Vadakkekara, R. and Hajoori, M. 

(2014). Green synthesis of iron nanoparticlesusing 

plant extracts. Int. 

J. Biol. Pharm. Res. 5, 549–552. 

Shankar, S.S. Rai, A. Ahmad, A. and 

Sastry, M. (2004). Rapid synthesis 

of Au, Ag, and bimetallic Au core- 

Ag shell nanoparticles using Neem 

(Azadirachta indica) leaf broth. J. 

Colloid Interface Sci. 275, 496–502. 

Sharma, G. Sharma, A.R. Bhavesh, R. 

Park, J. Ganbold, B. Nam, J.S. 

and Lee, S.S. (2014). Biomoleculemediated 

synthesis of selenium 

nanoparticles using dried Vitis 

vinifera (raisin) extract. 

Molecules. 19(3), 2761-70. 

Sheny, D.S. Mathew, T. and Philip, D. 

(2011). Phytosynthesis of Au, Ag 

and Au-Ag bimetallic nanoparticles 

using aqueous extract and dried leaf 

of Anacardium occidentale. Spectrochim. 

Acta A. 79, 254–262. 

Shikuo, L. Yuhua, S. Anjian, X. 

Xuerong, Y. Xiuzhen, Z. Liangbao, 

Y. and Chuanhao, L. (2007) 

Rapid, room-temperature synthesis 

of amorphous selenium/protein 

composites using Capsicum annuum 

L extract. Nanotechnology. 18, 40. 

Shukla, V.K. Singh, R.P. and Pandey, 

A.C. (2010). Black Pepper Assisted 

Biomimetic Synthesis of Silver Nanoparticles. 

Journal of Alloys and 

Compounds. 507, L13-L16. 

Siegel, R.W. Hu, E. and Roco, M.C. 

(1999). Nanostructure Science and 

Technology: R & D Status and 

Trends in Nanoparticles, Nanostructured 

Materials, and Nanodevices. 

Springer Science & Business Media, 

Kluwer Academic Publishers, Boston, 

336. 

Simon-Deckers, A. Loo, S. Mayne- 

L’hermite, M.N. Herlin-Boime, N. 

Menguy, N. Reynaud, C. Gouget, 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 183




B. and Carrière, M. (2009). Size 

composition and shape dependent 

toxicological impact of metal oxide 

nano-particles and carbon nanotubes 

toward bacteria. Environ. Sci. 

Technol. 43, 8423–8429. 

Singh, A. Jain, D. Upadhyay, M.K. 

Khandelwal, N. and Verma, H.N. 


nanoparticles using Argemone mexicana 

leaf extract and evaluation of 

their antimicrobial activity. Dig. J. 

Nanomater. Biostruct. 5, 483–489. 

Singh, P.P. and Bhakat, C. (2012). 

Green synthesis of gold nanoparticles 

and silver nanoparticles from 

leaves and bark of Ficus carica for 

nanotechnological applications. Int. 

J. Sci. Res. Publ. 2, 1–4. 

Singh, R. Gautam, N. Mishra, A. and 

Gupta, R. (2011). Heavy metals 

and living systems: An overview. 

Indian J. Pharmacol. 43, 246–253. 

Sondi, I. and Salopek-Sondi, B. (2004). 

Silver nanoparticles as antimicrobial 

agent: A case study on E. coli as a 

model for Gram-negative bacteria. 

J. Colloid Interface Sci. 275, 177– 

182. 

Song, J.Y. Kwon, E.Y. and Kim, B.S. 

(2010). Biological synthesis of platinum 

nanoparticles using Diopyros 

kaki leaf extract. Bioprocess. Biosyst. 

Eng. 33, 159–164. 

Sotiriou, G.A. and Pratsinis, S.E. 

(2010). Antimicrobial activity of 

silver nanosilver ions and particles. 

Environ. Sci. Technol. 44, 5649– 

5654. 

Soundarrajan, C. Sankari, A. 

Dhandapani, P. Maruthamuthu, 

S. Ravichandran, S. Sozhan, G. 

and Palaniswamy, N. (2012). Rapid 

biological synthesis of platinum 

nanoparticles using Ocimum sanctum 

for water electrolysis applications. 

Bioprocess Biosyst Eng. 

35,827–833. 

Sowndarya, P. Ramkumar, G. and 

Shivakumar, M.S. (2016). Green 

synthesis of selenium nanoparticles 

conjugated Clausena dentata plant 

leaf extract and their insecticidal 

potential against mosquito vectors. 

Artificial cells, Nanomedicine and 

Biotechnology.1-6. 

Subhankari, I. and Nayak, P.L. (2013). 

Antimicrobial Activity of Copper 

Nanoparticles Synthesised by Ginger 

(Zingiber officinale) Extract. 

World Journal of Nano Science & 

Technology. 2(1), 10–13. 

Sundrarajan, M. and Gowri, S. (2011). 

Green synthesis of titanium dioxide 

nanoparticles by nyctanthes arbortristis 

leaves extract. Chalcogenide 

Lett. 8, 447–451. 

Suresh, D. Nethravathi, P.C Udayabhanu, 

Rajanaika, H. Nagabhushana, 

H. and Sharma, S.C. 

(2015). Green synthesis of multifunctional 

zinc oxide (ZnO) nanoparticles 

using Cassia fistula plant 

extract and their photodegradative, 

antioxidant and antibacterial activities. 

Materials science in semiconductor 

processing, 31, 446-454. 

Tahir, K. Nazir, S. Li, B. Khan, A.U. 

and Khan, Z.U.H. (2015). Nerium 

oleander leaves extract mediated 

synthesis of gold nanoparticles and 

its antioxidant activity. Mater Lett. 

156, 198-201. 

Tamuly, C. Hazarika, M. and Bordoloi, 

M. (2013). Biosynthesis of Au nanoparticles 

by Gymnocladu sassamicus 

and its catalytic activity. 

Mater Lett. 108, 276-279. 

Tan, M. Wang, G. Ye Z. and Yuan, J. 

(2006). Synthesis and characterization 

of titania-based monodisperse 

fluorescent europium nanoparticles 

for biolabeling. Journal of luminescence. 

117(1), 20–28. 

Thema, F.T. Manikandan, E. Dhlamini, 

M.S. and Maaza, M. (2015). Green 

synthesis of ZnO nanoparticles 

via Agathosma betulina natural extract. 

Materials Letters. 161(15), 

124–127. 

Thirumurugan, A. Aswitha, P. 

Kiruthika, C. and Nagarajan, S. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 184




(2016). A. Nancy Christy Green 

synthesis of platinum nanoparticles 

using Azadirachta indica— an ecofriendly 

approach. Mater. Lett. 170, 

175–178. 

Thirunavukkarasu, S. Abdul, A.R. 

Chidambaram, J. Govindasamy, 

R. Sampath, M. Arivarasan, V.K. 

Kanayairam, V. (2014). Green synthesis 

of titanium dioxide nanoparticles 

using Psidium guajava extract 

and its antibacterial and antioxidant 

properties. Asian Pacific Journal of 

Tropical Medicine. 968-976. 

Tripathi, A. Chandrasekaran, N. Raichur, 

A.M. and Mukherjee, A. 

(2009). Antibacterial Applications 

of Silver Nanoparticles Synthesized 

by Aqueous Extract of Azadirachta 

indica (Neem) Leaves. Journal of 

Biomedical Nanotechnology. 5, 93- 

98. 

Valentin, V.M. Svetlana, S.M. Andrew, 

J.L. Olga, V.S. Anna, O.D. Igor, 

V.Y. Michael, E.T. and Natalia, 

O.K. (2014). Biosynthesis of Stable 

Iron Oxide Nanoparticles in 

Aqueous Extracts of Hordeum 

vulgare and Rumex acetosa Plants. 

Langmuir. (20), 5982–5988. 

Valodkar, M. Jadeja, R.N. 

Thounaojam, M.C. Devkar R.V. 

and S. Thakore (2011). Biocompatible 

synthesis of peptide capped 

copper nanoparticles and their biological 

effect on tumor cells. Materials 

Chemistry and Physics. 128(1- 

2), 83–89. 

Vasudev, K.B. and Pramod, K. (2013). 

Green Synthesis of Copper Nanoparticles 

Using Ocimum Sanctum 

Leaf Extract. International Journal 

of Chemical Studies. 1(3), 1-4. 

Veera, B. N. Jahnavi, A. Rama, K. 

Manisha, R. D. Rajkiran, B. and 

Pratap, R.M.P. (2013). Green synthesis 

of plant mediated silver nanoparticles 

using Withania somnifera 

leaf extract and evaluation of their 

antimicrobial activity. International 

Journal of Advanced Research. 1(9), 

307-313. 

Velayutham, K. Rahuman, A.A. Rajakumar, 

G. Santhoshkumar, T. 

Marimuthu, S. Jayaseelan, C. 

Bagavan, A. Kirthi, A.V. Kamaraj, 

C. and Zahir, A.A. (2011). 

Evaluation of Catharanthus roseus 

leaf extract-mediated biosynthesis 

of titanium dioxide nanoparticles 

against Hippobosca maculata and 

Bovicola ovis. Parasitol. Res. 111, 

2329–2337. 

Velmurugan, P. Shim, J. Kim, K. and 

Oh, B.T. (2016). Prunus × yedoensis 

tree gum mediated synthesis of 

platinum nanoparticles with antifungal 

activity against phytopathogens, 

Materials letters. 174, 61-65. 

Venu, R. Ramulu, T.S. Anandakumar, 

S. Rani, V.S. and Kim, C.G. 

(2011). Bio-directed synthesis of 

platinum nanoparticles using aqueous 

honey solutions and their catalytic 

applications. Colloids and Surfaces 

A: Physicochemical and Engineering 

Aspects. 384(1-3), 733- 738. 

Vidya, C. Shilpa, H. Chandraprabhab, 

M.N. Lourdu Antonyraja, M.A. 

Indu, V.G. Aayushi, J. and Kokil, 

B. (2013). Green synthesis of ZnO 

nanoparticles by Calotropis Gigantea. 


Engineering and Technology, 1. 

Vijaya, K. S. (2012). Silver nanoparticles 

synthesized by in-vitro derived 

plants and Callus culture of Clitoriaternatea; 

evaluation of antimicrobial 

activity. Research in Biotechnology. 

3 (5), 26-38. 

Vijaykumar, P.P.N. Pammi, S.V.N. 

Kollu, P. Satyanarayana, K.V.V. 

and Shameem, U. (2014). Green 

Synthesis and Characterization of 

Silver Nanoparticles Using Boerhaavia 

diffusa Plant Extract and 

Their Antibacterial Activity. Industrial 

Crops and Products. 52, 562- 

566. 

Vinod, V.T.P. Saravanan, P. Sreedhar, 

B. Keerthi, D.D. and Sashidhar, 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 185



R.B. (2011). A facile synthesis and 

characterization of Ag, Au and Pt 

nanoparticles using a natural hydrocolloid 

gum kondagogu (Cochlospermum 

gossypium). Colloids and 

Surfaces B: Biointerfaces. 83(2), 

291-298. 

Wang, S. Lawson, R. Ray, P.C. and Yu, 

H. (2011). Toxic effects of gold nanoparticles 

on Salmonella typhimurium 

bacteria. Toxicology and 

industrial health 27(6), 547-554. 

Wang, Y. and Herron, N. (1991). Nanometer-sized 

semiconductor clusters: 

materials synthesis, quantum 

size effects, and photophysical 

properties. The Journal of Physical 

Chemistry. 95(2), 525–532. 

Wu, W. Huang, J. Wu, L. Sun, D. and 

Lin, L. (2013). Two-step size- and 

shape-separation of biosynthesized 

gold nanoparticles. Sep. Purif. 

Technol. 106, 117-122. 

Yang, N. WeiHong, L. and Hao, L. 

(2014). Biosynthesis of Au nanoparticles 

using agricultural waste mango 

peel extract and its in vitro cytotoxic 

effect on two normal cells. 

Mater. Lett.134, 67-70. 

Yang, X. Li, Q. Wang, H. Huang, J. 

Lin, L. Wang, W. Sun, D. Su, Y. 

Opiyo, J.B. Hong, L. Wang, Y. 


He, N. and Jia, L. (2010). Green 

synthesis of palladium nanoparticles 

using broth of Cinnamomum camphora 

leaf. J. Nanopart Res. doi: 

12:1589–1598. 

Yen, P.Y. Kamyar, S. Mikio, M. 

Noriyuki, K. Nurul, B. Ahmad, K. 

Shaza, E. Mohamad,B.and 

Kar,X.L (2016). Green Synthesis of 

Magnetite (Fe 3 O 4 ) Nanoparticles 

Using Seaweed (Kappaphycus 

alvarezii) Extract. Nanoscale 

Research Letters. 11, 276. 

Zahir, A.A. Chauhan, I.S. Bagavan, A. 

Kamaraj, C. Elango, G. Shankar, 

J. Arjaria, N. Roopan, S.M. Rahuman, 

A.A. and Singh, N. (2015). 

Green synthesis of silver and titanium 

dioxide nanoparticles using Euphorbia 

prostrate extract shows 

shift from apoptosis to G0/G1 arrest 

followed by necrotic cell death in 

Leishmania donovani. Antimicrob. 

Agents Chemother. 59, 4782–4799. 

Zheng, B. Kong, T. Jing, X. Wubah, 

T.O. Li, X. Sun, D. Lu, F. Zheng, 

Y. Huang, J. and Li, Q. (2013). 

Plant-mediated synthesis of platinum 

nanoparticles and its bioreductive 

mechanism Journal of Colloid 

and Interface Science. 396, 138- 

145. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 186



Production of Secondary Metabolites Using a 

Biotechnological Approach 

Produtur Chandramati Shankar 1, * and Senthilkumar Rajagopal 2 

1 Department of Biotechnology, Yogi Vemana University, Kadapa-516003, AP, India; 

2 Department of Biochemistry, Rayalaseema University, Kurnool-518002, AP, India; 

email: senthilanal@yahoo.com; *Correspondence: pchandra20@gmail.com; Tel: +91- 

8562-225426 

Abstract: Secondary metabolites produced by Medicinal plants are useful for the production 

of life saving drugs. The use of natural phytochemicals has its own advantage 

as it has no side effects. Because of sustainability, attention on in vitro plant materials 

as potential factories for secondary phytochemical products is increasing. More than 

80% of the world‟s population relies on traditional medicine as it has negligible side 

effects for their primary health care needs. Annually, about 95% of the medicinal 

plants‟ used as a raw material is growing at the rate of more than 40%. However, majority 

of medicinal plants are collected from their wild habitats, especially from forests. 

Repeated use of these medicinal plants has resulted in depletion and extinction of many 

important medicinal plant species from their natural habitats. The other most common 

reason is poor regeneration capacity of plant under natural habitats due to improper environmental 

factors like weather conditions, seasonal change, soil erosion etc. Due to 

these reasons, the need of hour is to protect and conserve valuable important medicinal 

plants otherwise many of these plants will be lost from natural vegetation forever. In 

this article, biotechnological approaches such as plant cell culture and hairy root culture 

is described as alternative methods for the production of secondary metabolites. The 

advantages of these methods and enhancements of secondary metabolites production by 

different methods are also discussed. 

Keywords: Biotransformation; cell suspension culture; hairy root culture; immobilization; 

medicinal plants 


Plants produce a wide variety of 

chemical molecules that play important 

roles in its development and its adaptation 

to the environment. These molecules based 

on their functions and role in plants development 

have been grouped into two types 

namely, primary metabolites such as carbohydrates, 

lipids and amino acids etc and 

secondary metabolites which are low molecular 

weight compounds which have no 

recognized role in the maintenance of fundamental 

life processes in the plants that 

synthesize them but are known to play a 

major role in the adaptation of plants to 

their environment. Many higher plants are 

major sources of natural products used as 

pharmaceuticals, agrochemicals, flavor 

and fragrance ingredients, food additives, 

and pesticides (Balandrin and 

Klocke, 1988). The search for new plantderived 

chemicals should thus be a priority 

in current and future efforts toward sustainable 

conservation and rational utilization 

of biodiversity (Phillipson, 1990). It 

is estimated that about 100,000 plant secondary 

metabolites or natural products 

have been identified. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 187


Production of Secondary Metabolites Using a Biotechnological Approach 

Shankar and Rajagopal 

Over one quarter of new drugs that 

have been approved in the last 30 years are 

based on a lead from a molecule from 

plant origin. Moreover, 9 of the top 20 

selling drugs are derived from knowledge 

of plant secondary metabolites (Harvey, 

2000; Tulp and Bohlin, 2002). Higher 

plants are rich source of bioactive constituents 

or phyto-pharmaceuticals used in 

pharmaceutical industry. Some of the plant 

derived natural products include drugs 

such as morphine, codeine, cocaine, quinine 

etc; anti-cancer Catharanthus alkaloids, 

belladonna alkaloids, colchicines, 

phytostigminine, pilocarpine, reserpine 

and steroids like diosgenin, digoxin and 

digitoxin. Many of these pharmaceuticals 

are still in use today and often no useful 

synthetic substitutes have been found that 

possess the same efficacy and pharmacological 

specificity. Currently one fourth of 

all prescribed pharmaceuticals in industrialized 

countries contain compounds that 

are directly or indirectly, via semisynthesis, 

derived from plants. Studies on 

plant secondary metabolites have been increasing 

over the last 50 years. 

There are three potential pathways 

for primary metabolism: the Embden 

Meyerhof-Parnas Pathway (EMP), the 

Entner-Dourdorof pathway, and the hexose 

monophosphate (HMP) pathway. Due to 

rapid deforestation and depletion of genetic 

stocks, various efforts were made to 

implement new methods for several plant 

species conservation and also yielding in 

high amounts of secondary metabolites, 

with photosynthetic efficiency, pest resistant 

and disease resistant plants. To 

overcome these problem new improved 

methods of plant tissue culture and transformation 

through molecular approaches 

presents an alternative option for multiplication, 

development and conservation of 

elite plant species. Biotechnological approaches 

such as plant tissue culture hold 

great promise for controlled production of 

useful secondary metabolites on demand. 

The current yield and productivity cannot 

fulfill the commercial goal of plant cellbased 

bioprocess for the production of 

most secondary metabolites. The recent 

advances, new directions, and opportunities 

in plant cell-based processes are being 

critically examined. Such as biotransformation 

using an exogenous supply of biosynthetic 

precursors, genetic manipulation, 

and metabolic engineering may improve 

the accumulation of compounds. Use of 

biotic and a biotic elicitors, can also be 

used for triggering the formation of secondary 

metabolites. The possible use of 

plant cell cultures for the specific biotransformation 

of natural compounds has been 

demonstrated (Cheetham, 1995; Scragg, 

1997; Krings and Berger, 1998; Ravishankar 

and Ramachandra Rao, 2000). In the 

search for alternatives for production of 

desirable medicinal compounds from 

plants, biotechnological approaches, specifically, 

plant tissue cultures, are found 

to have potential as a supplement to traditional 

agriculture and also in the industrial 

production of bioactive plant metabolites 

(Ramachandra Rao and Ravishankar, 

2000). Cell suspension culture systems 

could be used for large scale culturing of 

plant cells from which secondary metabolites 

could be extracted. Due to these 

advances, research in the area of tissue 

culture technology for production of plant 

chemicals has bloomed beyond expectations. 

The oncogenic strains of Agrobacterium 

rhizogenes is used to transform a 

range of plant species, which induces hairy 

roots. The hairy roots induced from Agrobacterium 

rhizogenes transformation has 

shown to have attractive properties for 

secondary metabolite production as compared 

to differential cell cultures (Kim et 

al., 2002). While research to date has succeeded 

in producing a wide range of valuable 

secondary photochemical in unorganized 

callus or suspension cultures, in 

other cases production requires more differentiated 

micro plant or organ cultures 

(Davioud et al., 1989). In this chapter we 

will be describing the popular types of biotechnological 

approaches for production of 

secondary metabolites. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 188




Figure 1: Schematic diagram of secondary metabolite production process. 

2. Biotechnological approaches for production 

of secondary metabolites 

The increased appeal of natural 

products for medicinal purposes coupled 

with the low product yields and supply 

concerns of plant harvesting has renewed 

interest in large-scale plant cell and hairy 

root culture technology. Secondary metabolites 

can be produced by using different 

biotechnological approaches (Table1). In 

this chapter, some techniques have been 

described. 

2.1. Plant cell culture 

Plant cell and tissue cultures can be 

established routinely under sterile conditions 

from explants, such as plant leaves, 

stems, roots, and meristems for multiplication 

and extraction of secondary metabolites. 

The explants selected from high 

yielding mother plants are usually selected 

as the starting material ((Figure 1)). Following 

the standard sterilization procedures 

the cultures are inoculated on a suitable 

plant tissue culture medium for induction 

of callus which is the base for all further 

production work (Figure 2). Callus 

cultures, can be divided into one of two 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 189




Table 1: Bioactive secondary metabolites produced using plant tissue cultures (Table courtesy: 

Vanisree and Tsay, 2004) 

Plant name Active ingredient Culture type Reference 

Agave amaniensis Saponins Callus Andrijany et al., 1999 

Ailanthus altissima Alkaloids Suspension Anderson et al., 1987 

Ailanthus altissima Canthinone alkaloids Suspension Anderson et al., 1986 

Allium sativum L. Alliin callus Malpathak and David, 

1986 

Aloe saponaria Tetrahydroanthracene glucosides suspension Yagi et al., 1983 

Ambrosia tenuifolia Altamisine Callus Goleniowski and Trippi, 

1999 

Anchusa officinalis Rosmarinic acid Suspension De-Eknamkul and 

Ellis, 1985 

Brucea javanica (L.) Merr. Canthinone alkaloids Suspension Liu et al., 1990 

Bupleurum falcatum Saikosaponins Callus Wang and Huang, 

1982 

Bupleurum falcatum L. Saikosaponins Root Kusakari et al., 2000 

Camellia sinensis Theamine, γ-glutamyl derivatives suspension Orihara and Furuya, 

1990 

Canavalia ensiformis L-Canavanine Callus Ramirez et al., 1992 

Capsicum annuum L. Capsaicin Suspension Johnson et al., 1990 

Cassia acutifolia Anthraquinones Suspension Nazif et al., 2000 

Catharanthus roseus Indole alkaloids Suspension Moreno et al., 1993 

Catharanthus roseus Catharanthine Suspension Zhao et al., 2001b 

Cephaelis ipecacuanha A. Emetic alkaloids Root Teshima et al., 1988 

Richard 

Chrysanthemum cinerariaefolium 

Pyrethrins Callus Rajasekaran et al., 

1991 

Chrysanthemum cinerariaefolium 

Chrysanthemic acid and pyethrins Suspension Kueh et al., 1985 

Cinchona L. Alkaloids Suspension Koblitz et al., 1983 

Cinchona robusta Robustaquinones Suspension Schripsema et al., 

1999 

Cinchona spec. Anthraquinones Suspension Wijnsma et al., 1985 

Cinchona succirubra Anthraquinones Suspension Khouri et al., 1986 

Citrus sp. Naringin, Limonin Callus Barthe et al., 1987 

Coffea arabica L. Caffeine Callus Waller et al., 1983 

Cruciata glabra Anthraquinones Suspension Dornenburg and 

Knorr, 1996 

Cryptolepis buchanani 

Roem. & shult 

Cryptosin Callus Venkateswara et al., 

1987 

Digitalis purpurea L. Cardenolides Suspension Hagimori et al., 1982 

Dioscorea deltoidea Diosgenin Suspension Heble and Staba, 1980 

Dioscorea doryophora Diosgenin Suspension Huang et al., 1993 

Hance 

Duboisia leichhardtii Tropane alkaloids Callus Yamada and Endo, 

1984 

Ephedra spp. L- Ephedrine, D Pseudoephidrin Suspension O‟Dowd et al., 1993 

Eriobotrya japonica Triterpenes Callus Taniguchi et al., 2002 

Eucalyptus tereticornis SM. Sterols and Phenolic compounds callus Venkateswara et al., 

1986 

Fumaria capreolata Isoquinoline alkaloids Suspension Tanahashi and Zenk, 

1985 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 190






Gentiana sp. Secoiridoid glucosides Callus Skrzypczak et al., 

1993 

Ginkgo biloba Ginkgolide A Suspension Carrier et al., 1991 

Glehnia littoralis Furanocoumarin Suspension Kitamura et al., 1998 

Glycyrrhiza echinata Flavanoids Callus Ayabe et al., 1986 

Glycyrrhiza glabra var. Triterpenes callus Ayabe et al., 1990 

glandulifera 

Hyoscyamus niger Tropane alkaloids Callus Yamada and Hashimoto, 

1982 

Isoplexis isabellina Anthraquinones Suspension Arrebola et al., 1999 

Linum flavum L. 5-Methoxypodophyllotoxin Suspension Uden et., al 1990 

Lithospermum erythrorhizon Shikonin derivatives Suspension Fujita et al., 1981 

Lithospermum erythrorhizon Shikonin derivatives Suspension Fukui et al., 1990 

Lycium chinense Cerebroside Suspension Jang et al., 1998 

Mentha arvensis Terpenoid Shoot Phatak and Heble, 

2002 

Morinda citrifolia Anthraquinones Suspension Zenk et al., 1975 

Morinda citrifolia Anthraquinones Suspension Bassetti et al., 1995 

Mucuna pruriens L-DOPA Suspension Wichers et al., 1993 

Mucuna pruriens L-DOPA Callus Brain, 1976 

Nandina domestica Alkaloids Callus Ikuta and Itokawa, 

1988 

Nicotiana rustica Alkaloids Callus Tabata and Hiraoka, 

1976 

Nicotiana tabacum L. Nicotine Suspension Mantell et al., 1983 

Ophiorrhiza pumila Camptothecin related alkaloids Callus Kitajima et al., 1998 

Panax ginseng Saponins and Sapogenins Callus Furuya et al., 1973 

Panax notoginseng Ginsenosides Suspension Zhong and Zhu, 1995 

Papaver bracteatum Thebaine Callus Day et al., 1986 

Papaver somniferum L. Alkaloids Callus Furuya et al., 1972 

Papaver somniferum Morphine, Codeine Suspension Siah and Doran, 1991 

Peganum harmala L. β-Carboline alkaloids Suspension Sasse et al., 1982 

Phytolacca americana Betacyanin Suspension Sakuta et al., 1987 

Picrasma quassioides Bennett 

Quassin Suspension Scragg and Allan, 

1986 

Podophyllum hexandrum Podophyllotoxin Suspension Uden et al., 1989 

royle 

Polygala amarella Saponins Callus Desbene et al., 1999 

Polygonum hydropiper Flavanoids Suspension Nakao et al., 1999 

Portulaca grandiflora Betacyanin Callus Schroder and Bohm, 

1984 

Ptelea trifoliata L. Dihydrofuro [2,3-b] quinolinium Callus Petit-Paly et al., 1987 

Rauwolfia sellowii Alkaloids Suspension Rech et al., 1998 

Rauwolfia serpentina Benth. Reserpine Suspension Yamamoto and Yamada, 

1986 

Rauvolfia serpentina x Rhazya 

stricta 

3-Oxo-rhazinilam Callus Gerasimenko et al., 

2001 

Rhus javanica Gallotannins Root Taniguchi et al., 2000 

Ruta sp. 

Acridone and Furoquinoline alkaloids 

and cumarins 

Callus Baumert et al., 1992 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 191






Salvia miltiorrhiza 

Lithospermic acid B and rosmarinic 

Callus Morimoto et al., 1994 

acid 

Salvia miltiorrhiza Cryptotanshinone Suspension Miyasaka et al., 1989 

Scopolia parviflora Alkaloids Callus Tabata et al., 1972 

Scutellaria columnae Phenolics Callus Stojakowska and 

Kisiel, 1999 

Solanum chrysotrichum Spirostanol saponin Suspension Villarreal et al., 1997 

(Schldl.) 

Solanum laciniatum Ait Solasodine Suspension Chandler and Dodds, 

1983a 

Silybum marianum Flavonolignan Root Alikaridis et al., 2000 

Solanum paludosum Solamargine Suspension Badaoui et al., 1996 

Tabernaemontana divaricata 

Alkaloids Suspension Sierra et al., 1992 

Taxus spp. Taxol Suspension Wu et al., 2001 

Taxus baccata Taxol baccatin III Suspension Cusido et al., 1999 

Thalictrum minus Berberin Suspension Kobayashi et al., 1987 

Thalictrum minus Berberin Suspension Nakagawa et al., 1986 

Torreya nucifera var. radicans 

Diterpenoids Suspension Orihara et al., 2002 

Trigonella foenumgraecum Saponins Suspension Brain and Williams, 

1983 

Withaina somnifera Withaferin A Shoot Ray and Jha, 2001 

. 

Figure 2: Schematic diagram showing 

the process of callus induction and cell 

suspension culture establishment. 

types depending on the nature of callus 

i.e., compact or friable. In compact callus 

the cells are densely aggregated, whereas 

in friable callus the cells are only loosely 

associated with each other and the callus 

become soft and breaks apart easily. Friable 

callus are a good source to establish a 

cell-suspension cultures. The friability of 

callus is improved by manipulating the 

medium components or by frequent subculturing 

or use of semi solid medium. 

When friable callus is placed into a suitable 

plant tissue culture liquid medium 

(usually the same composition as the solid 

medium used for the callus culture) and 

then agitated, single cells and/or small 

clumps of cells are released into the medium. 

Under the correct conditions, these 

released cells continue to grow and divide, 

eventually producing a cellsuspension 

culture. Use of large inoculum 

for initiation of cell suspensions cultures 

release more cell numbers into the medium 

quickly. Cell suspensions can be maintained 

just simply as batch cultures in conical 

flasks. They are continually cultured 

by repeated sub culturing into fresh medi- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 192




um. This results in dilution of the suspension 

and the initiation of another batch 

growth cycle. The degree of dilution during 

subculture should be determined empirically 

for each culture. Too great a degree 

of dilution will result in a greatly extended 

lag period or, in extreme cases, 

death of the transferred cells. After subculture, 

the cells divide and the biomass of 

the culture increases in a characteristic 

fashion, until nutrients in the medium are 

exhausted and/or toxic byproducts build up 

to inhibitory levels this is called the „stationary 

phase‟. If cells are left in the stationary 

phase for too long, they will die 

and the culture will be lost. Therefore, 

cells should be transferred as they enter the 

stationary phase. It is therefore important 

that the batch growth-cycle parameters are 

determined for each cell-suspension culture. 

Strain improvement, methods for the 

selection of high-producing cell lines, and 

medium optimizations can lead to an enhancement 

in secondary metabolite production. 

Compared to whole plant cultivation 

system cell culture system has the advantages 

as follows: 

i. Useful compounds can be produced 

under controlled conditions 

independent of climatic change or 

soil conditions; 

ii. Cultured cells will be free of pathogens 

and pests; 

iii. Less space is required and production 

is uniform. 

2.2. Hairy root culture 

Agro bacterium rhizogenes a gram 

negative soil borne bacteria belonging to 

the family Rhizobiaceaae, induces hairy 

root formation at the site of infection 

(Mugnier, 1988). Hairy roots are adventitious 

roots with lateral branching, growing 

rapidly and showing plagiotrophic growth 

with high branching and independent of 

plant hormones in the medium, these roots 

often posses the capacity to grow even 

when removed from the mother plant. The 

transformed roots generated show high 

differentiation and can cause stable and 

extensive production of secondary metabolites. 

The secondary metabolites produced 

by hairy roots arising from the infection of 

plant material by A. rhizogenes are the 

same as those usually synthesized in intact 

parent roots, with similar or higher yields. 

This feature, together with genetic stability 

and generally rapid growth in simple media 

lacking phytohormones, makes them 

especially suitable for biochemical studies 

not easily undertaken with root cultures of 

an intact plant. The hairy roots are normally 

induced on aseptic, wounded parts of 

plants by inoculating them with A. rhizogenes. 

2.3. Biotransformation using precursors 

Commercial production of secondary 

metabolites requires a reproducible and 

standardized protocol for cultivation of 

plant cells/organs on a large scale. Employing 

precursor feeding, transformation 

methods, and immobilization techniques. 

The treatment of plant cells with biotic 

and/or abiotic elicitors has been a useful 

strategy to enhance secondary metabolite 

production in cell cultures (Karuppusamy, 

2009). The most frequently used elicitors 

are fungal carbohydrates, yeast extract, 

Methyl Jasmonate (MJ) and chitosan. MJ, 

a proven signal compound, is the most effective 

elicitor of taxol production in Taxus 

chinensis Roxb (Wink et al., 2008) and 

gonsenoside production in P. ginseng C.A. 

Meyer cell/organ culture (Xu et al., 2008; 

Yamanaka et al., 1996). 

2.4. Immobilization of cells for secondary 

metabolite production 

One of the major limiting factors in 

the development of a commercial production 

system using plant cell culture has 

been the production cost of phytopharmaceuticals. 

The use of high biomass levels 

for extended periods would be one method 

of increasing productivity and hence reducing 

the costs. This can be achieved by 

the immobilization of plant cells. Immobilization 

is the newest culture technology 

of plant cell, and considered as to be the 

most “natural”. It has been defined as a 

technique, which confines to a catalytical- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 193




ly active enzyme or to a cell within a reactor 

system and prevents its entry into the 

mobile phase, which carries the substrate 

and product. The first successful immobilization 

of plant cells was reported by 

Brodelius et al., (1979) and they entrapped 

Catharathus roseus and Daucus carota 

cells in alginate beds. Following success 

with enzymatic and microbial process, 

immobilization has been suggested as a 

strategy to enhance the overall productivity 

of secondary metabolite in plant cell 

culture. The ability to immobilize plant 

cells has been reported for a large number 

of plant cells and protoplasts by using a 

variety of polymers. Immobilization of 

plant cells has been used for a wide range 

of reactions, which can be divided into 

three groups. (1) Biotransformation or bioconversion, 

(2) synthesis from precursors 

and (3) the De Novo synthesis of compounds. 

Some of the advantages of immobilization 

are, retention of biomass enables 

its continuous reutilization as a production 

system. The immobilization of cells allows 

the use of a higher biomass level compared 

to cell suspension culture, Separation of 

cells from medium and the product is extra 

cellular, which will simplify downstream 

processing compared to extract from tissue. 

Immobilization allows a continuous 

process, which increase volumetric 

productivity and allows the removal of 

metabolic inhibitors. Reduces problems 

such as aggregate, growth and foaming 

.Some of the disadvantage is the microenvironment 

favoring optimal production can 

be unfavorable for released secondary metabolites 

and cause their degradation or 

metabolization. 

2.4.1. Different types of immobilization 

i. Direct intracellular binding due to 

natural affinity (adsorption, adhesion 

and agglutination). 

ii. Covalent coupling on otherwise inert 

matrices. 

iii. Intracellular connection via bi or 

poly functional reagent (crosslinking). 

iv. Mixing with suitable materials, 

changing their consistency with 

temperature (embedding). 

v. Physical retention within the 

framework of diverse pore size and 

permeability (entrapment, micro 

encapsulation). 

2.4.1.1. Selection of immobilization system 

The choice of a suitable immobilization 

system is determined by the following 

requirements. 

i. The polymer material used for immobilization 

must be available in 

large quantities; it must be inert, 

non-toxic and cheap. 

ii. It must be able to carry large quantities 

of biomass and its fixing potential 

must be high. 

iii. The immobilization process must 

not diminish enzymatic activity of 

biological catalyst. 

iv. Manipulation of the biological catalyst 

must be as simple as possible. 

2.4.2. Methods for immobilization 

2.4.2.1. Gel entrapment by polymerization 

A monomer or a mixture of monomers 

is polymerized in the presence of a 

cell suspension, which is entrapped inside 

the lattice of the polymer. 

2.4.2.2. Gel entrapment by ionic net work 

formation 

In this method, polymerization of 

polyelectrolyte is achieved by addition of 

multivalent ions. The most common method 

is the entrapment in calcium alginate. 

This is a non-toxic process in which sodium 

alginate solution containing the cell 

suspension is dropped into a mixture of 

counter ion solution such as calcium chloride. 

A uniform, spherical and highly microspores 

structure results, which retains 

the 

cell. 

2.4.2.3. Gel entrapment formation by precipitation 

Gels may be formed by precipitation 

of some natural and synthetic poly- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 194




mers by changing one or more parameters 

in the solution, such as temperature, salinity 

or PH of solvent. Several materials can 

be used for entrapment. The examples include 

methods involving thermal treatment. 

Some disruption of viability can occur 

naturally. 

2.4.2.4. Entrapment in preformed structures 

Hollow fiber reactors can be used 

to immobilize plant cells by entrapment. 

The cells are placed on the shell side of the 

hollow fibre cartridge and nutrient medium 

is rapidly re-circulated through the fibers. 

This may have important applications in 

large-scale. 

2.4.2.5. Surface immobilization 

Surface immobilization may occur on both 

natural and other matrices. Examples of 

natural matrices are deeper callus layers 

and cellulose, while synthetic one includes 

nets of steel and nylon. 

2.4.2.6. Immobilization by embedding 

The temperature dependent solubility of 

macromolecules like agarose, agar and 

carrageenan or the differing solubility of 

the sodium and calcium salts in the case of 

alginate are utilized to form polymeric gels 

or gel combination. Insoluble are formed 

under cold conditions (Agar) or in aqueous 

CaCl 2 solutions (Alginate). Their structure 

is non-uniform, with differing pore diameters 

at the surface and in deeper layers. 

The size and form of the beds can be determined 

in part by stirring speed and using 

alginate, by the viscosity of the solution 

and dropping aperture. 

2.4.2.7. Types of bioreactors used for immobilization 

of plant cells 

The following types of reactors are 

generally used for immobilized plant cell: 

(1) Packed bed reactors (2) Well mixed 

reactor (3) Fluidized bed reactors (4) 

Membrane reactors 

2.5. Metabolic engineering and production 

of secondary metabolites 

Metabolic engineering involves the 

targeted and purposeful alteration of metabolic 

pathways found in an organism to 

achieve better understanding and use of 

cellular pathways for chemical transformation, 

energy transduction, and supramolecular 

assembly (Lessard, 1996). This 

technique applied to plants will permit endogenous 

biochemical pathways to be manipulated 

and results in the generation of 

transgenic crops in which the range, scope, 

or nature of a plant's existing natural products 

are modified to provide beneficial 

commercial, agronomic, and/or postharvest 

processing characteristics (Kinney, 

1998). 

Several genes in the biosynthetic 

pathways for scopolamine, nicotine, and 

berberine have been cloned, making the 

metabolic engineering of these alkaloids 

possible. Expression of two branchingpoint 

enzymes was engineered: putrescine 

N-methyltransferase (PMT) in transgenic 

plants of Atropa belladonna and Nicotiana 

sylvestris and (S)-scoulerine 9-Omethyltransferase 

(SMT) in cultured cells 

of C. japonica and Eschscholzia californica. 

Over expression of PMT increased the 

nicotine content in N. sylvestris, whereas 

suppression of endogenous PMT activity 

severely decreased the nicotine content 

and induced abnormal morphologies. Ectopic 

expression of SMT caused the accumulation 

of benzylisoquinoline alkaloids 

in E. californica (Sato et al., 2001). 

3. Few commercial products obtained 

by tissue cultures 

Some of the commercial products 

obtained by plant tissue culture are listed 

below: 

3.1. Taxol 

Taxol (paclitaxel), a complex 

diterpene alkaloid found in the bark of the 

Taxus tree, is an anticancer agent. At present; 

production of taxol by various Taxus 

species cells in cultures has been one of 

the most extensively explored areas of 

plant cell cultures. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 195




3.2. Morphine and codeine 

Latex from the opium poppy, Papaver 

somniferum, is a commercial source 

of the analgesics, morphine, and codeine. 

Callus and suspension cultures of P. somniferum 

are being investigated as an alternative 

means for the production of these 

compounds Biotransformation of codeinone 

to codeine with immobilized cells 

of P. somniferum has been reported by Furuya 

et al., (1972). The conversion yield 

was 70.4%, and about 88% of the codeine 

converted was excreted into the medium. 

3.3. l-Dopa 

L-3, 4-dihydroxyphenylalanine, is 

an important intermediate of secondary 

metabolism in higher plants and is known 

as a precursor of alkaloids, betalain, and 

melanine, isolated from Vinca faba, 

(Daxenbichler et al., 1971) Mucuna, Baptisia, 

and Lupinus (Brain and Lockwood, 

1976). 

3.4. Diosgenin 

Tal et al. (1983) reported on the 

use of cell cultures of Dioscorea deltoidea 

for the production of diosgenin. They 

found that carbon and nitrogen levels 

greatly influenced diosgenin accumulation 

in one cell line. Ishida (1988) established 

Dioscorea immobilized cell cultures, in 

which reticulated polyurethane foam was 

shown to stimulate diosgenin production, 

increasing the cellular concentration by 

40% and total yield by 25%. 

3.5. Capsaicin 

Capsaicin, an alkaloid, is used 

mainly as a pungent food additive in formulated 

foods (Ravishankar et al., 2003). 

It is obtained from fruits of green pepper 

(Capsicum spp.). Capsaicin is also used in 

pharmaceutical preparations as a digestive 

stimulant and for rheumatic disorders 

(Sharma et al., 2008). Suspension cultures 

of Capsicum frutescens produce low levels 

of capsaicin, but immobilizing the cells in 

reticulated polyurethane foam can increase 

production approximately 100-fold. 

3.6. Camptothecin 

Campothecin, a potent antitumor 

alkaloid, was isolated from Camptotheca 

acuminate (Padmanabha et al., 2006; Sakato 

and Misawa, 1974) induced C. acuminata 

callus on MS medium containing 

0.2 mg/l 2, 4-D and l mg/l kinetin and developed 

liquid cultures in the presence of 

gibberellin, l-tryptophan, and conditioned 

medium, which yielded camptothecin at 

about 0.0025% on a dry weight basis. 

When the cultures were grown on MS medium 

containing 4 mg/l NAA, accumulation 

of camptothecin reached 0.998 mg/l 

(Thengane et al., 2003). 

3.7. Berberine 

Berberine is an isoquinoline alkaloid 

found in the roots of Coptis japonica 

and cortex of Phellondendron amurense. 

This antibacterial alkaloid has been identified 

from a number of cell cultures, notably 

those of C. japonica, (Vanisree and 

Tsay, 2004; Breuling et al., 1985; Sato et 

al., 2001) Thalictrum spp., (Nakagawa et 

al., 1984; Suzuki et al., 1988) and Berberis 

spp. (Breuling et al., 1985). The productivity 

of berberine was increased in cell cultures 

by optimizing the nutrients in the 

growth medium and the levels of phytohormones 

(Sato and Yamada 1984; Nakagawa 

et al., 1986; Morimoto et al., 1988). 

By selecting high-yielding cell lines, Mitsui 

group produced berberine on a large 

scale with a productivity of 1.4 g/l over 2 

weeks. Other methods for increasing 

yields include elicitation of cultures with a 

yeast polysaccharide elicitor, which has 

been successful with a relatively lowproducing 

Thalictrum rugosum culture 

(Funk et al., 1987). 


Industrial compounds obtained and 

prepared from plant sources are in high 

demand. This is because it is safer with no 

side effects and cheaper compared to syn- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 196




thetic products. Many lead molecules in 

drugs are from plant origin; hence, large 

amount of natural resources are destroyed 

for the production of these compounds. 

The alternate means of production of industrially 

important compounds is needed 

for the sustainability of natural resources 

and preserve the diversity in the ecosystem. 

There are many advanced approaches 

which can be used for the production of 

secondary metabolites; but, only limited 

methods have been highlighted in this article. 

Biotechnology can play an important 

role in production of useful secondary metabolites 

and will help in sustained development 

of plant diversity. 

References 

Balandrin, M., and Klocke, J. (1988). 

Medicinal, aromatic and industrial 

materials from plants, Vol. 4, 

Springer-Verlag, Berlin, Heidelberg. 

Brain, K., and Lockwood, G. (1976). 

Hormonal control of steroid levels in 

tissue cultures from Trigonella 

foenumgraecum, Phytochemistry 15, 

1651-1654. 

Breuling, M., Alfermann, A., and 

Reinhard, E. (1985). Cultivation of 

cell cultures of Berberis wilsonae in 

20l airlift bioreactors, Plant Cell 

Reproduction 4, 220-223. 

Brodelius, P., Deus, B., Mosbach, K., & 

Zenk, M. H. (1979). Immobilized 

plant cells for the production and 

transportation of natural products. 

Febs Letters 103(1), 93-97. 

Cheetham, P. (1995). Biotransformations: 

new routes to food ingredients, 

Chemistry and Industry 4, 265-268. 

Davioud, E., Kan, C., Hamon, J., 

Tempe, J., and Husson, H. (1989). 

Production of indole alkaloids by in 

vitro root cultures from 

Catharanthus trichophyllus, 

Phytochemistry 28, 2675–2680. 

Daxenbichler, M., VanEtten, C., 

Hallinan, E., Earle, F., and 

Barclay, A. (1971). Seeds as sources 

of L-DOPA, Journal of MEdicinal 

Chemistry 14, 463-465. 

Funk, C., Gugler, K., and Brodelius, P. 

(1987). Increased secondary product 

formation in plant cell suspension 

cultures after treatment with a yeast 

carbohydrate preparation (elicitor) 

Phytochemistry 26, 401-405. 

Harvey, A. (2000). Strategies for 

discovering drugs from previously 

unexplored natural products, Drug 

Discovery Today 5, 294-300. 

Karuppusamy, S. (2009). A review on 

trends in production of secondary 

metabolites from higher plants by in 

vitro tissue, organ and cell cultures, 

Journal of Medicinal Plant Research 

3, 1222-1239. 

Kim, Y., Wyslouzil, E., and Pamela, J. 

(2002). Secondary metabolism of 

hairy root cultures in bioreactors, In 

vitro Cell Developmental Biology 

and Plant 38, 1-10. 

Kinney, A. (1998). Manipulating flux 

through plant metabolic pathways, 

Current Opinion in Plant Biology 1, 

173-178. 

Krings, U., and Berger, R. (1998). 

Biotechnological production of 

flavours and fragrances, Applied 

Microbiology and Biotechnology 49, 

1-8. 

Lessard, P. (1996). Metabolic 

engineering: the concept coalesces, 

Nature Biotechnology 14, 1654- 

1655. 

Morimoto, T., Hara, Y., Kato, Y., 

Hiratsuka, J., Yoshioka, T., and 

Fujita, Y. (1988). Berberine 

production by cultured Coptis 

japonica cells in one-stage culture 

using medium with a high copper 

concentration, Agricultural 

Biological chemistry 52, 1835-1836. 

Mugnier, J. (1988). Establishment of new 

axenic hairy root lines by inoculation 

with Agrobacterium rhizogenes, 

Plant Cell Reproduction 7, 9-12. 

Nakagawa, K., Konagai, A., Fukui, H., 

and Tabata, M. (1984). Release and 

crystalization of berberine in the 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 197




liquid medium of Thalictrum minus 

cell suspension cultures, Plant Cell 


Nakagawa, K., Fukui, H., and Tabata, 

M. (1986). Hormonal regulation of 

berberine production in cell 

suspension cultures of Thalictrum 

minus, Plant Cell Reproduction 5, 

69-71. 

Padmanabha, B., Chandrashekar, M., 

Ramesha, B., Hombe Gowda, H., 

Rajesh, P., and Suhas, S. (2006). 

Patterns of accumulation of 

camptothecin, an anti-cancer 

alkaloids in Nothapodytes 

nimmoniana Graham, in the Western 

Ghats, India: Implications for 

identifying high-yielding sources of 

the alkaloid, Current Science 90, 95- 

100. 

Phillipson, J. (1990). Plants as source of 

valuable products, Clarendon Press, 

Oxford.Ramachandra Rao, S., and 

Ravishankar, G. (2000). 

Biotransformation of protocatechuic 

aldehyde and caffeic acid to van illin 

and capsaicin in freely suspended 

and immobilized cell cultures of 

Capsicum frutescens, Journal of 


Ravishankar, G., and Ramachandra 

Rao, S. (2000). Biotechnological 

production of phyto-pharmaceuticals, 

Journal of Biochemistry and 

Molecular Biology and Biophysics 4, 

73-102. 

Ravishankar, G., Suresh, B., Giridhar, 

P., Rao, S., and Johnson, T. (2003). 

Biotechnological studies on 

capsicum for metabolite production 

and plant improvement, Harwood 

Academic Publishers, United 

kingdom. 

Sakato, K., and Misawa, M. (1974). 

Effects of chemical and physical 

conditions on growth of 

Camptotheca acuminata cell 

cultures, Agricultural Biological 

chemistry 38, 491-497. 

Sato, F., and Yamada, Y. (1984). High 

berberine producing cultures of 

Coptis japonica cells, 

Phytochemistry 23, 281-285. 

Sato, F., Hashimoto, T., Hachiya, A., 

Tamura, K., Choi, K., and 

Morishige, T. (2001). Metabolic 

engineering of plant alkaloid 

biosynthesis, Proceedings of Natural 


372. 

Scragg, A. (1997). The production of 

aromas by plant cell cultures, Vol. 

55, Springer-Verlag, Berlin. 

Sharma, A., Kumar, V., Giridhar, P., 

and Ravishankar, G. (2008). 

Induction of in vitro flowering in 

Capsicum frutescens under the 

influence of silver nitrate and cobalt 

chloride and pollen transformation, 

Plant Biotechnology Journal 11, 1-8. 

Suzuki, M., Nakagawa, K., Fukui, H., 

and Tabata, M. (1988). Alkaloid 

production in cell suspension 

cultures of Thalictrum flavum and T. 

dipterocarpum, Plant Cell 


Thengane, S., Kulkarni, D., Shrikhande, 

V., Joshi, S., Sonawane, K., and 

Krishnamurthy, K. (2003). 

Influence of medium composition on 

callus induction and camptothecin(s) 

accumulation in Nothapodytes 

foetida, Plant Cell Tissue and Organ 

Culture 72, 247-251. 

Tal, B., Rokem, J.S., Gressel, J., 

Goldberg, I. (1983). Diosgenin 

production by Dioscorea deltoidea 

cell suspension culture. IAPTC 

Newslett 40, 15. 

Tulp, M., and Bohlin, L. (2002). 

Unconventional natural sources for 

future drug discovery, Drug 

Discovery Today 9, 450-458. 

Vanisree, M., and Tsay, H. (2004). Plant 

Cell Cultures - An Alternative and 

Efficient Source for the Production 

of Biologically Important Secondary 

Metabolites, International Journal of 

Applied Science and Engineering 2, 

29-48. 

Wink, M., Alfermann, A., Franke, R., 

Wetterauer, B., Distl, M., and 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 198




Windhovel, J. (2008). Sustainable 

bioproduction of phytochemicals by 

plant in vitro cultures: anticancer 

agents, Plant Genetic Resources 12, 

113-123. 

Xu, H., Kim, Y., Suh, S., Udin, M., Lee, 

S., and Park, S. (2008). Deoursin 

production from hairy root culture of 

Angelica gigas, Journal of Korean 

Society and Applied Biological 

Chemistry 51, 349-351. 

Yamanaka, M., Ishibhasi, K., 

Shimomura, K., and Ishimaru, K. 

(1996). Polyacetylene glucosides in 

hairy root cultures of Lobelia 

cardinalis, Phytochemistry 41, 183- 

185. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 199



Potential of Marine Algae Derived Extracts as a Natural 

Biostimulant to Enhance Plant Growth and Crop 

Productivity 

Lakkakula Satish and Manikandan Ramesh* 

Department of Biotechnology, Science Campus, Alagappa University, Karaikudi, Tamil 

Nadu, India; *Correspondence: pandu.pine@gmail.com / mrbiotech.alu@gmail.com; Tel: 

+91 4565 225215 

Abstract: Photobioreactor (PBR) is a reactor which exploits a light supply to farm phototrophic 

microorganisms which can generate biomass using light, water and carbon dioxide. 

Macro- and microalgae are developed naturally in fresh water as well as marine water 

(highly economical) or brackish water. Universal production of marine algal monocultures 

is basically limited to a selective species such as Scenedesmus spp., Nannochloropsis spp., 

and Chlorella spp., and some extremophiles like Arthrospira spp., and Dunaliella spp.,. 

Owing to their inborn resistance to predators and competitors, algal species can be cultivated 

naturally in open systems akin to ponds, stirred tanks and bubble columns. Nowadays, 

numerous research groups are focusing on PBRs (viz., selective closed type or open systems) 

to produce marine algae (cyanobacteria or seaweeds) through utilizing a light source 

with lavish biomass accumulation. The cultivation of marine algae has distinguished a new 

extension in numerous fields crucial for humanity, especially energy, food, health and environment. 

Currently, marine algae and algae-based extracts remain predominantly unexploited 

despite their huge potential applications. This chapter highlights the present and future 

prospects of marine plants and their derived aqueous extracts for crop improvement 

and enhancement of plant biomass production. 

Keywords: Crop protection; cyanobacteria; marine seaweed; photobioreactor; plant biostimulants; 

plant growth hormones 


As per previously published data 

about micro algae biotechnology, the 

foremost concern of external mass farming 

has been planned at obtaining successful 

consumption of prominent light 

potency (Masojidek et al., 2003). The 

word algae cover a wide choice of diverse 

organisms which can be normally illustrated 

as eukaryotic protests (a complicated 

group to describe), that are diverse 

from plants but are naturally aquatic and 

photosynthetic. They can also be microscopic 

single celled micro algae or larger, 

more complex multi cellular seaweeds. 

They can be found all-inclusive in both 

fresh water and marine environments 

across a wide range of habitats. Micro 

algae, comprise of cyanobacteria and 

seaweeds (macroalgae) are a resource of 

valuable bioactive compounds including 

vitamins, pigments, secondary metabolites, 

plant growth hormones and other 

food appurtenances representing extremely 

discriminatory pharmacological activities 

(Masojidek et al., 2009; Satish et al., 

2015, 2016, Rency et al., 2017). Marine 

algae (macro and micro) is the vital resource 

for bio-fuel production, since they 

can collect a large quantity of lipid content 

within their cells and contain very 

huge biomass productive capacity (Wu 

and Merchuk, 2004; Yang et al., 2014). In 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 200


Potential of Marine Algae Derived Extracts 

case of filamentous micro algae, a variety 

of strains are recognized to make intra- or 

extracellular metabolites by dissimilar 

biological activities (Glombitza and 

Koch, 1989). However, numerous research 

groups focusing on photobioreactors 

(PBRs) for making of prominent algal 

biomass production, is not nearly as 

superior as requisite by the worlds desires, 

particularly in the pharmaceutical, 

for human utilization as well as fish and 

animal feed, and for field level agricultural 

applications. Still, there is inert question 

as to whether algal based cultures are 

able to make plant growth stimulants and 

concerning their mechanisms of action 

towards elevated yielding, biotic and abiotic 

stress resistance and high biomass 

production of crops in agriculture through 

regulation of growth succession such as 

cell division and in the sensitivity of environmental 

changes. 

2. Recent developments for cultivation 

of marine algae using photobioreactors 

The majority of marine algae use 

photosynthesis to confine light energy to 

change inorganic substances into useful 

sugars and then other molecules which is 

similar to plants. The limitations of development 

forced by the constraint and 

saturation of light and results have impelled 

algal biotechnologists to come upon 

for resolution to convince this outcome 

in outdoor cultures (Masojidek et al., 

2003). Due to the significance of light 

saturation, information on the behavior of 

cyanobacteria cultures showed to very 

high illumination is comparatively sparse 

and most of it has been acquired in laboratory 

experiments. The possible development 

of algal biotechnology is moving 

towards the field of high charge commodities 

grown in well controlled cultivation 

conditions, such as temperature, nutrient 

supply, pH, light, and CO 2 (Masojidek et 

al., 2009). Aquaculture has turned into 

extremely important in the salvation of 

food and nutrition for the growing world 

Lakkakula and Manikandan 

population over the foregoing few decades. 

In the year 2012, an incredible 90.4 

million tonnes of marine based food and 

beverages were farmed and this amount is 

expected to increase until about 2030, at 

which moment it is indefinite that capture 

fisheries and aquaculture will distribute 

equal quantities (FAO, 2013; Taelman et 

al., 2015). Nevertheless, an entire control 

on culture conditions is sufficient only 

through closed systems, only PBR. 

Commonly, PBR is a bioreactor 

which uses a light source to grow phototrophic 

microorganisms which can produce 

biomass through light and CO 2 and 

include plants, macro and micro algae, 

cyanobacteria, purple bacteria and mosses. 

The first step was taken by Richmond 

and group in 1978 as the introduction of 

external algal biotechnology in Israel 

(Richmond and Vonshak, 1978; 

Masojidek et al., 2003, 2009). Compared 

to open culture systems like ponds and 

raceways, PBR systems have several advantages 

viz., reproducible farming with 

regard to environmental changes, maintenance 

of dissolved oxygen concentration, 

sufficient mixing of the culture with the 

appropriate flow rate, the opportunity of 

temperature regulation with low CO 2 

losses and making it feasible to focus and 

regulate the irradiance for augmentation 

of algal cultures with reduced risk of culture 

contamination (Masojidek et al., 

2009). Rorrer et al. (2004) showed the 

bioprocess engineering method, especially 

for the marine algae process through 

the development of cell and tissue culture 

systems and his group described a diversity 

of techniques to grow phototropic suspension 

cultures appropriate for the cultivation 

in PBR systems. Also, the same 

group compared three foremost PBR configurations 

for macroalgal suspension culture 

systems viz., bubble-column or airlift, 

tubular recycle and stirred tank, and 

considered the major factors that limit 

their development and cultivation performance 

(Rorrer et al., 2004). Later on, a 

novel tubular PBR with linear Fresnel 

lenses depends on solar concentrators dis- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 201



covered and studied the correlation between 

changes in physiological and photochemical 

characters in central Europe 

(Masojidek et al., 2009). Nevertheless, a 

number of guideline ideologies about the 

best possible design of PBRs have been 

established (refer for recent reviews, Pulz, 

2001; Carvalho et al., 2006). But, still the 

worldwide monoculture of algae is largely 

limited to some unique species, including 

extremophiles, easy and quick growers 

and this slow growing spp., have to be 

cultivated using PBR systems in order to 

certify their control, although the cultivation 

of subtle spp., needs unusual protection 

to keep away from forces which 

could give stress to the cells, centrifugal 

forces, mainly surface and shear stress 

(Masojidek et al., 2009). 

3. A long history of algal use 

Over the past two decades a great 

covenant of literature has been focused on 

the PBR potential of algal commercial 

applications, due to space limitations we 

account a few of them in this chapter. 

Kelps are cost-effectively precious and 

primary producers, as a result, numerous 

studies on algal cultivation through breeding 

have considered enhancing its quality 

and productivity (Sato et al., 2017). However, 

most cultivation tests have been performed 

in the ocean, thereby limiting the 

development of new cultivars. Macroalgae 

was being eaten at least 1,500 years 

ago in Japan and it remains an important 

food in many cultures where it is valued 

for its high mineral content (i.e., Nori and 

Laverbread). Closer to home in Europe, 

kelp was farmed extensively from the 17 th 

to 19 th centuries for processing into soda 

for the linen industry and into iodine for 

medicinal purposes. Micro algae have 

been used for decades as a food supplement 

(i.e., spirulina) and as a feedstock 

for farmed shellfish and finfish. Compounds 

extracted from both micro- and 

macroalgae today find their way into everyday 

foods, health, cosmetics and pharmaceutical 

industries, agricultural fields, 


energy and environment (Muller-Feuga et 

al., 2012). Greater attention to several 

enterprises and businesses has to be the 

reality that there is plenty of other value 

added product opportunities that exist 

based on algae. The recombinant pharmaceutical 

protein production began to be 

improved more than 25 years before and 

today above 300 protein commodities are 

in the market or in late clinical stages 

(Decker and Reski, 2008). Though, a few 

years back, the moss Physcomitrella patens 

were suggested and merchandised as 

substitute production host that convene 

this concern through providing a special 

amenability for accurate genetic engineering 

together with economic cultivation 

(Decker and Reski, 2008). However, the 

production cost of algae in large scale is 

not yet competitive, predominantly because 

of the prevalence of the PBR technology 

worldwide. As well the major 

progress in classic PBR design, a number 

of novel configurations have been anticipated 

in the last two decades to advance 

their management expressed in terms of 

biomass productivity, photosynthetic efficiency, 

light absorption and light to biomass 

acquiesce. 

4. Advantages of PBRs in algal cultivation 

and exploring the power of 

algae 

The novel advantages of PBR 

have been recommended in the last two 

decades as a substitute to the open type 

cultures and they are widely used in food, 

cosmetics and pharmaceutical industries 

to produce algal biomass in large scale. In 

another way, the cost of nutrient media 

for algal (cyanobacteria) cultivation is 

greatly cheaper than to heterotrophic bacteria 

and the inorganic nutrients at low 

concentration limit the contamination 

through other microorganisms (Chetsumon 

et al., 1994). The harvesting cost can 

be controlled through the production of 

bioactive substances from the marine algae 

cultivated by PBRs. An evidence of 

the rising interest in PBR applications is 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 202



the large number of research and review 

articles published over the past few years 

showing novel PBR developments 

(Olivieri et al., 2014; reviewed in this 

chapter). An interesting application of 

micro algal cultures in PBRs concerns the 

welfare of space mission teams. A PBR 

intended to produce algae protein along 

with oxygen and to remediate devastate 

and CO 2 can form a closed ecological life 

support system where it is elemental for 

astronauts concerned in long term investigation 

task and on space stations on other 

planets like the Moon and Mars 

(Olivieri et al., 2014). Recently, Cao et al. 

(2012) patented a method for micro algal 

biomass cultivation by 100% CO 2 to prolong 

the life support system in such a 

harsh environment. 

Algal products are again being 

commercially explored and developed 

with the help of a growing global industry 

using the latest algal biotechnologies. 

This involves the mass cultivation of micro- 

and macroalgae and conversion of 

the harvested biomass into a range of value-added 

products. The EnAlgae project 

aims to develop technologies that will be 

both economically-viable and environmentally-friendly 

ways so that the production 

of algal biomass can be rolled out 

on industrial scales. Since macroalgae 

generates energy through photosynthesis, 

algae biomasses are situated in the aquatic 

euphotic zone upper layers. Algal photosynthetic 

systems are comparable to that 

of plants growing on terrestrial lands, 

however, usually, they are highly capable 

of converting sunlight into biomass since 

less complex cellular structure and their 

direct access to water, nutrients and CO 2 

(Kilinc et al., 2013; Taelman et al., 2015). 

Chetsumon et al. (1994) produced antibiotic 

through the immobilized cyanobacterium, 

Scytonema spp., in a seaweed type 

PBR. European Union imports of 

macroalgae have conventionally been 

used as the products for agriculture or in 

the food, pharmaceutical and cosmetic 

industries for their useful extracts and are 

very less frequently used for direct human 


consumption (Ngo et al., 2011; Taelman 

et al., 2015). 

5. Extraction technology of bioactive 

compounds from marine algae 

Extraction technology is critical 

for the complete use of marine algae 

(seaweeds and cyanobacteria) raw materials 

to reducing time, with high yields at 

low costs and with low consumption of 

solvents (Caamal-Fuentes et al., 2013). 

Natural bioactive composite consists of 

an extensive range of functionalities and 

structures which give an outstanding pool 

of molecules for the assembly of functional 

foods, nutraceuticals and food additives. 

A few of those natural compounds 

can be originated in nature at an elevated 

level eg., polyphenols but other compounds 

can only be available at very 

small concentrations, so that enormous 

harvesting is essential to acquire adequate 

amounts. The structural complexity of the 

compounds makes chemical synthesis unsuccessful 

and the intrinsic complications 

in producing and screening of these compounds 

include led to the improvement of 

advanced technologies (Gil-Chavez et al., 

2012). Marine algae derived bioactive 

compound production may be regulated 

by the assortment of suitable cultivation 

conditions and making these algae accurate 

natural bioreactors (Ibanez et al., 

2012). For extraction of novel compounds 

from algae, it is essential to estimate how 

efficient ingredients are acquired. In this 

view, there is a need to unite relevant, 

cost-effective, choosy and environmentally 

friendly extraction methods with the 

permitting requirements about the use of 

food grade reagents/solvents and progressions. 

The application of nature friendly, 

advanced and clean extraction methods 

allows for the accomplishment of the 

aimed compounds of significance with 

high efficient extraction techniques, 

whereas, at the similar moment in time, 

reducing the utilize of organic toxic solvents. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 203



The “Green Chemistry” association 

has been investigating traditions to 

ease the risk of harmful chemical contact 

to the environment and humans. Ibanez et 

al. (2012) showed a schematic diagram 

which stated about the changes in the 

waste prevention hierarchy. The innovation 

and progress of marine algae based 

bioactive compounds is a quite new area 

when compared to the invention of bioactive 

compounds from other global resources. 

As a result, in the growth of this 

field, novel, eco-friendly and sustainable 

trends should be followed (reviewed by 

Ibanez et al., 2012). 

6. Application of algal extracts for 

plant growth and development 

Worldwide, the policy drivers 

supporting the application of agricultural 

biostimulants derived marine algae in agriculture 

and are also emphasised in crop 

management through soil drenches, seed 

priming, hydroponic treatments and foliar 

sprays (Sharma et al., 2014). The majority 

of the research works associated to 

crop improvement and protection has attracted 

in recent times an extraordinary 

awareness in order to widen safer and 

new control methods as an alternative of 

these methods depending on chemical 

pesticides. One approach might be the 

generation of the self-defenses in plants 

through natural extracts (Terry and Joyce, 

2004; Walters et al., 2005). Marine micro- 

and macroalgae are the abundant for 

several bioactive compounds, including 

algal polysaccharides, and the composition 

and structure of these compounds 

have greatly contributed to their potential 

on activating signaling pathways and enhancing 

defense mechanisms in a variety 

of plants (El Modafar et al., 2012; Arman 

and Qader, 2012; Abouraicha et al., 2015; 

de Freitas and Stadnik, 2015). In the late 

1940’s, the first seaweed liquid extract for 

agricultural utilize has been urbanized 

and sold as Maxicrop (Satish et al., 2015). 

All through the past two decades, β-(1,4)- 

d-polyglucuronic acids (glucuronans) 


synthesized from marine algae have been 

industriously illustrated in literature for 

their biological and physico-chemical 

characteristics in various models (Caillot 

et al., 2012). To give an instance, a linear 

β-1,3-glucan polymer known as laminarin 

derived from brown algae provoked the 

development of antifungal compounds in 

alfalfa (Medicago sativa) cotyledons 

(Kobayashi et al., 1993) and a number of 

defense responses in rice (Oryza sativa) 

(Inui et al., 1997), tobacco (Nicotiana 

tabacum) cell suspension cultures 

(Klarzynski et al., 2000), and grapevine 

(Vitis vinifera) (Aziz et al., 2003). The 

effectiveness of two different algal saccharides, 

glucuronan and oligoglucuronans 

against postharvest gray mold 

caused by Botrytis cinerea and blue mold 

caused by Penicillium expansum on apple 

fruit, and the associated defense responses 

implicated were assessed (Abouraicha et 

al., 2015, 2016). Ulvan (algae derived 

glyco product) activated a large set of related 

defense enzymes and accumulated a 

variety of resistance substances induced 

the resistance to anthracnose caused by 

Colletotrichum lindemuthianum in beans 

(Phaseolus vulgaris) (Paulert et al., 2009; 

de Freitas and Stadnik, 2012), and protected 

from Fusarium wilt in seedlings of 

tomato (Lycopersicon esculentum) (El 

Modafar et al., 2012). Pre-treatment of 

wheat (Triticum aestivum) and barley 

(Hordeum vulgare) plants with ulvan obtained 

from green macroalgae Ulvan fasciata 

significantly reduced the symptom 

severity of Blumeria graminis infection 

(Paulert et al., 2010). 

The application of algae derived 

agricultural biostimulants on crop plants 

produced numerous benefits in the midst 

of reported effects including enhanced 

seed germination, increased shooting proliferation, 

enhanced rooting (Hernandez- 

Herrera et al., 2013; Satish et al., 2015, 

2016), higher crop and fruit yields, enhanced 

photosynthetic activity, salinity, 

drought and freezing tolerance, and resistance 

to various bacteria, fungi and viruses 

(Sharma et al., 2014). We examined 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 204



the various marine macroalgae extracts in 

bioassays of Solanum trilobatum and Eleusine 

coracana seed germination, 

growth assays and in vitro regeneration 

including Bacopa monnieri (Satish et al., 

2015, 2016; Rency et al., 2017). In the 

past, marine algae extracts have been successfully 

affianced as biostimulatns in 

numerous plants, such as Arabidopsis thaliana 

(Khan et al., 2011) and Nicotiana 

tobaccum (Sanderson and Jameson, 1986) 

and Glycine max (Stirk and Van Staden, 

1997) for the detection of cytokinin-like 

activity. Marine algae or their substances 

are applied successfully for the improvement 

of vegetables like Brassica oleracea 

(Abetz and Young, 1983), Lactuca sativa 

(Abetz and Young, 1983; Crouch et al., 

1990), Beta vulgaris (Featonby-Smith and 

Van Staden, 1983b), Cucumis sativus 

(Nelson and Van Staden, 1984; Jayaraman 

et al., 2011), Daucus carota (Jayaraj 

et al., 2008), Lycopersicon esculentum 

(Featonby-Smith and Van Staden, 1983a; 

Finnie and Van Staden, 1985; Kumari et 

al., 2011; Zadope et al., 2011; Basher et 

al., 2012; Vinoth et al., 2012, 2014; Hernandez-Herrera 

et al., 2013; Briceno- 

Dominguez et al., 2014), Brassica rapa 

(Chinese cabbage) (Sharma et al., 2012), 

Abelmoschus esculentus (Papenfus et al., 

2013) and Brassica napus plants (Ferreira 

and Lourens, 2002). Other than this, pulses 

such as Phaseolus vulgaris (Featonby- 

Smith and Van Staden, 1984; Beckett et 

al., 1994), Phaseolus acutifolius (Beckett 

el al., 1994), Vigna sinensis (Sivasankari 

et al., 2006), Glycine max (Rathore et al., 

2009) and Vigno mungo (Sharma et al., 

2012; Selvam and Sivakumar, 2013; Briceno-Dominguez 

et al., 2014), in cereals 

like Zea mays (Jeannin, 1991), Hordeum 

vulgare (Steveni et al., 1992) and Triticum 

aestivum (Beckett and Van Staden, 

1989; Kumar and Sahoo, 2011), horticultural 

crops like Fragaria ananassa (Spinelli 

et al., 2010) and in Malus domestica 

(Spinelli et al., 2009) seaweed extracts 

applied successfully in different forms. 

Marine algae extracts elicit a wide 

range of responses in the different plants 


spp., through increased chlorophyll content, 

enhanced nutrient uptake, augmented 

flower and fruit set leading to elevated 

yields, delayed senescence and longer 

shelf life of fruits (Briceno-Dominguez et 

al., 2014). In addition, the chemical 

breakdown of marine algae and their bioactive 

compounds has made known the 

standpoint of a wide range of substances 

such as gibberellins, auxins and cytokinins 

which are stimulating plant shoot and 

root growth, maturation and production 

(Hernandez-Herrera et al., 2013; Satish et 

al., 2015). The supplementation of algae 

based extracts showed positive response 

to the growth of various vegetables, fruits 

and other food crops since micro (Cu, Zn, 

B, Mn, Co and Mo), macro (Ca, K and P) 

nutrients, amino acids, organic acids, vitamins, 

antioxidants and complex minerals 

are available in their extracts (Hernandez- 

Herrera et al., 2013; Briceno-Dominguez 

et al., 2014; Satish et al., 2015, 2016; 

Rency et al., 2017). More or less, around 

15 million metric tons of marine plant 

products are emerging every year (FAO, 

2006), a substantial percentage of which 

distributed for nutrient supplementation 

and as biostimulants or biofertilizers in 

worldwide agriculture. At present, the 

advanced crop growing is paying attention 

for existing biotechnologies where it 

would consent for a concession in the application 

of chemicals without toxic effects 

on crop growth and yield as well as 

the farmers' earnings (Hernandez-Herrera 

et al., 2013). These biostimulants can be 

useful as an alternative to herbs and pesticides, 

or used in conjunction through synthetic/artificial 

crop protection goods and 

as plant growth stimulants (Satish et al., 

2015, 2016), and they play a role in sustaining 

the crop production levels, high 

yield, health and quality (Sharma et al., 

2014), and marine algae or their derivatives 

are remaining largely unexploited 

universally. 

7. Future prospects 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 205



Although a little information on 

the effects of glucuronan and its oligomer 

is already available on biological activities 

in plants (El Modafar et al., 2012; 

Caillot et al., 2012; reviewed above) there 

is a lack of studies regarding plantpathogen 

models (Abouraicha et al., 

2017). Research outputs to decrease use 

of synthetic fertilizers and chemical pesticides 

for growing agricultural crops 

through the invention of novel natural 

compounds is immediately required in 

order to assemble the needs of sustainable 

agriculture and to counter to the increasing 

demand for pesticide-free food 

(Abouraicha et al., 2017). Worldwide environmental 

demonstration in relation to 

the exhaustion of natural resources and 

industrialized pollution had led to the improvement 

of more renewable resources 

such as algal biomass, which is translated 

in elevated aquaculture assembly rates of 

6.4 million tonnes in 2000 where it was 

20.8 million tonnes fresh weight in 2012. 

Since the potential ecological and economic 

payment becomes evident, the research 

institutions, government and industries 

have been showing special interest 

towards developing the algal biomass 

(Kilinc et al., 2013; Taelman et al., 2015). 

For the past few years, PBR systems 

have been improved through adopting 

photosynthesis in marine algae and it 

is a positive application of a photovoltaic 

PBR system mimicking a plant. The efficiency 

and miniaturization of algal PBRs 

are a different and novel challenge and 

the PBR culture systems are extraordinarily 

scaled up with respect to the open cultures. 



The author L. Satish sincerely 

thanks the University Grants Commission, 

New Delhi, India for financial support 

in the form of UGC-BSR SRF (UGC 

order no. F.4-1/2006 (BSR)/7-326/2011- 

BSR). Also the authors gratefully 

acknowledge the computational and bioinformatics 

facility provided by the Alagappa 

University Bioinformatics Infrastructure 

Facility (funded by Department 

of Biotechnology, Government of India; 

Grant No.BT/BI/25/001/2006). 

References 

Abetz, P. and Young, C. L. (1983). The 

effect of seaweed extract sprays 

derived from Ascophyllum nodusum 

on lettuce and cauliflower 

crops. Botanica Marina 26, 487– 

492. 

Abouraicha, E. F., Alaoui-Talib, Z., 

Boutachfaiti, R. E., Petit, E., 

Courtois, B., Courtois, J. and 

Modafar, C. E. (2015). Induction 

of natural defense and protection 

against Penicillium expansum and 

Botrytis cinerea in apple fruit in response 

to bioelicitors isolated from 

green algae. Scientia Horticulturae 

181, 121–128. 

Abouraicha, E. F., Alaoui-Talibi, Z. E., 

Tadlaoui-Ouafi, A., Boutachfaiti, 

R. E., Petit, E., Douira, A., Courtois, 

B., Courtois, J. and Modafar, 

C. E. (2017). Glucuronan and oligoglucuronans 

isolated from green 

algae activate natural defense responses 

in apple fruit and reduce. 

Journal of Applied Phycology 29, 

471–480. 

Arman, M. and Qader, S. A. U. (2012). 

Structural analysis of kappacarrageenan 

isolated from Hypneamusciformis 

(red algae) and evaluation as 

an elicitor of plant defense mechanism. 

Carbohydrate Polymers 88, 

1264–1271. 

Basher, A. A., Mohammed, A. J. and 

Teeb, A. I. H. (2012). Effect of 

seaweed and drainage water on 

germination and seedling growth of 

tomato (Lycopersicon spp.). Euphrates 

Journal of Agriculture Science 4, 

24–39. 

Beckett, R. P. and Van Staden, J. 

(1989). The effect of seaweed concentrate 

on the growth and yield of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 206




potassium stressed wheat. Plant Soil 

116, 29–36. 

Beckett, R. P., Mathegka, A. D. M. and 

Van Staden, J. (1994). Effect of 

seaweed concentrate on yield of nutrient-stressed 

tepary bean 

(Phaseolus acutifolius Gray). Journal 

of Applied Phycology 6, 429– 

430. 

Briceno-Dominguez, D., Hernandez- 

Carmona, G., Moyo, M., Stirk, W. 

and Van Staden, J. (2014). Plant 

growth promoting activity of seaweed 

liquid extracts produced from 

Macrocystispyrifera under different 

pH and temperature conditions. 


2203–2210. 

Caamal-Fuentes, E., Chale-Dzul, J., 

Moo-Puc, R., Freile-Pelegrin, Y. 

and Robledo, D. (2013). Bioprospecting 

of brown seaweed 

(Ochrophyta) from the Yucatan 

Peninsula: cytotoxic, antiproliferative, 

and antiprotozoal activities. 


1009–1017. 

Caillot, S., Rat, S., Tavernier, M. L., 

Michaud, P., Kovensky, J., 

Wadouachi, A., Clement, C., 

Baillieul, F. and Petit, E. (2012). 

Native and sulfated oligoglucuronans 

as elicitors of defencerelated 

responses inducing protection 

against Botrytis cinerea of Vitis 

vinifera. Carbohydrate Polymers 87, 

1728–1736. 

Cao, G., Concas, A., Corrias, G., Licheri, 

R., Orru, R. and Pisu, M. 

(2012). A process for the production 

of useful materials to sustain 

manned space missions on Mars 

through in-situ resources utilization. 

Patent PCT/IB2012/053754 

Carvalho, A. P., Meireles, L. A. and 

Malcata, F. X. (2006). Microalgal 

reactors: a review of enclosed system 

designs and performances. Biotechnology 

Progress 22, 490–1506. 

Chetsumon, A., Maeda, I., Umeda, F., 

Yagi, K., Miura, Y., and Mizoguchi, 

T. (1994). Antibiotic production 

by the immobilized cyanobacterium, 

Scytonema sp. TISTR 8208, 

in a seaweed-type photobioreactor. 

Journal of applied phycology, 6(5), 

539-543. 

Crouch, I. J., Beckett, R. P. and Van 

Staden, J. (1990). Effect of seaweed 

concentrate on the growth and 

mineral nutrition of nutrient-stressed 

lettuce. Journal of Applied Phycology 

2, 269–272. 

de Freitas, M. B. and Stadnik, M. J. 

(2012). Race-specific and ulvaninduced 

defense responses in bean 

(Phaseolus vulgaris) against Colletotrichum 

lindemuthianum. Physiological 

and Molecular Plant Pathology 

78, 8–13. 

de Freitas, M. B. and Stadnik, M. J. 

(2015). Ulvan-induced resistance in 

Arabidopsis thaliana against Alternaria 

brassicicola requires reactive 

oxygen species derived from 

NADPH oxidase. Physiological and 

Molecular Plant Pathology 90, 49– 

56. 

Decker, E. L. and Reski, R. (2008). 

Current achievements in the production 

of complex biopharmaceuticals 

with moss bioreactors. Bioprocess 

and Biosystems Engineering 31, 3– 

9. 

El Modafar, C., Elgadda, M., Elboutachfaiti, 

R., Abouraicha, E., 

Zehhar, N., Petit, E., El Alaoui- 

Talibi, Z., Courtois, B. and Courtois, 

J. (2012). Induction of natural 

defence accompanied by salicylic 

acid-dependant systemic acquired 

resistance in tomato seedlings in response 

to bioelicitors isolated from 

green algae. Scientia Horticulturae 

138, 55–63. 

FAO. (2012). Yearbook of fishery and 

aquaculture statistics, in: FAO (Ed.), 

Dataset global aquaculture production 

1950–2012. 

http://www.fao.org/fishery/statistics 

/global-aquacultureproduction/query/en. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 207




FAO. (2013). Agriculture and environmental 

services discussion paper 03, 

in: FAO (Ed.), Fish to 2030 prospects 

for fisheries and aquaculture, 

World Bank report number 83177- 

GLB (Washington DC, United 

States). 

Featonby-Smith, B. C. and Van 

Staden, J. (1983a). The effect of 

seaweed concentrate on the growth 

of tomato plants in nematode infected 

soil. Scientia Horticulturae 20, 

137–146. 


Staden, J. (1983b). The effect of 

seaweed concentrate and fertilizer 

on the growth of Beta vulgaris. 

Zeitschrift für Pflanzenphysiologie 

112, 155–162. 


Staden, J. (1984). The effect of 

seaweed concentrate and fertilizer 

on growth and endogenous cytokinin 

content of Phaseolus vulgaris. 

South African Journal Botany 3, 

375–379. 

Ferreira, M. I. and Lourens, A. F. 

(2002). The efficacy of liquid seaweed 

extract on the yield of canola 

plants. South African Journal of 

Plant and Soil 19, 159–161. 

Finnie, J. F. and Van Staden, J. (1985). 

Effect of seaweed concentrate and 

applied hormones on in vitro cultured 

tomato roots. Journal of Applied 

Phycology 3, 215–222. 

Gil-Chavez, G. J., Villa, J. A., Ayala- 

Zavala, J. F., Heredia, J. B., 

Sepulveda, D., Yahia, E. M. and 

Gonzalez-Aguilar, G. A. (2013). 

Technologies for extraction and 

production of bioactive compounds 

to be used as nutraceuticals and 

food ingredients: An overview. 

Comprehensive Reviews in Food 

Science and Food Safety 12, 5–23. 

Glombitza, K. W. and Koch, M. 

(1989). Secondary metabolites of 

pharmaceutical potential. In Cresswell 

RC, Rees TAV, Shah N (Eds), 

Algal and cyanobacterial biotechnology. 

Longman Scientific & 

Technical, New York. pp. 161–238. 

Hernandez-Herrera, R. M., Santacruz- 

Ruvalcaba, F., Ruiz-Lopez, M. A., 

Norrie, J. and Hernandez- 

Carmona, G. (2013). Effect of liquid 

seaweed extracts on growth of 

tomato seedlings (Solanum lycopersicum 

L.). Journal of Applied Phycology 

26, 619–628. 

Ibanez, E., Herrero, M., Mendiola, J. 

A. and Castro-Puyana, M. (2012). 

Extraction and characterization of 

bioactive compounds with health 

benefits from marine resources: 

Macro and micro algae, cyanobacteria, 

and invertebrates. Hayes M. 

(Ed.) Marine bioactive compounds: 

Sources, characterization and applications. 

pp. 55–98. 

Jayaraj, J., Wan, A., Rahman, M. and 

Punja, Z. K. (2008). Seaweed extract 

reduces foliar fungal diseases 

on carrot. Crop Protection 27, 1360– 

1366. 

Jayaraman, J., Norrie, J. and Punja, 

Z. K. (2011). Commercial extract 

from the brown seaweed Ascophyllum 

nodosum reduces fungal diseases 

in greenhouse cucumber. Journal 

of Applied Phycology 23, 353–361. 

Jeannin, I., Lescure, J. C. and Morot- 

Gaudry, J. F. (1991). The effect of 

aqueous seaweed sprays on the 

growth of maize. Botanica Marina 

34, 141–145. 

Khan, W., Hiltz, D., Critchley, A. T. 

and Prithiviraj, B. (2011). Bioassay 

to detect Ascophyllum nodosum 

extract-induced cytokinin-like activity 

in Arabidopsis thaliana. Journal 


Kilinc, S., Cirik, B., Turan, G., 

Tekogul, H. and Koru, E. (2013). 

Seaweeds for food and industrial 

applications, in: Innocenzo Muzzalupo 

(Ed.), Agricultural and biological 

sciences, food industry. In 

Tech. ISBN: 978-953-51-0911-2 

Kumar, G. and Sahoo, D. (2011). Effect 

of seaweed liquid extract on 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 208




growth and yield of Triticum aestivum 

var. Pusa Gold. Journal of 

Applied Phycology 23, 251–255. 

Kumari, R., Kaur, I. and Bhatnagar, 

A. K. (2011). Effect of aqueous extract 

of Sargassum johnstonii Setchell 

& Gardner on growth, yield and 

quality of Lycopersicon esculentum 

Mill. Journal of Applied Phycology 

23, 623–633. 

Masojidek, J., Papacek, S., Sergejevova, 

M., Jirka, V., Cerveny, J., 

Kunc, J., Korecko, J., Verbovikova, 

O., Kopecky, J., Stys, D. and 

Torzillo, G. (2003). A closed solar 

photobioreactor for cultivation of 

microalgae under supra-high irradiance: 

basic design and performance. 


239–248. 

Masojidek, J., Sergejevova, M., Rottnerova, 

K., Jirka, V., Korecko, J., 

Kopecky, J., Zatkova, V., Torzillo, 

G. and Stys, D. (2009). A two-stage 

solar photobioreactor for cultivation 

of microalgae based on solar concentrators. 

Journal of Applied Phycology 

21, 55–63. 

Muller-Feuga, A., Lemar, M., Vermel, 

E., Pradelles, R., Rimbaud, L. and 

Valiorgue, P. (2012). Appraisal of a 

horizontal two-phase flow photobioreactor 

for industrial production of 

delicate microalgae species. Journal 


Nelson, W. R. and Van Staden, J. 

(1984). The effect of seaweed concentrate 

on growth of nutrientstressed 

greenhouse cucumbers. 

Hortscience 19, 81–82. 

Ngo, D. H., Wijesekaraa, I., Voa, T. S., 

Van Taa, Q. and Kima, S. K. 

(2011). Marine food-derived functional 

ingredients as potential antioxidants 

in the food industry: an 

overview. Food Research International 

44, 523–529. 

Olivieri, G., Salatino, P. and Marzocchella, 

A. (2014). Advances in 

photobioreactors for intensive microalgal 

productions: configurations, 

operation strategies and applications. 

Journal of Chemical Technology 

and Biotechnology 89, 178– 

195. 

Papenfus, H. B., Kulkarni, M. G., 

Stirk, W. A., Finnie, J. F. and Van 

Staden, J. (2013). Effect of a commercial 

seaweed extract (Kelpak ® ) 

and polyamines on nutrientdeprived 

(N, P and K) okra seedlings. 

Scientia Horticulturae 151, 

142–146. 

Paulert, R., Talamini, V., Cassolato, J., 

Duarte, M., Noseda, M., Smania, 

A. J. and Stadnik, M. (2009). Effects 

of sulfated polysaccharide and 

alcoholic extracts from green seaweed 

Ulva fasciata on anthracnose 

severity and growth of common 

bean (Phaseolus vulgaris L.). Journal 

of Plant Diseases and Protection 

116, 263–270. 

Pulz, O. (2001). Photobioreactors: production 

systems for phototrophic 

microorganisms. Applied Microbiology 

and Biotechnology 57, 287– 

293. 

Rathore, S. S., Chaudhary, D. R., 

Boricha, G. N. and Ghosh, A. 

(2009). Effect of seaweed extract on 

the growth, yield and nutrient uptake 

of soybean (Glycine max) under 

rainfed conditions. South African 

Journal of Botany 75, 351–355. 

Rency, A. S., Satish, L., Pandian, S., 

Rathinapriya, P. and Ramesh, M. 

(2017). In vitro propagation and genetic 

fidelity analysis of alginateencapsulated 

Bacopa monnieri 

shoot tips using Gracilaria salicornia 

extracts. Journal of Applied 

Phycology 29, 481–494. 

Richmond, A. and Vonshak, A. (1978). 

Spirulina culture in Israel. Biological 

constraints in algal biotechnology 

11, 274–279. 

Rorrer, G. L. and Cheney, D. P. 

(2004). Bioprocess engineering of 

cell and tissue cultures for marine 

seaweeds. Aquacultural Engineering 

32, 11–41. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 209



Sanderson, K. J. and Jameson, P. E. 

(1986). The cytokinins in a liquid 

seaweed extract: could they be active 

ingredients?. Acta Horticulturae 

179, 113–116. 

Satish, L., Rameshkumar, R., 

Rathinapriya, P., Pandian, S., 

Rency, A. S., Sunitha, T. and 

Ramesh, M. (2015). Effect of seaweed 

liquid extracts and plant 

growth regulators on in vitro mass 

propagation of brinjal (Solanum 

melongena L.) through hypocotyl 

and leaf disc explants. Journal of 


Satish, L., Rathinapriya, P., Rency, A. 

S., Ceasar, S. A., Pandian, S. and 

Rameshkumar, R. (2016). Somatic 

embryogenesis and regeneration using 

Gracilaria edulis and Padina 

boergesenii seaweed liquid extracts 

and genetic fidelity in finger millet 

(Eleusine coracana). Journal of Applied 

Phycology 28, 2083–2098. 

Sato, Y., Yamaguchi, M., Hirano, T., 

Fukunishi, N., Kawano, T. and 

Kawano, S. (2017). Effect of water 

velocity on Undaria pinnatifida and 

Saccharina japonica growth in a 

novel tank system designed for 

macroalgae cultivation. Journal of 


Selvam, G. G. and Sivakumar, K. 

(2013). Effect of foliar spray from 

seaweed liquid fertilizer of Ulva reticulate 

(Forsk.) on Vigna mungo L. 

and their elemental composition using 

SEM – energy dispersive spectroscopic 

analysis. Asian Pacific J 

Reproduction 2, 119–125. 

Sharma, H. S. S., Fleming, C., Selby, 

C., Rao, J. R. and Martin, T. 

(2014). Plant biostimulants: a review 

on the processing of macroalgae 

and use of extracts for crop 

management to reduce abiotic and 

biotic stresses. Journal of Applied 

Phycology 26, 465–490. 

Sharma, S. H. S., Lyons, G., 

McRoberts, C., McCall, D., Carmichael, 

E., Andrews, F., Swan, 


R., McCormack, R. and Mellon, 

R. (2012). Biostimulant activity of 

brown seaweed species from 

Strangford Lough: compositional 

analyses of polysaccharides and bioassay 

of extracts using mung bean 

(Vigno mungo L.) and pakchoi 

(Brassica rapachinensis L.). Journal 

of Applied Phycology 24, 1081– 

1091. 

Sivasankari, S., Venkatesalu, V., Anantharaj, 

M. and Chandrasekaran, 

M. (2006). Effect of seaweed 

extracts on the growth and biochemical 

constituents of Vigna sinensis. 

BioresourceTechnology 97, 1745– 

1751. 

Spinelli, F., Fiori, G., Noferini, M., 

Sprocatti, M. and Costa, G. 

(2009). Perspectives on the use of a 

seaweed extract to moderate the 

negative effects of alternate bearing 

in apple trees. The Journal of Horticultural 

Science and Biotechnology 

84, 131–137. 

Spinelli, F., Fiori, G., Noferini, M., 

Sprocatti, M. and Costa, G. 

(2010). A novel type of seaweed extract 

as a natural alternative to the 

use of iron chelates in strawberry 

production. Scientia Horticulturae 

125, 263–269. 

Steveni, C. M., Norrington-Davies, J. 

and Hankins, S. D. (1992). Effect 

of seaweed concentrate on hydroponically 

grown spring barley. 


173–180. 

Stirk, W. A. and Van Staden, J. (1997). 

Comparison of cytokinin- and auxin-like 

activity in some commercially 

used seaweed extracts. Journal of 


Taelman, S. E., Champenois, J., Edwards, 

M. D., Meester, S. D. and 

Dewulf, J. (2015). Comparative environmental 

life cycle assessment of 

two seaweed cultivation systems in 

North West Europe with a focus on 

quantifying sea surface occupation. 

Algal Research 11, 173–183. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 210




Terry, L. A. and Joyce, D. C. (2004). 

Elicitors of induced disease resistance 

in postharvest horticultural 

crops: a brief review. Postharvest 

Biology and Technology 32, 1–13. 

Vinoth, S., Gurusaravanan, P. and 

Jayabalan, N. (2012). Effect of 

seaweed extracts and plant growth 

regulators on high-frequency in 

vitro mass propagation of Lycopersicon 

esculentum L (tomato) 

through double cotyledonary nodal 

explant. Journal of Applied Phycology 

24, 1329–1337. 

Vinoth, S., Gurusaravanan, P. and 

Jayabalan, N. (2014). Optimization 

of somatic embryogenesis protocol 

in Lycopersicon esculentum L. using 

plant growth regulators and seaweed 

extracts. Journal of Applied Phycology 

26, 1527–1537. 

Walters, D., Walsh, D., Newton, A. and 

Lyon, G. (2005). Induced resistance 

for plant disease control: maximizing 

the efficacy of resistance elicitors. 

Phytopathology 95, 1368– 

1373. 

Wu, X. and Merchuk, J. C. (2004). 

Simulation of algae growth in a 

bench scale internal loop airlift reactor. 

Chemical Engineering Science 

59, 2899–2912. 

Yang, Z., del Ninno, M., Wen, Z. and 

Hu, H. (2014). An experimental investigation 

on the multiphase flows 

and turbulent mixing in a flat-panel 

photobioreactor for algae cultivation. 

Journal of Applied Phycology 

26, 2097–2107. 

Zadope, S. T., Gupta, A., Bhandari, S. 

C., Rawat, U. S., Chaudhary, D. 

R., Eswaran, K. and Chikara, J. 

(2011). Foliar application of seaweed 

sap as biostimulant for enhancement 

of yield and quality of 

tomato (Lycopersicon esulentum 

Mill.). Journal of Scientific and Industrial 

Research 70, 215–219. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 211



Biotransformation of Various Wastes into a Nutrient 

Rich Organic Biofertilizer - a Sustainable Approach 

towards Cleaner Environment 

Geetha Karuppasamy 1 , Michael Antony D’Couto 1, 2 , Sangeetha Baskaran 1, 3 and 

Anant Achary 1, * 

1 Department of Biotechnology, Centre for Research, Kamaraj College of Engineering and 

Technology, K.Vellakulam-625701, Near Virudhunagar, Madurai District, Tamil Nadu, 

India; 2 JLL-Jones Lang LaSalle, TVH Belicia Towers, Thandavarayan Street, Mandavelipakkam, 

Raja Annamalai Puram, Chennai-600028, Tamil Nadu, India; 3 Department of 

Biotechnology, St. Joseph's College Of Engineering, Old Mamallapuram Road, Semmencherry, 

Kamaraj Nagar, Semmancheri, Chennai-600119, Tamil Nadu, India; * Correspondence: 

achyanant@yahoo.com; Tel: 91-4549-278-171 

Abstract: Environmental degradation is one of the main threats confronting the world and 

the widespread use of chemical fertilizers contributes essentially to the degeneration of the 

environment through exhaustion of fossil fuels, generation of carbon dioxide (CO 2 ) and 

contamination of water bodies. Excessive use of fertilizers has adversely affected agricultural 

productivity causing soil degradation. Biotransformation through vermicomposting 

implies the production of nutrient-rich excreta of worms. After earthworms digest organic 

matter, they excrete a high-nutrient product known as Castings. Sustainable development 

can be achieved by providing adequate quantity of food for which agricultural land management 

is an important aspect. Earthworms are known to consume all types of organic 

wastes including vegetable waste, wastes generated by pulse and rice processing industries 

and other organic wastes. The food passes through the digestive tract and the worms secrete 

chemicals that break down organic matter into sustainable nutrition. Vermicompost is a 

peat like material consisting of excellent porosity, structure, aeration and moisture holding 

capacity that makes it a good organic manure for growing plants. Another important property 

of vermicompost is its vast surface area that provides strong absorbability and nutrient 

retention ability. Vermicompost increases soil fertility, enhance plant growth and suppress 

the population of plant pathogens and pests. As a soil conditioner, vermicompost is healthier 

to traditional compost for its capability to improve and enhance soil configuration and its 

water-holding capacity. Thus vermicomposting can be proposed as a cost effective and environment 

friendly method for efficient utilization of various organic wastes. This will 

promote healthy plant growth and aid in sustainable management of agricultural lands with 

cleaner environment. 

Keywords: Biotransformation; earthworms; organic waste; vermicomposting; vermiwash 


With the increase in the population 

there has been a tremendous increase 

in generation of a variety of wastes. Although 

various measures are in place to 

handle the wastes generated by domestic 

sector and industrial sector, the disposal 

of wastes is still a prime concern. There is 

an estimate that India produces approximately 

3000 million tons of wastes per 

annum and among that more than 60% is 

found to be decomposable. Vegetable 

wastes are one of the major sources of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 212


Conversion of Wastes into Organic Biofertilizer for Sustainability 

municipal wastes. The disposal of biodegradable 

solid wastes from domestic, 

agricultural and industrial sources has 

caused ever-increasing environmental and 

economic problems (Garg et al., 2006). 

Moreover, an additional key threat 

is the environmental degradation due to 

the extensive use of chemical fertilizers 

that has led to deterioration of the environment 

through generation of carbon 

dioxide (CO 2 ), exhaustion of fossil fuels, 

and water resources being contaminated. 

Agricultural productivity also has taken a 

heavy toll due to disproportionate use of 

fertilizers leading to soil degradation. 

Recycling of wastes through biotransformation 

of various biodegradable 

wastes can reduce the problem of nonutilization 

of wastes. Locally available 

organic wastes of anthropogenic nature, 

domestic waste and agricultural lignocellulosic 

waste products can be used as biofertilizer 

as an alternative to chemical fertilizers. 

Vermicomposting employing 

earthworm as decomposers, for degradation 

and recycling, may be used to enhance 

the production of crops which are 

free from pollution and health hazard 

(Bakthvathsalam and Ramakrishnan, 

2004). Preserving the quantity and quality 

of soil is one of the main objectives of 

current efforts to make agriculture more 

"sustainable". 

2. Current waste disposal practices & 

its impact 

Currently, waste is a major concern 

worldwide becoming exceedingly 

significant in developing countries like 

India, China as well as in Europe. Waste 

can be classified into industrial, agricultural, 

sanitary and solid urban residues on 

the basis of their origin. There might be a 

significant change in their distribution 

depending on the country. Waste generated 

by various food processing industries 

is a good example of globally generated 

waste on a large scale. Waste of this type 

has become a great source of concern as 

in certain cases it is found to be over 50% 

Karuppasamy et al. 

of the total waste produced in countries. It 

has also been noted that 60% of food processing 

industry waste belongs to organic 

matter. 

Global urbanization has led to increase 

in the volume of solid wastes. According 

to a survey conducted in 1990, 

about 1.3 billion metric tons of municipal 

solid waste was generated globally 

(Beede and Bloom, 1995). In today‟s scenario, 

the generation of solid waste per 

year equals to 1.6 billion metric tons approximately. 

A significant amount of capital 

is being invested into managing such 

huge volumes of solid waste suggesting 

that solid waste management (SWM) has 

become a large, complex and costly service. 

The solid waste and its management 

at various stages are chiefly affected 

by the ever growing population. The 

numbers of households owing to the increase 

in population also has an important 

role in generation and collection of the 

solid waste. Solid waste is mainly collected 

by municipalities and the uncollected 

waste, approx. 31% to 49%, is left on 

street or road corners, open spaces like 

vacant plots that pollute the environment 

on continuous basis. The collected waste 

is usually disposed of within or outside 

the municipal limits into low lying areas 

like ponds etc, without any treatment except 

separation of recyclable waste by 

scavengers. 

Extensive research around the 

world today is being carried out to find a 

solution for utilization of various agricultural 

residues as energy sources. A simple 

example is the sugarcane processing industries. 

Sugarcane bagasse and sugarcane 

agriculture residues are the two main 

residues of sugar and ethanol production 

process. Sugarcane bagasse is the fibrous 

waste that remains after recovery of sugar 

juice via crushing and extraction. It is one 

of the principal fuels used around the 

world in the sugarcane agro-industry because 

of its well-known energy properties. 

However, the bagasse management 

and disposal practices employed by the 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 213



sugar agro-industry have, in most cases, 

remained the same as those used back in 

the early 19th century leading to enormous 

amount of bagasse being disposed 

(Faria et al., 2012). This bagasse can be 

an excellent material for biotransformation 

by vermicomposting. A similar 

scenario is also found in industries dealing 

with pulses and grains. The amount of 

waste generated as husk and bran is 

enormous. Although the husk and bran 

are being widely used in various industrial 

applications like industrial fuel, activated 

carbon, as pet food fiber, substrate 

for various fermentation processes etc, 

there is still lot of waste that is left unused. 

Consumption of fruits and vegetables 

has radically increased in the various 

nations by more than 30% during the past 

few years. It is also projected that approximately 

20% of all the fruits and vegetables 

produced is lost each year due to 

spoilage. Also, vegetable markets in various 

cities & towns are known to produce 

significant amount of non-edible vegetable 

wastes. As mentioned earlier, solid 

waste management of spoilt fruits and 

vegetables is one of the biggest problems 

faced today by all the cities including collection, 

transportation and disposal of the 

waste. These wastes are usually discarded 

in the market itself and allowed to rot. 

However, this discarding leads to production 

of hazardous ecological impacts 

(Kumari, 2013). 

The poultry industry is one of the 

biggest and fastest growing livestock production 

systems in the world (Edwards 

and Daniel, 1992). As per the reports of 

Foreign Agricultural Service in 1992, internationally, 

approximately 40 million 

metric tons of poultry meat and 600 billion 

eggs were produced. Although reasonably 

flourishing, the poultry industry 

is at present facing a challenging environmental 

problem. From an agricultural 

standpoint, the role of poultry wastes in 

the contamination of soil and groundwater, 

the eutrophication of surface waters 

by nitrogen and phosphorus, and the fate 


of pesticides, heavy metals, and pathogens 

polluting the soil via poultry wastes 

are the central environmental issues at the 

present time. 

3. Role of chemical fertilizers in environmental 

degradation 

3.1. Nutrient requirements for plant 

growth 

For the survivability and growth 

of a plant, 16 essential nutrients are required 

including carbon, hydrogen, oxygen, 

nitrogen, phosphorous, potassium, 

magnesium, calcium and sulphur, iron, 

zinc, copper, manganese, boron, chlorine 

and molybdenum. Air, water and sunlight 

provide the necessary oxygen, carbon, 

hydrogen and energy. 

3.2. Chemical fertilizers – a threat 

Current practices mainly use the 

chemical fertilizer to accomplish the nutrients 

requirement of a growing plant 

which has a serious impact on the soil and 

water bodies. Excessive utilization of 

chemical fertilizer leads to increased salinity 

of soil which is one of the major 

threats causing lower productivity in the 

soil. Some of the serious impacts of using 

chemical fertilizers are: 

Pollution of ground & surface water 

Soil fertility is reduced leading to 

reduced food production 

Depletion in the soil microbial 

ecosystem 

Ground water pollution and destruction 

of the aquatic life 

Loss of terrestrial and aquatic biodiversity 

Depletion of the Ozone leading to 

global warming 

Exhaustive cropping involving 

continuous use of high levels of chemical 

fertilizers (CF) often leads to nutritional 

disparity in soil and decline in crop 

productivity (Nambiar, 1994). Numerous 

properties characterizing the status of soil 

microbial biomass, activity and nutrient 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 214




content have been suggested as indicators 

of soil quality (Doran and Parkin, 1994). 

Although microbial biomass only forms a 

small fraction of soil organic matter, it 

contributes to agricultural sustainability 

because its high turnover rate is responsible 

for nutrient release and therefore promotes 

plant uptake (Smith et al., 1993). 

Conventional agro-ecosystems 

have been characterized by high input of 

chemical fertilizer instead of organic 

amendments, leading to deterioration of 

soil quality due to reductions in soil organic 

matter. With increasing global concerns 

about energy crisis and environmental 

protection, it is becoming more 

important to rely on locally abundant agricultural 

bioresources than on chemical 

fertilizers. Recent studies have focused on 

re-considering traditional fertilization 

practices to enhance soil organic input by 

amendment of organic fertilizers like 

vermicompost. 

4. Biological decomposition of wastes 

The breakdown of raw organic 

materials to a finished compost - a process 

known as decomposition - is a complex 

gradual process wherein both chemical 

and biological processes take place in 

order to mineralize the organic matters. 

Generally, biological degradation of organic 

material takes place through two 

distinct pathways: 

i) Anaerobic digestion: Anaerobic 

digestion may be defined as the 

breakdown of organics in the absence of 

oxygen under controlled conditions. Here, 

fermentation of waste results in the formation 

of ammonia-like substances and 

hydrogen sulfide. Organic wastes with 

high degradability are easily degraded 

through anaerobic digestion. Bacterial 

species capable of degrading targeted organics 

are involved in this process along 

with thorough mixing for efficient substrate 

conversion (Figure 1). The carbon 

content released as biogas containing methane, 

carbon dioxide and energy is represented 

using the equation: 

C 6 H 12 O 6 3CO 2 + 2CH 4 + 393kJ … 1 

The drawback of this technique is that it 

cannot be used for mixed domestic waste 

composting. 

Figure 1: Schematic representation of 

anaerobic digestion process for composting 

of agricultural wastes. 

ii) Aerobic digestion: Aerobic digestion 

may be defined as a biological 

degradation process where vigorous humification 

and pasteurization of organic 

residues takes place. The process requires 

air breathing microbes like bacteria, fungi, 

actinomycetes, mesophilic-exothermic 

microbes and thermophilic microorganisms 

that flourish in elevated temperature 

of greater than 60°C. Mineralization of 

biodegradable organic matter takes place 

leading to release of carbon dioxide, water 

and energy (Figure 2). Conversion of 


aerobic digestion process for composting 

of agricultural wastes. 

residual organic components to humic 

acids results in stabilization of the process. 

As the end result, the heterogeneous 

waste is transformed into a homogeneous 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 215



and valuable organic fertilizer that is rich 

in humus. 

C 6 H 12 O 6 + 6O 2 6CO 2 + 6H 2 O + 2840kJ 

… 2 

Compared to chemical fertilizers, 

compost is an excellent product that retains 

most of the original nutrients beneficial 

for the increase of soil‟s organic and 

nutrient constituents. Application of compost 

can improve structure and fertility of 

the soil. There are basically four important 

parameters that must be considered 

for the evaluation of quality of compost 

and process performance. They are: 

volatile solids, respiration rate, germination 

tests and pathogen indicators. 

5. Vermicomposting 


Vermicomposting is an ecological 

stabilization process involving the breakdown 

of organic waste by the joint action 

of earthworms and mesophilic microorganisms. 

Earthworms require an environment 

conducive for microbial degradation 

and maintenance of biochemical processes 

for enhanced microbial decomposition. 

Various intestinal microflora of 

earthworms are transferred to the compost 

matrix along with their gut enzymes that 

play an important role (Whiston and Seal, 

1988). Furthermore, earthworms are also 

known to augment the microbial activities 

by improvement of the environment necessary 

for their growth (Syers et al., 1979; 

Mulongoy and Bedoret, 1989). Through 

the process of vermicomposting, wastes 

are converted to a better homogenized, 

nutrient rich and well stabilized product. 

Several studies reveal that vermicomposting 

can be used as an effective technique 

for the treatment of wastes rich in pathogens 

(Eastman et al., 2001). Vermicomposting 

gives a better end product than 

composting due to the enzymatic and microbial 

activity that takes place during the 

process (Bajsa et al., 2003). Various studies 

indicate that vermicomposting can attain 

harmless pathogen levels which may 

be aided by the microbial and enzymatic 

activity. An added advantage of this process 

is the conversion of important plant 

nutrients into a more soluble state (Nair et 

al., 2006). 

Several parameters can be used 

for the evaluation of the vermicomposting 

process, like, survival of the worms, biomass 

growth, and increase in worm population. 

Since earthworms are known to 

feed on the pathogens present in the waste 

used, vermicomposting process guarantees 

pathogen removal. Hence, as per 

USEPA, Vermicomposting has been recognized 

as „Class A‟ stabilization process 

(Eastman et al., 2001). Another advantage 

of biotransformation of waste through 

vermicomposting is the loss of moisture 

to yield a drier product. High concentration 

of moisture in the compost can lead 

to process failure. In vermicomposting, 

the worm burrows act as channels for air 

passage, hence, this can support higher 

humidity. 

The main problems encountered 

with composting of many organic wastes 

are their high moisture content, need of 

bulking substrate and components undesirable 

for worm consumption. As a result, 

composting of raw organic wastes 

requires constant monitoring of moisture 

level, composition of the waste, C/N ratio 

and the composting period. This has been 

overcome by the process of pre-digestion 

of organic waste before it can be utilized 

for vermicomposting. Geetha et al., 

(2016) and Nair et al., (2006) have carried 

out pre-digestion of the non-edible 

vegetables waste and kitchen wastes respectively, 

in order to achieve a better 

quality of vermicompost. Pre-digestion 

prior to vermicomposting was helpful in 

pH, moisture and waste stabilization. Predigestion 

was also found to effectively 

reduce the pathogen load from the vermicompost. 

5.1. Earthworms 

Earthworms belong to kingdom 

Animalia, phylum Annelida, class Clitelatta 

and subclass Oligochaetae. They are 

known as soil forming organisms inhabiting 

almost all part of the earth. They can 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 216



be found in different groups of varying 

size, shape, colouration, life span, feeding 

habit and depth in the soil where they are 

found. The shape of the worm is cylindrical 

with segmented body tapering off at 

both ends. The segments are separated by 

fluid-filled compartment that surrounds a 

central digestive tract. 

More than 1800 known species of 

earthworm have been found all over the 

world (Minnich, 1977) and they can be 

subdivided in three basic groups: 

Epigeics, Endogeics and Anecics. Although 

there are probably many species of 

earthworms available, only a few have 

been utilized for biotransformation of organic 

waste processing. The most commonly 

used species of earthworms include 

Eisenia foetida (Red wiggler), 

Lumbricus rubellus (Red worm), Eisenia 

Andrei (Red tiger), Perionyx excavates 

(Blue worm), Eudrilus eugeniae (African 

night crawler), Enchytraeids (White 

worm), Dendroba enaveneta and Perionyx 

hawayana. Although all the above 

mentioned species may be used for vermicomposting, 

Eisenia foetida and Lumbricus 

rubellus are the most commonly 

used as it is easy to replicate the suitable 

environmental condition for their growth 

and regeneration. Among the two species, 

Eisenia foetida has been proved best for 

vermicomposting of any organic waste 

(Edwards and Bater, 1992) because the 

growth and reproduction of these worms 

is quite rapid. Eisenia foetida, commonly 

known as Red wiggler, is an epigenic 

worm that favours living in organic manure 

or compost. It has the ability to process 

large quantities of organic matter as 

under ideal conditions; it is known to 

consume food as much as its body weight 

each day. 

5.2. Vermicomposting process 

The process of vermicomposting 

can be carried out either in batch or in 

continuous modes. The time taken for the 

completion of decomposition process may 

vary depending on the time taken by the 

earthworms to ingest the feed, digest and 


excrete. The following steps are involved 

in vermicomposting process (Abbasi et 

al., 2008): 

i. Ingestion and breaking down of the ingested 

organic waste by the action of 

earthworm‟s gizzard, located next to 

the mouth of the worm. 

ii. Digestion of the broken down particles 

by the action of enzymes and microbes 

while it passes through the earthworm‟s 

body. 

iii. Exit of the digested matter as “Vermicast” 

after few hours of ingestion. 

Vermicomposting bins (Figure 3) 

are set up using Vermitech pattern 

(Geetha et al., 2016). The plastic or ceme- 


design of vermicompost bin (Geetha et 

al., 2016). 

-nt bins are placed in a shaded elevated 

area on a pedestal of bricks for effective 

water drainage. The basal layer of the 

vermibed comprises of broken bricks and 

stones above which a layer of sand up to 

the height of 10 cm is set up to ensure 

proper filtration and drainage. This is followed 

by first a 10 cm high layer of cow 

dung (3kg) and 5cm high layer of predigested 

organic waste. A 6~8 cm layer of 

straw is added to cover the bedding material 

that helps in retaining moisture. Ap- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 217



proximately 60-75 earthworms are inoculated 

in the composting bins. The vermicomposting 

units are kept in shade and 

covered with a mesh. Water is sprinkled 

to maintain sufficient moisture. For operative 

collection of wormy wash, a small 

hole is drilled near the base of composting 

bin and a tube is attached to it. Turning 

of the vermicomposting pile must be 

carried out periodically to ensure proper 

aeration. 

5.3. Advantages of waste management by 

vermicomposting 

Composting is known as an effective 

biotransformation technique for treating 

organic wastes, following nature‟s 

way of recycling. Vermicomposting 

requires very less resources like water, 

energy and land/space required for treatment 

of per unit of bio-waste as compared 

to aerobic composting. It is a rapid, cost 

effective and sustainable alternative for 

transformation of organic waste, carried 

out by earthworms, leading to formation 

of a product rich in plant nutrients and 

humic acids. The compost thus generated 

has the ability to hold nutrients for a 

longer period without unfavorably impacting 

the environment. No curing is 

necessary for vermicompost as it is highly 

rich in beneficial microorganisms. The 

overall time required for processing of 

waste is therefore reduced to a great extent, 

resulting in non-toxic by-products. 

Gardeners, all over the world, highly prefer 

the application of vermicompost over 

chemical fertilizers; hence, vermicompost 

is finding a significant market value 

(Sudhakar et al., 2002). 

Application of vermicompost has 

been proved to improve the texture, structure, 

aeration, fertility and water holding 

capacity of soil. This way most of the 

valuable nutrients that are taken out of the 

soil during crop cultivation are replenished. 

Also, vermicompost addition is 

known to enhance plant-root developments 

that help in control of soil erosion. 

Vermicompost are also known to be rich 


in various enzymes, high quality organics 

(humus) and plant growth regulators. 

6. Application of biotransformed waste 

for better agricultural productivity 

One of the biggest environmental 

challenges that the world is facing today 

is solid waste management. A sustainable 

approach towards management of this 

problem may be the use of biotransformation 

technique to treat and convert organic 

waste into vermicompost. Composting 

is the most cost-effective and ecological 

option for management of various organic 

wastes since it is easily operable 

and can be carried out in a small scale 

level with proper management of the process 

to obtain a good quality product. A 

list of various wastes converted to vermicompost 

is given in Table 1. Although, 

husk is abundantly available as lignocellulosic 

waste, the problem with using 

husks as vermicomposting feedstock is its 

high initial carbon (lignin and cellulose) 

contents, which impedes the composting 

process (Kumar et al., 2013). Hence, the 

substrate requires amendments to achieve 

an optimum range of C/N ratio to attain 

optimal process efficiency (Goyal et al., 

2005). This can be achieved with the help 

of pre-digestion of husks along with other 

organic wastes (such as non-edible vegetable 

waste) as amendment, prior to vermicomposting. 

This will help in conversion 

of husk into biofertilizer within few 

months. 

Integrated use of fly ash along 

with organic wastes has been effectively 

used in increasing the yield of crops when 

compared to continuous use of chemical 

fertilizers alone (Rautaray et al., 2003). 

This may be due to beneficial effects on 

rice and residual effects on mustard. According 

to Rautaray et al. (2003) greater 

crop yield was related to higher uptake of 

nutrient. Further to the yield advantage, a 

better soil chemical properties were also 

noted namely pH, organic carbon and 

available N, P and K as compared to the 

soils where chemical fertilizers were con- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 218




tinuously used. When fly ash was used in 

combination with organic waste during 

vermicomposting, the end product obtained 

gave better plant growth than with 

chemical fertilizers alone. 

Vigna radiata (Green gram) when 

grown in soil amended with a combination 

of vermicompost and wormy wash 

were found to develop nodes and new 

leaves at a significantly higher rate when 

compared to control (Geetha et al., 2016). 

This might be due to increase in bioavailability 

of macro and micronutrients from 

vermicompost and wormy wash (Erich et 

al., 2002). Vermicompost and wormy 

wash augments crop growth and yield 

when added to soil (Lalitha et al., 2000). 

According to the studies conducted by 

Atiyeh et al. (2001), and Suthar (2009), 

addition of vermicompost in bedding media 

enhanced seed germination and 

growth leading to overall increase in plant 

productivity. 

Table 1: List of organic wastes converted to vermicompost 

No. Organic 

waste 

Earthworm employed 

Outcome 

1. Non-edible Eisenia foetida Enhanced growth of Vigna 

vegetables 

radiata (green gram) 

from market 

2. Kitchen 

waste 

3. Industrial 

sludge 

4. Vegetable 

wastes 

Eisenia fetida & 

Lumbricus rubellus 

Eisenia foetida 

Eisenia foetida 

5. Farm garbage Eisenia fetida, Eudrilus 

eugeniae & 

Peronyx excavates 

6. Agriculture 

waste from 

market yard 

Eisenia Foetida & 

Eudrilus euginiae 

7. Fruit waste Eisenia Foetida & 

Eudrilus euginiae 

8. Coconut Eudrilus euginiae 

waste 

Effect of thermocomposting 

prior to vermicomposting 

Removal of the heavy metals 

from electronic industrial 

sludge 

Composting with minimum 

resources leading to zero waste 

generation 

Odourless product with high 

nutrient status. 

Simplest, scientific, economic 

and environmental friendly 

way to transform waste materials 

into compost 

through vermicomposting by 

using an exotic species of 

earthworm 

Degradation strategy of organic 

waste 

Efficient method to convert 

coconut waste into valuable 

by-product 

Reference 

Geetha et 

al., 2016 

Nair et al., 

2006 

Shaymaa et 

al., 2010 

Sibi and 

Manpreet, 

2011 

Indrajeet et 

al., 2010 

Mane and 

Raskar, 

2012 

Seetha et 

al., 2012 

Tahir and 

Hamid, 

2012 

Lim et al., 

2012 

Eisenia fetida Nutrient rich vermicompost Ansari and 

Rajpersaud, 

2012 

9. Rice husk Eudrilus eugeniae Bio-transforming RH into value-added 

material, 

10. Water hyacinth 

and 

Grass clippings 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 219




Environmental degradation is a 

major cause of concern challenging the 

world. Owing to improper waste management 

amenities and treatment, discarding 

of organic wastes from various sectors 

like domestic, agricultural and industrial 

sources has become a source of distress 

causing serious environmental complications. 

As a result of fast growing population, 

there is also significant increase in 

the generation of various wastes all over 

the world. Vermicomposting, being an 

environmental-friendly technique, is an 

attractive method for conversion of waste 

into wealth. In today‟s world of organic 

products, people are becoming increasingly 

aware of the importance of converting 

our waste into valuable product that 

will not only solve the problem of waste 

disposal but also help the agriculturalists 

in acquiring a safe and cost-effective crop 

promotion technique. 


The authors wish to thank Management 

and Department of Biotechnology, 

Kamaraj College of Engineering and 

Technology, Virudhunagar, Tamil Nadu, 

India, for their constant support and encouragement. 

References 

Abbasi, T., Gajalakshmi, S. and Abbasi, 

S. A. (2009). Towards modeling 

and design of vermicomposting 

systems: Mechanisms of composting/vermicomposting 

and their implications. 

Indian Journal of Biotechnology 

8, 177-182. 

Ansari, A. A. and Rajpersaud, J. 

(2012). Physicochemical Changes 

during Vermicomposting of Water 

Hyacinth (Eichhornia crassipes) and 

Grass Clippings. International Scholarly 

Research Network, ISRN Soil 

Science 2012, 1-6. 


Atiyeh, R. M., Arancon, N. Q., Edwards, 

C. A. and Metzger, J. D. 

(2001). The influence of earthwormprocessed 

pig manure on the growth 

and productivity of marigolds. Bioresource 

Technology 81(2), 103-108. 

Bajsa, O., Nair, J., Mathew, K. and Ho, 

G. E. (2003). Vermiculture as a tool 

for domestic wastewater management. 

Water Science and Technology 

48(11–12), 125–132. 

Bakthvathsalam, R. and Ramakrishnan, 

G. (2004). Culture of earthworm 

using different organic wastes 

of agricultural importance. Environment 

& Ecology 22(Spl-2), 386-391. 

Beede, D. N. and Bloom, D. E. (1995). 

The Economics of Muni- cipal Solid 

Waste. World Bank Research Observer 

10(2), 113-150. 

Doran, J. W. and Parkin, T. B. (1994). 

Defining and assessing soil quality. 

In: Defining Soil Quality for a Sustainable 

Environment. Doran, J.W., 

Coleman, D. C., Bezdicek, D.F. and 

Stewart, B.A. (eds.). SSSA, Inc., 

Madison, Wisconsin, USA. 

Eastman, B. R., Kane, P. N., Edwards, 

C. A., Trytek, L., Gunadi, B., 

Stermer, A. L. and Mobley, J. R. 

(2001). The effectiveness of vermiculture 

in human pathogen reduction 

for USEPA biosolids stabilization. 

Compost Science and Utilization 

9(1), 38-49. 

Edwards, C. A. and Bater, J. E. (1992). 

The use of earthworms in environmental 

management. Soil Biology 

and Biochemistry 24(12), 1683-1689. 

Edwards, D. R. and Daniel, T. C. 

(1992). Environmental Impacts of 

On-Farm Poultry Waste Disposal - A 

Review. Bioresource Technology 41, 

9-33. 

Erich, M. S., Fitzgerald, C. B. and Porter, 

G. A. (2002). The effect of organic 

amendments on phosphorus 

chemistry in a potato cropping system. 

Agriculture, Ecosystems & Environment 

88, 79-88. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 220



Faria, K. C. P., Gurgel, R. F. and Holanda, 

J. N. F. (2012). Recycling of 

sugarcane bagasse ash waste in the 

production of clay bricks. Journal of 

Environmental Management 101, 7- 

12. 

Garga, P., Guptaa, A. and Satya, S. 

(2006). Vermicomposting of different 

types of waste using Eisenia foetida: 

A comparative study. Bioresource 

Technology 97(3), 391-395. 

Geetha, K., Michael A. D. & Anant A. 

(2016). Bioconversion of Non Edible 

Vegetables from Market into Biofertilizer 

for Crop Improvement. Journal 

of Agricultural Science 8(6), 71- 

83. 

Goyal, S., Dhull, S. K. and Kapoor, K. 

K. (2005). Chemical and biological 

changes during composting of different 

organic wastes and assessment of 

compost maturity. Bioresource Technology 

96, 1584–1591. 

Indrajeet, Rai, S. N. and Singh, J. 

(2010). Vermicomposting of Farm 

Garbage in different combination. 

Journal of Recent Advances in Applied 


Kumar, S., Sangwan, P., Dhankhar, R., 

Mor, V. and Bidra, S. (2013). Utilization 

of Rice Husk and Their Ash: 

A Review. Research Journal of 

Chemical and Environmental Sciences 

1(5), 126-129. 

Kumari, S. (2013). Solid Waste Management 

by Vermicomposting. International 

Journal of Scientific & Engineering 

Research 4(2), 1-5. 

Lalitha, R., Fathima, K. and Ismail, S. 

A. (2000). Impact of biopesticides 

and microbial fertilizers on productivity 

and growth of Abelmoschus esculentus. 

Vasundhara the Earth, 

1&2, 4-9. 

Lim, S. L., Wu, T. Y., Sim, E. Y. S., 

Lim, P. N. and Clarke, C. (2012). 

Biotransformation of rice husk into 

organic fertilizer through vermicomposting. 

Ecological Engineering 41, 

60-64. 


Mane, T. T. and Raskar Smita, S. 

(2012). Management of Agriculture 

Waste from Market yard through 

Vermicomposting. Research Journal 

of Recent Sciences 1, 289-296. 

Minnich, J. (1977). The Earthworm 

book. Rodale press. Emmaus, P. A. 

pp. 372. 

Mulongoy, K. and Bedoret, A. (1989). 

Properties of worm casts and surface 

soil under various plant covers in the 

humid tropics. Soil Biology and Biochemistry 

21, l97-203. 

Nair, J., Sekiozoic, V. and Anda, M. 

(2006). Effect of pre-composting on 

vermicomposting of kitchen waste. 

Bioresource Technology 97, 2091– 

2095. 

Nambiar, K. K. M. (1994). Soil Fertility 

and Crop Productivity under Longterm 

Fertilizer Use in India. ICAR 

Publication, New Delhi. 

Rautaray, S. K., Ghosh, B. C. and Mittra, 

B. N. (2013). Effect of fly ash, 

organic wastes and chemical fertilizers 

on yield, nutrient uptake, heavy 

metal content and residual fertility in 

a rice–mustard cropping sequence 

under acid lateritic soils. Bioresource 

Technology 90, 275–283. 

Seetha Devi, G., Karthiga, A., Susila, S. 

and Muthunarayanan, V. (2012). 

Bioconversion Of Fruit Waste Into 

Vermicompost By Employing Eudrillus 

eugeniae And Eisenia fetida. 

International Journal of Plant, Animal 

and Environmental Sciences 

2(4), 245-252. 

Shaymaa, M., Ahmed, H., Norli, I., 

Morad, N. and Hakimi, I. M. 

(2010). Removal of Aluminium, 

Lead and Nickel from Industrial 

Sludge via Vermicomposting Process. 

World Applied Sciences Journal 

10(11), 1296-1305. 

Sibi, G. and Manpreet, K. (2011). Management 

of vegetable waste by Vermicomposting 

Technology. American-Eurasian 

Journal of Agricultural 

& Environmental Sciences 10(6), 

983-987. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 221




Smith, D. H., Wells, M. A., Porter, D. 

M. and Fox, F. R. (1993). Peanuts. 

In: Nutrient deficiencies & toxicities 

in crop plants, Bennett, W. F. (eds.). 

St. Paul, M. N. The American Phytopathological 

Society. pp. 105-110. 

Sudhakar, G., Lourduraj, A. C., 

Rangasamy, A., Subbian, P. and 

Velayutham, A. (2002). Effect of 

Vermicompost Application On The 

Soil Properties, Nutrient Availability, 

Uptake And Yield Of Rice - A Review. 

Agricultural Reviews 23(2), 

127 – 133. 

Suthar, S. (2009). Vermicomposting of 

vegetable-market solid waste using 

Eisenia fetida: Impact of bulking material 

on earthworm growth and decomposition 

rate. Ecological Engineering 

35, 914-920. 

Syers, J. K., Sharpley, A. N. and Keeney, 

D. R. (1979). Cycling of nitrogen 

by surface-casting earthworms in a 

pasture ecosystem. Soil Biology and 

Biochemistry 11, 181-185. 

Tahir, T. A. and Hamid, F. S. (2012). 

Vermicomposting of two types of coconut 

wastes employing Eudrilus eugeniae: 

a comparative study. International 

Journal of Recycling of Organic 

Waste in Agriculture 2, 1-7. 

Whiston, R. A. and Seal, K. J. (1988). 

The occurrence of cellulases in the 

earthworm Eisenia foetida. Biological 

Wastes 25, 239-242. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 222



Bacterial Endophytes as Biofertilizers and Biocontrol 

Agents for Sustainable Agriculture 

Amrutha V. Audipudi 1, *, Bhaskar V. Chakicherla 2 and Shubhash Janardhan Bhore 3 

1 Department of Microbiology, Acharya Nagarjuna University, Nagarjuna Nagar 522510, 

Andhra Pradesh, India; 2 Department of Botany, V.R College, Affiliated to V.S. University, 

Nellore 524002, A.P, India; 3 Department of Biotechnology, Faculty of Applied Sciences, 

AIMST University, Bedong-Semeling Road, 08100 Bedong, Kedah Darul Aman, Malaysia; 

*Correspondence: audipudiamrita@gmail.com; Tel.: +91 9440995842 

Abstract: Plant health and development promoted through microbial interactions have been 

the main motif for sustainable agriculture. To find new and beneficial endophytic microorganisms 

from plants of different ecosystems is highly considerable because endophytic bacteria 

are not restricted to a specific species but showed a wide range of host diversity. Endophytes 

colonize an ecological niche similar to that of phytopathogens and baffle disease 

development through endophyte-mediated de novo synthesis of novel compounds and antifungal 

metabolites. Seedling emergence, plant growth and plant’s establishment under adverse 

conditions can be accelerated by endophytes. Endophytic Pseudomonas sp. and Bacillus 

sp. recorded a significant improvement in morphological characters and ISR in the 

plantlets. Endophyte fortifies plant cell wall strength, alters host physiology and metabolic 

responses thereby enhance the defense mechanism by the synthesis of different metabolites 

such as phenolic compounds, pathogenicity related protein (PR-1, PR-2, PR-5), oxidative 

stress enzymes (chitinases, peroxidases, polyphenyoxidase, phenyl alanine ammonia 

lyase, Oxidase and/or chalcone synthase, phytoalexins etc. The contribution of endophytes 

as biological fertilizers is highly significant because of their metabolic acclimatization in 

the host plant with mutualism. Endophytic microbes must be properly selected, combined 

and formulated with respect to the environmental conditions for development of efficient 

endophytic biofertilizers that can very much contribute to the enhanced food production in 

the world. This review aims to provide an overview on bacterial endophytes and their potential 

application for sustainable agriculture. 

Keywords: Agriculture; biocontrol; endophytes; endophytic bacteria; induced systemic resistance; 

phytopathogens; plant growth promotion; sustainable development 


Agriculture and agri-food sector is 

expected to move towards environmentally 

sustainable development by increasing 

the productivity and protecting the natural 

resource base for future generations. 

Greater productivity and competitiveness 

are anticipated to come from increased 

efficiency through the acquisition and 

management of new biotechnologies and 

crop production strategies (Stutz et al., 

1999). Plant–microbe interactions that 

promote plant health and development 

have been the subject of considerable 

study for sustainable agriculture. A renewed 

interest in the internal colonization 

of healthy plants by non- rhizobium bacteria 

and exploitation of their potential in 

agriculture becomes apparent (Fahey et 

al., 1991; Kloepper et al., 1992; Turner et 

al., 1993). Rhizosphere bacteria which 

can easily colonize the internal roots and 

stems are major source of endophytes 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 223


Bacterial Endophytes as Bio fertilizers and Bio control Agents… 

(Figure 1) and often phyllosphere bacteria 

may also be a source of endophytes 

(Hallmann et al., 1997; Germaine et al., 

2004). Though each individual plant exists 

on the earth is a host to one or more 

endophytes (Strobel et al., 2004), only a 

few of these plants have ever been completely 

studied to their endophytic biology 

and to find novel beneficial endophytic 

microorganisms. 

2. Endophytic bacterial diversity in the 

host plants 

Endophytic bacteria were isolated 

from both monocotyledonous and dicotyledonous 

plants (Table 1) ranging from 

woody trees to herbaceous crops such as 

prairie plants, agronomic crops, tuberous 

crops and grasses (McInroy and 

Kloepper, 1995; Gutiérrez-Zamora and 

Martínez-Romero, 2001; Germida and 

Audipudi et al. 

Siciliano, 2001; Zinniel et al., 2002; Sessitsch 

et al., 2002; Dent et al., 2004; Sun 

et al., 2008). More than 200 bacterial 

genera from 16 phyla have been reported 

as endophytes since the first report of endophytic 

bacteria (Samish et al., 1963a) 

and include both culturable and unculturable 

bacteria (Sun et al., 2008; Berg 

and Hallmann, 2006; Mengoni et al., 

2009; Manter et al., 2010; Sessitsch et al., 

2012). Most predominantly studied endophytes 

belong to three major phyla (Actinobacteria, 

Proteobacteria and Firmicutes) 

and include members of Streptomyces 

(Suzuki et al., 2005), Azoarcus (Krause et 

al., 2006), Acetobacter (renamed as Gluconobacter) 

(Bertalan et al., 2009), Pseudomonas, 

Serratia, Stenotrophomonas 

(Ryan et al., 2009), Enterobacter (Pedrosa 

et al., 2011). Bacteria which are 

ubiquitous in the soil/ rhizosphere represent 

the main source of endophytic 

Figure 1: Plant colonization routes by endophytic bacteria. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 224




Table 1: Monocotyledonous and dicotyledonous plants that harbor bacterial endophytes 

(Sudhir and Audipudi, 2014) 

Plant species Endophytes Reference 

Rice (Oryza sativa L.) 

Potato (Solanum tuberosum 

L.) tuber 

Red clover (Trifolium 

pratense L.) 

Rough lemon (Citrus 

jambhiri Lush.) 

Rhizobium leguminosarum, 

Pseudomonas 

Azorhizobium caulinodans, 

Sphingomonas paucimobilis 

Chromobacterium violaceum, 

Sphingobacterium sp. 

Bradyrhizobium japonicum 

Serratia marcescens 

Serratia sp. 

Agrobacterium, Azorhizobium, 

Azospirillum, Bacillus, 

Actinomyces, Agrobacterium, 

Alcaligenes, Arthrobacter, 

Bacillus, Capnocytophaga, 

Cellulomonas, Clavibacter, 

Comamonas, Corynebacterium, 

Curtobacterium, Deleya, 

Enterobacter, Erwinia, 

Flavobacterium, Kingella, 

Klebsiella, Leuconostoc, Micrococcus, 

Pantoea, Pasteurella, 

Photobacterium, Pseudomonas, 

Psychrobacter, Serratia, 

Shewanella, Sphingomonas, 

Vibrio, Xanthomonas, Sinorhizobium 

meliloti, Paenibacillus 

odorifer, Enterobacter asburiae 

Acidovorax, Agrobacterium, 

Arthrobacter, Bacillus, Bordetella, 

Cellulomonas, Comamonas, 

Curtobacterium, Deleya, 

Enterobacter, Escherichia, 

Klebsiella, Methylobacterium, 

Micrococcus, 

Pantoea, Pasteurella, 

Phyllobacterium, Pseudomonas, 

Psychrobacter, Rhizobium, 

Serratia, Sphingomonas, Variovorax, 

Xanthomonas 

Achromobacter, Alcaligenes 

Moraxella, Acinetobacter, 

Actinomyces, Arthrobacter, Bacillus, 

Citrobacter, Corynebacterium, 

Enterobacter, Flavobacterium, 

Klebsiella, Providencia, 

Pseudomonas, Serratia, Vibrio, 

Yersinia, Rickeltsia-like 

Yanni et al., 1997, 

Engelhard et al., 2000, 

Phillips et al., 2000, 

Chantreuil et al., 2000 

Gyaneshwar et al., 2001 

Sandhiya et al., 2005 

Reddy et al., 1997; 

Stoltzfus et al., 1997 

Hollis, 1951; de Boer I 

Copeman, 1974; 

Sturz, 1995; Sturz 

& Matheson, 1996; 

Sturz et al., 1998 

Reiter et al., 2003 

Asis and Adachi, 2003 



Feldman et al., 1977; 

Gardner et al., 1982 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 225





Grapevine (Vitis spp.) 

Soybean 

Maize 

Citrus plants 

Carrot 

Sugar cane (Saccharum 

officinarum L.) 

Corn (Zea mays L.) 

Bacillus, Clavibacter, Comamonas, 

Curtobacterium, Enterobacter, 

Klebsiella, Moraxella, 

Pantoea, Pseudomonas, Rahnella, 

Rhodococcus, Staphylococcus, 

Xanthomonas 

Enterobacter sakazakii 

Enterobacter agglomerans 

Erwinia sp. 

Klebsiella oxytoca 

Klebsiella pneumoniae 

Pseudomonas citronellolis 

Burkholderia pickettii 

Enterobacter spp. 

Arthrobacter globiformis 

Microbacterium testaceum 

Bacillus spp. 

Curtobacterium flaccumfaciens 

Nocardia sp. 

Methylobacterium mesophilicum 

Pseudomonas putida 

Pseudomonas fluorescens 

Staphylococcus saprophyticus 

Klebsiella terrigena 

Herbaspirillum rubrisulbalbicans, 

Acetobacter, Herbaspirillum 

Bacillus, Burkholderia, 

Corynebacterium, Enterobacter, 

Klebsiella, Pseudomonas 

Bell et al., 1995a; 1995b 

Kuklinsky-Sobral et al., 

2004 

McInroy and Kloepper, 

1995 

Chelius and Triplett, 2000a 

Zinniel et al., 2002 

Araujo et al., 2001, 2002 

Surette et al., 2003 

Olivares et al., 1996 

Cavalcante & Döbereiner, 

1988; 

Gillis et al., 1989; 

Boddey et al., 1991; 

Dong et al., 1994; 

Ohvares et al., 1997 

Lalande et al., 1989; 

Fisher et al., 1992; 

Mclnroy & Kloepper, 1995; 

Palus et al., 1996 

Wheat Streptomyces Coombs and Franco, 2003a 

Alfalfa (Medicago sativa Erwinia-likc, Pseudomonas Gagne et al., 1987 

L.) 

Coffee (Coffea arabica Acetobacter Jimenez-Salgado et al., 

L.); Cameroon grass 

1997; Reis et al., 1994 

(Pennisetum purpureum 

Schumach) 

Cotton (Gossypium hirsutum 

L.) 

Cucumber (Cucumis sativis 

L.) 

Agrobacterium, Bacillus, 

Burkholderia, Clavibacter 

Erwinia, Serratia, 

Xanthomonas 

Agrobacterium, Arthrobacter, 

Bacillus, Burkholderia, 

Misaghi & Donndelinger, 

1990; 

Mclnroy & Kloepper, 

1995 

Mclnroy & Kloepper, 

1995 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 226





Hybrid spruce {Picea 

glauca x Engelmannii) 

Kallar grass (Leptochloa 

fusca [L.] Kunth) root 

Lodgepole pine (Pinus 

contorta Dougl. Ex Loud) 

root 

Sorghum bicolor L. 

Moench shoot 

Sugar beet (Beta vulgaris 

L.) 

Teosinte (Zea luxurians 

Itins 

and Doebley) stem 

Banana 

Chryseobacterium, Enterobacter, 

Pseudomonas, Stenotrophomonas 

Bacillus, Pseudomonas, 

Phyllobacterium, actinomycetes, 

O'Neill et al., 1992; 

Chanway et al., 1994 

Staphylococcus 

Azoarcus Reinhold et al., 1986; 

Reinhold-Hurek et al., 

1993 

Bacillus Shishido et al., 1995 

Herbaspirillum James et al., 1997 

Bacillus, Corynebacterium, Erwinia, 

Jacobs et al., 1985 

Lactobacillus; Pseudomonas, 

Xanthomonas 

Klebsiella Palus et al., 1996 

Azospirillum brasilense 

Citrobacter sp. 

Weber et al., 1999 

Martínez et al., 2003 

Marigold 

Kocuria varians Sturz and Kimpinski, 2004 

Microbacterium esteraromaticum 

Banana, pineapple Azospirillum amazonense Weber et al., 1999 

Sugarcane, coffee 

Gluconacetobacter diazotrophicus 

Cavalcante and Döbereiner, 

1988; Jiménez-Salgado et 

al., 1997 

Scots pine, citrus plants Methylobacterium extorquens Araujo et al., 2002; Pirttilä 

et al., 2004 

Carrot, rice 

Rhizobium (Agrobacterium) Surette et al., 2003 

radiobacter 

Kallar grass, rice Azoarcus sp. Reinhold-Hurek et al., 1993, 

Engelhard et al., 2000; 

Yellow lupine, citrus Burkholderia cepacia Araujo et al., 2001; Barac et 

plants 

al., 2004 

Banana, pineapple, rice Burkholderia sp. Araujo et al., 2001; Barac et 

al., 2004 

Sugarcane, rice, maize, 

sorghum, banana 

Herbaspirillum seropedicae Olivares et al., 1996; Weber 

et al., 1999 

Banana, rice, maize, sugarcane 

Klebsiella variicola Rosenblueth et al., 2004. 

Citrus plants, sweet potato 

Pantoea agglomerans Araujo et al., 2001, 2002; 

Asis and Adachi 2003 

Rice, soybean Pantoea sp. Kuklinsky-Sobral et al., 

2004; Verma et al., 2004 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 227





Marigold (Tagetes spp.), 

carrot 

Alfalfa, carrot, radish, 

tomato 

Dune grasses (Ammophila 

arenaria and 

Elymus mollis) 

Maize, carrot, citrus 

plants 

Pseudomonas chlororaphis Sturz and Kimpinski, 2004; 


Salmonella enterica Cooley et al., 2003; Guo et 

al., 2002; Islam et al., 2004 

Stenotrophomonas Dalton et al., 2004 

Bacillus megaterium Araujo et al., 2001; McInroy 

and Kloepper, 1995; Surette 

et al., 2003 

Clostridium Miyamoto et al., 2004 

Grass Miscanthus sinensis 

Wheat, Scots pine Mycobacterium sp. Conn and Franco 2004; Prittilä 

et al., 2005 

Citrus plants, maize Enterobacter cloacae Araujo et al. 2002; Hinton et 

al., 1995 

Lettuce Escherichia coli Ingham et al., 2005 

Wheat, sweet potato, rice Klebsiella sp. Engelhard et al., 2000; 

Iniguez et al., 2004; 

colonizers (Hallmann and Berg, 2006). 

Earlier studies reported that endophytic 

population of Bacillus polymyxa inside 

the root is highly specific and less diverse 

than the root surface population, though 

there are different populations of Bacillus 

polymyxa in soil, rhizosphere, and rhizoplane 

of wheat field. It clearly indicates 

that endophytes appear to be originated 

from rhizosphere or rhizoplane (Mavingui 

et al., 1992; Germida et al., 1998). 

Endophytic genera isolated from 

the interior of ginseng roots cultivated in 

three different areas showed marked differences 

in microbial community (Cho et 

al. 2007). Ryan et al., (2008) reported 

that endophytic bacteria in a single plant 

host not restricted to a single species but 

comprise several genera and species. Endophytic 

bacteria belong to one genera 

was not only restricted a specific host but 

also showed wide range of host diversity. 

Bacteria belonging to the genera Bacillus 

and Pseudomonas were identified as predominantly 

occurring endophytes (Seghers 

et al., 2004). 

Jha and Kumar (2007) isolated 

and characterized 10 endophytic diazotrophic 

bacteria from surface-sterilized 

roots and culm of Typha australis. Long 

et al., (2008) isolated 77 endophytic bacteria 

from roots, stems and leaves of Solanum 

nigrum grown in two different native 

habitats of Jena and Germany. Aravind 

et al., (2009) isolated 80 endophytic 

bacteria from different varieties of Piper 

nigrum L. cultivated in different regions 

of India. Bhore and Sathisha (2010) isolated 

115 putative cultivable endophytic 

bacteria from leaves of 72 different plant 

species collected from northern part of 

Peninsular Malaysia. Magnani et al., 

(2010) isolated 32 endophytic bacteria 

from Brazilian sugarcane. A total of 264 

colonies of endophytic bacteria were reported 

from leaves and roots of young 

radish (Seo et al., 2010). 

Pereira et al., (2011) investigated 

endophytic bacterial diversity associated 

with the roots of maize through culturedependent 

and culture-independent methods. 

Enterobacter, Erwinia, Klebsiella, 

Pseudomonas, and Stenotrophomonas 

genera belong to γ-Proteobacteria were 

reported as predominant group. Based on 

culturable component of the bacterial 

community genus Bacillus belong to Firmicutes 

was identified as another predominant 

group, while Achromobacter, 

Lysinibacillus, and Paenibacillus genera 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 228



were rarely found in association with the 

roots. 

Yang et al. (2011) isolated 45 

strains from stems and 27 strains from 

leaves of tomato as endophytic bacteria 

and reported that endophytic efficiency of 

bacteria in stem was higher than leaves. 

Out of 72 endophytic bacteria isolated 

from tomato on of the W4 was identified 

as Brevibacillus brevis W4 on the bases 

of 16S rDNA gene analysis and Biolog 

systemic analysis. Patel et al. (2012) isolated 

and characterized bacterial endophytes 

from root and stem of Lycopersicon 

esculentum. Out of 18 endophytic 

bacteria selected from tomato only one 

isolate HR7 was identified as Pseudomonas 

aeruginosa by 16S rDNA analysis. 

Fisher et al. (1992) found that endophytic 

bacteria appear to be preferentially 

located in the lower part of the stems of 

corn, with a declining gradient running 

from the base to the top of the plant. 

Roots and other below ground tissues 

tend to yield the highest numbers of CFU 

of bacteria compared with above-ground 

tissues is an indicative of the upward path 

of bacteria (Table 2) from the roots into 

the stem during plant development 

(McInroy and Kloepper, 1994; Sturz et 

al., 1997a; Rosenblueth and Martínez- 

Romero, 2004; Gagné et al. (1987). 


Internal colonization of plant tissues 

by bacteria is primarily intercellular and 

xylem vessels act as reservoirs of internal 

populations of bacteria (Gardner et al., 

1982; Jacobs et al., 1985; Sumner, 1990; 

Frommel et al., 1991; Kloepper et al., 

1992; Bell et al., 1995; Sprent and James 

1995; Reinhold-Hurek and Hurek 1998a). 

Intracellular endophytic bacteria have also 

been found in the cytoplasm and vacuoles 

of epidermal cells (Quadt - Hallman 

and Kloepper, 1996), root hairs (Vance, 

1983), and parenchyma cells (Jacobs et 

al., 1985). 

3. Endophytes in agriculture 

The endophyte-plant interaction is one 

of the least studied biochemical systems 

in nature. Plants host to one or more endophytic 

microorganisms include fungi, 

bacteria and actinomycetes. Endophytes 

reside in the tissues beneath the epidermal 

cell layers (Stone et al., 2000) are transiently 

symptomless and inconspicuous 

with several beneficial effects on plants 

(Hallmann et al., 1997). More than thousand 

endophytic bacteria were reported in 

the last decade (Table 3). Like plant 

growth promoting Rhizobacteria (PGPR), 

Table 2: Population densities of endophytic bacteria in host tissues 

Part of the plant Colony forming Unit (CFU) Reference 

Alfalfa xylem tissue 

6.0 × 10 3 to 4.3 × 104 per g Gagné et al., 1987 

Cotton xylem tissue 1 × 102 to 11 × 103 per g Misaghi and Donndelinger, 1990 

Sugar beet tissue 3.3 × 103 to 7.0 × 105 per g Jacobs et al., 1985 

Potato tubers 0 to 1.6 × 104 per g De Boer and Copeman, 1974 

Table 3: Endophytic strains reported in between 2001-2007 (Sudhir, 2014) 

Source No of endophytes Reference 

Indian sugarcane 81 endophytic bacterial strains Suman et al., 2001 

agronomic crop species 853 endophytic bacteria Lodewyckx et al., 2002 

prairie plant species 27 endophytes Zinniel et al., 2002 

Daucus carota 

and Agrobacterium 

360 endophytic strains of 

Pseudomonas and Staphylococcus 


soybean 35 endophytic bacteria Hung et al., 2007 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 229



endophytes also influence the growth of 

plant directly by the production of plant 

growth promoting traits such as IAA production, 

Phosphate solubilization, siderophore 

production, ammonia production, 

nitrogen fixation antagonism against phytopathogens 

and indirectly by induced 

systemic resistance (ISR). Endophytic 

bacteria colonize an ecological niche similar 

to that of phytopathogens, which 

makes them suitable as biocontrol agents 

(Berg et al., 2005a). Endophytic microorganisms 

control plant pathogens (Sturz & 

Matheson, 1996; Duijff et al., 1997; 

Krishnamurthy & Gnanamanickam, 

1997), insects (Azevedo et al., 2000) and 

nematodes (Hallmann et al., 1997, 1998) 

through endophyte-mediated de novo synthesis 

of novel compounds and antifungal 

metabolites. Endophytes can also accelerate 

seedling emergence, promote plant 

establishment under adverse conditions 

(Chanway, 1997) and enhance plant 

growth (Bent & Chanway, 1998). 

Bacterial endophytes stimulate the 

growth of host plant by nitrogen fixation, 

enhancement in the availability of minerals 

and the production of phytohormones 

(Hurek et al. 2002; Iniguez et al. 2004; 

Sevilla et al. 2001). Endophyte mediated 

de novo synthesis of antifungal or antibacterial 

metabolites, siderophores and 

competition for nutrients induce systematic 

resistance in the host to check the 

progress of disease (Sessitsch et al. 

2002a; Sturz et al. 2000). 

4. Endophytic bacteria as bio fertilizers 

Research has been revealed that 

endophyte increase plant growth through 

the improved cycling of nutrients and 

minerals such as phosphate solubilisation 

(Verma et al., 2001; Wakelin et al., 

2004), indole acetic acid production (Lee 

et al., 2004) production of siderophore 

(Costa and Loper, 1994) and supply of 

essential vitamins to plants (Pirttila et al., 

2004). Compant et al. (2005) reported 

that endophytes also influence other beneficial 

effects of host include osmotic 


adjustment, stomatal regulation, modification 

of root morphology, enhanced uptake 

of minerals and alteration of nitrogen 

accumulation and metabolism. Adhikari 

et al.( 2001) reported that endophytic bacterial 

strains are potential in controlling 

the seedling disease of rice and promote 

growth in rice. Application of endophytic 

bacterial strains significantly increased 

the growth parameters viz., pseudostem 

height, girth, number of leaves and physiological 

parameters viz., chlorophyll stability 

index, stomatal resistance and transpiration 

in banana plants both under 

greenhouse and field conditions (Harish, 

2005). 

4.1. IAA production 

According to earlier studies IAA 

production by endophytes can vary 

among different species and isolates and 

it is also influenced by culture condition, 

growth stage and substrate availability. 

Out of 65 endophytes of soybean, 15 isolates 

were positive for IAA production 

produced more than 25 μg/ml of IAA and 

Acetobacter diazotrophicus and Herbaspirillum 

seropedicae found to produce 

IAA in chemically defined culture media. 

Seven out of 10 endophytic isolates of 

Typha australis were positive for IAA 

production (Hung and Annapurna 2004; 

Chen et al., 1998; Jha and Kumar, 2007). 

Two bacterial endophytes of Capsicum 

annuum L. also reported to show plant 

growth promotion and defense against 

phytopathogens along with IAA production. 

Long et al. (2008) reported 1.1 to 

154μg/ml of IAA production by the endophytic 

bacteria isolate from Solanum 

nigrum. Gangawar and Kaur (2009) reported 

15 endophytic isolates of sugarcane 

produced 4 to 19.3 μg/ml of IAA. 

Chilli endophytes are observed to be more 

potential in IAA production than sugarcane 

and similar to Solanum nigrum reported 

(Sudhir 2014). Amrutha et al. 

(2012) reported that Pseudomonas aureginosa 

CEFR3, Bacillus sp CEFR19, 

Curtobacterium oceanosedimentum 

AVSCE3 and Bacillus cereus AVSCE 5 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 230



isolated from different parts of chilli produced 

23µg/ml, 21µg/ml, 111.5µg/ml 

and 125μg/ml of IAA, respectively. It 

was much higher than that of found in 

other reports (Long et al. 2008). 

Harish et al., (2008) assessed the 

plant growth promotion efficacy of 45 

endophytic bacteria isolated from corm 

and root of banana. 12 strains isolated 

from gingseng plant, endophytic P. fluorescens 

WCS365 as biocontrol s bacteria 

isolated from Lycopersicon esculentum 

produced significant amounts of IAA 

(Thamizh Vendan et al., 2010, Patel et 

al., 2012). Three strains isolated from 

sugar beet roots produced indole-3-acetic 

acid (IAA) promote plant growth significantly 

increased plant height, fresh and 

dry weights and number of leaves per 

plant (Long et al., 2008; Yingwu Shi et 

al., 2009). Vetrivelkalai et al., 2010 also 

reported significant enhancement in the 

germination percentage, shoot and root 

length and vigour index of bhendi seedlings 

by seed bacterization. 

4.2. Phosphate solubilization 

Earlier reports revealed endophytic 

bacteria also solubilize phosphate from 

organic or inorganic bound phosphates 

and type of organic acid produced during 

phosphate solubilizaton depends on the 

carbon source utilized as substrate. Highest 

P solubilization has been observed 

when glucose, sucrose or galactose has 

been used as sole carbon source in the 

medium (Khan et al., 2009; Park et al., 

2010). Endophytic bacteria able to solubilize 

inorganic phosphate and extracellular 

tricalcium phosphate effectively in 

presence of glucose (Kuklinsky - Sobral 

et al., 2004; Long et al., 2008; Thamizh 

Vendan et al., 2010; Patel et al., 2012). 

Endophytic Bacillus, Pseudomonas, 

Klebsiella and Acinetobacter are also reported 

as potential phosphate solubilizers 

(Huang et al., 2010). Endophytic Bacillus 

cereus and B. megaterium isolated of 

Ginseng plant also showed significantly 

high P solubilization (ThamizhVendan et 

al., 2010). Endophytic and rhizosphere 


Pseudomonas spp. Isolated from Serbia 

able to solubilize TCP (Stajković et al., 

2011; Djuric et al., 2011; Josic et al., 

2012b). Amrutha et al. (2012) reported 

that Pseudomonas aureginosa CEFR3 

(198ppm/ml), Bacillus sp CEFR19 

(1354ppm/ml) isolated from ripened fruit 

and isolated from green fruit and Bacillus 

cereus AVSCE 5(137ppm/ml) isolated 

from leaf of chilli are able to solubilize 

inorganic phosphate efficiently. Increase 

in the yield of canola by endophytic 

Bacillus sp. was reported by de Freitas 

et al. (1997). Sundara et al. (2002) reported 

that enhancement in available phosphorus 

and yield of sugarcane by application 

endophytic bacteria. Pseudomonas 

spp. are able to increase the growth and 

phosphorus content of maize by endophytes 

(Vyas and Gulatti, 2009). 

4.3. Siderophore 

Researchers reported that endophytic 

fungal siderophore have lower affinity 

than bacteria to sequester iron and deprive 

pathogenic fungi (Whipps, 2001; Loper 

and Henkels 1999). Endophytic bacterial 

isolates associated with hyacinth and 

Genseng plants produce siderophore 

(Jafra et al., 2009; Thamizh Vendan et 

al., 2010). Rajkumar et al., (2010) reported 

that the siderophore play a pivotal role 

in nitrogen fixation under iron deficiency. 

4.4. Nitrogen fixation 

According to earlier studies endophytic 

bacteria better express their nitrogen 

fixation potential inside plant tissues 

due to the lower competition for nutrients 

and protection against high levels 

of O 2 present on the root surface. Many 

diazotrophic bacteria are able to establish 

a symbiotic relationship with plants for 

biological nitrogen fixation (Robson et 

al., 1986; Chisnell et al., 1988; Dekas et 

al., 2009). Earlier studies reveled that endophytic 

diazotrophs constitute only a 

small proportion of total endophytic bacteria 

include Azospirillum lipoferum, 

Klebsiella pnemoniae and Azorhizobium 

caulinadans (Schloter et al., 1994; Bar- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 231



raquio et al,. 1997; Martínez et al., 2003). 

Unlike symbiotic diazotrophes, endophytic 

bacteria are unable to form nodules. 

Gluconacetobacter diazotrophicus was 

identified as first N 2 -fixing endophytic 

bacteria associated with sugarcane stem 

(Cavalcante and Dobereiner, 1988) and 

confirmed by other scientists in USA, 

UK, and Germany and two more N 2 - 

fixing endophytes Herbaspirillum seropedicae 

and H. rubrisubalbicans were 

reported by Boddey et al., (1995). James 

(2000) reported Herbaspirillum sp. as endophytic 

diazotroph in sugarcane and 

rice. Azoarcus sp. in rice and Kallar grass 

and endophytic Klebsiella sp. Kp342 

strain of wheat identified as nitrogen fixers 

(Iniguez et al., 2004). Silva-Froufe 

(2009) reported Glucanoacetobacter diazotrophicus 

as endophytic diazotrophic 

bacteria in sugarcane, sweet potato, and 

pineapple. Endophytic Bacillus species of 

soybean nodule showed potential N fixing 

and reported to improve root growth and 

function, (Bai et al., 2002; Asis et al., 

2004; Matiru and Dakora, 2004). 23 endophytic 

bacteria are identified as potential 

ammonia producers in chilli (Amrutha 

et al., 2012) and reported that Pseudomonas, 

Curtobacterium and Bacillus sp isolated 

from chilli were found to be maximum 

producers of Ammonia. 

4.5. Volatile compounds 

Earlier literature reveled that endophytic 

bacteria can produce a wide 

range of volatiles. Biological function of 

most of these volatiles is not yet understood. 

It is assumed that volatile compounds 

involved in a number of processes 

including cell-cell signaling, inter-species 

signaling, promote plant growth and act 

as microbial inhibiting agents (Wheatley, 

2002; Vesperman et al., 2007; Kai et al., 

2009). Ryu et al., (2003) reported that 

Bacillus sp. produce 2, 3 butanediol and 

acetoin and promote plant growth in Arabidopsis 

thaliana. 38 volatile compounds 

were reported from rhizobacteria (Farag 

et al., 2006). Blom et al. (2011) screened 

42 strains in four different growth media 


and studied growth response of A. thaliana. 

Each strain showed significant volatile-mediated 

plant growth modulation 

and also reported that Burkholderia pyrrocinia 

as significant plant growthpromoter. 

The volatiles indole, 1-hexanol 

and pentadecane promotes growth only 

under stress conditions 

5. Endophytes as biological control 

agents (BCA) 

Endophytes are potential biocontrol 

agents like other biocontrol agents 

such as associative nitrogen fixing PGPB 

on sugarcane (Boddy, 2003) or Burkholderia 

phytofirmans PsJN, non-symbiotic 

endophytic bacteria (Sharma and Nowak, 

1998) endophyte holds potential of BCA 

may be due to self-perpetuating nature of 

endophytes inside the host by colonization 

and being transfer to progeny (Boddy, 

2003). According to Backman et al. 

(1997), the effectiveness of endophytes as 

biological control agents (BCA) is dependent 

on many factors. These factors 

include: host specificity, the population 

dynamics, pattern of host colonization, 

ability to move within host tissues and the 

ability to induce systemic resistance. Certain 

endophytic bacteria trigger induced 

systemic resistance (ISR) which is phenotypically 

similar to systemic-acquired resistance 

(SAR). SAR develops when 

plants successfully activate their defense 

mechanism in response to primary infection 

by a pathogen and induces a hypersensitive 

reaction in the form of a local 

necrotic lesion of brown desiccated tissue 

(van Loon et al., 1998). ISR is effective 

against different types of pathogens bu 

differs from SAR because in ISR the inducing 

bacterium does not cause visible 

symptoms on the host plant (van Loon et 

al., 1998). 

The first record of an endophyte 

affecting a plant disease was reported by 

Shimanuki (1987) who showed that Phleum 

pratense plants infected with the 

Epichloe typhina were resistant to the 

fungus Cladosporium phlei. In some cas- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 232



es, endophytes also accelerate seedling 

emergence and promote plant establishment 

under adverse conditions and enhance 

plant growth and development (Pillay 

and Nowak, 1997). Several endophytic 

bacterial species including Achromobacter 

sp., Acinetobacter baumannii, A. 

lwoffii, Alcaligenes, Moraxella sp., Alcaligenes 

sp., Arthrobacter sp., Bacillus sp., 

Burkholderia cepacia, Citrobacter freundii, 

Corynebacterium sp., Curtobacterium 

flaccumfaciens, Enterobacter cloacae, E. 

aerogenes, Methylobacterium extorquens, 

Pantoea agglomerans, Pseudomonas aeruginosa, 

and Pseudomonas sp. isolated 

from the xylem of lemon roots (Citrus 

jambhiri) have been reported as antagonistic 

bacteria against root phytopathogens 

(Araújo et al., 2001; Lima et al., 

1994). 

Cabbage treated with endophytes 

did not reach the economic threshold for 

the disease approximately 50 days after 

inoculation with Xanthomonas campestris 

pv. Campestris due to the induction of 

defense mechanisms (Jetiyanon, 1994). 

Several bacterial endophytes have been 

reported to support growth and improve 

the health of plants (Hallmann et al., 

1997). Erwinia caratovora, for example, 

is inhibited by numerous endophytic bacteria, 

including several strains of Pseudomonas 

sp., Curtobacterium luteum, and 

Pantoea agglomerans (Sturz et al., 1999). 

Wilhelm et al. (1997) demonstrated 

that Bacillus subtilis strains isolated 

chestnut trees exhibit antifungal effects 

against Cryphonectria parasitica causing 

chestnut blight. The ability of endophytic 

bacteria to inhibit pathogen has been decreased 

in potato tubers due to deep colonization 

interior to the plant host (Struz et 

al., 1999). Barka et al. (2002) demonstrated 

the ability endophytes to colonize 

in divergent hosts. Pseudomonas sp. an 

onion endophyte colonized in grape vine 

inhibited Botrytis cinerea Pers. and promoted 

growth in grapevines. Colonization 

of multiple hosts has also been observed 

with other species of endophytes 

such as Pseudomonas putida 89B-27 and 


Serratia marcescens 90-166 reduced Cucumber 

Mosaic Virus (CaMV) in tomatoes 

and cucumbers (Raupach et al., 

1996), anthracnose and Fusarium wilt in 

cucumber (Liu et al., 1995). 

Sixty one (61) endophytic bacteria 

isolated from potato stem tissues were 

identified as effective biocontrol agents 

against Clavibacter michiganensis subsp. 

Sepedonicus (Sturz et al., 1999). Bacillus 

mycoides BacJ and Bacillus pumilis 203-7 

isolates from different host plants suppressed 

Cercospora leaf spot in sugar 

beet (Bargabus et al., 2002: 2004). Araujo 

et al. (2002) reported Curtobacterium 

flaccumfaciens, citrus endophyte help 

citrus plants to better resist against the 

pathogenic infection of Xylella fastidiosa. 

Endophytes isolated from potato 

plants produce antibiotics and siderophore 

and showed antagonismic against fungal 

pathogens (Berg et al., 2005a; Sessitsch 

et al., 2004) and bacterial pathogens Erwinia 

and Xanthomonas (Sessitsch et al., 

2004). Of 2,648 bacterial isolates from 

the rhizosphere, phyllosphere, endosphere, 

and endorhiza, only Serratia 

plymuthica root endophyte was a highly 

effective against Xanthomonas sp. in 

Brassica seeds (Berg et al., 2005b). 

Endophytic actinobacteria also 

show effective antagonism against the 

pathogenic fungus Gaeumannomyces 

graminis of wheat (Coombs et al. 2004).. 

A number of endophytic actinobacteria 

isolated by culture dependent methods 

belong to the genera of Streptomyces, Microbispora, 

Micromonospora, and Nocardioides 

(Coombs and Franco, 2003a) 

were capable of suppressing Rizoctonia 

solani, Pythium spp., and Gaeumannomyces 

graminis var tritici, fungal pathogens 

of wheat in vitro and in planta, (Coombs 

et al., 2004). 

Aravind et al. (2009) reported against. 

Native endophytes IISRBP 35, IISRBP 25 

and IISRBP 17 isolated from black pepper 

exhibited 70% disease suppression of 

Phytophthora capsici in black pepper in 

greenhouse trials. The intimate relationship 

between endophytic bacteria and 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 233



their hosts make the endophytes as natural 

candidates for selection as biocontrol 

agents with high level of competition 

(Chen et al., 1995; Van Buren et al., 

1993). 

Twenty two (22) endophytic bacteria 

isolated from different parts of chilli 

plant showed antagonism against Colletotrichum 

capsici, C. gloeosporioides and 

C. acutatum (Amrutha et al., 2014). Antagonism 

of endophytic bacteria against 

Clavibacter michiganensis subsp. 

sepedonicum cause rot on tomato(Van 

Buren et al., 1993), Pseudomonas chlororaphis, 

P. fluorescens, P. graminis, P. 

putida, P. tolaasii and P. veronii agaist 

bacterial pathogens (Chen et al., 1995; 

Adhikari et al., 2001) Invitro inhibition of 

various fungal pathogens by Bacillus subtilis 

ME488nd (Chung et al., 2008) hace 

also been reported. 

6. Induced systemic resistance (ISR) 

Endophytes have a natural and intimate 

association with plants because 

the internal tissues of plant provide relatively 

uniform and protected environment 

and favors endophytic bacteria to be a 

potential agent of ISR (Chen et al. 1995). 

Viswanathan (1999) reported that application 

of endophytic strain is more beneficial 

in vegetatively propagated crops like 

banana, sugarcane and tapioca for inducing 

systemic resistance and also reported 

that endophytic P.fluorescens strain EP1 

isolated from stalk tissues of sugarcane 

induced systemic resistance against red 

rot caused by Colletotrichum falcatum 

(Viswananthan and Samiyappan ,1999a). 

van Loon (2007) reported that the phenomenon 

of ISR has been noted to be exhibited 

both associative and endophytic 

bacteria. ISR triggered by endophytes fortifies 

plant cell wall strength, alters host 

physiology and metabolic responses and 

enhance synthesis of plant defense chemicals 

such as phenolic compounds, pathogenicity 

related protein (PR-1, PR-2, PR- 

5), ROS enzymes (chitinases, peroxidases, 

polyphenyoxidase, phenyl alanine 


ammonia lyase, , oxidase and/or chalcone 

synthase) and phytoalexins to protect the 

host plant from future infections (Nowak 

and Shulaev, 2003). Elicitors include lipopolysaccharides, 

flagella, siderophore, 

antibiotics, VOCs or quorum-sensing signals 

produced by endophytic bacteria 

elicit ISR in plants (Van Loon, 2007). 

ISR activated by PGPB is mediated by 

jasmonate or ethylene in majority of 

plants. Thickening of the outer tangential 

and outermost part of the radial side of 

the first layer of cortical cell walls was 

also noticed in tomato roots with the 

treatment of endophytic P. fluoresces 

WCS417 (Duijff et al.,1997). 

Application of endophytic bacteria 

by stem injection in cotton plants reduced 

the root rot caused by Rhizoctonia solani 

and vascular wilt caused by F.oxysporum 

f. sp. vasinfectum (Chen et al., 1995). 

Pleban et al. (1995) reported that endophytic 

bacteria move upward and downward 

from the point of application before 

colonizing the internal tissues and check 

the entry of F. solani in cotton and Sclerotium 

rolfsii in beans. Application of 

Pseudomonas florescence prevented the 

entry Pythium ultimum in the roots of pea, 

P. florescence restrict the growth of 

Fusarium oxysporum f. sp. pisi and 

P.ultimum in pea plant (Benhamou et al., 

1996b, 1998 ). Pseudomonas strain 63-28 

also induced resistant in tomato against F. 

oxysporum f. sp. radicislycopersici 

(M’Piga et al., 1997). Inoculation of endophytic 

Pseudomonas denitrificans 

strain 1-15 and P.putida strain 5-48 protected 

the oak tree against Ceratocystis 

fagacearum (Brooks et al., 1994) .Recent 

reports have been reviewed mechanisms 

of ISR induced by endophytic Psuedomonas 

(Jankiewicz and Koltonowicz, 2012). 

Bacillus cereus AR156 in A.thaliana 

(Niu et al., 2011) and different endophytic 

bacillus sp. from vegetable crops induced 

systemic resistance . Combination 

of three endophytic isolates resulted in 

significant growth promotion and ISR in 

tomato, bell pepper, cucumber and tobacco 

(Kloepper et al., 2004). Mixed formu- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 234



lation of B. subtilis GB03, B. amyloliquefaciens 

IN937a and B. subtilis IN937b 

together with chitosan in tomato and different 

endophytic bacillus sp. isolated 

from vegetable crops induced systemic 

resistance in Theobroma cacao seedlings. 

Harish et al. (2008) reported mixed formulation 

of rhizobacterial and endophytic 

bacterial (EPB5 + EPB22 + Pf1 + CHA0) 

was significantly effective in suppressing 

Banana Bunchy top virus under field conditions. 


Microorganisms with phytopathogenic 

antagonism act as Biocontrol agents 

(BCA). Most of the biocontrol agents 

have not fulfilled their initial promise because 

of poor rhizosphere competence. 

The failure of BCA being attributed the 

difficulties in long-term culture. It would 

obviate the need for selecting bacterial 

types with high levels of rhizosphere 

competence and successful seed or root 

bacterization treatments before or at 

planting (Schroth et al., 1984; Weller, 

1988). The intimate relationship between 

endophytic bacteria and their hosts make 

the endophytes as natural candidates as 

biocontrol agents with high level of competence 

(Chen et al., 1995; Van Buren et 

al., 1993). 

Selection of endophytic bacteria that 

can elicit plant growth promotion and ISR 

in plants and research on such endophytes 

in understanding plant responses that occur 

during the signal transduction pathways 

that culminate in disease protection 

is essential to delineate the pathosystems. 

In many cases, elicitation of ISR by endophytic 

bacilli is associated with increased 

plant growth, and the relationship 

between ISR and growth promotion 

should be further investigated. Elucidation 

of specific bacterial determinants that 

account for elicitation of ISR is just beginning, 

and further work is needed to 

understand why one strain of a given bacterial 

species can elicit ISR while another 

strain of the same species cannot. The 


principle behind the plant and endophytic 

interaction to elicit ISR or plant growth 

promotion and development of endophytic 

bioformulations will be an index of 

growing scientific knowledge in agriculture 

and horticulture. 


According to the research conducted 

so far, the use of chemical fertilization 

is necessary because biological fertilization 

has not yet proven to be good 

enough in providing complete plant nutrient 

requirements. It has been indicated 

that about less than 50% of chemical fertilizers 

is absorbed by plant and the rest 

would not be accessible by plant as it is 

subjected to leaching, runoff, and emission 

from the soil surface. Hence, the use 

of biological fertilizers as supplementary 

fertilization to chemical fertilization is 

necessary with the above mentioned advantages. 

Accordingly, the right and 

proper application of chemical and biological 

fertilization is very much dependent 

on realizing the interactions between 

soil, plant and microorganisms. Endophytic 

bacteria a big help to plant and the 

environment as they own great abilities 

that collectively enhance plant growth 

and ISR. Among such abilities, enhanced 

nutrient uptake by plant is also of great 

importance; in the presence of soil microbes, 

plant absorbs higher amounts of 

nutrients and less risk of environmental 

pollution is likely. Some of the most important 

functions of endophytic bacteria 

were reviewed in this article. However, 

the particular emphasis has been on the 

use of endophytic bacteria for biological 

fertilization and biological control of phytopathogens. 

PGPR bio-fertilization is a 

very common method because of its rapid 

effects on plant growth and yield production. 

But there are issues regarding the 

use of PGPR fertilizers, as their competence 

in soil and interaction with plant 

vary with the rhizosphere environment. 

Since the endophyte is metabolically acclimatized 

to the host plant with mutual- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 235



ism indicate the contribution of endophytes 

as biological fertilizer is better option 

in comparison to other fertilizers. For 

the development of efficient endophytic 

bacterial biofertilizers, the microbes must 

be properly selected, combined and formulated 

with respect to the agricultural 

and environmental conditions. 


Authors are thankful to UGC New 

Delhi, India for financial assistance for 

research project, F. No. 40-132-2011(SR). 

Authors are also thankful to the central 

instrumentation centre of Acharya Nagarjuna 

Univeristy, Guntur, Andhra Pradesh, 

India. 

References 

Adhikari, T. B., Joseph, C. M., Yang, 

G., Phillips, D. A. and Nelson, 

L. M. ( 2001). Evaluation of bacteria 

isolated from rice for plant 

growth promotion and biological 

control of seedling disease of rice. 

Can. J. Microbiol., 47, 916-924. 

Agrios, G. N. (2005). Plantpathology. 5th 

ed. New York, Academic Press. 

Alschul, S. F., Gish, W., Miller, W., 

Myers, E. W. and Lipman, D. J. 

(1990). Basic local alignment 

search tool. J. Mol. Biol., 215, 

403-410. 

Amrutha V. Audipudi, Sudhir, A., 

Pradeep Kumar, N. and Chowdappa, 

P. (2014). Plant growth 

promoting potential of a novel endophytic 

Curtobacterium CEG: 

Isolation, evaluation and formulation. 

Annals of Biological Research, 

5, 15-21. 

Aneja, K. R. (2006) Experiments in 

Microbiology”. Plant Pathology 

and Biotechnology, 4th Edition, 

New Delhi, 245-275. 

Aravind,R., Kumar,A. and Eapen, 

S.J. (2012). Pre-plant bacterization: 

a strategy for delivery of 

beneficial endophytic bacteria and 


production of disease free plantlets 

of black pepper (Piper nigrum 

L.). Arch. Phytopathol. Plant Protection., 

48,1–12. 

Arshad,M. and Frankenberger, W. T. 

(1991).Microbial production of 

plant hormones. In: Keister BD, 

Cregan PB (eds) The rhizosphere 

and plant growth. Kluwer Academic 

Publishers, Dordrecht, The 

Netherlands, 327-334. 

Asis C. A.J. R., Adachi, K. and 

Shoichiro Akao. (2004).N2 fixation 

in sugarcane and population 

of N2 fixing endophytes in stem 

apoplast solution. Philippine J. 

Crop Sci., 29, 45-58. 

Astchul, S. F., Gish, W., Miller, W., 

Myers, E. V. and Lipman, D. 

(1990). Basic lcal alignment tooll. 

J. Mol. Biol., 215, 403-410. 

Azevedo,J.L., Maccheroni, J. Jr., Pereira, 

O. and Ara, W. L. (2000). 

Endophytic microorganisms: a review 

on insect control and recent 

advances on tropical plants”. 

Electr. J. Biotech., 3, 40-65. 

Backman,P.A.,Wilson, M. and Murphy, 

J. F. (1997).Bacteria for biological 

control of plant diseases. 

Environmentally Safe Approaches 

to Plant Disease Control, CRC/ 

Lewis Press, Boca Raton, F. L., 

95-109. 

Bacon, C. W. and White, J. F. 

(2000).Microbial endophytes. 

Marcel Dekker Inc, New York, 

USA, 199. 

Bacon, C.W., Yates, I. E., Hinton, D. 

M. and Meredith, F. (2001). Biological 

control of Fusarium moniliforme 

in maize. Environ. Health 

Perspect., 109, 325-332. 

Bai, Y. M., D’Aoust, F., Smith, D. L. 

and Driscoll, B.T. 

(2002).Isolation of plant growth 

promoting Bacillus strains from 

soybean root nodules. Can. J. Microbiol., 

48, 230-238. 

Bailey, J. A. 1987. “Phytoalexins: A 

genetic view of their signifi- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 236



cance”. In day P.R, Jellis, G. J 

(Eds), Genetics and plant pathogenesis, 

Black well oxford, 13-26. 

Bailey, J. A. and Jeger, M. J. (1992). 

Colletotrichum: Biology, Pathology 

and Control”. Common wealth Mycological 

Institute, Wallingford, 

388 . 

Bano, N.and Musarrat,J.(2004). 

Characterization of a novel carbofuran 

degrading Pseudomonas sp. 

withcollateral biocontrol and plant 

growth promoting potential. 

FEMS Microbiol. Lett., 231, 13- 

17. 

Bargabus, R. L., Zidack, N. K., 

Sherwood, J. E. and Jacobsen, 

B. J. (2002) Characterization of 

systemic resistance in sugar beet 

elicited by a nonathogenic, phyllosphere 

colonizing Bacillus mycoides 

biological control agent. 

Physiol. Mol. Plant Pathol., 61, 

289-298. 

Bargabus, R. L., Zidack, N. K., 

Sherwood, J. E. and Jacobsen, 

B. J.( 2004). Screening for the 

identification of potential biological 

control agents that induce systemic 

acquired resistance in sugar 

beet. Biol. Control., 30, 342-350. 

Barka, E. A., Gognies, S., Nowak, J., 

Audran, J. C. and Belarbi, A. 

(2002).Inhibitory effect of endophytic 

bacteria on Botrytis cinerea 

and its influence to promote the 

grapevine growth. Biol. Control., 

24, 135-142. 

Barraquio,W.L., Revilla, L. and 

Ladha, J. K. (1997). Isolation of 

endophytic diazotrophic bacteria 

from wetland rice. Plant Soil, 194, 

Berg, G. T., Tesoriero, L. and Hailstones, 

D. L. (2005b). PCR-based 

detection of Xanthomonas campestris 

pathovars in Brassica seed. 

Plant Pathol., 54, 416-427. 

Berg, G. and Hallmann, J. (2006). 

Control of plant pathogenic fungi 

with bacterial endophytes. In: 

Schulz, B. J. E., Boyle, C. J. C., 


15-24. 

Benhamou, N., Bea langer, R. R. and 

Paulitz, T. C. (1996a) Ultrastructural 

and cytochemical aspects of 

the interaction between Pseudomonas 

fluorescens and Ri T-DNA 

transformed pea roots: host response 

to colonization by Pythium 

ultimum Trow. Planta, 199, 105- 

117. 

Benhamou, N., Bea langer, R. R. and 

Paulitz, T. C. (1996b). Induction 

of differential host responses by 

Pseudomonas fluorescens in Ri-T 

DNA transformed pea roots upon 

challenge with Fusarium oxysporum 

f. sp. pisi and Pythium 

ultimum. Phytopathology, 86, 

1174-1185. 

Benhamou, N., Kloepper, J. W. and 

Tuzun, S. (1998). Induction of 

resistance against Fusarium wilt 

of tomato by combination of chitosan 

with an endophytic bacterial 

strain: ultrastructure and cytochemistry 

of the host response. 

Planta, 204, 153-168. 

Bent, E. and Chanway, C. P. (1998). 

The growth promoting effects of a 

bacterial endophyte on lodgepole 

pine are partially inhibited by the 

presence of other rhizobacteria. 

Can. J. Microbiol., 44,980-988. 

Berg, G., Krechel, A., Ditz, M., Sikora, 

R. A., Ulrich, A.and 

Hallmann, J. (2005a). Endophytic 

and ectophytic potato associated 

bacterial communities differ in 

structure and antagonistic function 

against plant pathogenic fungi 

FEMS Microbiol. Ecol., 51, 215- 

229. 

Sieber, T. N., (eds) Microbial 

Root Endophytes. Springer- 

Verlag, Berlin, 53-69. 

Bertalan, M., Albano, R., de Padua, V., 

Rouws, L., Rojas, C., Hemerly, A. 

and Teixeira,K. ( 2009). Complete 

genome sequence of the 

sugarcane nitrogen fixing endophyteGluconacetobacter 

diazo- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 237



trophicus Pal5 BMC Genomics, 10, 

450. 

Bhore, S. J. and Sathisha, G. (2010). 

Screening of endophytic colonizing 

bacteria for cytokinin-like 

compounds: crude cell-free broth 

of endophytic colonizing bacteria 

is unsuitable in cucumber cotyledon 

bioassay. World J. Agri. Sci., 

6, 345-352. 

Bhutto, M., Aqeel, Dahot M. and 

Umar. (2010). Effect of Alternative 

Carbon and Nitrogen Sources 

on Production of Alpha-amylase 

by Bacillus megaterium. World 

Applied Sciences Journal(Special 

Issue of Biotechnology & Genetic 

Engineering), 8, 85- 90. 

Blom, D., Fabbri, C., Connor, E. C., 

Schiestl, F. P., Klauser, D. R., 

Boller, T.,Eberl, L.and 

Weisskopf, L. (2011). Production 

of plant growth modulating volatiles 

is widespread among rhizosphere 

bacteria and strongly depends 

on culture conditions. Environ. 

Microbiol., 13, 3047-3058. 

Boddey, R. M., Oliveira, O. C., Urquiaga, 

S., Reis, V. M., Olivares, 

F. L., Baldani, V. L.D. and 

Dobereiner, J. (1995). Biological 

nitrogen fixation associated with 

sugar cane and rice: Contributions 

and prospects for improvement. 

Plant Soil, 174, 195-209. 

Boddey, R. M., Urquiaga, S., Alves, 

B. J. R. and Reis, V.( 2003). Endophytic 

nitrogen fixation in sugarcane: 

present knowledge and future 

applications. Plant Soil, 249, 

165- 179. 

Bosland, P. W. and Votava, E. J. 

(2003). Peppers: Vegetable and 

Spice Capsicums. CAB International, 

England, 233. 

Brooks, D. S., Gonzalez, C. F., Appel, 

D. N. and Filer, T. H. (1994). 

Evaluation of endophytic bacteria 

as potential biological control 

agents for oak wilt. Biol. Control., 

4, 373-381. 


Brunel, B., Périssol, C., Fernandez, 

M., Boeufgras, J. M. and Le 

Petit, J. (1994). Occurrence of 

Bacillus species on evergreen oak 

leaves. FEMS Microbiol. Ecol., 

14, 331-342. 

Byung K. K., Ok-Sun, K., Eun 

Young, M. and Jongsik, C. 

(2009). Proposal to transfer Flavobacteriumoceanosedimentum 

CartyandLitchceld1978 to the genus 

Curtobacterium as Curtobacterium 

oceanosedimentum comb. 

Nov. FEMS Microbiol. Lett., 296, 

137- 141. 

Cavalcante, V. A. and Dobereiner, J. 

(1988). A new acid tolerant nitrogen-fixing 

bacterium associated 

with sugar cane. Plant Soil, 108, 

23-31. 

Chanway, C. P. (1997). Inoculation of 

tree roots with plant growth promoting 

soil bacteria: an emerging 

technology for reforestation. Forest 

Sci., 43, 99-112. 

Chen, C., Bauske, E. M., Musson, G., 

Rodriguez Kabana, R.and 

Kloepper. J. W. (1995). Biological 

control of Fusarium wilt on 

cotton by use of endophytic bacteria. 

Biol. Control., 5, 83-91. 

Chen, R., Hilson, P., Sedbrook, J., 

Rosen, E., Caspar, T. and Masson, 

P.H. (1998). The Arabidopsis 

thaliana Agravitropic gene encodes 

a component of the polarauxin- 

transport efflux carrier. 

Proc. Natl. Acad. Sci., USA, 95, 

15112-15117. 

Cho, K. M., Hong, S. Y., Lee, S. M., 

Kim, Y. H., Kahng, G. G., Lim, 

Y. P., Kim H. and Yun H.D. 

(2007).Endophytic bacterial 

communities in ginseng and their 

antifungal activity against pathogens. 

Microbial Ecol., 54, 341- 

351. 

Chung, B. S., Aslam, Z., Kim, S. W., 

Kim, G. G., Kang, H. S., Ahn, J. 

W. and Chung, 

Y.R. (2008). A bacterial endophyte, 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 238



Pseudomonas brassicacearum 

YC5480 isolated from the root of 

Artemisia sp. producing antifungal 

and phytotoxic compounds. 

The Plant Pathology Journal, 24, 

461-468. 

Compant, S., Reiter, B., Sessitsch, A., 

Nowak, J., Clément, C. and Ait 

Barka, E. (2005). Endophytic 

colonization of Vitis vinifera L. by 

plant growth promoting bacterium 

Burkholderia sp. strain PsJN. 

Appl. Environ. Microbiol., 71, 

1685-1693. 

Coombs, J. T. and C. M. M. Franco. 

(2003a). Isolation and identification 

of actinobacteria isolated 

from surface-sterilized wheat 

roots. Appl. Environ. Microbiol., 

69, 5303-5308. 

Coombs, J. T. and Franco, C. M. 

M. ( 2003b). Visualization of an 

endophytic 

Streptomyces species in wheat seed. Appl. 

Environ. Microbiol., 69, 4260-4262. 

Coombs, J. T., Michelsen, P. P.and 

Franco, C. M. M. (2004). Evaluation 

of endophytic actinobacteria 

as antagonists of Gaeumannomyces 

graminis var. tritici in wheat. 

Biol. Control, 29, 359-366. 

Costa, J. M. and Loper, J. E. (1994). 

Characterization of siderophore 

production by the biological control 

agent Enterobacter cloacae. 

Mol. Plant Microbe. Interact., 7, 

440- 448. 

Datta, S., Kim, C. M. and Pernas, M. 

(2011). Root hairs: development, 

growth and evolution at the plant 

soil interface. Plant Soil, 346, 1- 

14. 

Dekas, A. D., Poretsky, R. S. and Orphan, 

V. J.( 2009). Deep-sea archaea 

fix and share nitrogen in 

methane-consuming microbial 

consortia. Science, 326, 422-426. 

Dent, K. C., Stephen, J. R. and Finchsavage, 

W. E. (2004). Molecular 

profiling of microbial communities 

associated with seeds of Beta 


vulgaris subsp. vulgaris (sugar 

beet). J. Microbiol. Methods, 56, 

17-26. 

Djuric, S., Pavic, A., Jarak, M., Pavlovic, 

S., Starovic, M., Pivic, R. 

and Josic, D. (2011). Selection of 

indigenous fluorescent pseudomonad 

isolates from maize 

rhisospheric soil in Vojvodina as 

possible PGPR. Romanian Biotechnological 

Letters, 16, 6580- 

6590. 

Elbeltagy, A., Nishioka, K., Suzuki, 

H., Sato, T., Sato, Y. I., Morisaki, 

H., Mitsui, H. and 

Minamisawa, K. (2000). Isolation 

and characterisation of endophytic 

bacteria from wild and traditionally 

cultivated rice varieties. 

Soil Sci. Plant Nutrition. 46, 617- 

629. 

Fahey, J. W., Dimock, M. 

B.,Tomasino, S. F., Taylor, J. 

M. and Carlson, P. S. (1991). 

Genetically engineered endophytes 

as biocontrol agents : a 

case study from industry. In: Microbial 

Ecology of Leaves. Andrews, 

J. H., and Hirano, S. S., 

Eds., Springer-Verlag, London, 

UK, 401-411. 

Farag, M. A., Ryu, C. M., Summer, 

L. W. and Pare, P. W. ( 2006). 

GC-MS SPMEprofiling of rhizobacterial 

volatiles reveals prospective 

inducers of growth promotion 

and induced resistance in plants. 

Phytochem., 67, 2262-2268. 

Fritze, D. (2004). Taxonomy of the genus 

Bacillus and related genera: 

the aerobic endospore-forming 

bacteria. Phytopathology, 94, 

1245-1248. 

Gangwar, M. and Kaur, G. (2009). 

Isolation and characterization of 

endophytic bacteria from endorhizosphere 

of sugarcane and rye 

grass. Internet. J. Microbiol., 7. 

Germaine, K., Keogh, E., Garcia- 

Cabellos, G., Borremans, B., 

van Der-Lelie D., Barac, T., 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 239



Oeyen, L., Vangronsveld, J., 

Moore, F. P., Moore, E. R. B., 

Campbell, C. D., Ryan,D. and 

Dowling, D. N. (2004). Colonisation 

of poplar trees by gfp expressing 

bacterial endophytes. 

FEMS Microbiol. Ecol., 48, 109- 

118. 

Germida, J. and Siciliano, S. (2001). 

“Taxonomic diversity of bacteria 

associated with the roots of modern, 

recent and ancient wheat cultivars”. 

Biol. Fertil. Soil. 33, 410- 

415. 

Gnanamanickam, S. S. (2006). Plant- 

Associated Bacteria. Springer 

www.springer.com, 195-218. 

Gutierrez-Zamora, M. L. and Martínez-Romero, 

E. (2001). Natural 

endophytic association between 

Rhizobium etli and maize (Zea 

mays L.). J. Biotechnol. 91, 117- 

126. 

Hallmann, J., Quadt-Hallmann, A., 

Mahaffee, W. F. and Kloepper, 

J. W. (1997).Bacterial endophytes 

in agricultural crops. Can. J. Microbiol., 

43, 895-914. 

Hallmann, J., Quadt-Hallmann, A., 

Rodriguez-Kabana, R. and 

Kloepper, J. W. (1998). Interactions 

between Meloidogyne incognita 

and endophytic bacteria in 

cotton and cucumber. Soil Biol. 

Biochem., 30, 925-937. 

Hallmann, J. and Berg, G. (2006). 

Spectrum and population dynamics 

of bacterial root endophytes”. 

In: Microbial root endophytes. 

Schulz, B., Boyle, C., Sieber, T., 

eds. Springer, Heidelberg, 15-31. 

Harish, S., Kavino, M., Kumar, N., 

Saravanakumar, D., Soorianathasundaram, 

K. and 

Samiyappan, R. (2008). Biohardening 

with plant growth promoting 

rhizosphere and endophytic 

bacteria induces systemic resistance 

against banana bunchy 

top virus. Appl. Soil Ecol., 39, 

187-200. 


Harish, S., Kavino, M., Kumar, N., 

Balasubramanian, P. and 

Samiyappan, R.( 2009). Induction 

of defense-related proteins by 

mixtures of plant growth promoting 

endophytic bacteria against 

Banana bunchy top virus. Biol. 

Control., 51, 16-25. 

Huang, Z. J., Yang, R. Y., Guo, Z. Y., 

She, Z. G. and Lin, Y. C. (2010). 

A new naphtho-γ- pyrone from 

mangrove endophytic fungus 

ZSU-H26. Chem. Nat. Compd., 

46, 15-18. 

Hung, P. Q., Senthil Kumar, M., Govindsamy, 

V. and Annapurna, 

K. (2007). Isolation and characterization 

of endophytic bacteria 

from wild and cultivated soybean 

varieties. Biol. Fertil. Soils., 44, 

155-162. 

Husen, E. (2003). Screening of soil bacteria 

for plant growth promoting activities 

in vitro. 

Indones . J. Agric. Sci., 4, 27-31. 

Idris, R., Trifonova, R., Puschenreiter, 

M. and Wenze, L. W. W. 

(2004). Bacterial communities associated 

with flowering plants of 

the Ni hyperaccumulator Thlaspi 

goesingens. Appl. Environ. Microbiol., 

70, 2667-2677. 

Idriss, E. E., Makarewicz, O., Farouk, 

A., Rosner, K., Greiner, R., 

Bochow, H., Richter,T and Borriss, 

R. (2002). Extracellular 

phytase activity of Bacillus amyloliquefaciensFZB45 

contributes to its 

plant-growth-promoting effect. Microbiol., 

148, 2097-2109. 

Igual, J. M., Valverde, A., Cervantes, 

E. and Velaquez, E. (2001). 

Phosphate solubilizing bacteria as 

inoculants for agriculture: use of 

updated molecular techniques in 

their study. Agronomie., 21, 561- 

568. 

Iniguez, A. L., Dong, Y. and Triplett, 

E. W. (2004). Nitrogen fixation in 

wheat provided by Klebsiella 

pneumoniae 342. Mol. Plant Mi- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 240



crobe Interact., 17, 1078-1085. 

Jafra, S., Przysowa, J., Gwizdek- 

Wisniewska, A. van Der-wolf, J. 

M. (2009). Potential of bulb associated 

bacteria for biocontrol of 

hyacinth soft rot caused by Dickeya 

zeae J. Appl. Microbiol., 

106,268-277. 

James, E. K. (2000). Nitrogen fixation 

in endophytic and associative 

symbiosis. Field Crops Res. 65, 

197-209. 

Jha, P. N. and Kumar, A. (2007). Endophytic 

colonization of Typha 

australis by a plant growth promoting 

bacterium Klebsiella oxytoca 

strain GR-3. J. Appl. Microbiol., 

103, 1311-1320. 

Josic, D., Delic, D., Rasulic, N., Stajkovic, 

O., Kuzmanovic, D., 

Stanojkovic, A. and Pivic, R. 

(2012b). Indigenous Pseudomonads 

from Rhizosphere of 

Maize grown on Pseudogley Soil 

in Serbia. Bulgarian Journal of 

Agricultural Science, 18, 197-206. 

Josic, D., Pivic, R., Miladinovic, M., 

Starovic, M., Pavlovic, S., 

Duric, S. and Jarak, M. (2012a). 

Antifungal activity and genetic 

diversity of selected Pseudomonas 

spp. from maize rhizosphere in 

Vojvodina. Genetika, 44, 377- 

388. 

Jung, H. W., Kim, W., Hwang, B. K. 

(2003). Three pathogen inducible 

genes encoding lipid transfer protein 

from pepper are differentially 

activated by pathogens, abiotic, 

and environmental stresses. Plant 

Cell Environ., 26, 915-928. 

Kai, M., Haustein, M., Molina, F., Petri, 

A., Scholz, B. and Piechulla, 

B. (2009). Bacterial volatiles and 

their actionAppl. Microbiol. Biotechnol., 

81, 1001-1012. 

.Khan, M. S., Zaidi, A., Wani, P. A. 

and Oves, M. (20090. “Role of 

plant growth promoting rhizobacteria 

in the remediation of metal 

contaminated soils”. Environ. 


Chem. Lett., 7, 1- 19. 

Kim, K. Y., Jordan, D. and McDonald, 

G. A. (1998). Enterobacter 

agglomerans, phosphate solubilizing 

bacteria and microbial activity 

in soil: effect of carbon sources. 

Soil Biol. Biochem., 30, 995-1003. 

Kloepper, J. W. (1992). Plant growth 

promoting rhizobacteria as biological 

control agents”. In: Soil 

Microbial Ecology: Applications 

in Agricultural and Environmental 

Management, Metting, F. B., 

Jr., Ed., Marcel Dekker, New 

York, 255-274. 

Kloepper, J. W., Ryu, C. M. and 

Zhang, S. (2004). Induced systemic 

resistance and promotion of 

plant growth by Bacillus sp. Phytopathology, 

94, 1259-1266. 

Krause, A., Ramakumar, A., Bartels, 

D., Battistoni, F., Bekel, T., 

Boch, J. and Bohm, M. (2006). 

Complete genome of the mutualistic, 

N2 fixing grass endophyte 

Azoarcus sp. strain BH72. Nature 

Biotech., 24, 1385-1391. 

Krishnamurthy, K. and Gnanamanickam, 

S. S. (1997). Biological 

control of sheath blight of rice: 

induction of systemic resistance in 

rice by plant associated Pseudomonas 

sp. Curr. Sci., 72, 331-334. 

Lalande, R., Bissonnette, N., Coutlee, 

D. and Antoun, H. (1989). Identification 

of rhizobacteria from 

maize and determination of their 

plant growth promoting potential . 

Plant Soil, 115, 7-11. 

Lee, S., Flores-Encarnacion, M., Contreras-Zentella, 

M., Garcia- 

Flores, L., Escamilla, J.E. and 

Kennedy, C.(2004). Indole-3-acetic 

acid biosynthesis is deficient in 

Gluconacetobacter diazotrophicus 

strains with mutations in cytochrome 

C biogenesis genes. J. Bacteriol., 

186, 5384-5391. 

Lipton, D. S., Blanchar, R. W. and 

Blevins, D. G. (1987). Citrate, 

malate and succinate concentra- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 241



tion in exudates from P sufficient 

and P stressed Medicago sativa L. 

seedlings. Plant Physiol., 85,315- 

317. 

Liu, L., Kloepper, W. and Tuzun, S. 

(1995). Induction of systemic resistance 

in cucumber against 

Fusarium wilt by plant growth 

promoting rhizobacteria. Phytopathology, 

85, 695-698. 

Long, H. H., Schmidt, D. D. and 

Baldwin, I. T. (2008). Native 

bacterial endophytes promote host 

growth in a species specific manner 

phytohormone manipulations 

do not result in common growth 

responses. PLoS ONE, 3, 2702. 

Lugtenberg, B. and Kamilova, F.( 

2009). Plant rowth promoting rhizobacteria. 

Annu. Rev. Microbiol., 

63, 541-556. 

Manandhar, J. B., Hartman, G. L. 

and Wang, T. C. 

(1995).Anthracnose development 

on pepper fruits inoculated with 

Colletotrichum gloeosporioide. 

Plant Disease, 79, 380- 383. 

Manter, D. K., Delgado, J., Holm, D. 

G. and Stong, R. (2010). Pyrosequencing 

reveals a highly diverse 

and cultivar specific bacterial endophyte 

community in potato 

roots. Microb. Ecol., 60, 157-166. 

Marques, A. P. G., Pires, C., Moreira, 

H., Rangel, A. O. S. S. and Castro, 

P. M. L. (2010). Assessment 

of the plant growth promotion 

abilities of six bacterial isolates 

using Zea mays as indicator plant. 

Soil Biology Biochemistry, 42, 

1229-1235. 

Martinez, C., Michaud, M., Belanger, 

R. R. and Tweddell, R. J. 

(2002). Identification of soils 

suppressive against Helminthosporium 

solani, the causal 

agent of potato silver scurf. Soil 

Biol. Biochem, 34, 1861-1868. 

Mayak, S., Tirosh, T. and Glick, B. R. 

(2004). Plant growth-promoting 

bacteria that confer resistance to 


water stress in tomatoes and peppers. 

Plant Sci., 166, 525-530. 

McInroy, J. A. and Kloepper, J.W. 

(1994). Studies on indigenous endophytic 

bacteria of sweet corn 

and cotton. In: Molecular ecology 

of rhizosphere microorganisms: 

Biotechnology and the release of 

GMOs. F. O’Gara, D. N. 

Dowling, and B. Boesten eds. 

VCH, New York, 19-28. 

M’Piga, P., Belanger, R. R., Paulitz, 

T. C. and Benhamou, N. (1997). 

Increased resistance to Fusarium 

oxysporum f. sp. radicislycopersici 

in tomato plants treated with 

the endophytic bacterium Pseudomonas 

fluorescens strain 63-28. 

Physiol. Mol. Plant Pathol., 50, 

301-320. 

Naik, P. R., Raman, G., Narayanan, 

K. B. and Sakthivel, N. (2008). 

Assessment of genetic and functional 

diversity of phosphate solubilizing 

fluorescent pseudomonads 

isolated from rhizospheric 

soil”. BMC Microbiology, 8, 

230. 

Ngoma, L., Babalola, O. O. and Ahmad, 

F. (2012). Ecophysiology of 

plant growth promoting bacteria. 

Science Research Essays, 7, 4003- 

4013. 

Niu, D., Liu, H., Jiang, C., Wang, Y., 

Jin, H. and Guo, J. (2011). The 

plant growth promoting rhizobacterium 

Bacillus cereus AR156 induces 

systemic resistance in Arabidopsis 

thaliana, by simultaneously 

activating salicylate and 

jasmonate/ethylene- dependent 

signaling pathways. Mol. Plant. 

Microbe., 24, 533-542. 

Nowak, J. and V. Shulaev. (2003). Priming 

for transplant stress resistance in 

in vitropropagation. In Vitro Cell. 

Dev. Biol. Plant., 39, 107-124. 

Park, K. H., Lee, O. M., Jung, H. I., 

Jeong, J. H., Jeon, Y. D., 

Hwang, D. Y., Lee, C. Y. and 

Son, H. J. (2010). Rapid solubili- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 242



zation has of insoluble phosphate 

by a novel environmental stresstolerant 

Burkholderia vietnamiensis 

M6 isolated from ginseng 

rhizospheric soil. Appl. Microbiol. 

Biotechnol., 86, 947-955. 

Patel, H. A., Patel, R. K., Khristi, S. 

M., Parikh, K. and Rajendran, 

G. (2012). Isolation and characterization 

of bacterial endophytes 

from Lycopersicon esculentum 

plant and their plant growth promoting 

characteristics . Nepal J. 

Biotech., 2, 37-52. 

Payne, S. M. (1994). Detection, isolation 

and characterization of siderophores, 

in Methods Enzymology. 

edited by Clark, V. L. and Bovil, 

P. M. Academic press New York, 

235, 329-344. 

Pereira, P., Ibanez, F., Rosenblueth, 

M., Etcheverry, M. and Martinez-Romero, 

M. (2011). Analysis 

of the bacterial diversity associated 

with the roots of maize 

(Zea mays L.) through culturedependent 

and cultureindependent 

methods. ISRN Ecology, 

doi:10.5402/2011/938546. 

Pillay, V. K. and Nowak, J. (1997), 

Inoculum density, temperature 

and genotype effects on in vitro 

growth promotion and epiphytic 

and endophytic colonization of 

tomato (Lycopersicon esculentum 

L.) seedlings inoculated with a 

pseudomonad bacterium. Can. J. 

Microbiol., 43, 354-361. 

Pleban, S., Ingel, F. and Chet, I. 

(1995). Control of Rhizoctonia 

solani and Sclerotium rolfsii in 

the greenhouse using endophytic 

Bacillus sp. Eur. J. Plant Pathol., 

101, 665 - 672. 

Provorov, N. A., Borisov, A. Y. and 

Ikhonovich, I. A. (2002). Developmental 

genetics and evolution 

of symbiotic structures in nitrogen 

fixing nodules and arbuscular mycorrhiza”. 

J. Theor. Biol., 214, 

215-232. 


Rajkumar, M., Prasad, M. N. V. and 

Freitas, H. (2010). Potential of 

siderophoreproducing bacteria for 

improving heavy metal phytoextraction. 

Trends Biotechnol., 28, 

142-149. 

Ramamoorthy, V. and Samiyappan, 

R. (2001). Induction of defense 

related genes in Pseudomonas 

fluorescens treated chilli plants in 

response to infection by Colletotrichum 

capsic”. Journal of Mycology 

and Plant Pathology. 31, 

146-155. 

Ramamoorthy, V., Viswanathan, R., 

Raguchande, Tr., Prakasam, V. 

and Samiyappan, R. 2001. Induction 

of systemic resistance by 

plant growth promoting rhizobacteria 

in crop plants against pests 

and diseases. Crop Protection, 2, 

1-11. 

Raupach, G.S., Liu, L., Murphy, J.F., 

Tuzun, S. and Kloepper, J.W. 

(1996). Induced systemic resistance 

in cucumber and tomato 

against cucumber mosaic cucumovirus 

using plant growth 

promoting rhizobacteria (PGPR)”. 

Plant Dis., 80, 891-894. 

Robson, R. L., Eady, R. R., Richardson, 

T. H., Miller, R. W., Hawkins, 

M. and Postgate,J. R. 1986. 

The alternative nitrogenase of Azotobacter 

chroococcum is a vanadium 

enzyme. Nature, 322, 388-390. 

Rodriguez, H., Fraga, R., Gonzalez, T. 

and Bashan, Y. (2006). Genetics of 

phosphate solubilisation and its potential 

applications for improving 

plant growth promoting bacteria. 

Plant Soil, 287, 15-21 

Rosenblueth, M. and Martinez 

Romero, E. (2004). Rhizobium 

etli maize populations and their 

competitiveness for root colonization. 

Arch. Microbiol., 181, 337- 

344. 

Ryan, R. P. Germaine, K., Franks, A., 

Ryan, D. J. and Dowling, D. N. 

(2008). Bacterial endophytes: re- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 243



cent developments and applications. 

FEMS Microbiol. Lett. 278, 

1-9. 

Ryan, R. P., Monchy, S., Cardinale, 

M., Taghavi, S., Crossman, L., 

Avison, M. B., Berg, G., van der 

Lelie, D. and Dow, J. M. (2009). 

The versatility and adaptation of 

bacteria from the genus Stenotrophomona. 

Nat. Rev. Microbiol., 

7, 514-525. 

Samish, Z. and Etinger-Tulczynska, 

R. (1963b). Distribution of bacteria 

within the tissue of healthy 

tomatoes. Appl. Environ. Microbiol., 

11, 7-10. 

Schloter, M., Kirchhof, G., Heinzmann, 

U., Dobereiner, J. and 

Hartmann, A. (1994). Immunological 

studies of the wheat root 

colonization by the Azospirillum 

brasilense strains Sp7 and Sp 245 

using strain specific monoclonal 

antibodies”. In: Hegazi, N., Fayez, 

M., and Monib, M., (eds.) Nitrogen 

Fixation with nonlegumes. 

The American University in Cairo 

Press, Cairo, Egypt, 290-295. 

Schloter, M. and Hartmann, A. 

(1998). Endophytic and surface 

colonization of wheat roots (Triticum 

aestivum) by different 

Azospirillum brasilense strains 

studied with strain- specific monoclonal 

antibodies. Symbiosis., 25, 

159-179. 

Schroth, M. N. and Hancock, J. G. 

(1981). Selected topics in biological 

control”. Annu. Rev. Microbiol., 

35, 453-476. 

Seghers, D., Wittebolle, L., Top, E. 

M., Verstraete, W. and Siciliano, 

S. D. (2004). Impact of agricultural 

practice on the Zea mays 

L. endophytic community. Appl. 

Environ. Microbiol., 70, 1475- 

1482. 

Sessitsch, A., Howieson, J. G., Perret, 

X., Antoun, H. and Martinez- 

Romero, E. (2002). Advances in 

Rhizobium research. Crit. Rev. 


Plant Sci., 21, 323-378. 

Sessitsch, A., Reiter, B. and Berg, G. 

(2004). Endophytic bacterial 

communities of field- grown potato 

plants and their plant growth 

promoting and antagonistic abilities. 

Can. J. Microbiol., 50, 239- 

249. 

Sessitsch, A., Hardoim, P., Doring, J., 

Weilharter, A., Krause, A., 

Woyke, T. and Mitter,B.(2012). 

Functional characteristics of an endophyte 

community colonizing rice 

roots as revealed by metagenomic 

analysis. Mol. Plant Microbe Interact., 

25, 28-36. 

Sharma, V. K. and Nowak, J. (1998). 

Enhancement of verticillium wilt 

resistance in tomato transplants by 

in vitro co culture of seedlings 

with a plant growth promoting 

rhizobacterium (Pseudomonas sp. 

strain PsJN). Can. J. Microbiol., 

44: 528-536. 

Sharma, P. N., Kaur, M., Sharma, 

O.P., Sharma, P. and Pathania, 

A. (2005). Morphological, Pathological 

and molecular variability 

in Colletotrichum capsici the 

cause of fruit rot of chillies in the 

substropical region of north western 

India. J. Phytopathol., 153, 

232-237. 

Sharma, A., Wray, V. and Johri, B. 

N. (2007). Molecular characterization 

of plant growth promoting 

rhizobacteria that enhance peroxidase 

and phenylalanine ammonia 

lyase activities in chile (Capsicum 

annuum L.) and tomato (Lycopersicon 

esculentum Mill.). Arch Microbiol., 

188, 483-494. 

Shimanuki, T. (1987). Studies on the 

mechanisms of the infection of 

timothy with purple spot disease 

caused by Cladosporium (Gregory) 

de Vries. In: Res. Bull. Hokkaido 

Natl. Agric. Exp. Sta., 148, 

1-56. 

Silva, F. S. (2009). Quantification of 

natural populations of Glu- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 244



conacetobacter diazotrophicus 

and Herbaspirillum sp. in sugar 

cane (Saccharum sp.) using different 

polyclonal antibodies. Braz. 

J. Microbiol., 40, 4. 

Stajkovic, O., Delic, D., Josic, D., Kuzmanovic, 

D., Rasulic, N. and 

Knezevic Vukcevic, J. (2011). 

Improvement of common bean 

growth by co-inoculation with Rhizobium 

and plant growth promoting 

bacteria. Romanian Biotechnological 

Letters, 16, 5919-5926. 

Stone, J. K., Bacon, C. W. and White, 

J. F. (2000). An overview of endophytic 

microbes: Endophytism 

defined. In: C. W. Bacon, and J. 

F. White (eds.) Microbial Endophytes. 

Marcel Dekker, New 

York, 3, 30. 

Strobel, G., Daisy, B., Castillo, U. and 

Harper, J. (2004). Natural products 

from endophytic microorganisms. J. 

Nat. Prod., 67, 257-268. 

Sturz, A. V. and Christie, B. R. 

(1995). Endophytic bacterial systems 

governing red clover growth 

and development. Ann. Appl. Biol., 

126, 285-290. 

Sturz, A. V. and Matheson, B. G. 

(1996). Populations of endophytic 

bacteria which influence hostresistance 

to Erwinia induced bacterial 

soft rot in potato tubers. 

Plant Soil., 184, 265-271. 

Sturz, A. V., Christie, B. R., Matheson, 

B. G. and Nowak, J. (1997). 

Biodiversity of endophytic bacteria 

which colonize red clover 

nodules, roots, stems and foliage 

and their influence on host 

growth. Biol. Fertil. Soils, 25, 13- 

19. 

Sturz, A. V., Christie, B. R., Matheson, 

B. G., Arsenault, W. J. and 

Buchanan, N. A. (1999). Endophytic 

bacterial communities in 

the epiderm of potato tubers and 

their potential to improve resistance 

to soil borne plant pathogens. 

Plant Pathol., 48, 360- 369. 


Sturz, A. V., Christie, B. R. and 

Nowak, J. (2000). Bacterial endophytes: 

Potential role in developing 

sustainable systems of crop 

production. Crit. Rev. Plant Sci., 

19, 1-30. 

Sudhir, A., and Amrutha V. A. 

(2014). Ph D thesis entitled Bioformulations 

from endophytic 

bacteria isolated from capsicum 

fruitescence US341 against anthracnose 

caused by Colletotrichum 

sp .Acarya Nagarjuna 

University ,Guntur ,AP INDIA 

Sudhir, A., Pradeep Kumar, N. and 

Amrutha V. A. (2014). Isolation, 

Biochemical and PGP characterization 

of endophytic Pseudomonas 

aeruginosa isolated from chilli 

red fruit antagonistic against 

chilli anthracnose disease. 

Int.J.Curr.Microbiol.App.Sci. 3, 

318-329. 

Suman, A., Shasany, A. K., Singh, M. 

and Shahi, H. N. (2001). Molecular 

assessment of diversity 

among endophytic diazotrophs 

isolated from subtropical Indian 

sugarcane. World J. Microbiol. 

Biotechnol., 17, 39-45. 

Surette, M. A., Sturz, A. V., Lada, R. 

R. and Nowak, J. (2003). Bacterial 

endophytes in processing carrots 

(Daucus carota L. var. sativus): 

their localization, population 

density, biodiversity and their 

effects on plant growth. Plant 

Soil., 253, 381-390. 

Suzuki, T., Shimizu, M., Meguro, A., 

Hasegawa, S., Nishimura, T. 

and Kunoh, H. (2005). Visualization 

of infection of an endophytic 

Actinomycete Streptomyces 

galbus in leaves of tissue cultured 

Rhododendron. Actinomycetologica, 

19, 7-12. 

Thamizh Vendan, R., Yu, Y. J., Lee, 

S. H. and Rhee, Y. H. (2010). 

Diversity of endophytic bacteria 

in ginseng and their potential for 

plant growth promotion. J. Mi- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 245



crobiol. 48, 559-565. 

Than, P. P., Jeewon, R., Hyde, K. D., 

Pongsupasamit, S., Mongkolporn, 

O. and Taylor, P.J.( 2008a). Characterization 

and pathogenicity of 

Colletotrichum species associated 

withanthracnose disease on chilli 

(Capsicum spp.) in Thailand. Plant 

Pathology, 57, 562-572. 

Ting, A. S. Y., Meon, S., Kadir, J., 

Radu, S. and Singh, G. (2008). 

Endophytic microorganisms as 

potential growth promoters of banana. 

BioControl, 53, 541-553. 

Turner, J. T., Kelly, J. L. and Carlson, 

P. S. (1993). Endophytes: an 

alternative genome for crop improvement”. 

In: International 

Crop Science I. International 

Crop Science Congress, Ames, 

Iowa, July 14-22, 1992. Buxton, 

D. R., Shibles, R., Forsberg, R. 

A., Blad, B. L., Asay, K. H., 

Paulsen, G. M. and Wilson, R. F., 

Eds., Crop Science Society of 

America, Madison, WI, 555-560. 

van Buren, A. M., Andre, C. and 

Ishimaru, C. A. (1993). Biological 

control of the bacterial ring rot 

pathogen by endophytic bacteria 

isolated from potato. Phytopathology, 

83, 1406. 

van Loon, L. C. (2007).Plant responses 

to plant growth-promoting rhizobacteria. 

Eur. J. Plant Pathol., 

119, 243-254. 

van Loon, L. C., Bakker, P. A. and 

Pieterse, C. M. J. (1998). Systemic 

resistance induced by rhizosphere 

bacteria. Ann. Rev. Phyto., 

36, 453-483. 

van Peer, R., Punte, H. L. M., de Weger, 

L. A. and Schippers, B. 

(1990). Characterization of root 

surface and endorhizosphere 

pseudomonads in relation to their 

colonization of roots,Appl. Environ. 

Microbiol., 56,2462-2470. 

.Verma, S. C., Ladha, J. K. and 

Tripathi, A.K. (2001). Evaluation 

of plant growth promoting 


and colonization ability of endophytic 

diazotrophs from deep water 

rice. J. Biotechnol. 91, 127- 

141. 

Vesperman, A., Kai, M. and 

Piechulla, B. (2007). Rhizobacterial 

volatiles affect the growth of 

fungi and Arabidopsis thaliana. 

Appl. Environm. Microbiol., 73, 

5639-5641. 

Vetrivelkalai, P., Sivakumar, M. and 

Jonathan, E. I.( 2010). Biocontrol 

potential of endophytic bacteria 

on Meloidogyne incognita and 

its effect on plant growth in bhendi. 

J. Biopest. 3, 452-457. 

Viswanathan, R. and Samiyappan, 

R.( 1999a). Induction of systemic 

resistance by plant growth promoting 

rhizobacteria against red 

rot disease in sugarcane. Sugar 

Tech, 1, 67-76. 

Viswanathan, R. and Samiyappan, R. 

(1999b). Identification of antifungal 

chitinase from sugarcane. IC- 

AR News, 5, 1-2. 

Vyas, P. and Gulati, A. (2009). Organic 

acid production in vitro and 

plant growth promotion in maize 

under controlled environment by 

phosphate solubilizing fluorescent 

Pseudomonas. BMC Microbiol., 

9, 174-189. 

Wakelin, S., Warren, R., Harvey, P. 

and Ryder, M. (2004). Phosphate 

solubilization by 

Penicillium sp. closely associated with 

wheat roots. Biol. Fertil. Soils, 40, 

36-43. 

Weller, D. M. (1988). Biological control 

of soil borne plant pathogens 

in the rhizosphere with bacteria. 

Ann. Rev. Phytopathol., 26,379- 

407. 

Wheatley, R. E. (2002). The consequences 

of volatile organic compound 

mediated bacterial and 

fungal interactions. Antonie Van 

Leeuwenhoek, 81, 357-364. 

Whipps, J. M. (2001). Microbial interactions 

and biocontrol in the rhi- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 246



zosphere. J. Exp. Bot., 52, 487- 

511. 

Whitesides, S. K. and Spotts, R. A. 

(1991). Frequency, distribution, 

and characteristics of endophytic 

Pseudomonas syringe in pear 

trees. Phytopathology, 81, 453- 

457. 

Wilhelm, E., Arthofer, W. and 

Schafleitner, R. (1997). Bacillus 

subtilis, an endophyte of chestnut 

(Castanea sativa), as antagonist 

against chestnut blight 

(Cryphonectria parasitica), In A. 

C. Cassells (ed.), Pathogen and 

microbial contamination management 

in micropropagation. 

Kluwer Academic Publishers, 

Dortrecht, The Netherlands, 331- 


337. 

Yang, C., Xang, Z., Shi, G., Zhao, H., 

Chen, L., Tao, K. and Hou, T. 

(2011). Isolation and identification 

of endophytic bacterium W4 

against tomato Botrytis cinerea 

and antagonistic activity stability”. 

African J. Microbiol., 5, 131- 

136. 

Zinniel, D. K., Lambrecht, P., Harris, 

N. B., Feng, Z., Kuczmarski, D., 

Higley, P., Ishimaru, C. A., 

Arunakumari, A., Barletta, R. 

G. and Vidaver, A. K. (2002). 

Isolation and characterization of 

endophytic colonizing bacteria 

from agronomic crops and prairie 

plants. Appl. Environ. Microbiol., 

68, 2198-2208. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 247



Biotech Sustainability (2017), P248--261 

Microbial Metabolic Engineering: A Key Technology to 

Deal with Global Climate and Environmental Challenges 

Meerza Abdul Razak 1 , Pathan Shajahan Begum 2 , Senthilkumar Rajagopal 3, * 

1 Department of Biotechnology, Rayalaseema University, Kurnool, Andhra Pradesh, 

India; 2 Department of Zoology, KVR Women’s degree college, Kurnool, Andhra Pradesh, 

India; 3 Department of Biochemistry, Rayalaseema University, Kurnool, Andhra 

Pradesh, India;*Correspondence: senthilanal@yahoo.com; Tel: +91 9566860390 

Abstract: Global climate change and green house effects are very serious and controversial 

problems that have severe negative impacts on environment, society, energy industry 

and government policies and sustainability. Global environment and climate 

challenges are directly connected to the accumulation of green house gases which has 

caused concerns related to the usage of traditional and fossil fuels as the key energy resource. 

To mitigate climate and environment changes, one solution is to utilize the potential 

of metabolic engineering of microbes for biofuels production from renewable 

sources. For long-term economic sustainability of the energy, industries and transportation 

sectors should adopt renewable and sustainable fuels produced by metabolically 

engineered microorganisms. The biofuels produced from renewable sources by metabolically 

engineered microbes carry good energy contents with minimal emission of 

greenhouse gases and causes minimal impact on the environment, food chain, water 

supply and land use. Toxic organic and inorganic chemicals are also one of the main 

reasons for environment contamination and also present major risk for climate change. 

Avoiding of upcoming contamination from these chemicals poses a huge technical 

challenge. Currently, metabolically engineered microbes have been explored only for 

selective and high capacity bioremediation of heavy toxic metals and chemicals. This 

chapter will shed light on current trend and developments in metabolic engineering of 

microbes for biofuel and bio-based chemicals production from renewable resources. 

This chapter also highlights the potential of metabolically engineered microbes for bioremediation, 

a possible futuristic solution for sustainable development for energy and 

reduction of global climate change and green house effects. 

Keywords: Biofuels; bioremediation; metabolic engineering; synthetic biology; systems 

biology 


Since the past some decades, 

constantly increasing greenhouse gases in 

environment such as carbon dioxide, nitrous 

oxide, methane have been linked to 

global environmental and climate concerns 

(O’Neill and Oppenheimer, 2002; 

Stocker, 2013). Some of the effects of 

global climate and environment change 

are abolition of coral reef, rising of sea 

level and weakening of thermohaline circulation. 

The atmospheric carbon dioxide 

concentration is 400 parts per million 

(ppm) and the carbon dioxide released 

from fossil fuels worldwide is 7 Gt of 

carbon per year (Pacala and Socolow, 

2004; Lewis and Nocera, 2006). If the 

present upward trend of carbon dioxide 

continues, by the end of year 2050 the 

carbon dioxide release rate will be doubled. 

It is estimated that the carbon diox- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 248


Microbial Metabolic Engineering for Sustainability 

Meerza et al. 

ide concentration will reach to 500 ppm 

by doubling of the carbon dioxide emission 

rate without remediation. The carbon 

dioxide concentration of 500 ppm will 

lead to a global warming of around 2°C 

above the level in year 1900 (Pacala and 

Socolow, 2004). This level of increase in 

temperature would raise the threat of disintegration 

of the West Antarctic Ice 

Sheet (WAIS) along with other negative 

effects. It is estimated that increase in 

temperature by 2°C would lead to disruptive 

rise of sea level by 4–6 meters 

(Stocker, 2013). 

The atmospheric CO 2 concentration, 

CO 2 emission and global temperature 

are having severe negative effects on 

the environment, climate, economy and 

society and we need to deal with it. To 

avoid the rise in the temperature, it is 

necessary that we should reduce the carbon 

emission from the fossil fuel usage 

and increase the sources of renewable 

energy and remove the toxic chemicals 

and metals present in the environment. 

When compared with different renewable 

energy sources, biofuels are well-suited 

with present infrastructure and have an 

advanced energy density. Thus there has 

been much curiosity in establishing biorefineries 

for the production of fuels and 

chemicals from renewable resources. 

We are in the modern age of 

microbial metabolic engineering which 

comprises of progressively more efforts at 

cell, and pathway design. One of the reasons 

for the more success of microbial 

metabolic engineering is development of 

additional “omics” tools which provide 

both temporally and spatially analyzing 

opportunity for cellular systems at the 

level of protein, metabolite, RNA and 

DNA (Peralta-Yahya et al., 2012). Microbial 

metabolic engineering have been 

evolved to solve crucial international 

problems such as global warming, bioremediation, 

food and human health. If 

properly utilized microbial metabolic engineering 

can play an important role in 

facing the global challenges. By development 

of novel technological innovation 

in combination with rising global crises 

provides the platform for microbial metabolic 

engineering applications and innovation 

on a large scale. Scientists from 

industries and academics strongly believe 

that microbial metabolic engineering in 

combination with other technologies such 

as synthetic biology and systems biology 

can help to solve the growing concerns of 

climate and environment (Zhang et al., 

2011). 

From the beginning microbial 

metabolic engineering had aimed the production 

of fuels and chemicals as chief 

goals of the rising field. The metabolic 

engineering of yeast Saccharomyces 

cerevisiae and E. coli are the classic examples 

of metabolic application in the 

field of biofuel production (Kuyper et al., 

2003, Bro et al., 2006, Ingram et al., 

1987). Microbial metabolic engineering 

applications have expanded in the past 

few years because of the growing attention 

on biofuels and chemical production 

through biomass conversion. There are 

many reports stating that the combination 

of microbial metabolic engineering along 

with combinatorial approaches supported 

by high throughput systems had given 

good results in biofuel production. The 

key advantage of utilizing microorganism 

for the production of biofuels from renewable 

sources is the metabolic diversity 

of fungi, algae and bacteria facilitate us 

the use of diverse substrates as the starting 

point for biofuel generation. 

Bioremediation is a very cost 

effective and eco-friendly method and is 

steadily making inroads for environmental 

clean-up applications. Bioremediation 

depends on enhanced detoxification and 

degradation of toxic metals and degradation 

of toxic pollutants either through enzymatic 

transformation or intracellular 

accumulation to less or non-toxic compounds. 

There are several physiochemical 

processes for treating toxic pollutants 

in environment, but these processes 

are non specific, very costly and sometimes 

they may introduce secondary contamination. 

Microbes naturally have the 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 249



Meerza et al. 

capability to transform, degrade and chelate 

several toxic chemicals. But the microbial 

bioremediation process is having 

relative slow transformation rates. By 

metabolically engineering microbes it is 

possible to remove the toxic inorganic 

and organic chemicals from the environment 

(Shailendra et al., 2008). The better 

understanding of microbes’ natural transformation 

ability at genetic level and advance 

of novel genetic tools are very essential 

for metabolic engineering of microorganism 

for bioremediation. There 

are several metabolically engineered microorganisms 

with superior biotransformation 

capacity and more accumulation 

of toxic wastes. In this chapter we discuss 

metabolic engineering strategies and successful 

examples of metabolically engineered 

microorganisms for production of 

biofuels and chemicals and for bioremediation 

(Brar et al., 2006). 

2. Biofuels production by metabolically 

engineered microorganisms 

With the increasing costs of 

energy and the challenges of global 

warming that arise due to the usage of 

petroleum based feedstock, the scientific 

community throughout the globe is 

searching for energy substitutes without 

adding up to the existing carbon footprint. 

Biofuels can be an exciting substitute to 

solve both the climate and environmental 

issues since they are produced from the 

renewable resources. Microbial production 

of hydrogen as future fuel is a hopeful 

possibility as an alternative for petroleum 

based fuels. Hydrogen is a more 

energy dense source and its conversion to 

power or heart is very simple and clean. 

Hydrogen when combusted with oxygen 

only H 2 O is formed without the formation 

of toxic pollutants (Kim and Lee 2010, 

Panagiotopoulos et al., 2009). 

2.1. Hydrogen production 

Large number of microbes such as 

Sporoacetigenium mesophilum, Clostridium 

beijerinckii and Thermoanaerobacterium 

thermosaccharolyticum has been 

used for the hydrogen production. The 

drawback of these microbes is low production 

of hydrogen (Cai et al., 2011, Oh 

et al., 2011). Kim et al. overcome the major 

obstacle of low hydrogen production 

by metabolically engineered E. coli 

strains (Kim et al., 2009). In one of the 

investigation a high volumetric productivity 

of 2.4 H2/L/h was produced using 

immobilized cells of a metabolically engineered 

E. coli which had deletion mutation 

(Seol et al., 2011). Even though, biological 

hydrogen production was considerably 

increased by metabolically engineered 

E. coli strain, several vital obstacles 

involved in the productivity, yield 

and metabolic robustness are still not up 

to the mark that would permit commercialization. 

2.2. Bioethanol production 

Bioethanol is the major renewable 

liquid energy source comprising 90% of 

the global world Biofuel market. It is estimated 

that annual production of bioethanol 

throughout the world is more than 

105 billion liters. Most of the Bioethanol 

production is by yeast and it is based on 

the sugarcane and starch, this type of production 

competes with feed and food 

(Geddes et al., 2011b). In one of the study 

E. coli was metabolically engineered to 

efficiently convert glycerol to ethanol. 

This strain was able to convert 40 g/L 

glycerol to ethanol in 48 h with 90% of 

the ethanol yield (Trinh and Srienc, 

2009). By introducing the adhB and pdc 

genes from Zymomonas mobilis which 

encodes alcohol dehydrogenase and pyruvate 

decarboxylase into E. coli redirected 

the carbon flux into the ethanol 

production and the obtained metabolically 

engineered E. coli produced ethanol upto 

1.28% (v/v) using xylose as carbon 

source within 36 hour fermentation (Sanny 

et al., 2010). Metabolic engineering 

approaches were implemented to engineer 

E. coli for ethanol production from mixed 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 250



Meerza et al. 

sugars, glucose and xylose (Sanny et al., 

2010, Wang et al., 2008). 

2.3. Isopropanol production 

E. coli do not have some of the 

necessary pathways which are very much 

necessary pathways for the production of 

advanced fuels, metabolic engineering 

has provided the chance to produce nontraditional 

biofuels by the construction of 

non native biosynthesis pathways (Atsumi 

et al., 2008b). The microorganisms such 

as Clostridium can naturally produce Isopropanol 

which is one of the secondary 

alcohols. Isopropanol has several diverse 

applications. In Clostridium several attempts 

have been made to enhance the 

production ability, but product inhibition 

and low titer. As a result, metabolic engineering 

of E. coli for Isopropanol production 

become a promising substitute for 

industrial production of Isopropanol. 

Metabolically engineered strain of E. coli 

was constructed that produced a 13.6 g/L 

isopropanol from glucose under vigorous 

aerobic culture conditions (Jojima et al., 

2008). Isobutanol has same physical 

properties like isopropanol, except it has 

higher ocatane number. Metabolic engineering 

of E. coli resulted in the production 

of isobutanol using non-fermentative 

pathways through 2-ketoisovalerate as 

precursor. 

2.4. 1- butanol and 1- Propanol production 

1-Butanol is very attractive and 

alternative biofuel with high energy density 

and good compatibility with the ability 

to completely substitute gasoline. 

Generally, C. acetobutylicum produces 1- 

butanol along with butyrate, ethanol and 

acetone. But due to absence of genetic 

information, complex physiology and unavailability 

of genetic tools the C. acetobutylicum 

had not been improved to the 

level of usage in large scale industrial 

production. The complete information of 

the 1-butanol metabolic pathway, it become 

possible to construction of butanol 

pathway in E. coli. Atsumi et al. developed 

an E. coli strain which can fermentatively 

produce 1- butanol by transferring a 

group of six genes from C. acetobutylicum 

1- butanol pathway and removal of 

the competing pathway. This metabolically 

engineered E. coli strain produced 552 

mg/L of 1- butanol under semi-anaerobic 

conditions from a rich medium (Atsumi 

and Liao, 2008a). 1- Propanol is universal 

solvent with several industrial applications 

which can be converted to propylene 

and diesel and it is a promising gasoline 

substitute. The wild strain organisms cannot 

produce the 1-propanol is considerable 

amounts (Shen and Liao, 2008). 2- 

ketobutyrate is a precursor of 1-propanol 

and isoleucine. It is also can be converted 

to 2-methy 1-butanol and 1- butanol 

through several multi steps enzymes reactions. 

E. coli was metabolically engineered 

to produce 1-propanol and 1- 

butanol through 2-ketobutyrate (Shen and 

Liao, 2008). 

Metabolically engineered 

strain was developed by overexpression 

of the genes such as thrAfbBC, 

leuABCD, ilvA from E. coli, kivd 

from L. lactis and adh2 from S. 

cereviasiae followed by deletion of 

the competing genes such as adhE, 

ilvB, metA and tdh. This engineered 

strain produced 2 g/L propanol and 

butanol in 1: 1 ratio. The drawback 

this developed strain is it production 

of unwanted fermentative by products. 

In another study, a more direct 

pathway to produce 2-ketobutyrate 

through citramalte pathway was engineered 

in Methanococcus jannaschii 

by directed evolution to produce 1- 

butanol and 1- propanol. CimA specific 

activity was enhanced by the 

DNA shuffling and error prone PCR 

in E. coli strain. This E. coli strain 

with enhanced CimA activity produced 

more than 524 mg/L and 

3.5g/L propanol. The production of 

other side products is drawback of 

this strain. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 251



2.5. 2-Methyl-1-butanol and 3-methyl- 

1-butanol production 

The five carbon alcohols such as 

3-methyl-1-butanol and 2-Methyl-1- 

butanol have characteristics like high energy 

density and lower vapor pressure. 

When compared with ethanol, 3-methyl- 

1-butanol and 2-Methyl-1-butanol are 

more suitable to replace gasoline and 

more well-suited for the present fuel infrastructure. 

3-methyl-1-butanol and 2- 

Methyl-1-butanol can be naturally produced 

by as byproducts and some metabolic 

approaches have been tried to 

enhance the production of 2-Methyl-1- 

butanol in S. cerevisiae (Abe and 

Horikoshi, 2005). Table 1 illustrates the 

biofuels production by metabolically engineered 

E. coli. 

3. Bioremediation by metabolically 

engineered microorganisms 

3.1. Bioremediation of organic pollutants 

The extensive use of extremely 

toxic compound oraganophosphates 

(OPs) in agriculture as pesticide had led 

to a serious environmental pollution. Oraganophosphates 

are used in the insecticides; 

generally oraganophosphates are in 

the form of phosphoric acid. The bacteria 

growing in the soil had naturally acquired 

the ability to degrade oraganophosphates 

with the help of enzyme called organophosphate 

hydrolase (McDaniel et al., 

1988; Kulkarni and Chaudhari 2006). P-O 

linkage is hydrolysed by the organophosphate 

hydrolase, releasing p-nitrophenol 

as leaving group. As the toxicity of oraganophosphates 

is extensively diminished 

by hydrolysis of phoshoester bonds, several 

scientists have concentrated on the 

primary hydrolysis by organophosphate 

hydrolase. Even though oraganophosphates 

are transformed into dialkyl phosphates 

and p-nitrophenol (PNP) by initial 

hydrolysis. Still these degraded products 

are resistant to biodegradation and they 

are toxic. One solution to remove even 

Meerza et al. 

the less toxic dialkyl phosphates and p- 

nitrophenol is by metabolic engineering 

of microbes with the required degradation 

pathways (Cook et al., 1980; de la Pena 

Mattozzi et al., 2006). 

The p-nitrophenol can be degraded 

by metabolically engineered P.putida 

and Moraxella species. In Moraxella species 

organophosphate hydrolase was expressed 

on the cell surfaces and this strain 

showed to degrade paraoxon, parathion 

and methyl parathion and their hydrolysis 

product p-nitrophenol. In P. putida 

strain, natural p-nitrophenol degrading 

operon was introduced. A synthetic operon 

for expression of alkaline phosphatase 

(PhoA), phosphodiesterase (Pde) and organophosphate 

hydrolase was introduced 

to improve the diethyl phosphate (DEP) 

mineralization and hydrolysis of oraganophosphates. 

The resulting metabolically 

engineered P.putida strain was able to 

totally degrade p-nitrophenol in 78 hours, 

paraoxon in 24 hours and diethyl phosphate 

within 142 hours (de la Pena Mattozzi, 

et al., 2006). This research study 

presents an effective novel approach to 

create an artificial metabolite pathway for 

total mineralization. 

The present hydrodesulfurization 

method for decreasing the level of organic 

sulfur compounds is not capable to please 

the strict government regulations. The 

present advances in desulfurization have 

therefore been focused on biological processes 

using microbes. Dibenzothiophene 

which is a popular organosulfur compound 

is not removed by the hydrodesulfurization 

from fossil fuels. The desulfurization 

of dibenzothiophene requires a 

group of enzymes (Kilbane, 2006). 

Dibenzothiophene monooxygenase 

(DszC) is the first enzyme that converts 

dibenzothiophene to dibenzothiophene 

sulfone. The dibenzothiophene sulfone is 

converted into 2-hydroxybiphenyl-2- 

sulfinate by the catalysis of dibenzothiophene-5,5-dioxide 

monooxygenase. 2- 

hydroxybiphenyl-2-sulfinate is converted 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 252



Table 1: Biofuels produced by metabolically engineered E. coli 

Biofuels Fermentation 

process 

Engineering 

strategy 

Carbon 

source 

Titer and 

yield 

Hydrogen Immobilized 

recombinant 

cells 

Bioethanol 

1-Propanol 

1-Butanol 

3-Methyl- 

1-butanol 

10-L bioreactor 

batch anaerobic 

cultivation 

Shake flasks and 

IPTG induction 

1-L Bioreactor 

with aerobic– 

anaerobic dual 

phase fermentation 

Shake flask with 

two-phase fermentation 

Isopropanol Fed-batch fermentation, 

gasstripping-based 

recovery process 

Isobutanol 

Screw-cap conical 

flasks and 

IPTG induction 

Deletion of negative 

regulator and 

competing carbon 

metabolic pathways 

Minimized metabolic 

functionality 

for conversion of 

xylose and glucose 

into ethanol 

by multiple-gene 

knockout 

Improving specific 

activity and 

releasing feedback 

inhibition of the 

key enzyme by 

directed evolution 

Construction of 

modified clostridial 

1-butanol 

pathway in E. coli 

strain and enhancement 

of 

driving forces 

Random mutagenesis 

and selection 

combined 

with overexpression 

of key genes 

Heterologous expression 

of target 

product pathway 

from various 

sources in E. coli 

host 

Introducing nonfermentative 

synthetic pathway 

in E. coli and 

elimination of 

pathways competing 

for pyruvate 

and cofactors 

Formate 1.0 mol 

H 2 /mol 

formate; 

2.4 l 

H 2 /L/ha; 

formate 

Xylose 


glucose 

38.81 g/L; 

0.49 g/g 

glucose 

or xylose 

Glucose 3.5 g/L; 

0.049 

g/gb 

Glucose 30 g/L; 

88% of 

the 

theoretical 

yield 

Glucose 9.5 g/L; 

0.11 g/g 

glucose 


67.4% 

(mol/mol) 


86% of 

the theoretical 

maximum 

Meerza et al. 

Industrial 

applications 

Fuel and energy 

carrier 

Fuel, solvent, 

food, beverage 

Gasoline additive, 

allround solvent 

Bulk material, 

gasoline additive 

or fuel 

Advanced 

fuel, alternative 

gasoline 

Biodiesel, 

precursor of 

polypropylene 

Gasoline 

blend stock, 

precursor of 

butenes 

into 2-hydroxybiphenyl (HBP) and sulfite 

by the enzyme2-hydroxybiphenyl- 2- 

sulfinate sulfinolyase (Reichmuth et al., 

2004). By mutating at the 50 untranslated 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 253



Meerza et al. 

region of dibenzothiophene monooxygenase 

and overexpressing it enhanced the 

desulfurization rate by 9 fold increase 

when compared with the unmutated 

dibenzothiophene monooxygenase 

(Reichmuth et al., 2004). 

Nitrotoluene and nitrobenzene 

which are extensively used as pesticides, 

some polymers dyes and explosives are 

one of the most commonly seen pollutants. 

Naturally microorganisms can 

transform nitroaromatic compounds into 

amines with the help of redox enzymes. 

But the degradation is very slow because 

of electron loosing effect of nitro groups 

and toxicity. Through metabolic engineering 

and site directed mutagenesis at 

the position of 258 of 2-nitrotoluene dioxygenase 

which is responsibe for the 

oxidation of nitrotoluene to 3-methyl catechol 

and nitrite changed the enantiospecificity 

and resulted in more degradation of 

nitroaromatic compounds (Lee et al., 

2005). By metabolic engineering of 

Sphingobium chlorophenolicum ATCC 

39723 the rapid degradation of another 

toxic pesticide pentachlorophenol was 

achieved. Three rounds of genome shuffling 

in Sphingobium chlorophenolicum 

resulted in a strain with more resistance to 

pentachlorophenol, increased growth and 

rapid pentachlorophenol removal (Dai 

and Copley, 2004). 

3.2. Bioremediation of inorganic pollutants 

Generally microorganisms when 

they come in contact with heavy metals, 

they synthesize metal binding peptides 

such as metallothionein and phytochelatins. 

These peptides which are rich in thiol 

group bind to different heavy metals 

and by sequestration they reduce the toxicity. 

Moreover, these peptides produced 

in different sub cellular locations of microbes 

have been very useful for them to 

enhance their metal accumulation ability. 

E. coli was metabolically engineered by 

over expression of phytochelatin synthase 

of Arabidopsis thaliana which is responsible 

for synthesis of phytochelatins, this 

resulted in 20 fold higher heavy metal 

accumulation (Sauge-Merle, et al., 2003). 

Metallothionein and phytochelatins have 

restriction such as non selective binding 

to diverse heavy metals. Specific heavy 

metal transporters support to enhance uptake 

and accumulation of particular toxic 

heavy metals. By expression of Cd transporter 

MntA, selective Cd accumulation 

can be achieved (Kim, et al., 2005). The 

Fucus Metallothionein which is obtained 

from the arsenic resistant marine alga Fucus 

vesiculosus is used in metabolic engineering 

to create superior strains of E. 

coli. The co-expression of specific arsenic 

transporter and Fucus Metallothionein in 

E. coli resulted in 45 fold increase in arsenic 

accumulation. It is possible that the 

same strategies can be applied for other 

heavy metals also. Table 2 elucidates the 

environmental pollution creating inorganic 

heavy metals and radionuclides. 

The over expression of phytochelatin 

synthase responsible for enzymatic 

phytochelatins synthesis by enzymatic 

method in symbiotic Rhizobia bacterium 

showed good results. From this 

study it was demonstrated that phytochelatins 

can be successfully used for 

heavy metal accumulation, but the limitations 

in this approach are the minimum 

supply of the precursor glutathione in 

phytochelatin production and other heavy 

metal accumulation. This limitation can 

be overcome by co-expression of the enzyme 

phytochelatin synthase and the enzymes 

responsible for precursor glutathione 

production Sriprang, et al., (2002). 

The co-expression of phytochelatin synthase 

with feedback resistant glutathione 

synthase resulted in 10 fold increases in 

phytochelatin production. The overexpression 

of Cadmium transporter increased 

the final cadmium accumulation 

by 31.6 mmol/g dry weight (Kang et al., 

2007). 

3.3. Bioremediation of radionuclide 

In addition to organic and inorganic 

pollutants, radionuclide contamination 

occurred through nuclear weapons or 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 254



Meerza et al. 

Table 2: Environmental pollution creating inorganic heavy metals and radionuclides 

Contaminant MCLG 

(mg/L) 

MCL 

(mg/L) 

Potential health effects 

from ingestion of wa- 

Sources of contaminant 

in drinking 

ter 

Arsenic 0 0.010 Skin damage or problems 

with circulatory 

systems, and may 

have increased risk of 

getting cancer 

water 

Erosion of natural 

deposits; runoff from 

orchards, runoff from 

glass and electronic 

production wastes 

Cadmium 0.005 0.005 Kidney damage Corrosion of galvanized 

pipes; erosion 

of natural deposits; 

discharge from metal 

refineries; runoff 

from waste batteries 

and paints 

Lead 0 0.015 Infants and children: 

delays in physical or 

mental development; 

children could show 

slight deficits in attention 

span and learning 

abilities. Adults: kidney 

problems; high blood 

pressure 

Mercury 

(inorganic) 

Radium 

226/228 

Corrosion of household 

plumbing systems; 

erosion of natural 

deposits 

0.002 0.002 Kidney damage Erosion of natural 

deposits; discharge 

from refineries and 

factories; runoff from 

landfills and 

croplands 

0 5 pCi/L Increased risk of cancer Erosion of natural 

deposits 

Uranium 0 30 mg/L Increased risk of cancer, 

kidney toxicity 

Erosion of natural 

deposits 

MCLG: maximum contaminant level goal; MCL: Maximum contaminants limit; Source: 

EPA safe water 

nuclear plant leakage is one of the major 

environmental problems. Naturally occurring 

bacteria which are more resistant to 

radiation are ideal metabolic engineering 

candidates for enhanced radionuclide removal. 

Metabolic engineering of thermophilic 

bacterium Deinococcus geothemalis 

was done by over expressing of mer 

operon from E. coli coding for Hg2+ reduction, 

which resulted in radiation resistant 

bacterium (Brim et al., 2003). The 

metabolically engineered bacteria Deinococcus 

geothemalis is able to decrease 

mercury at higher temperature and ionizing 

radiation, it is also able to reduce 

Cr(VI), U(VI) and Fe(III). This study reported 

the possibility of using metabolically 

engineered microorganisms for removal 

of versatile radioactive wastes at 

high temperatures. In P. aeruginosa, radionuclide 

precipitation can be achieved as 

metal phosphate by over expressing of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 255



Meerza et al. 

exopolyphosphatases and polyphosphate 

kinase (Renninger et al., 2004). In radiation 

resistant bacterium D. radiodurans, 

non-specific phosphatases phoN was expressed 

which resulted bioprecipitation of 

uranium from dilute nuclear waste (Appukuttan 

et al., 2006). The enzymes uranyl 

reductases and chromate are subjected 

to directed evolution and these engineered 

enzymes are used for metabolic engineering 

of P.putida and E. coli. These metabolically 

engineered strains of P.putida 

and E. coli showed more resistant against 

radiation and further enhanced the radionuclide 

precipitation efficiency (Barak et 

al., 2006). 

4. Metabolic engineering strategies 

Generally the metabolic engineering 

strategies are based on genetic engineering 

techniques. Some of the essential 

requirements for metabolic engineering 

are 1. Complete information of the biosynthetic 

pathway of the interested chemical. 

2. Particular of the genes coding related 

enzyme. 3. Regulating factors of 

enzymes involved in pathway. 4. Expression 

or deletion of required enzyme in 

host bacteria. 5. Gene mutation effects on 

the enzyme properties 6. Assembly of 

group of genes and their co-expression. 

Now a day’s along with bacteria and 

yeast, plant cell, animal cells and fungi 

are also used for metabolic engineering 

(Kell et al., 2005). 

Some of the strategies of metabolic 

engineering for the achievement of 

production of required biochemical are 

discussed below. 1. One of the most 

commonly used strategies is over expressing 

of the gene encoding the rate-limiting 

enzyme of the biosynthetic pathway of 

the required end product. 2. In this way, 

we can achieve the overproduction of the 

desired product by inhibiting or deleting 

the genes responsible for the competing 

metabolic reactions which use the same 

substrate. Through this way the substrate 

is metabolically channeled particularly 

towards the desired chemical. 3. There is 

possibility of production of the desired 

biochemical or product in the non-native 

organism. The genes can be isolated from 

the native organisms which can produce 

the required product and these genes can 

be expressed in another non-native organism 

(heterologous host organisms). It is 

mandatory that the required substrate 

should be available in the non-native organism. 

Multiple genes representing the 

group of enzymes of particular pathway 

can be expressed in the non-native host. 

Expressing the group of genes encoding 

the most capable enzymes from diverse 

organisms is another way to obtain the 

product which is produced in low quantities 

or not produced (Patil et al., 2005). 

Figure 1 explains the strategies for metabolic 

engineering for the production of a 

desired chemical. 

5. Tools for metabolic engineering 

Metabolic engineers use different 

protein engineering techniques and directed 

proteomic techniques to get useful 

information about protein levels in the 

microorganisms. Metabolic engineers believe 

that protein engineering and targeted 

proteomics are very valuable tools that fill 

the gap to engineer novel metabolic 

pathways for microorganisms. Synthetic 

biology had its application in the metabolic 

engineering for the alteration of microbes 

for the biorenewable production of 

biofuels and bioremediation (James, et 

al., 2016). Through synthetic biology we 

can design or redesign and construct new 

biological components such as cells, enzymes, 

proteins and pathways. The main 

goal of systems biology is the explaining 

the cell physiology and function through 

the integrated use of broad physiological 

and genomic data. Directed evolution and 

genetic engineering are the main tools of 

metabolic engineering which are very 

much responsible for the its success 

(Leber and Da Silva, 2014, Meerza et al., 

2016). The tools of metabolic engineering 

are shown in Figure 2. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 256



Meerza et al. 

Wild type strain 

A B C 

1 


A B C 

2 

Metabolically 

engineered strain 

A B C 

Metabolically 


A B C 

D 


A 

Metabolically 


A 

B 

B 

C 

D 

C 

D 

3 

Figure 1: Strategies for metabolic engineering 

for the production of a desired chemical; 

(1) overexpression of the rate-limiting enzyme; 

(2) inhibition of the competing pathway; 

(3) engineering a novel enzyme for the 

production of non-natural chemical. 

SYNTHETIC BIOLOGY 

Heterologous expression of 

natural pathways 

- de novo pathway design 

SYSTEMS BIOLOGY 

Metabolic models 

- Omics analysis 

METABOLIC 

ENGINEERING 

DIRECTED 

EVOLUTION 

GENETIC ENGINEERING 

Deletion or silencing 

- Transcriptional tuning 

- Translational tuning 

PROTEIN 

ENGINEERING 

Random mutagenesis 

- Rational modification 

Figure 2: Overview of tools for metabolic engineering. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 257



Meerza et al. 


Unquestionably, microbial metabolic 

engineering is the main tool for 

production of biofuels and for bioremediation. 

The scientific and technological 

progress in metabolic engineering has 

been estimated to make a good contribution 

to promote green and sustainable energy 

by biofuels production. The key advantages 

of biofuels produced by the 

metabolically engineered microbes over 

petroleum based fuels are they are very 

environmentally friendly, produced from 

the renewable feedstocks and very low 

emissions of carbon. From the microbial 

metabolic engineering standpoint, the 

discovery and design of novel pathways 

and enzymes for metabolic engineering 

of microorganisms coupled with efficient 

innovative and low cost processing technologies 

will contribute significantly to 

the success of production of biofuels and 

bioremediation through which we can 

meet the global challenges of environment 

and climate change. Metabolically 

engineered microbes could be used as 

platforms towards green energy and biochemical 

production, another crucial area 

of application promises to the human 

health. 

References 

Abe, F. and Horikoshi, K. (2005). 

Enhanced production of isoamyl 

alcohol and isoamyl acetate by 

ubiquitination-deficient Saccharomyces 

cerevisiae mutants. Cell 

Mol Biol Lett 10,383–8. 

Appukuttan, D. Rao, A. S. and Apte, 

S. K. (2006). Engineering of Deinococcus 

radiodurans R1 for bioprecipitation 

of uranium from 

dilute nuclear waste. Appl Environ 

Microbiol 72, 7873-7878. 

Atsumi, S. and Liao, J. C. (2008a). 

Directed evolution of Methanococcus 

jannaschii citramalate 

synthase for biosynthesis of 1- 

propanol and 1-butanol by Escherichia 

coli. Appl Environ Microbiol, 

74, 7802–8. 

Atsumi, S. Hanai, T. and Liao, J. C. 

(2008b). Non-fermentative 

pathways for synthesis of 

branched-chain higher alcohols 

as biofuels. Nature 451, 86–9. 

Barak, Y. Ackerley, D. F. Dodge, C. 

J. Banwari, L. Alex, C. Francis, 

A. J. and Martin, A. 

(2006). Analysis of novel soluble 

chromate and uranyl reductases 

and generation of an improved 

enzyme by directed evolution. 

Appl Env Microbiol 72, 7074- 

7082. 

Brar, S. K. Verma, M. Surampalli, 

R.Y. Misra, K. Tyagi, R. D. 

Meunier, N, and Blais, J. F. 

(2006). Bioremediation of hazardous 

wastes: a review. Pract 

Periodical Hazard, Toxic Radioactive 

Waste Manag, 

10, 59-72. 

Brim, H. Venkateshwaran, A. 

Kostandarithes, H. M. 

Fredrickson, J. K. and Daly, 

M. J. (2003). Engineering Deinococcus 

geothermalis for bioremediation 

of high-temperature 

radioactive waste environments. 

Appl Environ Microbiol, 69, 

4575-4582. 

Bro, C. Regenberg, B. Foerster, J. 

and Nielsen, J. (2006). In silico 

aided metabolic engineering of 

Saccharomyces cerevisiae for 

improved bioethanol production. 

Metab. Eng 8, 102–111. 

Cai, G. Jin, B. Monis, P. and Saint, 

C. (2011). Metabolic flux network 

and analysis of fermentative 

hydrogen production. Biotechnol 

Adv 29:375–87. 

Cook, A. M. Daughton, C. G. and 

Alexander, M. (1980). Desulfuration 

of dialkyl thiophosphoric 

acids by a Pseudomonad. Appl 

Environ Microbiol, 40, 463-465. 

Dai, M. H. and Copley, S. D. (2004). 

Genome shuffling improves deg- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 258



radation of the anthropogenic 

pesticide pentachlorophenol by 

Sphingobium chlorophenolicum 

ATCC 39723. Appl Env 

Microbiol 70, 2391-2397. 

de la Pena Mattozzi, M. Tehara, S. 

K. Hong, T. Keasling, J. D. 

(2006). Mineralization of 

paraoxon and its use as a sole C 

and P source by a rationally designed 

catabolic pathway in 

Pseudomonas putida. Appl Environ 

Microbiol, 72, 6699-6706. 

Geddes, C. C. Nieves, I. U. and Ingram, 

L. O. (2011b). Advances 

in ethanol production. Curr Opin 

Biotechnol 22, 312–9. 

Ingram, L. O. Conway, T. Clark, D. 

P. Sewell, G.W. and Preston, 

J.F. (1987). Genetic engineering 

of ethanol production in Escherichia 

coli. Appl. Environ. Microbiol 

53, 2420–2425. 

James, C. Luo, M. Sammy, P. and 

Shanshan, P. (2016). LuoFuelling 

the future: microbial engineering 

for the production of 

sustainable biofuels. Nature reviews 

14, 288-304. 

Jojima, T. Inui, M. and Yukawa, H. 

(2008). Production of isopropanol 

by metabolically engineered 

Escherichia coli. Appl Microbiol 

Biotechnol 77, 1219–24. 

Kang, S. H. Singh, S. Kim, J. Y. 

Lee, W. Mulchandani, A. and 

Chen, W, (2007). Bacteria metabolically 

engineered for enhanced 

phytochelatin production 

and cadmium accumulation. 

Appl Environ Microbiol 

73, 6317-6320. 

Kell, D. B. Brown, M. Davey, H. M. 

Dunn, W. B. and Spasic, I. 

(2005). Metabolic 

footprinting and systems biology: 

The medium is the message. 

Nat Rev Microbiol 3, 557-565. 

Kilbane, J. J. (2006). Microbial biocatalyst 

developments to upgrade 

Meerza et al. 

fossil fuels. Curr Opin Biotech 

17, 305-314. 

Kim, M. S. and Lee, D. Y. (2010). 

Fermentative hydrogen production 

from tofu processing waste 

and anaerobic digester sludge using 

microbial consortium. Bioresour 

Technol 101, 48–52. 

Kim, S. K. Lee, B. S. Wilson, D. B. 

and Kim, E. K. (2005). Selective 

cadmium accumulation using 

recombinant Escherichia 

coli. J Biosci Bioeng 99, 109- 

114. 

Kim, S. Seol, E. Oh, Y. K. Wang, G. 

and Park, S. (2009). Hydrogen 

production and metabolic flux 

analysis of metabolically engineered 

Escherichia coli strains. 

Int J Hydrogen Energy 34, 

7417–27. 

Kulkarni, M. and Chaudhari, A. 

(2006). Biodegradation of p- 

nitrophenol by P. putida. Bioresource 

Technol 97, 982-988. 

Kuyper, M. Harhangi, H. R. Stave, 

A. K. Winkler, A. A. Jetten, M. 

S. M. De Laat, W. T. A. M. 

Den Ridder, J. J. J., Op den 

Camp, H. J. M. Van Dijken, J. 

P. and Pronk, J. T. (2003). 

High-level functional expression 

of a fungal xylose isomerase: the 

key to efficient ethanolic fermentation 

of xylose by Saccharomyces 

cerevisiae?. FEMS Yeast 

Res. 4, 69–78. 

Leber, C. and Da Silva N. A. (2014). 

Engineering of Saccharomyces 

cerevisiae for the synthesis of 

short chain fatty acids. Biotechnol 

Bioeng 111, 347-358. 

Lee, K. S. Parales, J. V. Friemann, 

R. and Parales, R. E. (2005). 

Active site residues controlling 

substrate specificity in 2- 

nitrotoluene dioxygenase from 

Acidovorax sp. strain JS42. J Ind 

Microbiol Biotechnol 32, 465- 

473. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 259



Meerza et al. 

Lewis, S. N. and Nocera, G. D. 

(2006). Powering the planet: 

chemical challenges in solar energy 

utilization. Proc. Natl Acad. 

Sci. USA 103, 15729–15735. 

McDaniel, C. S. Harper, L. L. and 

Wild, J. R. (1988). Cloning and 

sequencing of a plasmid-borne 

gene (opd) encoding a phosphotriesterase. 

J Bacteriol 170, 

2306-2311. 

Meerza, A. R. Pathan, S. B. Rao D. 

M. and Viswanath, B. (2016). 

Metabolic Engineering to Improve 

the Activity of Aspartate 

kinase for Biotechnological Production 

of L-lysine by Corynebacterium 

glutamicum ATCC 

13032. American Journal of Biochemistry 

and microbiology 6, 

33-44. 

O’Neill, B. C. and Oppenheimer, M. 

(2002). Dangerous climate 

impacts and the Kyoto Protocol. 

Science 296, 1971–1972. 

Oh, Y. K. Raj, S. M. Jung, G. Y. 

and Park, S. (2011). Current 

status of the metabolic engineering 

of microorganisms for biohydrogen 

production. Bioresour 

Technol, 102, 8357–67. 

Pacala, S. and Socolow, R. (2004). 

Stabilization wedges: solving 

the climate problem for the next 

50 years with current 

technologies. Science 305, 968– 

972. 

Panagiotopoulos, I. A. Bakker, R. R. 

Budde, M. A. de Vrije, T. 

Claassen, P. A. and Koukios, 

E. G. (2009). Fermentative hydrogen 

production from pretreated 

biomass: a comparative study. 

Bioresour Technol 100, 6331- 

6338. 

Patil, K. R. Rocha, I. Forster, J. 

and Nielsen, J. (2005). Evolutionary 

programming as a platform 

for in silico metabolic engineering. 

BMC Bioinformatics 

6, 308. 

Peralta-Yahya, P. P. Zhang, F.Z. del 

Cardayre, S.B. and Keasling, 

J. D. (2012). Microbial engineering 

for the production of advanced 

biofuels. Nature 488, 

320-328. 

Reichmuth, D. S. Blanch, H. W. and 

Keasling, J. D. (2004). Dibenzothiophene 

biodesulfurization 

pathway improvement using diagnostic 

GFP fusions. Biotechnol 

Bioeng 88, 94-99. 

Renninger, N. Knopp, R. Nitsche, 

H. Clark, D. S. and Keasling, 

J. D. (2004). Uranyl precipitation 

by Pseudomonas aeruginosa 

via controlled polyphosphate 

metabolism. Appl Environ Microbiol 

70, 7404-7412. 

Sanny, T. Arnaldos, M. Kunkel, S. 

A. Pagilla, K. R. and Stark, B. 

C. (2010). Engineering of ethanolic 

E. coli with the Vitreoscilla 

hemoglobin gene enhances ethanol 

production from both glucose 

and xylose. Appl Microbiol Biotechnol, 

88,1103–1112. 

Sauge-Merle, S. Cuine, S. Carrier, 

P. Lecomte-Pradines, C. Luu, 

D. T. and Peltier, G. (2003). 

Enhanced toxic metal accumulation 

in engineered bacterial cells 

expressing Arabidopsis thaliana 

phytochelatin synthase. Appl Environ 

Microbiol 69, 490-494. 

Seol, E. Manimaran, A. Jang, Y. 

Kim, S. Oh, Y. K. and Park, S. 

(2011). Sustained hydrogen production 

from formate using immobilized 


coli SH5. Int J Hydrogen 

Energy 36, 8681–8686. 

Shailendra, S. Seung, H. K. Ashok, 

M. and Wilfred, C. (2008). Bioremediation: 

environmental 

clean-up through pathway engineering. 

Current Opinion in Biotechnology 

19, 437–444. 

Shen, C. R. and Liao, J. C. (2008). 

Metabolic engineering of Escherichia 

coli for 1-butanol and 1- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 260



propanol production via the ketoacid 

pathways. Metab Eng 10, 

312–320. 

Sriprang, R. Hayashi, M. Ono, H. 

Takagi, M. Hirata, K. and 

Murooka, Y. (2002). Enhanced 

accumulation of Cd2+ by a 

Mesorhizobium sp. transformed 

with a gene from Arabidopsis 

thaliana coding for 

phytochelatin synthase. Appl Environ 

Microbiol 69, 1791-1796. 

Stocker, T. F. In Climate Change 

(2013). The Physical Science 

Basis. Contribution of Working 

Group I to the Fifth Assessment 

Report of the Intergovernmental 

Panel on Climate Change (eds 

Stocker, T. F. et al.) pp 13–115. 

Meerza et al. 

Trinh, C. T. and Srienc, F. (2009). 

Metabolic engineering of Escherichia 

coli for efficient conversion 

of glycerol to ethanol. Appl 

Environ Microbiol 75, 6696– 

705. 

Wang, Z. Chen, M. Xu, Y. Li, S. Lu, 

W. and Ping, S. (2008). An ethanol-tolerant 


coli expressing Zymomonas 

mobilis pdc and adhB genes 

for enhanced ethanol production 

from xylose. Biotechnol Lett 30, 

657–63. 

Zhang, F. Rodriguez, S. and 

Keasling, J. D. (2011). Metabolic 

engineering of microbial 

pathways for advanced biofuels 

production. Curr Opin Biotechnol 

22, 775-783. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 261



Biodiesel Production for Sustainability: An Overview 

R. Meena Devi, R. Subadevi and M. Sivakumar* 

Energy Materials Lab, #120, School of Physics, Alagappa University, Karaikudi-630 004, 

Tamil Nadu, India; *Correspondence: susiva73@yahoo.co.in; Tel: +91 4565 223304 

Abstract: Diminishing petroleum reserves and increasing environmental regulations are the 

main driving forces to search for renewable fuel. Biodiesel is a renewable substitute fuel for 

petroleum diesel fuel and produced by transesterification in which oil or fat is allowed to 

react with a monohydric alcohol in the presence of a catalyst. This paper gives an overview 

of the work carried out by researchers in the field of biodiesel production from different 

types of oil. The different methods of biodiesel production techniques and factors affecting 

biodiesel production are also highlighted. 

Keywords: Biodiesel, catalyst, transesterification, vegetable oil, viscosity 


The demand for energy is 

increasing in the world due to the rapidly 

growing global population and 

urbanization. The depletion of world 

petroleum reserves and increased 

environmental concerns has stimulated 

the search for alternative renewable fuels 

that are capable of fulfilling an increasing 

energy demand in a sustainable manner 

(Narasimharao et al., 2007). In recent 

decades, research concerning and 

knowledge about the external benefits of 

renewable raw materials have intensified 

the efforts for sustainable energy sources. 

The various alternative fuel 

options tried in place of hydrocarbon oils 

are mainly biogas, producer gas, ethanol, 

methanol and vegetable oils. Out of all 

these, biodiesel offers an advantage 

because of their comparable fuel 

properties with that of diesel. The 

emissions produced from biodiesel are 

cleaner compared to petroleum-based 

diesel fuel. Biodiesel can be regarded as 

an alternative diesel fuel. 

Biodiesel is the colloquial name 

for “fatty acid alkyl ester” (FAAE). 

According to the American Society for 

Testing and Materials (ASTM), biodiesel 

is defined as the monoalkyl esters 

derivative from lipid feedstocks, such as 

vegetable oils or animal fats (Avhad and 

Marchetti, 2016). The dominant biodiesel 

production process, namely 

transesterification, typically involves the 

reaction of an alkyl-alcohol with a long 

chain ester linkage in the presence of a 

catalyst to yield mono-alkyl esters 

(biodiesel) and glycerol (Verma et al., 

2016). 

Due to the recent increased 

awareness and development in this area, 

the objective of this review is to give 

fundamental insight into the production of 

biodiesel by different raw materials. Also, 

this paper, reviews the factors affecting 

biodiesel production process such as 

temperature, reaction time, methanol to 

oil molar ratio, type and amount of 

catalyst, mixing intensity and fuel 

properties of biodiesel. 

2. Merits and demerits of biodiesel 

Some of the advantages of using 

biodiesel as a replacement for diesel fuel 

are (Knothe, 2006; Romano et al., 2006): 

Renewable fuel, obtained from 

vegetable oils or animal fats. 

Low toxicity, in comparison with 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 262


Biodiesel Production for Sustainability 

Meena Devi et al. 

 

 

 

 

 

 

 

 

diesel fuel. 

Degrades more rapidly than diesel 

fuel, minimizing the environmental 

consequences of biofuel spills. 

Lower emissions of contaminants: 

carbon monoxide, particulate matter, 

polycyclic aromatic hydrocarbons, 

aldehydes. 

Lower health risk, due to reduced 

emissions of carcinogenic substances. 

No sulfur dioxide (SO 2 ) emissions. 

Higher flash point (100 °C minimum). 

May be blended with diesel fuel at 

any proportion; both fuels may be 

mixed during the fuel supply to 

vehicles. 

Excellent properties as a lubricant. 

It is the only alternative fuel that can 

be used in a conventional diesel 

engine, without modifications. 

Used cooking oils and fat residues 

from meat processing may be used as 

raw materials. 

There are certain disadvantages of 

using biodiesel as a replacement for diesel 

fuel that must be also taken into 

consideration: 

Slightly higher fuel consumption due 

to the lower calorific value of 

biodiesel. 

Slightly higher nitrous oxide (NOx) 

emissions than diesel fuel. 

Higher freezing point than diesel fuel. 

This may be inconvenient in cold 

climates. 

It is less stable than diesel fuel, and 

therefore long-term storage (more 

than six months) of biodiesel is not 

recommended. 

May degrade plastic and natural 

rubber gaskets and hoses when used 

in pure form, in which case 

replacement with Teflon components 

is recommended. 

It dissolves the deposits of sediments 

and other contaminants from diesel 

fuel in storage tanks and fuel lines, 

which then are flushed away by the 

biofuel into the engine, where they 

can cause problems in the valves and 

injection systems. In consequence, the 

cleaning of tanks prior to filling with 

biodiesel is recommended. It must be 

noted that these disadvantages are 

significantly reduced when biodiesel 

is used in blends with diesel fuel. 

3. History of biodiesel 

Dr. Rudolf Diesel actually 

invented the diesel engine to run on a 

myriad of fuels including coal dust 

suspended in water, heavy mineral oil, 

and, vegetable oil. Dr. Diesel’s first 

engine experiments were catastrophic 

failures. But by the time he showed his 

engine at the World Exhibition in Paris in 

1900, his engine was running on 100% 

peanut oil. Dr. Diesel was visionary. In 

1911, he stated that “the diesel engine can 

be fed with vegetable oils and would help 

considerably in the development of 

agriculture of the countries which use it’. 

In 1912, Diesel said, “The use of 

vegetable oils for engine fuels may seem 

insignificant today. But such oils may 

become in course of time as important as 

petroleum and the coal tar products of the 

present time”. No doubt, this statement 

has come to stay. Since Dr. Diesel’s 

untimely death in 1913, his engine has 

been modified to run on the polluting 

petroleum fuel we now know as “diesel.” 

Nevertheless, his ideas on agriculture and 

his invention provided the foundation for 

a society fueled with clean, renewable, 

locally grown fuel. Today throughout the 

world, countries are returning to using 

this form of fuel due to its renewable 

source and reduction in pollution 

(Owolabi et al., 2012). 

4. Oil crops in India 

The various oil sources are 

classified as edible and non-edible. The 

edible sources like groundnut, peanut etc 

are primarily used to meet the food 

requirement. India is not using vegetable 

oils derived from rapeseed & mustard, 

soybean or oil palm for the production of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 263



biodiesel. It is because; India is not selfsufficient 

in edible oils production and 

depends upon imports of palm oil and 

other vegetable oils in large quantities to 

meet the domestic demand. However, 

utilization of non-edible seed oils 

extracted from trees and forest sources 

does not interfere with food security 

directly if the trees are grown on 

marginal/waste land that does not 

compete with food production. Every year 

around 1.2 million tonnes of tree borne 

non-edible seed oils are produced in the 

country (Dwivedi et al., 2011). In India, 

biodiesel is produced mostly from the 

non-edible oils extracted from the seeds 

of plants like Jatropha, Pongamia, Mahua, 

Neem etc. Depending on climate and soil 

conditions, different nations are looking 

for different vegetable oils as substitute of 

diesel fuel for example soybean oil in 

USA, rapeseed and sunflower oils in 

Europe, palm oil in south East Asia and 

coconut oil in Philippines are being 

considered as substitutes for diesel. 

Table 1 summaries the non-edible 

oil producing plants that can be cultivated 

for oil production on suitable land and 

consequently the oil can be used for 


biodiesel production (Agarwal, 2007). 

The non-edible oil seed plant given in the 

above table has potential to produce oil 

and subsequent conversion to biodiesel 

apart from their uses for illumination, 

burning, soap making, candle making etc. 

It is estimated that the potential 

availability of such oils in India is about 2 

million tons per year. The most abundant 

oil sources are Sal, Mahua, Neem, 

Pongamia and Jatropha oil. Based on 

extensive research, Jatropha and 

Pongamia have been identified as the 

potential feed stocks for biodiesel 

production in India. 

The future demand for biodiesel in 

India is given in Table 2. The above table 

indicates that by the year 2020–2021, 

about 24.61 MT of diesel could be saved 

if B20 blend is utilized. This will ensure 

sustainable fuel availability with secured 

environmental conditions. As per the 

report of the committee on biofuel, the 

estimated demand of diesel in 2011–2012 

was 64.19 MT, requiring 12.84MT of 

biodiesel and plantation of Jatropha 

curcas over about 13.69 million hectare 

of land. As per Government of India 

survey, out of total land area, 124.7 mill- 

Table 1: Production of non-edible oils in India 

No 

Botanical 

Local Annual Productivity 

Name 

Name 

(Tons) 

1. Jatropha curcas Ratanjyot 45,000 

2. Pongamia pinnata Karanja 135,000 

3. Schleichera oleosa Kusum 25,000 

4. Azadirachta indica Neem 1,00,000 

5. Shorea robusta Sal 1,80,000 

6. Modhuca indica Mahua 1,80,000 

Table 2: Projections of biodiesel demand and corresponding Jatropha area required for 

meeting the blending targets in India (Area in Mha, Demand in Mt) 

Year 

For 5% blending For 10 % blending For 20 % blending 

Diesel 

Biodiesel Jatropha Biodiesel Jatropha Biodiesel Jatropha 

demand 

demand area demand area demand area 

2011- 64.19 3.21 3.42 6.42 6.85 12.84 13.69 

12 

2016- 92.15 4.61 4.91 9.21 9.83 18..43 19.66 

17 

2020- 

21 

123.06 6.15 6.56 12.31 13.13 24.61 26.25 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 264



-ion hectare is classified as waste and 

degraded land (Dwivedi et al., 2014). 

4.1. Typical oil crops useful for biodiesel 

production 

The main characteristics of typical 

oil crops that have been found useful for 

biodiesel production are summarized in 

the following paragraphs. 

4.1.1. Castor seed 

The castor oil plant grows in 

tropical climates, with temperatures in the 

range 20–30◦C; it cannot endure frost. It 

is important to note that once the seeds 

start germinating, the temperature must 

not fall below 12 ◦C. The plant needs a 

warm and humid period in its vegetative 

phase and a dry season for ripening and 

harvesting. It requires plenty of sunlight 

and adapts well to several varieties of 

soils. The total rainfall during the growth 

cycle must be in the range 700–1,400 

mm; although it is resistant to drought, 

the castor oil plant needs at least 5 months 

of rain during the year. Castor oil is a 

triglyceride, ricinolenic acid being the 

main constituent (about 90%). The oil is 

non-edible and toxic owing to the 

presence of 1–5% of ricin, a toxic protein 

that can be removed by cold pressing and 

filtering. The presence of hydroxyl groups 

in its molecules makes it unusually polar 

as compared to other vegetable oils. 

4.1.2. Jojoba 

Although jojoba can survive 

extreme drought, it requires irrigation to 

achieve an economically viable yield. 

Jojoba needs a warm climate, but a cold 

spell is necessary for the flowers to 

mature. Rainfall must be very low during 

the harvest season (summer). The plant 

reaches its full productivity 10 years after 

planting. The oil from jojoba is mainly 

used in the cosmetics industry; therefore, 

its market is quickly saturated. 

4.1.3. Jatropha 

Jatropha is a shrub that adapts 

well to arid environments. Jatropha 


curcas is the most known variety; it 

requires little water or additional care; 

therefore, it is adequate for warm regions 

with little fertility. Productivity may be 

reduced by irregular rainfall or strong 

winds during the flowering season. Yield 

depends on climate, soil, rainfall and 

treatment during sowing and harvesting. 

Jatropha plants become productive after 3 

or 4 years, and their lifespan is about 50 

years. Oil yield depends on the method of 

extraction; it is 28–32% using presses and 

up to 52% by solvent extraction. Since the 

seeds are toxic, jatropha oil is nonedible. 

The toxicity is due to the presence of 

curcasin (a globulin) and jatrophic acid 

(as toxic as ricin). 

4.1.4. Microalgae 

Microalgae have great potential for 

biodiesel production, since the oil yield 

(in liters per hectare) could be one to two 

orders of magnitude higher than that of 

other raw materials. Oil content is usually 

from 20 to 50%, although in some species 

it can be higher than 70%. However, it is 

important to note that not all microalgae 

are adequate for biodiesel production. 

High levels of CO2, water, light, nutrients 

and mineral salts are necessary for the 

growth of microalgae. Production 

processes take place in raceway ponds 

and photobiological reactors. 

5. Biodiesel production techniques 

There are different processes which 

can be applied to synthesize biodiesel 

such as direct use and blending, micro 

emulsion process, thermal cracking 

process and the most conventional way is 

transesterification process (Gashaw et al., 

2015). 

5.1. Direct use and blending 

The direct use of vegetable oils in 

diesel engine is not favorable and 

problematic because it has many inherent 

failings. Even though the vegetable oils 

have familiar properties as biodiesel fuel, 

it required some chemical modification 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 265



before can be used into the engine. It has 

only been researched extensively for the 

past couple of decades, but has been 

experimented with for almost hundred 

years. Although some diesel engine can 

run pure vegetable oils, turbocharged 

direct injection engine such as trucks are 

prone to many problems. 

5.2. Microemulsion process 

A micro emulsion is defined as the 

colloidal equilibrium dispersion of 

optically isotropic fluid microstructures 

with dimensions generally in the range of 

1–150 nm formed spontaneously from 

two normally immiscible liquids and one 

or more ionic or non-ionic. The problem 

of the high viscosity of vegetable oils was 

solved by micro-emulsions with solvents 

such as methanol, ethanol, and 1-butanol. 

The components of a biodiesel microemulsion 

include diesel fuel, vegetable 

oil, alcohol, surfactant and cetane 

improver in suitable proportions. 

Alcohols such as methanol and ethanol 

are used as viscosity lowering additives, 

higher alcohols are used as surfactants 

and alkyl nitrates are used as cetane 

improvers. Microemulsions can improve 

spray properties by explosive 

vaporization of the low boiling 

constituents in the micelles. Microemulsion 

results in reduction in viscosity 

increase in cetane number and good spray 

characters in the biodiesel. However, 

continuous use of microemulsified diesel 

in engines causes problems like injector 

needle sticking, carbon deposit formation 

and incomplete combustion. 

5.3. Thermal cracking (pyrolysis) 

Pyrolysis is defined as the 

conversion of one substance into another 

by means of heat or heating with the aid 

of a catalyst. Pyrolysis involves heating in 

absence of air or oxygen and cleavage of 

chemical bonds to yield small molecules. 

The pyrolysis of vegetable oil to produce 

biofuels has been studied and found to 

produce alkanes, alkenes, alkadienes, 

aromatics and carboxylic acids in various 


proportions. The equipment for thermal 

cracking and pyrolysis is expensive for 

modest biodiesel production particularly 

in developing countries. Furthermore, the 

removal of oxygen during the thermal 

processing also removes any 

environmental benefits of using an 

oxygenated fuel. Another disadvantage of 

pyrolysis is the need for separate 

distillation equipment for separation of 

the various fractions. Also the product 

obtained is similar to gasoline containing 

Sulphur which makes it less ecofriendly. 

The pyrolyzed material can be vegetable 

oils, animal fats, natural fatty acids and 

methyl esters of fatty acids. 

5.4. Transesterification 

Generally, biodiesel is produced 

by means of transesterification. 

Transesterification is the reaction of a 

lipid with an alcohol to form esters and a 

byproduct, glycerol. It is, in principle, the 

action of one alcohol displacing another 

from an ester, referred to as alcoholysis 

(cleavage by an alcohol). In 

Transesterification mechanism, the 

carbonyl carbon of the starting ester 

(RCOOR 1 ) undergoes nucleophilic attack 

by the incoming alkoxide (R 2 O−) to give 

a tetrahedral intermediate, which either 

reverts to the starting material, or 

proceeds to the transesterified product 

(RCOOR 2 ). Transesterification consists of 

a sequence of three consecutive reversible 

reactions. The first step is the conversion 

of triglycerides to diglycerides, followed 

by the conversion of diglycerides to 

monoglycerides, and finally 

monoglycerides into glycerol, yielding 

one ester molecule from each glyceride at 

each step. The reaction is represented in 

equation 1. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 266



There are different 

transesterification processes that can be 

applied to synthesize biodiesel: (a) basecatalyzed 

transesterification, (b) acidcatalyzed 

transesterification, (c) enzymecatalyzed 

transesterification, and (d) 

supercritical alcohol transesterification. 

5.4.1. Catalysts: acid catalyst 

The use of an acid catalyst is 

observed to be more effective than alkali 

catalysts when the concentration of free 

fatty acids is high. Also the performance 

of the acid catalyst is not strongly 

affected by the presence of FFAs in the 

feedstock. In fact, acid catalysts can 

simultaneously catalyze both 

esterification and transesterification. 

Thus, a great advantage with acid 

catalysts is that they can directly produce 

biodiesel from low cost lipid feedstocks, 

generally associated with high FFA 

concentrations (low-cost feedstocks, such 

as used cooking oil and greases, 

commonly have FFAs levels of >6%)3. 

However, Homogeneous acid catalyzed 

reaction is about 4000 times slower than 

the homogeneous base-catalyzed reaction. 

Acids used in the catalysis of the 

transesterification of biodiesels are 

usually either hydrochloric acid or 

sulfuric acid. Though these two acids are 

the most common, any Bronsted acid can 

also be used in this reaction. 

5.4.2. Base catalyst 

Transesterification reaction can be 

catalyzed by both homogeneous (alkalies 

and acids) and heterogeneous catalysts. 

The used alkali catalysts are NaOH, 

CH 3 ONa, and KOH for producing 

biodiesel (Wang et al., 2007). The alkali 

catalyzed transesterification of vegetable 

oils proceeds faster than the acid 

catalyzed. But the use of base catalyzed 

transesterification is only limited to oil 

having low water and FFA content. This 

reaction is the most widely used process 

for production of biodiesel worldwide. To 

keep check on the water and FFA content 

of the oil, they are first pretreated with an 


acid catalyzed transesterification process, 

which converts the FFA to esters (Leung 

and Guo, 2006). 

5.4.3. Enzyme catalysts 

Lipase enzymes can also catalyze 

methanolysis of triglycerides. The most 

promising results were obtained by using 

immobilized Candida Antarctica lipase 

(Novozym 435). Shimada et al., (1999), 

found that Novozym435 was inactivated 

by shaking it in a mixture containing 

more than 1.5 M eq. of methanol to oil. 

Above this concentration, methanol is 

partially present as small droplets in the 

oil phase. These droplets are believed to 

cause enzyme deactivation. Therefore, 

methanol was added stepwise; after the 

addition of the third methanol equivalent, 

conversion to methyl esters was almost 

complete. The enzyme could be reused 50 

times without loss of activity. The 

occurrence of free fatty acids did not 

affect the enzyme catalyst. Before the 

inlet of every reactor,1 M eq. was added 

to the feed. Samukawa et al. (2000) 

reported a dramatic increase of the lipase 

efficiency when it was pretreated by a 

consecutive incubation in methyl ester 

and oil prior to reaction. The use of 

Novozym435 in methanolysis of 

triglycerides is also reported in 

supercritical carbon dioxide at 24.1 MPa 

and 50 ◦C. High yields (90–95%) of fatty 

acid methyl esters could be obtained 

when the reaction was carried out at 

molar methanol/oil ratios of 25:1. 

5.4.4. Supercritical transesterification 

Saka and Kusdiana (2001) have 

developed a catalyst free method for 

biodiesel fuel production by employing 

supercritical methanol. The supercritical 

treatment at 350 ◦C, 43 MPa, and 240 s 

with a molar ratio of 42:1 in methanol is 

the optimum condition for 

transesterification of rapeseed oil to 

biodiesel fuel. The great advantage of this 

method was that free fatty acids present in 

the oil could be simultaneously esterified 

in the supercritical solvent. Variables such 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 267



as the molar ratio of alcohol to vegetable 

oil and reaction temperature were 

investigated during the transesterification 

within this supercritical media. Increasing 

the reaction temperature within the 

supercritical regime resulted in increased 

ester conversion. 

6. Previous work done on production of 

biodiesel from edible oil 

Leung and Guo (2006) compared 

the transesterification reaction conditions 

for fresh canola oil and used frying oil. 

Higher molar ratio (7:1, methanol/used 

frying oil), higher temperature (60° C) 

and higher amount of catalyst (1.1 wt% 

NaOH) was maintained in used frying oil 

when compared to fresh canola oil where 

optimal conditions maintained were 315- 

318 K, 1.0 wt% NaOH and 6:1 

methanol/oil molar ratio. However, less 

reaction time (20 min) was observed for 

used frying oil when compared to fresh 

canola oil reaction time (60 min). Ying et 

al. (2011), developed a new method 

catalyst, benzyl bromide-modified 

calcium oxide (CaO) for production of 

biodiesel from rapeseed. The improved 

catalytic activity was obtained by better 

fat diffusion to the surface of the benzyl 

bromide-modified CaO. Further, a 99.2% 

yield of fatty acid methyl esters in 3h was 

obtained in comparison to by better fat 

diffusion to the surface of the benzyl 

bromide-modified CaO. Wakil et al. 

(2012), chosen Cottonseed oil, Mosna oil 

and Sesame oil for producing biodiesel. 

7. Previous work done on production of 

biodiesel from non-edible oil 

Mohibbe et al. (2005), found that 

FAME of Jatropha curcas were most 

suitable for use as biodiesel and met the 

major specification of bio-diesel 

standards of the European, Germany and 

USA Standards Organization. 

Chakrabarti and Ahmad (2008) presented 

work on extraction of oil from castor bean 

and converting it into biodiesel. It was 


found that reaction mixture containing 

65ml of methanol along with 2.4 g of 

catalyst (KOH) took a good start in half 

an hour at30°C. In this reaction, amount 

of glycerine removed as well as ester 

content produced was considerably 

increased with rise in temperature of 

mixture up to 70°C by extending time 

period (180-360 minutes). The removal of 

glycerine increased by two and half times 

and ester content by four times, 

respectively. When castor oil was 

subjected to acid esterification, prior to 

transesterification (a separate 

investigation), it was found that ester 

contents up to 95% could be obtained. 

Hasan et al. (2013) produced biodiesel 

from neem seeds, its properties were 

close to diesel. The methodology of 

esterification process was selected and 

carried out by 1000 ml raw neem oil, 

300ml methanol and sodium hydroxide 

on mass basis as a catalyst usually kept in 

oven to form methyl ester, and initially to 

reach equilibrium condition at 

temperature 55-66°C. The ester and 

glycerine were separated by stimulating 

continuously and allow settling under 

gravity for 24 h. Thus the separated ester 

contains 3% to 6% methanol and soap 

agents. The methanol was removed by 

vaporization. The biodiesel had some 

catalyst; it was removed by warm water 

mix with ester. Kinematic viscosity lay 

between 1.9 and 6.0 according to the 

ASTM D6751 specification. It was 

reported that, 0.95 L biodiesel was 

produced from 1 L neem oil. 

8. Factors affecting biodiesel 

production 

The yield of biodiesel in the 

process of transesterification is affected 

by several process parameters which 

include; reaction time, reaction 

temperature, catalyst and molar ratio of 

alcohol and oil and mixing intensity 

(Gashaw et al., 2015). 

8.1. Temperature 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 268



Reaction temperature is the 

important factor that will affect the yield 

of biodiesel. For example, higher reaction 

temperature increases the reaction rate 

and shortened the reaction time due to the 

reduction in viscosity of oils. However, 

the increase in reaction temperature 

beyond the optimal level leads to decrease 

of biodiesel yield, because higher reaction 

temperature accelerates the saponification 

of triglycerides and causes methanol to 

vaporize resulting in decreased yield. 

Usually, the transesterification reaction 

temperature should be below the boiling 

point of alcohol in order to prevent the 

alcohol evaporation. The range of optimal 

reaction temperature may vary from 50°c 

to 60°c depends upon the oils or fats used. 

Therefore, the reaction temperature near 

the boiling point of the alcohol is 

recommended for faster conversion by 

various literatures. At room temperature, 

there is up to 78% conversion after 60 

minutes, and this indicated that the 

methyl esterification of the FFAs could be 

carried out appreciably at room 

temperature but might require a longer 

reaction time. 

8.2. Reaction time 

The increase in fatty acid esters 

conversion observed when there is an 

increase in reaction time. The reaction is 

slow at the beginning due to mixing and 

dispersion of alcohol and oil. After that 

the reaction proceeds very fast. However 

the maximum ester conversion was 

achieved within < 90 min. Further 

increase in reaction time does not increase 

the yield product i.e. biodiesel/mono alkyl 

ester. Besides, longer reaction time leads 

to the reduction of end product (biodiesel) 

due to the reversible reaction of 

transesterification resulting in loss of 

esters as well as soap formation. 

8.3. Methanol to oil molar ratio 

One of the most important 

parameters affecting the yield of biodiesel 

is the molar ratio of alcohol to 

triglyceride. Stoichiometrically, 3 moles 


of alcohol and 1 mole of triglyceride are 

required for transesterification to yield 3 

moles of fatty acid methyl/ethyl esters 

and 1 mole of glycerol is used. In order to 

shift the reaction to the right, it is 

necessary to either use excess alcohol or 

remove one of the products from the 

reaction mixture. The second option is 

usually preferred for the reaction to 

proceed to completion. The reaction rate 

is found to be highest when excess 

methanol is used (Gashaw and Lakachew, 

2014). Methanol, ethanol, propanol, 

butanol and amyl alcohol can be used in 

the transesterification reaction, amongst 

these alcohols methanol is applied more 

frequently as its cost is low and it is 

physically and chemically advantageous 

(polar and shortest chain alcohol) over the 

other alcohols. In contrast, ethanol is also 

preferred alcohol for using in the 

transesterification process compared to 

methanol since it is derived from 

agricultural products and is renewable 

and biologically less offensive in the 

environment. 

8.4. Type and amount of catalyst 

Biodiesel formation is also 

affected by the concentration of catalyst. 

Most commonly used catalyst for 

biodiesel production is sodium hydroxide 

(NaOH) or Potassium hydroxide (KOH). 

The type and amount of catalyst required 

in the transesterification process usually 

depend on the quality of the feedstock 

and method applied for the 

transesterification process. For a purified 

feedstock, any type of catalyst could be 

used for the transesterification process. 

However, for feedstock with high 

moisture and free fatty acids contents, 

homogenous transesterification process is 

unsuitable due to high possibility of 

saponification process instead of 

transesterification process to occur. 

8.5. Mixing intensity 

Oils and alcohols are not totally 

miscible, thus reaction can only occur in 

the interfacial region between the liquids 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 269



and transesterification reaction is a 

moderately slow process. So, Mixing is 

very important in the transesterification 

process, adequate mixing between these 

two types of feedstock is necessary to 

promote contact between these two feed 

stocks, therefore enhance the 

transesterification reactions to occur 

(Jagadale and Jugulkar, 2012). 

9. Thermo-kinetics of transesterification 

The feasibility of a reaction is 

determined from the thermodynamic 

parameters. Since both reactants and 

products are liquids, entropy change will 

tend to zero, hence equilibrium constant 

will be low (Owolabi et al., 2012). 

Kinetics of transesterification reaction has 

at least 3 main reactions as shown in the 

equations stated below (Noureddini and 

Zhu, 1997). 

Activation energy for the reverse 

reaction is higher than that for the 

forward reaction, which again should 

confirm the low possibility of reverse 

reactions. Some of the few kinetics 

studies that have been performed in recent 


times include esterification of free fatty 

acids in sunflower oil and oleic acid 

(Berrios et al., 2007 and Kraai et al., 

2008). Transesterification kinetics of 

soybean oil with five different catalysts 

has also been studied (Singh and 

Fernando, 2007). 

10. Fuel properties of biodiesel 

The fuel properties of biodiesel 

are discussed below (Owolabi et al., 

2012; Gopal and Karupparaj, 2015). The 

limits of ASTM D 6751standard are listed 

in Table 3. 

10.1. Specific gravity and density 

Density is the mass of unit volume 

of a material at a specific temperature. A 

more useful unit used by the petroleum 

industry is specific gravity, which is the 

ratio of the weight of a given volume of a 

material to the weight of the same volume 

of water measured at the same 

temperature. Specific gravity is used to 

calculate the mass of oils. Density 

influences the efficiency of the fuel 

atomization for airless combustion 

system. It has some effect on the break-up 

of fuel injected into the cylinder. In 

addition, more fuel is injected by mass as 

the fuel density increases. 

Table 3: ASTM D 6751 standard for biodiesel 

Parameters Units ASTM D 6751 

Limits 

ASTM D 6751 

Methods 

Acid number 

mg 

0.50 max D 664 

KOH/g 

FFA % - - 

Specific gravity g/cm 3 0.87 - 0.90 D 1250 - 08 

Kinematic viscosity at cSt 1.9 - 6 D 445 

40ºC 

Peroxide value meq/kg - - 

Calorific value MJ/kg - D 240 - 02 

Sulphated ash % 0.02 D 874 

Water & Sediments % 0.05 D 2709 

Copper corrosion - No. 3 max D 130 

Carbon residue % 0.05 D 4530 

Flash point ºC 130 min D 93 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 270



10.2. Lower calorific value 

It is a measure of the energy 

produced when the fuel is burnt 

completely which also determines the 

suitability of methyl ester as an 

alternative to diesel fuel. The calorific 

value of methyl ester is normally lower 

than diesel due to oxygen content of 

methyl ester. 

10.3. Iodine value 

Iodine value or iodine number is 

defined as the number of grams of iodine 

taken up by 100 g of oil or fat. In this 

case, addition reaction takes place across 

the double bonds of unsaturated fatty 

acids present in the fat by the addition of 

a halogen, such as iodine. Thus, the 

iodine number gives the indication of the 

degree of unsaturation of fats. 

10.4. Molecular weight 

The average molecular weight of 

the methyl ester is calculated by 

considering the weight percent of each 

fatty acid and their corresponding 

molecular weights. 

10.5. Cetane number 

It is the measure of the ignition 

quality of diesel fuel; higher this number 

the easier it is to start a standard (directinjection) 

diesel engine. It denotes the 

percentage (by volume) of cetane 

(chemical name Hexadecane) in a 

combustible mixture (containing cetane 

and 1-Methylnapthalene) whose ignition 

characteristics match those of the diesel 

fuel being tested. 

10.6. Flash point 

It is the minimum temperature of 

the fuel at which the fuel gives flash when 

it comes to contact with testing flame. It 

is an important parameter from the safety 

point of view such as safe for transport, 

handling, storage purpose and safety of 

any fuels. This is higher than petrol diesel 

which has flash point of 70°C. A fuel with 

high flash point may cause carbon 

deposits in the combustion chamber. 


10.7. Pour point, cloud point 

The pour point of a crude oil or 

product is the lowest temperature at 

which oil is observed to flow under the 

conditions of the test. Handling and 

transporting oils and heavy fuels is 

difficult at temperatures below their pour 

points .Often, chemical additives known 

as pour point depressants are used to 

improve the flow properties of the fuel. 

The temperature at which wax crystals 

begin to form on the surface of the 

biodiesel is the cloud point. 

10.8. Kinematic viscosity 

This is the resistance to flow of 

oil. Ease of starting depends on viscosity. 

Glycerin contamination may cause 

biodiesel viscosity to increase. It is the 

most important fuel features and this 

factor affects the operation of fuel 

injection, blending formation and 

combustion process. The high viscosity 

interferes with the injection process and 

leads to insufficient atomization. 


Biodiesel, of the family of biofuel, 

has been described in this review as a fuel 

with necessary potentials to replace fossil 

diesel in future. The trials biodiesel and 

its blend have undergone is a 

confirmatory test to all advantages 

including environmental benefits accrued 

to it thereby plays a vital role in meeting 

future fuel requirements. The availability 

of major feedstock namely oil from 

biosources and simplicity of the 

transesterification technology that ensures 

its conversion to biodiesel are added 

advantage in terms of the future needs of 

biodiesel. The use of inedible oil and 

waste frying/cooking oil has equally 

assisted in establishing a balance between 

energy and food security. However, 

serious efforts have to be intensified on 

design of large scale bio-refineries for 

future biodiesel production. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 271



References 

Agarwal, A.K. (2007). Biofuels (alcohols 

and biodiesel) applications as fuels 

for internal combustion engines, 

Progress in Energy and Combustion 

Science 33, 233–271. 

Avhad, M.R. and Marchetti, J.M. 

(2016). Innovation in solid 

heterogeneous catalysis for the 

generation of economically viable 

and ecofriendly biodiesel: A review, 

Catalysis Reviews 58, 2: 157-208. 

Berrios, M. Siles, J. Martin, M.A and 

Martin A.A (2007). Kinetic Study of 

the Esterification of Free Fatty Acids 

(FFA) in Sunflower Oil. Fuel 86, 

2383-2388. 

Chakrabarti, M. and Rafiq Ahmad 

(2008). Transesterification studies on 

castor oil as a first step towards its 

use in bio diesel production. Pakistan 

Journal of Botany 40, 1153-1157. 

Dwivedi, G. Sharma, M.P. and Jain, S. 

(2011). Impact analysis of biodiesel 

on engine performance- A Review. 

Renewable and Sustainable Energy 

Review 15, 4633-4641. 

Dwivedi, G. Sharma, M.P. Kumar, M 

(2014). Status and Policy of 

Biodiesel Development in India. 

International Journal of Renewable 

Energy Research 4 (2), 246-254. 

Gashaw, A. and Lakachew, A (2014). 

Production of biodiesel from nonedible 

oil and its Properties. 

International Journal of Science, 

Environment and Technology, 3(4), 

1544 – 1562. 

Gopal, K. and Karupparaj, R (2015). 

Effect of pongamia biodiesel on 

emission and combustion 

characteristics of DI compression 

ignition engine, Ain Shams 

Engineering Journal 6, 297-305. 

Hasan, Ali Md. Mashud, M. Rubel, 

Md. R. and Ahmad R.H. (2013). 

Biodiesel from Neem oil as an 

alternative fuel for Diesel engine. 

Procedia Engineering 56, 625- 630. 

Jagadale,S.S. and Jugulkar, L.M 


(2012). Review of Various Reaction 

Parameters and Other Factors 

Affecting on Production of Chicken 

Fat Based Biodiesel. Int. J. Mod. 

Eng. Res., 2(2), 407–411. 

Knothe,G.(2006). Analyzing biodiesel: 

Standards and Other Methods, 

JAOCS., 83, 823 – 833. 

Kraai, G.N. Winkelman, J.G.M. De 

Vries, J.G. and Heeres, H.J (2008). 

Kinetic Studies on the Rhizomucor 

Miehei Lipase Catalyzed 

Esterification Reaction of Oleic Acid 

with 1-Butanol in Biphasic System. 

Biochem Engg. J. 41, 87–94. 

Kusdiana, D. and Saka, S (2001). 

Biodiesel fuel from rapeseed oil as 

prepared in supercritical methanol. 

Fuel 80, 225-231. 

Leung, Y. Guo (2006). 

Transesterification of neat and used 

frying oil: Optimization for biodiesel 

production. Fuel Processing 


Mohibbe, A.M. Waris, A. and Nahar, 

M. N (2005). Prospects and Potential 

of Fatty Acid Methyl Esters of Some 

Non-Traditional Seed Oils for Use as 

Biodiesel in India. J. Biomass and 

Bioenergy, 29, 293-302. 

Narasimharao, K. Lee, A. and Wilson, 

K. (2007). Catalysts in Production of 

Biodiesel: A Review, Journal of 

Biobased Materials and Bioenergy 1, 

1–12. 

Noureddini, H and Zhu, D (1997). 

Kinetics of transesterification of 

soybean oil. J Am Oil Chem Soc 

74(11),1457–1463. 

Owolabi, R.U. Adejumo, A.L.and 

Aderibigbe, A.F (2012). Biodiesel: 

Fuel for the Future (A Brief Review). 

International Journal of Energy 

Engineering, 2(5), 223-231. 

Samukawa, T. Kaieda, M. 

Matsumoto,T. Ban, K. Konda, A. 

Shimada, Y. Noda, H. and 

Fududa, H (2000). Pretreatment of 

immobilized Candida antarctica 

lipase for biodiesel fuel production 

from plant oil, Journal of Bioscience 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 272



and Bioengineering 90 (2), 180–183. 

Shimada, Y. Watanabe, Y. Samukawa, 

T. Sugihara, A. Noda, H. Fukuda, 

H. and Tominaga, Y (1999). 

Conversion of vegetable oil to 

biodiesel using immobilized Candida 

antarctica lipase. J Am Oil Chem Soc 

76(7),789–793. 

Singh, A. K. and Fernando, S.D (2007). 

Reaction Kinetics of Soybean Oil 

Transesterification 

using 

Heterogenous Metal Oxide Catalysts. 

Chem. Eng. Technol. 30, 1716– 1720. 

Verma, D. Raj, J. Pal, A. and Jain M. 

(2016). A critical review on 

production of biodiesel from various 

feedstocks, Journal of Scientific and 

Innovative Research 5, (2): 51-58. 

Wakil, Md. Abdul, Ahmed, Z.U. 


Rahman, Md. Hasibur, and Md. 

Arifuzzaman (2012). Study on fuel 

properties of various vegetable oil 

available in Bangladesh and 

biodiesel production. International 

Journal of Mechanical Engineering 

2(5),10-17. 

Wang, Y. Ou, S. Liu, P. and Zhang, Z 

(2007). Preparation of biodiesel from 

waste cooking oil via two-step 

catalyzed process. Energy 

Conversion and Management 

48,184–188. 

Ying, T. Gang, C. Zhang, J. and Lu, Y 

(2011). Highly active CaO for the 

transesterification to biodiesel 

production from rapeseed oil. 

Bulletin of the Chemical Society of 

Ethiopia 25(1), 37-42. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 273



Biotech Sustainability (2017), P274--286 


Narayanan Kannan 1, *, Poorani Krishnan 2 and Ahmad Zaharin Aris 3 

1 Postgraduate Research and Innovation and Strategic Development, Taylor's University 

(Lakeside Campus), No. 1, Jalan Taylor's, 47500, Subang Jaya, Selangor 

Darul Ehsan, Malaysia; 2 Department of Medical Microbiology and parasitology, 

Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, 

Selangor, Malaysia; 3 Faculty of Environmental studies, Department of Environmental 

Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia;*Correspondence: 

Kannan.Narayanan@taylors.edu.my; Tel: +603 5629 5463 

Abstract: Most fast developing countries in Asia face severe pollution problems due to industrialization 

and modernization. Analytical chemical measurements are common in pollution 

monitoring. However, the ecological (biological) effects of those chemicals are difficult 

to understand. The latest generation of bioanalytical tools, such as in vitro transactivation 

bioassays, show great promise as water and sediment quality monitoring tools. A battery 

of in vitro cell bioassays has been developed in recent years that are effective, specific, 

fast, less expensive and easy to handle. One example is, Aryl hydrocarbon Receptor (AhR) 

mediated CYP1A1 protein expression that has been utilized effectively as a biomarker of 

pollution stress to marine biota. Hence, AhR binding potential, subsequent induction, genotoxic 

expression and cytotoxic potential of pollutants are reviewed. The list of in vitro cell 

bioassays reviewed will hopefully be utilized in ecotoxicological studies in developing 

countries in Asia. 

Keywords: Developing countries; ecotoxicology; in vitro cell bioassays; monitoring; policy; 

pollution 


There is a growing concern over persistent 

organic pollutants (POPs) that are 

ubiquitous, persistent and toxic (Kannan 

et al. 1988, 1989 a.b,c, 1997, 2016; Kaw 

and Kannan 2016). Hence, monitoring 

these chemicals in the environment is important. 

Conventional monitoring of targeted 

chemicals relies on analytical 

methods for measurements in the matrix 

of interest (i.e. water or sediment). Monitoring 

can also be based on surveys on the 

health of individuals and populations of 

sentinel species (e.g. indigenous fish); or 

at a larger scale, the integrity of ecosystem 

health by measuring community 

composition, diversity and or function. 

Though these approaches are effective, 

they are not very fast, at times, cumbersome 

and usually expensive, as new 

chemicals keep entering the aquatic/terrestrial 

environment. Moreover, this 

approach does not address unknown 

chemicals such as photolytic/biological 

transformation products or complex 

chemical mixtures that sentinel species of 

interest are exposed to. 

Thus, chemical measurement of priority 

pollutants alone is not sufficient, as 

their biological impacts need to be assessed. 

Toxicity end-points such as survival 

and mortality of an organism are 

very useful indicators of biological impacts. 

However, most of the contaminants 

occur at low concentrations for any possible 

toxicological investigation. High 

throughput cell bioassay techniques have 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 274



recently been shown to effectively screen 

environmental contaminants based on 

their biological mode of action. In particular, 

a number of in vitro cell bioassays 

have been adopted to measure the integrated 

response of bioactive contaminants, 

such as estrogens, in recycled and 

surface waters as well as in wastewater 

effluents (van der Linden et al. 2008; 

Leusch et al. 2010; Escher et al. 2014; 

Mehinto et al. 2015). These emerging bioanalytical 

tools can indicate which classes 

of chemicals are of concern, thus narrowing 

the field of targeted chemical 

analysis needed and toxicity endpoints to 

evaluate (Brack et al. 2015; Maruya et al. 

2016). In general, in vitro bioassays are 

good markers of biological toxicity as 

they are sensitive, quick, less expensive 

(Mehinto et al. 1993; Qiao et al., 2006). 

The advantage of effect-based bioassay 

measurements is that they determine the 

integrated toxic potency of the complex 

mixture of micro contaminants in the environment. 

In addition, bioassays may 

detect mixture effects of compounds, 

even when the individual constituents of 

the mixture are present at concentrations 

too low to cause an effect or to be detected 

by chemical analysis (Hamers et al., 

2010). The impact of unmonitored contaminants 

(contaminants of emerging 

concern) remains largely uncharacterized. 

Considering these points, one can safely 

conclude that in-vitro bioassays are comprehensive 

and holistic and can act as early 

warning signals of environmental degradation. 

2. Significance of cell in vitro bioassays 

Kannan et al. 

In vitro cell bioassays utilizing either 

wild type cells or genetically engineered 

eukaryotic cells providing an assessment 

on the potency of contaminants extracted 

from environmental matrices. Applications 

of in vitro cell bioassays are highly 

effective in terms of cost and time. A notable 

advantage is that through in vitro 

bioassays the detection and assessment of 

toxicity of a complex mixture of pollutants 

that exist in environmental extracts 

is made possible. Moreover, in comparison 

to the limited identification of pollutants 

provided through instrumental analysis, 

in vitro techniques provide an assessment 

of the total biological impact 

exerted by complex mixtures of environmental 

contaminants that are mediated 

through a common mechanism of action. 

Various bioassays have been developed 

over the years to evaluate the toxic potency 

of environmental extracts with reference 

to a specific target receptor. Bioassays 

are reliable tools to measure the response 

of a cell in terms of protein expression 

and enzyme activity when biological 

organisms are exposed to environmental 

pollutants. These responses are 

reportedly triggered by specific genes that 

mediate the transcription of the particular 

target protein. Cell lines developed from 

mammals and fish utilizing CYP1A1 induction 

as a biomarker of exposure to 

Poly aromatic hydrocarbons (PAHs), Polychlorinated 

biphenyls (PCBs) and other 

Halogenated aromatic hydrocarbons 

(HAHs) are effective in vitro tools for 

cumulative impact of sediment extracts 

(Jung et al., 2012; Schnell et al. 2013). 

Thus, various cell lines developed from 

mammals (He et al., 2011; Willet et al., 

1997 a, b) and fish (Fernandes et al., 

2014; Schnell et al. 2013; Fent, 2001) inducing 

CYP1A monooxygenases enzyme 

belonging to the cytochrome P450 family 

have been utilized. 

3. Aryl hydrocarbon Receptor (AhR) 

mediated toxic response 

The most potent inducer of Aryl hydrocarbon 

Receptor (AhR) is 2,3,7,8- 

tetrachlorodibenzo-p-dioxin (TCDD). 

Thus chemicals that elicit response similar 

to TCDD are generally clustered as 

dioxin-like chemicals. The ability of these 

chemicals to cause hepatotoxicity, embryotoxicity, 

teratogenicity, immunotoxicity, 

dermal toxicity, lethality, carcinogenesis, 

and tumor promotion in many 

different species at low concentrations 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 275



have generated much concern (Giesy et 

al; 2002; Ahlborg et al., 1992; Peterson et 

al., 1993). These chemicals are largely 

mediated through AhR dependent mechanism 

of action in biological system (Poland 

and Knutson., 1982; Willet et al., 

1997, Yoo et al, 2006). Several reviews 

have been published on the potency of 

dioxin-like environmental xenobiotics 

(Gillesby and Zacharewski, 1998; Ankley 

et al., 1998; van den Berg et al., 1998; 

Giesy et al, 2002). 

AhR is described as a ligand dependent 

transcription factor located in the cytosol, 

chaperoned with heat shock proteins 

(Giesy et al., 2002). Increased toxicity, 

enhanced gene transcription and enzyme 

activity are indicators of the binding 

strength of congeners to the AhR (Giesy 

et al., 2002; Safe, 1995). The AhR mediated 

mechanism is initiated once the ligands 

bind to cytosolic AhR. The receptor 

ligand complex is then translocated into 

the nucleus leading to the dissociation of 

heat shock proteins followed by formation 

of a dimer by binding to Ah Receptor 

Nuclear Traslocator (ARNT) protein. 

The heteromeric ligand AhR:ARNT 

complex then binds to dioxin-responsive 

element (DRE) a specific DNA sequences. 

This binding in turn will stimulate 

transcriptional activation of adjacent responsive 

genes leading to production of 

specific protein such as CYP1A1 (Giesy 

et al., 2002; Denison and Heath-Pagliuso, 

1998; Hankinson, 1995; Celander et al., 

1996). CYP1A1, belonging to the superfamily 

of cytochrome P450 plays a significant 

role in the biotransformation of xenobiotic 

such as PAHs and PCBs in organisms. 

Induction of CYP1A has been 

the most useful biomarker for environmental 

contamination and had been applied 

in various pollution monitoring programs 

(Bucheli and Fent, 1995; Celander 

et al., 1996). 

Biotransformation is a complex process 

of excretion of hydrophobic substrate by 

converting them to hydrophilic metabolite 

through monooxygenation (Czeka, 2000). 

Bioassays that measure the activation of 

Kannan et al. 

AhR signaling pathway through the expression 

of the protein CYP1A and its 

enzymatic activity has been developed 

and constantly improved with modifications 

to evaluate and applied in environmental 

assessment. 

4. AhR active compounds 

AhR ligands are made by hydrophobic 

compounds such as polychlorinated 

dibenzo-p-dioxins and dibenzofurans 

(PCDDs and PCDFs), chlorinated azobenzenes 

and azoxybenzenes, polychlorinated 

biphenyls (PCBs), several polycyclic 

aromatic hydrocarbons (PAHs), polychlorinated 

naphthalenes (Giesy et al., 

2002; Blankenship et al., 2000; Jung et 

al., 2012) and various halogenated aromatic 

hydrocarbon (HAHs) (Willet et al., 

1997). Relatively weak AhR ligand has 

been identified for natural and synthetic 

compounds (Giesy et al., 1998; Denison 

and Heath-Pagliuso, 1998). 

5. Dioxin responsive (DR) CALUX bioassay 

The CALUX (chemically activated 

luciferase expression) is a reporter-gene 

based cell bioassay that is increasingly 

applied in screening of dioxin and dioxin 

like chemicals in environmental matrices 

and in food materials (Tsutsumi et al., 

2003; Cederberg et al., 2002). To perform 

this assay, recombinant mammalian cell 

lines that have been stably transfected 

with one of two different AhR-responsive 

luciferase reporter gene plasmids that 

responds to dioxin and related chemicals 

are utilized (Denison et al., 2004; 

Garrison et al., 1996). Briefly, these cells 

contains AhR, that when bound to 

activating ligands will innitiate the 

transcription of the luciferase genes and 

the induction of the luciferase activity that 

will be determined by measuring 

luminescence. Induction of luciferase 

activity is directly proportional to the 

amount and potency of inducing chemical 

to which the cells are exposed (Han et al., 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 276



2004; Denison et al., 2004). However, the 

results obtained will vary significantly 

depending on the type of cell lines used. 

This is due to the species and tissues 

specific differences and functionality of 

the AhR as well as the antagonistic and 

synergistic effects of some compounds 

that are cell line dependent. 

The analysis of the luminescence 

measured through this assay is converted 

to CALUX – TEQ toxic equivalents. The 

luminescence value given by a sample is 

compared to the dose response curve of a 

reference standard such as 2,3,7,8- 

tetrachlorodibenzo-p-dioxin. Briefly, the 

sample concentration that induce 

luciferase to 25% of the TCDD-induced 

maximum luciferase activity is designated 

as the EC 25 TCDD for that sample. The 

TEQ concentrations were calculated as; 

H4IIE-luc cells are among the most 

common cells utilised in luciferse based 

assay to characterize the induction 

potency of AhR active compounds such 

as halogenated aromatic hydrocarbon 

(HAH) mixtures containing 

polychlorinated biphenyls (PCBs), 

dibenzo-p-dioxins (PCDDs) in 

environmental matrices (Yoo et al., 2006; 

Giesy et al., 2002; Whyte et al., 2004; 

Willet et al., 1997 a,b; Schmitz et al. 

1995). H4IIE-luc cells have been studied 

and suggested as an alternative 

bioanalytical tool to the wild-type cells 

for the detection of AhR agonists in 

environmental samples (Sanderson et al., 

1996). 

Progressive studies in this particular 

assay lead to the development of a third 

generation (G3) CALUX cell bioassays. 

In such studies mouse hepatoma 

(hepa1c1c7) cells transfected with G3 

CALUX plasmids with increased 

numbers of dioxin response elements 

(DREs) are utilized. This development 

Kannan et al. 

provides a highly responsive and sensitive 

bioassay system for the detection and 

relative quantitation of very low levels of 

dioxin-like chemicals in sample extracts 

(He et al., 2011). This bioassay is an 

essential assay to characterize AhR active 

compounds in environmental matrices as 

it accounts for the total AhR mediated 

activities in the test samples including 

non-dioxin-like chemicals such as PAHs 

and HAHs. Several advantages of 

CALUX assay are discussed by Windal 

et al. (2005) such as the reliability of 

CALUX assay to analyzes the overall 

biological activity of all AhR ligands 

(agonists and antagonists) present in an 

extract, in comparison to chemical 

analyses that only focuses on a selected 

number of compounds. Besides, for 

enviromental screening CALUX and 

chemical analysis are complimentary 

reflecting the mangitude of dioxin like 

activity induced by other compounds that 

were undetected in chemical analaysis; 

particularly, when large numbers of environmental 

samples are to be screened. 

CALUX ensures a rapid and cost effective 

analysis for dioxin-like chemicals. 

6. EROD Assay 

Induction of 7-ethoxyresorufin-Odeethylase 

(EROD) activity has been applied 

in numerous environmental toxicology 

studies as to indicate pollution stress 

(Fernandes et al., 2014; Kim et al., 2013; 

Schnell et al., 2013; Huuskonen et al., 

2000). EROD activity is based on the 

deethylation of the synthetic model substrate 

7-ethoxyresorufin by CYP1A1, an 

enzyme that belong to the phase-1 group 

of biotransformation enzymes. This reaction 

will produce a fluorescent product 

resorufin that is quantified fluorometrically 

and normalized to the total protein content. 

In biological system, the induction of 

CYP1A1 is triggered when dioxin-like 

chemicals such as PAHs and HAHs bind 

to the AhR. As an immediate response, a 

binding enhanced gene expression of 

CYP1A1 protein occurs to detoxify the 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 277



system from the xenobiotics. A study 

conducted on ethoxyresorufin- O- 

deethylase (EROD) activity of 19 PAHs 

in the fish hepatoma cell line PLHC-1 has 

shown that it is a useful tool for the ecotoxicological 

evaluation of landfill leachates 

(Fent and Batscher, 2000). Previously, 

EROD assay was conducted mostly to 

study the traditional environmental pollutants 

such as PAHs, PCBs and HAHSs. 

Recently, EROD assay is being utilized 

for studying various new kinds of compounds/pollutants 

such as heterocyclic 

aromatic compounds containing nitrogen, 

sulfur or oxygen (NSO-HET) (Hinger et 

al., 2011). EROD assay is also reported to 

be used in determining the CYP1A induction 

potencies of Nitrated polycyclic aromatic 

hydrocarbons (NPAHs) and N- 

heterocyclic aromatic hydrocarbons 

(azaarenes) in fish hepatoma for the first 

time (Jung et al., 2001).These compounds 

are commonly found PAH-contaminated 

environmental samples. 

To reflect the induction potency in ecotoxicological 

evaluation, a concept of induction 

equivalency factors (IEFs) was 

developed based on half maximal effect 

concentration (EC 50 ). EC 50 is the concentration 

of substance that induces 50% of 

the maximum induction level. IEF values 

are calculated as follows; 

Kannan et al. 

IEQ Detected = Total Concentration of mixture 

× IEF of mixture 

(4) 

By relating the experimentally detected 

EC 50 value of the mixture to reference 

compound and multiplying this value by 

the total concentration of compounds present 

in the mixture, an IEQ det is determined 

and compared to the IEQ calc (Jung 

et al., 2001). The IEQ values are expressed 

in mg reference substance equivalents 

(Reference substance equivalents 

/L). 

The 2,3,7,8-tetrachlorodibenzo-p-dioxin 

(TCDD), the most potent ligand to the 

aryl hydrocarbon receptor and the strongest 

inducer of EROD activity in most test 

systems has been used as a reference substance 

in many studies (Huuskonen et al., 

2000; Celender et al., 1996), however, 

recent studies suggest that β- 

naphthoflavone is proved to be the most 

suitable reference for the routine in vitro 

EROD assay (Heinrich et al., 2014; Fernandes 

et al., 2014; Schnell et al., 2013). 

Many other alternatives have been developed 

to analyze the data generated 

through EROD assay. A formula developed 

by Sprague and Ramsay (1965) are 

also utilized in which the EROD activity 

is converted to toxic units (Schnell et al., 

2013). 

As for the analysis involving binary 

mixtures, the EC 50 of both single compounds 

and mixtures were determined 

and the IEF of the compounds in relation 

to a reference substance is determined. 

Induction equivalents (IEQs) for a mixture 

are then calculated to show the additive 

interactions of independent compound 

in a mixture. 

7. Enzyme-linked immunosorbent assay 

(ELISA) 

In the determination of CYP1A enzymatic 

activity through EROD assay, 

quantification of CYP1A protein induction 

level provides important and complementary 

information about the regula- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 278



tion of CYP1A and mechanism of toxic 

action. Enzyme-linked immunosorbent 

assay (ELISA) is a useful technique in 

immunologically detecting CYP1A protein. 

ELISA provides complementary information 

on the induction of CYP1A in 

the presence of inducing substances. Previously 

quantification of CYP1A protein 

content was made possible through the 

preparation of subcellular fractions involving 

scrapping of cells from culture 

flask, followed by sonication and centrifugation 

and eventually quantification 

through western blotting (Hahn et al., 

1993). This technique is rather time consuming. 

Hence, a much rapid and sensitive 

detection of CYP1A protein was developed 

in which the detection of the 

amount of CYP1A protein extracted was 

measured directly in the microwells (Bruschweiler 

et al., 1995). This technique 

allows a semiquantitave determination of 

the amount of immunoreactive CYP1A 

protein directly in the microwells in 

which the cells are cultured. The data of 

absolute absorption values are presented 

as the percent of maximal induction of 

CYP1A and EC 50 is determined. Studies 

have utilized ELISA to quantify CYP1A 

protein to provide a complementary quantification 

on CYP1A based EROD activity 

(Herrero and Castel, 1994; Jung et al., 

2001). 

Kannan et al. 

8.1. Comet assay 

Genotoxicity is of great interest because 

in the case of chronic exposure situation, 

the possibility of delayed consequences 

at the population level is high 

(Devaux et al., 2011). As such, the application 

of Comet assay provides an early 

and universal genotoxicity endpoint revealing 

the primary DNA damage occurred 

due to the exposure to environmental 

pollutants (Frenzilli et al., 2009). 

Genotoxicity assay such as Comet assay 

is a biomarker assay providing valuable 

information on the presence of potential 

carcinogens and mutagens in the environmental 

sample. Comet assay (single 

cell gel electrophoresis) is a technique of 

measuring DNA damage in eukaryotic 

cells or disaggregated tissues when exposed 

to hazardous agents (Azqueta and 

Collins, 2013). In this assay the DNA is 

drawn out towards the anode through 

electrophoresis, forming a comet-like image 

that is observed with fluorescence 

microscopy (Azqueta and Collins, 2013). 

In general, this assay refers to the relaxation 

of supercoiled DNA in agaroseembedded 

nucleoids (the residual bodies 

remaining after lysis of cells with detergent 

and high salt), which then allows the 

DNA to be drawn out towards the anode 

under electrophoresis, forming comet-like 

images as seen under fluorescence microscopy. 

DNA break frequency is indicated 

through the relative amount of DNA 

in the comet tail (Azqueta and Collins, 

2013). The extent of DNA migration are 

determined as a percentage of DNA in the 

tail (% tDNA) using an image analysis 

system. For statistical analysis, the induction 

factor (IF) was calculated using the 

following equation: 

(6) (Šrut et al., 2011) 

8. Genotoxicity assay 

Concentration dependent induction 

factor (CDI) index is then calculated by 

integrating all the important information. 

This forms the basis for a general comparison 

of the genotoxic potential of samples 

in the Comet assay. CDI is calculated 

according to the following equation: 

Recent advancement in this particular 

assay suggests that digestion with lesionspecific 

enzymes such as Formamidopyri- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 279



midine DNA glycosylase (FPG), provides 

a greater yield for strand breaks and increases 

the sensitivity of the assay in general 

(Azqueta and Collins, 2013). In the 

case of low level of strand breaks at noncytotoxic 

concentrations of genotoxic 

chemical, the yield of breaks was greatly 

enhanced after incubation with (FPG) 

(Azqueta et al., 2013). 

This is a sensitive, simple and versatile 

assay performed to visualize the single 

strand break in nuclear DNA of single 

cells (Wilkening et al., 2003). Besides 

providing the overall level of damage in 

the cells being analysed, comet assay also 

provides the data on how the individual 

cells respond to the xenobiotic. Comet 

assay has been applied in studies on 

PLHC-1 fish cell line (Šrut et al., 2011) 

and characterization of the genotoxicity 

of sediment extracts from the Baltic and 

North Sea on EPC (epithelioma papulosum 

cyprini) fish cell line (Kammann et 

al., 2001, 2004). 

Kannan et al. 

8.2. Cytotoxicity assay 

To assess the acute toxicity of pollutants 

on biological organisms, various cytotoxicity 

tests are usually performed. 

Among them, tetrazolium salt reduction 

(MTT) assay is widely used as an endpoint 

for the cytotoxicity measurement of 

chemicals/pollutants in monolayer cell 

cultures (Vakharia et al., 2001; Bruschweiler 

et al., 1995; Heinrich et al., 

2014). The MTT assay detects the reduction 

of soluble MTT tetrazolium salt to a 

blue insoluble MTT formazan product by 

mitochondrial succinate-dependent dehydrogenase 

(Bruschweiler et al., 1995). 

The percentage of viability was calculated 

relative to DMSO treatment control wells 

from triplicate observations that are designated 

as 100% viable cells. Only cells 

with active mitochondria are able to catalyze 

this reaction. Cellular stress determination 

is crucial when analyzing complex 

samples such as environmental samples 

because enzymatic assays such EROD 

assay requires an intact metabolism for 

the in-vitro live cell approaches. This intact 

metabolism ensures the existence of 

full biotransformation capacity within the 

cells that is being exposed to the samples. 

Recent research suggests that the results 

of in vitro EROD assay to be presented by 

reference to a truly physiological cytotoxicity 

assay, the 3-(4,5-dimethylthiazol-2- 

yl) 2,5-diphenyltetrazo-lium bromide 

(MTT) test replacing protein normalization 

which will enable an optimized in 

vitro EROD protocol to a reference compound 

(Heinrich et al., 2014). 

9. Conclusion and recommendations 

Pollution is a major setback in developing 

countries with heavy emphasis on 

industrialization and modernization. Constant 

and consistent pollution monitoring 

is crucial in order to maintain pollution 

level in control. In pursuit of the effort, 

extensive environmental toxicology studies 

are essential because they provide the 

actual impact situation before the expensive 

instrumental analysis is conducted. 

Ecotoxicological studies in Malaysia are 

extremely rare. Hence, there is a need for 

immediate exploration and application of 

these techniques and this will aid the relevant 

authorities to improve the environmental 

status monitoring. 


This work was supported by grants-inaid 

from Research University Grant 

Scheme by University Putra Malaysia 

(9331400) to Prof. Ahmad Zaharin Aris. 

References 

Ahlborg, U. G., Brouwer, A., Fingerhut, 

M. A., Jacobson, J. L., 

Jacobson, S. W., et al. (1992). Impact 

of polychlorinated dibenzo-pdioxins, 

dibenzofurans, and biphenyls 

on human and environmental 

health, with special emphasis 

on application of the toxic 

equivalency factor concept. Euro- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 280



pean Journal of Pharmacology 

228, 179–199. 

Ankley, G., Mihaich, E., Stahl, R., Tillitt, 

D., Colborn, T., et al. (1998). 

Overview of workshop on screening 

methods for detecting potential 

(anti-) estrogenic/androgenic 

chemicals in wildlife. Environmental 

Toxicological Chemistry 17, 

68–87. 

Azqueta, A., Arbillaga, L., Lopez de 

Cerain, A., Collins, A. (2013). 

Enhancing the sensitivity of the 

comet assay as a genotoxicity test, 

by combining it with bacterial repair 

enzyme FPG. Mutagenesis 28, 

271-277. 

Azqueta, A., and Collins, A. R. (2013). 

The essential comet assay: a comprehensive 

guide to measuring 

DNA damage and repair. Archives 

of Toxicology 87, 949–968. 

Blankenship, A. L., Kannan, K.., Villalobos, 

A., Falandysz, J., Giesy, J. 

P. (2000). Relative potencies of 

halowax mixtures and individual 

polychlorinated naphthalenes 

(PCNs) in H4IIe-Luc cell bioassay. 

Environmental Science and Technology 

34, 3153–3158. 

Brack, W., Altenburger, R., Schüürmann, 

G., Krauss, M., Herráez, 

D. L., et al. (2015). The SOLU- 

TIONS project: Challenges and responses 

for present and future 

emerging pollutants in land and 

water resources management. Science 

of the Total Environment 503- 

504: 22–31. 

Bruschweiler, B. J., Wurgle, F.E., Fent. 

K. (1995). An Elisa Assay For Cytochrome 

P4501A in Fish Liver 

Cells. Environmental Toxicology 

and Chemistry, 15 (4), 592–596. 

Bucheli, T. D., Fent K. (1995). Induction 

of cytochrome P450 as a biomarker 

for environmental contamination in 

aquatic ecosystems. Critical Reviews 

in Environmental Science 

and Technology 25, 201–268. 

Kannan et al. 

Burg, B. (2008). Detection of multiple 

hormonal activities in wastewater 

effluents and surface water, using a 

panel of steroid receptor CALUX 

bioassays. Environmental Science 

and Technology 42, 5814-5820. 

Cederberg, T., Laier, P., Vinggaard, A. 

M. (2002). Screening of food samples 

for dioxin levels Comparison 

of GC-MS determination with the 

CALUX bioassay. Organohalogen 

Compounds 58, 409-412. 

Celander, M., Hahn, M. E., Stegeman, 

J. J. (1996). Cytochromes P450 

(CYP) in the Poeciliopsis lucida 

Hepatocellular Carcinoma Cell 

Line (PLHC-1): Dose and Time- 

Dependent Glucocorticoid Potentiation 

of CYP1A Induction without 

Induction of CYP3A. Archives of 

Biochemistry and Biophysics. 329, 

113–122. 

Czeka, P. (2000). Phenobarbital-induced 

expression of cytochrome P450 

gene. Acta Biochimica Polonica 47 

(4), 149–159. 

Denison, M. S., Zhao, B., Baston, D. S., 

Clark, G. C., Murata, H., Han, 

D. H. (2004). Recombinant cell bioassay 

systems for the detection 

and relative quantitation of halogenated 

dioxins and related chemicals. 

Talanta 63, 1123-1133. 

Denison, M. S., Heath-Pagliuso. (1998). 

The Ah Receptor: a regulator of the 

biochemical and toxicological actions 

of structurally diverse chemicals. 

Bulletin of Environmental 

Contamination and toxicology 

61(5), 557-68. 

Devaux, A., Fiat, L., Gillet, C., Bony, S. 

(2011). Reproduction impairment 

following paternal genotoxin exposure 

in brown trout (Salmo trutta) 

and Arctic char (Salvelinus alpinus). 

Aquatic Toxicology 101, 

405–411. 

Escher, B. I., Allinson, M, Altenburger, 

R., Bain, P. A, Balaguer, P., et al. 

(2014). Benchmarking Organic 

Micropollutants in Wastewater, 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 281



Recycled Water and Drinking Water 

with In Vitro Bioassays . Environmental 


48 (3), 1940–1956. 

Fent, K., and Batscher, R. (2000). Cytochrome 

P4501A induction potencies 

of Polycyclic aromatic hydrocarbons 

in a fish Hepatoma cell 

line: Demonstration of additive interactions. 

Environmental Toxicology 

and Chemistry 19(8), 2047– 

2058. 

Fent, K. (2001). Fish cell lines as versatile 

tools in ecotoxicology: assessment 

of cytotoxicity, cytochrome 

P4501A induction potential and estrogenic 

activity of chemicals and 

environmental samples. Toxicology 

in Vitro 15, 477–488. 

Fernandes, D., Pujol, S., Albaladejo, E. 

P., Tauler, R., Bebianno, M. J., 

Porte, C. (2014). Characterization 

of the environmental quality of 

sediments from two estuarine systems 

based on different in-vitro bioassays. 

Marine Environmental 

Research 96, 127-135. 

Frenzilli, G., Nigro, M., Lyons, B. P. 

(2009). The Comet assay for the 

evaluation of genotoxic impact in 

aquatic environments. Reviews in 

Mutation Research 681, 80–92. 

Garrison, P. M., Tullis, K., Aarts, J. M. 

M. J. G., Brouwer, A., Giesy, J. 

P. and Denison, M. S. (1996). 

Species-specific recombinant cell 

lines as bioassay systems for the 

detection of 2,3,7,8- 

tetrachlorodibenzo-p-dioxin-like 

chemicals. Fundamentals of Applied 

Toxicology 30, 194–203. 

Giesy, J.P., Hilscherova, K., Jones, P. 

D., Kannan, K., Machala, M. 

(2002). Cell bioassays for detection 

of aryl hydrocarbon (AhR) and estrogen 

receptor (ER) mediated activity 

in environmental samples. 

Marine Pollution Bulletin 45, 3– 

16. 

Giesy, J.P., Kannan, K. (1998). Dioxinlike 

and non-dioxin-like toxic 

Kannan et al. 

effects of polychlorinated biphenyls 

(PCBs): implications from risk 

assessment. Critical Review in Toxicology 

28, 511–569. 

Gillesby, B. E., Zacharewski, T. R. 

(1998). Exoestrogens: Mechanisms 

of action and strategies for identification 

and assessment. Environmental 

Toxicological Chemistry 

17, 3–14. 

Hahn, M. E., Lamb, T. M., Schultz, 

Roxanna, M. E., Smolowitz, R. 

M., Stegeman, J. J. (1993). Cytochrome 

P4501A induction and inhibition 

by 3,3',4,4'- 

tetrachlorobiphenyl in an Ah receptor- 

containing fish hepatoma cell 

line (PLHC-1). Aquatic Toxicology 

26 185-208. 

Hamers, T., Leonards, P. E. G., Legler, 

J., Vethaak, A. D, Schipper, C. 

A. (2010). Toxicity Profiling: An 

integrated effect-based tool for 

site-specific sediment quality assessment. 

Integrated Environmental 

Assessment and Management 

6:761-773. 

Han, D., Nagy, S. R., Denison, M. S. 

(2004). Comparison of recombinant 

cell bioassays for the detection 

of Ah receptor agonists. Biofactor 

20, 11-22. 

Hankinson, O. (1995). The aryl hydrocarbon 

receptor complex. Annual 

Review of Pharmacology and Toxicology 

35, 307–340. 

He, G., Tsutsumi, T., Zhao, B., Baston, 

D. S., Zhao, J., et al. (2011). 

Third-generation Ah receptorresponsive 

luciferase reporter 

plasmids: amplification of dioxinresponsive 

elements dramatically 

increases CALUX bioassay sensitivity 

and responsiveness. Toxicological 

Science 123(2):511-22. 

Heinrich, P., Diehl, U., Förster, F., 

Braunbeck. T. (2014). Improving 

the in-vitro ethoxyresorufin-Odeethylase 

(EROD) assay with 

RTL-W1 by metabolic normalization 

and use of β-naphthoflavone as 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 282



the reference substance. Comparative 

Biochemistry and Physiology, 

Part C 164, 27–34. 

Herrero, M. E., and Castell, J. V. 

(1994). Quantification of CYP1A1 

and 2B1/2 in rat hepatocytes cultured 

in microwells by immunological 

methods. Toxicology In Vitro 

8(6), 1167-l 175. 

Hinger, G., Brinkmann, M., Bluhm, K., 

Sagner , A., Takner, H., et al. 

(2011). Some heterocyclic aromatic 

compounds are Ah receptor 

agonists in the DR-CALUX assay 

and the EROD assay with RTL-W1 

cells. Environmental Science and 

Pollution Research 18(8), 1297- 

1304. 

Huuskonen S. E, A. Tuvikene, M. 

Trapido, K. Fent, M. E. Hahn. 

(2000). Cytochrome P4501A Induction 

and Porphyrin Accumulation 

in PLHC-1 Fish Cells Exposed 

to Sediment and Oil Shale Extracts. 

Archives of Environmental Contamination 

and Toxicology 38, 59– 

69. 

Jung, D. K., Klaus, T., Fent, K. (2001). 

Some heterocyclic aromatic compounds 

are Ah receptor agonists in 

the DR-CALUX assay and the 

EROD assay with RTL-W1 cells. 

Environmental Toxicology and 

Chemistry 20(1), 149-159. 

Jung, J. H., Hong, S. H., Yim, U. H., 

Ha, S. Y., Shim, W. J., Kannan, 

N. (2012). Multiple In Vitro Bioassay 

Approach in Sediment Toxicity 

valuation: Masan Bay, Korea. Bulletin 


and Toxicology 89(1):32-37. 

Kammann, U., Biselli, S., Hühnerfuss, 

H., Reineke, N., Theobald, N., et 

al. (2004). Genotoxic and teratogenic 

potential of marine sediment 

extracts investigated with Comet 

assay and zebrafish test. Environmental 

Pollution 132, 279–287. 

Kammann, U., Bunke, M., Steinhart, 

H., Theobald, N. (2001). A permanent 

fish cell line (EPC) for 

Kannan et al. 

genotoxicity testing of marine sediments 

with the Comet assay. Mutation 

Research 498, 67–77. 

Kannan, K., Tanabe, S., Giesy, J.P., 

Tatsukawa, R. (1997). Organochlorine 

pesticides and polychlorianted 

biphenyls in foodstuffs 

from Asian and Oceanic countries. 

Reviews on Environmental Contamination 

and Toxicology 15, 1– 

55. 

Kannan, N., Tanabe,S., Tatsukawa, R. 

(1988). Toxic potential of monoortho 

and non-ortho coplanar PCBs 

in commercial PCB preparations: 

"2,3,7,8-T4CDD Toxicity Equivalence 

Factors Approach". Bulletin 


and Toxicology 41, 267-27. 

Kannan, N., Wakimoto, T., Tatsukawa, 

R. (1989a). Possible involvement 

of frontier (π) electrons in the metabolism 

of polychlorinated biphenyls 

(PCBs). Chemosphere 18, 

9-10. 

Kannan, N., Tanabe, S., Okamoto, T., 

Tatsukawa, R., Phillips, D. J. H. 

(1989b). Polychlorinated Biphenyls 

(PCBs) in Sediments in Hong 

Kong: A Congener-Specific Approach 

to the Study of Coplanar 

PCBs in Aquatic Ecosystems. Environmental 

Pollution 62, 223-235. 

Kannan, N., Tanabe, S., Ono, M. 

Tatsukawa, R. (1989c). Critical 

evaluation of PCB toxicity in Terrestrial 

and marne mammals: Increasing 

impact of non-ortho and 

mono-ortho coplanar PCBs from 

land to ocean. Archives of Environmental 

Contamination and Toxicology 

18, 850-857. 

Kaw, H. Y., Kannan, N. (2016). A review 

on Polychlorinated Biphenyls 

(PCBs) and Polybrominated Diphenyl 

Ethers (PBDEs) in South 

Asia with a focus on Malaysia. Reviews 


and Toxicology, 242, 153- 

181. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 283



Keiter, S., Grund, S., Bavel, B.V., Hagberg, 

J., Engwall, M., et al. 

(2008). Activities and identification 

of aryl hydrocarbon receptor 

agonists in sediments from the 

Danube River. Anals of Bioanal 

Chem 390: 2009–2019 

Kim, H. N., Park, C. I., Chae, Y. S., 

Shim, W. J., Addison, R. F., et al. 

(2013). Acute toxic responses of 

the rockfish (Sebastes schlegeli) to 

Iranian heavy crude oil: Feeding 

disrupts the biotransformation and 

innate immune systems. Fish and 

Shellfish Immunology 35, 357-365. 

Krishnan, P., Kannan, N., Aris, A. Z., 

Arulselvan, P., Fakurazi S. 

(2016). Possible Application of 

bio-analytical assays in the biological 

impact assessment of Persistent 

Organic Pollutants (POPs) in 

mangrove sediments in South East 

Asia with particular reference to 

Malaysia. In: Persistent Organic 

Chemicals in the Environment: 

Status and Trends in the Pacific 

Basin Countries II. ACS Symposium 

Series; American Chemical Society. 

Loganathan, B. G., Khim, J. 

S., Prasada Rao S. Kodavanti, P. R. 

S., Masunaga, S. (eds.) Publication 

Date (Web): December 7, 2016 | 

doi: 10.1021/bk-2016-1244.ch009 

Lapa, N., Barbosa, R., Morais, J., 

Mendes, B., Méhu, J., et al. 

(2002). Ecotoxicological assessment 

of leachates from MSWI bottom 

ashes. Waste Management 22, 

583–593 

Leusch, F. D, de Jager, C., Levi, Y., 

Lim, R., Puijker, L., et al. (2010). 

Comparison of five in vitro bioassays 

to measure estrogenic activity 

in environmental waters. Environmental 


44:3853-3860. 

Maruya KA, Dodder NG, Mehinto AC, 

Denslow ND, Schlenk D, et 

al.(2016). A tiered, integrated biological 

and chemical monitoring 

framework for contaminants of 

Kannan et al. 

emerging concern in aquatic ecosystems. 

Integrated Environmental 

Assessment Management 12:540- 

547. 

Mehinto, A. C, Jia, A., Snyder, S. A., 

Jayasinghe, B. S., Denslow, N. D., 

et al. (1993). Developmental and 

reproductive toxicity of dioxins 

and related compounds: Cross species 

comparisons. Critical Review 

in Toxicology 23, 283–335. 

Mehinto, A. C., Jia, A., Snyder, S. A., 

Jayasinghe, B. S., Denslow N. D., 

et al. (2015). Interlaboratory comparison 

of in vitro bioassays for 

screening of endocrine active 

chemicals in recycled water. Water 

Research 83, 303-9. 

Poland, A., Knutson, J. C. (1982). 

2,3,7,8-Tetrachlorodibenzo-pdioxin 

and related halogenated aromatic 

hydrocarbons: examination of the 

mechanism of toxicity. Annual Review 

of Pharmacology 22, 517– 

554. 

Qiao, M., Chen, Y., Zhang, Q., Huang, 

S., Ma, M., et al. (2006). Identification 

of Ah receptor agonists in 

sediment of Meiliang Bay, Taihu 

Lake, China. Environmental Science 

and Technology 40,1415– 

1419. 

Safe, S., Krishnan, V. (1995). Cellular 

and molecular biology of aryl hydrocarbon 

(AH) receptor mediated 

gene expression. Archives of. Toxicology. 

7, 99–115. 

Sanderson J. T., Aarts, M. M. J. G., 

Brouwer, A., Froese, K. I., Denison, 

M. S., et al. (1996). Comparison 

of Ah Receptor-Mediated Luciferase 

and EthoxyresorufinOdeethylase 

Induction in H4IIE 

Cells: Implications for Their Use as 

Bioanalytical Tools for the Detection 

of Polyhalogenated Aromatic 

Hydrocarbons. Toxicology and applied 

pharmacology 137, 316–325. 

Schmitz, H., Hagenmaier, A., Hagenmaier, 

H., Bock, K. W., 

Schrenk, D. (1995). Potency of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 284



mixtures of polychlorinated biphenyls 

as inducers of dioxin receptor-regulated 

CYP1A activity in 

rat hepatocytes and H4IIE cells. 


Schnell, S., Olivares, A., Piña, B., 

Erasun, B. E., Porte, C., et al. 

(2013). The combined use of the 

PLHC-1 cell line and the recombinant 

yeast assay to assess the environmental 

quality of estuarine and 

coastal sediments. Marine Pollution 

Bulletin 77, 282–289. 

Seitz, N., Böttcher, M., Keiter, S., 

Kosmehl, T., Manz, W., et al. 

(2008). A novel statistical approach 

for the evaluation of Comet assay 

data. Mutation Research 652, 38– 

45. 

Sprague, J. B., Ramsay, B. A. (1965). 

Lethal effects of mixed copper and 

zinc solutions for juvenile salmon. 

Journal of the Fisheries Research 

Board of Canada 22, 425–432. 

Šrut, M., Traven, L., Štambuk, A., 

Kralj, S., Roko , et al. (2011). 

Genotoxicity of marine sediments 

in the fish hepatoma cell line 

PLHC-1 as assessed by the Comet 

assay. Toxicology in Vitro 25, 308– 

314. 

Tsutsumi, T., Amakura, Y., Nakamura, 

M., Brown, D. J., Clark, G. C., et 

al. (2003). Validation of the 

CALUX bioassay for the screening 

of PCDD/Fs and dioxin-like PCB 

in retail fish. Analyst 128, 486-492. 

Vakharia, D. D., Liu, N., Pause, R., 

Fasco, M., Bessette E, et al. 

(2001). Polycyclic Aromatic Hydrocarbon/Metal 

Mixtures: Effect 

On PAH Induction Of CYP1A1 in 

Human HepG2 Cells. Drug Metabolism 

and Disposal 29(7), 999- 

1006. 

Van den Berg, D., Birnbaum, L., 

Bosveld, B. T. C., Brunstrom, B., 

Cook, P., et al. (1998). Toxic 

equivalency factors (TEFs) for 

PCBs, PCDDs, PCDFs for humans 

Kannan et al. 

and wildlife. Environmental Health 

Perspective 106, 775–790. 

Van der Linden, S. C., Heringa, M. B., 

Man, H. Y., Sonneveld, E., et al. 

(2008). Detection of multiple hormonal 

activities in wastewater effluents 

and surface water, using a 

panel of steroid receptor CALUX 

bioassays. Environmental Science 

and Technology 42(15), 5814-20. 

Whyte, J. J., Schmitt, C. J., Tillitt, D. 

E. (2004). The H4IIE cell bioassay 

as an indicator of dioxin-like chemicals 

in wildlife and the environment. 

Critical Reviews in Toxicology. 

34, 1–83. 

Wilke, B. M., Riepert, F., Kich, C., 

Kühne, T. (2008). Ecotoxicological 

characterization of hazardous 

wastes. Ecotoxicology and Environmental 

Safety 70, 283–293. 

Wilkening, S., Stahl, F., Bader, A. 

(2003). Comparison of primary 

human hepatocytes and hepatoma 

cell line HEPG2 with regard to 

their biotransformation properties. 

Drug Metabolism and Disposition 

31 (8) 1035-1042. 

Willett, K. L., Gardinali. P. R., Sericano, 

J. L., Wade, T. L., Safe, S. H. 

(1997a). Characterization of the 

H4IIE rat hepatoma cell bioassay 

for evaluation of environmental 

samples containing Polynuclear 

Aromatic Hydrocarbons (PAHs). 

Archives of Environmental Contamination 

and Toxicology 32, 

442–448. 

Willett, K. L., Randerath, K., Zhou, G. 

D., Safe, S. H. (1997b). Inhibition 

of CYP1A1-dependent activity by 

the Polynuclear Aromatic Hydrocarbons 

(PAH) Fuoranthene. Biochemical 

Pharmacology 55, 831– 

839. 

Windal, I., Denison, M. S., Birnbaum, 

L. S., Wouwe, N. V., Baeyens, 

W., et al. (2005). Chemically Activated 

Luciferase Gene Expression 

(CALUX) cell bioassay analysis 

for the estimation of Dioxin-like 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 285



Kannan et al. 

activity: Critical parameters of the 

CALUX procedure that impact assay 

results. Environmental Science 

and Technology 39, 7357-7364. 

Yoo, H., Khim, J. S., Giesy, J. P. (2006). 

Receptor-mediated in vitro bioassay 

for characterization of Ah-Ractive 

compounds and activities in 

sediment from Korea. Chemosphere 

62, 1261–1271. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 286



Lipopeptide Biosurfactants from Bioagent, Bacillus as a 

Weapon for Plant Disease Management 

Sampath Ramyabharathi, Balaraman Meena*, Lingan Rajendran and Thiruvengadam 

Raguchander 

Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricultural 

University, Coimbatore – 641-003, Tamil Nadu, India; *Correspondence: 

meepath@rediffmail.com; Tel: +91 422 6611226 

Abstract: Biosurfactants are surface active biological compounds that are produced by 

broad range of microorganisms. They are ecofriendly with low toxicity, no residual effects 

with high biodegradable properties and known to suppress the growth of pathogenic fungi. 

Bacillus genus is considered microbial factories for the production of a huge number of biologically 

active molecules that are inhibitory for plant pathogen growth. In rhizobacteria 

Bacillus subtilis, an average of 4 – 5% of its genome is dedicated to antibiotic synthesis and 

has the possibility to produce more than two dozen of structurally diverse antimicrobial 

compounds. Because of the surfactant properties, the antimicrobial peptide compounds or 

cyclic lipopeptide antibiotics of the surfactin, iturin and fengycin families are wellrecognized 

for their potential applications in biotechnology. Different groups of lipopeptides 

can give an advantage to the Bacillus strains in specific environmental niches. The 

ability to induce systemic resistance in plants and their use in the spreading of the bacterial 

cells that leads to rhizosphere colonization could open new fields of applications for their 

use in phytopharmaceutical products. In this review article, we are highlighting the role and 

functions of some major biocontrol lipopeptide biosurfactants present in the Bacillus species. 

Keywords: Biological control; Bacillus species; lipopeptide biosurfactants 


Biosurfactants are produced on the 

microbial cell surface and are capable of 

lowering surface and interfacial tensions. 

They are produced extracellularly and 

thus are potential substitutes for widely 

used synthetic surfactants. Biosurfactants 

are widely used in industries, pharmaceutical, 

agriculture, food, cosmetics, oil 

production industries and in bioremediation 

process. Till date a broad range of 

structurally different biosurfactants have 

been identified that includes lipopeptides, 

polysaccharides, proteins, lipoproteins 

and glycolipids. Lipopeptide biosurfactants 

are composed of a lipid tail connected 

to a short linear or cyclic oligopeptide. 

The hydrophobic portion of lipopeptides 

is made up of fatty acids and the hydrophilic 

portion is composed of peptides or 

polysaccharides (Georgiou et al., 1992). 

Hence the presence of hydrophobic and 

hydrophilic portions within a single molecule, 

the biosurfactants tend to migrate 

toward an interface with different degrees 

of polarity and hydrogen bonding (Desai 

and Banat, 1997). In Gram-positive Bacillus 

subtilis IAM1213 the production of 

lipopeptide biosurfactants was first reported 

(Arima et al., 1968) and the different 

types of lipopeptide biosurfactants 

with significant surface activity, antipathogenic 

(Ramyabharathi and Raguchander, 

2014; Ramyabharathi et al., 2016), 

antinematicidal (Ramyabharathi, 2015; 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 287


Lipopeptide Biosurfactants as a Weapon for Plant Disease Management 

Ramyabharathi et al. 

Sankari Meena et al., 2016) and antimicrobial 

activity have been reported 

from other Bacillus strains. 

2. Bacillus lipopeptides 

The lipopeptides in Bacillus are 

classified into three families namely Iturin, 

Surfactin and fengycin family. There 

are several Bacillus strains isolated and 

reported to produce all the three 

families of lipopeptide biosurfactants 

simultaneously (Ramyabharathi and Raguchander, 

2014). 

2.1. Iturin lipopeptide 

Iturins are a member of an antifungal 

lipopeptide group comprises iturin, 

bacillomycin and mycosubtilin which are 

cyclic lipoheptapeptides and are linked by 

beta amino acid residue. Iturin family 

contains iturins A-E, bacillomycins D, F, 

and L, and mycosubtilin. Members of this 

family have powerful antibiotic activity 

but moderate surfactant activity and enhanced 

swarming motility (Leclere et al., 

2006). Hence the presence of iturin in 

Bacillus is responsible for disease suppression 

and growth inhibition of wide 

number of phytopathogens. Bacillus subtilis 

produces a diversity of antibiotics 

that are effective against phytopathogenic 

fungi and bacteria (Phae et al., 1990). Iturin 

production seems to be constrained to 

B. subtilis and B. amyloliquefaciens 

(Bonmatin et al., 2003). In control of 

phyllosphere diseases, Podosphaera fusca 

infecting melon leaves the iturins and 

fengycins lipopeptides produced by 

B. subtilis contributed more in disease 

suppression. Vater (1986) reported that 

the iturin is produced, during slow, stationary 

growth phase. Sandrin et al. 

(1990) reported that iturin A, one member 

of the iturin group, shows a strong antibiotic 

activity with a broad antifungal activity, 

making it an ideal potential biological 

control agent with the aim of reducing the 

use of chemical pesticides in agriculture. 

The antimicrobial action of 

B. subtilis can be attributed, to a certain 

amount, to the production of iturin A 

(Tsuge et al., 2001). Gene clusters involved 

in iturin A production have been 

intensively investigated (Hiraoka et al., 

1992, Huang et al., 1993, Kunst et al., 

1997, Yao et al., 2003). The iturin A operon 

spans a region more than 38 kb long 

and it contains four open reading frames 

viz., ituD, ituA, ituB and ituC (Tsuge et 

al., 2001). The ituD gene encodes a putative 

malonyl coenzyme A transacylase, 

whose distraction results in deficiency of 

iturin A production (Kunst et al., 1997). 

Ramyabharathi and Raguchander (2014) 

reported that the B. subtilis strain EPCO 

16 contains ItuC, ItuD, BmyA, BacD, 

BacAB and FenD genes involved in the 

biosynthesis of Iturin, Bacillomycin, 

Bacilysin and Fengycin, respectively. Iturin 

and surfactin were detected in culture 

filtrates from isolate EPCO16 by thin layer 

chromatography that showed tremendous 

control over Fusarium oxysporum 

pathogen infecting tomato. 

Bacillomycin D which is a member 

of the Iturin family along with mycosubtilin 

and iturin A, is made of one β- 

amino fatty acid and seven α-amino acids 

exhibits a strong antifungal activity 

against a broad range of plant pathogenic 

fungi. Biosynthesis of bacillomycin D is 

independent of the ribosomal process and 

the enzymes responsible for 

bacillomycin D production are complex 

peptide synthetases. Bacillomycin D and 

fengycin jointly contributed to the inhibition 

of conidial germination of Monilinia 

fructicola and fengycin played a major 

role in suppressing mycelial growth of the 

fungal pathogen. Luo et al., 2011 performed 

bioassay of antifungal compound 

bacillomycin against Magnaporthe oryzae, 

Rhizoctonia solani and Botrytis cinerea. 

The hyphae of the pathogenic fungi 

treated with bacillomycin L showed 

abnormal growth, and conidia produced 

enlarged and constricted germ tube. When 

bacillomycin L used in high concentration 

there is a possibility for cellular leakage. 

Bacillomycin D was detected in 

B. subtilis (Moyne et al., 2001; Ramara- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 288




thnam et al., 2007) and 

B. amyloliquefaciens strains. B. subtilis 

AU195 exhibit antifungal and have an 

amino acid sequence identical to bacillomycin 

D. There is higher effect of bacillomycin 

D than of fengycin against various 

phytopathogenic fungi. (Koumoutsi, 

2006; Peypoux et al., 1984). 

2.2. Surfactin lipopeptide 

The biosurfactant surfactin is an 

acidic cyclic lipopeptide produced by 

strains of B. subtilis. Among the lipopeptide 

antibiotics produced by Bacillus spp 

surfactin and Iturin A was most common. 

The specific surface and membrane active 

properties of the surfactin help bacteria to 

form biofilm. It is thought to perform developmental 

functions rather than disease 

resistance mechanism in the environment. 

Surfactin also produces a sturdy membrane-destabilizing 

action at concentrations 

even below its critical micellar concentration 

and induces the arrangement of 

ion channels in lipid bilayers. Surfactins 

have been reported to be powerful surfactants 

due to their outstanding surface activities. 

Compared with conventional surfactants, 

surfactin also have significant 

biological activities, such as antiviral and 

antibacterial activity. Phae et al., (1990) 

reported that Iturin A and surfactin producing 

B. subtilis suppressed more than 

23 types of plant pathogens in vitro. 

Surfactin and lichenysin are structurally 

related lipopeptides produced by 

B. subtilis and B. licheniformis. Bonmatin 

et al., 2003 reported different forms of 

surfactin with amino acid variation at position 

2, 4, and 7. Surfactin bears powerful 

surfactant properties by declining the 

surface tension of water from 72 to 27 

mN/m at a critical micelle concentration 

(CMC) of 25–220 mg/L based on its variants 

and determined conditions. Surfactin 

is an inhibitor of fibrin clot formation. 

Surfactins (C12 to C16) were produced 

simultaneously to increase the antifungal 

activity of iturin A. Thus exhibits antiviral, 

anti-tumor, anti-microbial and hemolytic 

properties. It is required for biofilm 

formation of producing cells, swarming 

motility, and fruiting body formation. 

However, surfactin also inhibits biofilm 

formation of other bacteria by interfering 

with attachment of the cells to surfaces. 

Surfactin or closely related variants such 

as lichenysin have been isolated from B. 

coagulans, B. pumilus and B. licheniformis. 

Surfactins are not toxic for fungal 

pathogens by themselves but they maintain 

some synergistic effect on the antifungal 

activity of iturin A. The mode of 

action of surfactin is act on the phospholipids 

and is able to form selective ionic 

pores in lipid bilayers of cytoplasmic 

membranes. Both surfactin and iturinA 

are surfactants with a hydrophilic ring of 

seven amino acids and a long, hydrophobic 

hydrocarbon tail. Usually the amino 

acid end stays in the soil and the hydrocarbon 

tail penetrates inside the pathogen 

cell membranes. This action creates openings 

in cell membranes and restricting the 

growth of many phytopathogens (Ohno et 

al., 1995; Asaka and Shoda, 1996; Carrillo 

et al., 2003; Ongena and Jacques, 

2008). 

IturinA has antibiotic property 

while surfactin has extremely powerful 

surface-active property, making its separation 

much more difficult. Surfactin, 

which is produced early in the bacterium’s 

growth cycle, has a deep influence 

on B. subtilis colonization of the root surface. 

Surface motility can be increased in 

rapid manner and thereby surfactin accelerates 

the development of multicellular 

communities which brings colonization of 

the bacteria referred to as biofilms (Bais 

et al., 2004; Nagorska et al., 2007; Rudrappa 

et al., 2008). Biofilm formation in 

B. subtilis added a distinct advantage over 

many competing organisms in the rhizosphere 

soil. Poor root colonization by surfactin-deficient 

B. subtilis strains is associated 

with lack of biocontrol activity 

(Schippers et al., 1987; Bais et al., 2004). 

2.3. Fengycin lipopeptide 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 289




Fengycin is a biologically active 

lipopeptide produced by several Bacillus 

subtilis strains. The third family of 

lipopeptide biosurfactants includes 

fengycins A and B, which are also called 

plipastatins. The structure is composed of 

a β-hydroxy fatty acid linked to a peptide 

part comprising 10 amino acids. It is an 

anti-fungal antibiotic that inhibits filamentous 

fungi but is ineffective against 

yeast and bacteria. Fengycin production 

was identified in B. cereus and B. thuringiensis 

in addition to B. subtilis and B. 

amyloliquefaciens. It is also capable of 

inhibiting phospholipase A2 and biofilm 

formation of several bacteria. These types 

of lipodecapeptides are produced by various 

strains of Bacillus spp. and exhibit 

moderate surfactant activities. It shows 

antifungal activity and more specific for 

filamentous fungi. 

Fengycin produced by B. subtilis 

has antifungal activity against the filamentous 

fungus. The fungal cell membrane 

is the primary site for antimicrobial 

attack by antibiotics fengycin. Fengycins 

are fewer haemolytic than iturins and surfactins 

but maintain a strong fungitoxic 

activity, exclusively against filamentous 

fungi (Koumoutsi et al 2004; Vanittanakom 

et al., 1986). Mechanistically, the 

activity of fengycins is less well known 

compared with other lipopeptides but they 

also readily interact with lipid layers and 

to some extent hold the potential to alter 

cell membrane structure and permeability 

in a dose-dependent manner (Deleu et al., 

2005). The connection of iturins and 

fengycins was shown in the antibiosisbased 

biocontrol activity of Bacillus 

strains against various pathogens and in 

different plant species. In the case of soilborne 

diseases, iturin A produced by B. 

subtilis RB14 is involved in damping-off 

of tomato caused by Rhizoctonia solani 

(Asaka and Shoda, 1996). Bacillus subtilis 

S499 efficiently produces lipopeptides 

from the three families, and notably 

produces a wide variety of fengycins 

(Jacques et al., 1999). Mutant analyses in 

B. subtilis subsp. amyloliquefaciens strain 

FZB42 have indicated that fengycin and 

bacillomycin D act synergistically to inhibit 

the growth of Fusarium oxysporum 

under in vitro condition (Koumoutsi et 

al., 2004). 

3. Biocontrol potential of lipopeptide 

biosurfactants 

Antibiotic production by B. subtilis 

strains plays a major role in suppression 

of plant diseases (Kinsella et al., 

2009). B. subtilis EPCO16 has lipopeptide 

genes viz., ItuC gene, ItuD gene (Iturin); 

BmyA gene (Bacillomycin A), BacD 

gene (Bacillomycin D), BacAB gene 

(Bacilysin) and FenD gene (Fengycin). 

The presence of lipopeptide antibiotics in 

B. subtilis EPCO16 inhibited the mycelia 

growth (46.04%) of F. oxysporum f. sp. 

lycopersici (Fol) under in vitro (Ramyabharathi 

and Raguchander, 2014). B. subtilis 

strain Bbv 57 is positive for Iturin 

(ItuD gene), Surfactin (srfA gene; sfp 

gene), Bacilysin (bacAB gene; bacD 

gene), Bacillomycin D (bamD gene), 

Fengycin (fenB gene), Ericin (eriB gene), 

Mycosubtilin (mycC gene) and Subtilin 

(spaB gene) lipopeptides (Ramyabharathi 

and Raguchander 2014a). The inhibition 

of Fusarium oxysporum f. sp. gerberae 

might be due to the production of antimicrobial 

metabolites which are toxic to the 

pathogen. HPLC analysis for B. subtilis 

Bbv 57 showed 91.69 μg/μl of surfactin 

with the retention time of 2.304 min and 

0.453 μg/μl of Iturin with the retention 

time of 8.739 min at 205nm whereas the 

standard iturin and surfactin at 205nm 

recorded retention time of 8.5 min and 2.5 

min respectively. 

Crude lipopeptides extracted from 

the culture supernatant of B. subtilis 

strain, Bbv 57 treatment revealed least 

egg hatching of 7 juveniles / egg mass 

with highest juvenile mortality of 87 per 

cent of M. incognita with the 25 per cent 

concentration of the antibiotic after 72 h 

of exposure of nematodes in it. It also inhibited 

the mycelial growth of F. oxysporum 

(28.20 %) at 10 micro litre con- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 290




centrations. Presence of these diverse 

genes plays a crucial role in biological 

control of root knot nematode and 

Fusarium in gerbera both in poly house 

and in vitro through synergistic action of 

antimicrobial peptide genes (Ramyabharathi, 

2015). Among lipopeptides iturin 

have strong broad spectrum antifungal 

and hemolytic activity. B. subtilis isolate 

ME488 showed positive reaction for PCR 

detection of antimicrobial peptide gene 

viz., ituC, tuD, bacA, bacD, mrsAand 

mrsM (Chung et al. 2008). Ramarathnam 

et al. (2007) detected lipopeptides antibiotics 

genes bacillomycin and fengycin 

using specific primers in Bacillus spp. 

Several strains of Bacillus, has 

AMP biosynthetic genes bmyB, fenD, 

ituC, srfAA, and srfAB responsible for the 

suppression of plant pathogens (Gonzalez 

et al., 2010). Presence of antimicrobial 

peptide (AMP) biosynthetic genes srfA 

(surfactin), bacA (bacylisin), fenD 

(fengycin), bmyB (bacyllomicin), spas 

(subtilin), and ituC (iturin) in 184 isolates 

of Bacillus spp (Mora et al., 2011). Cadena 

et al., 2008 reported that B. amyloliquefaciens 

strain FZB42 produced 

lipopeptides, surfactins, bacillomycin D, 

and fengycins, which are secondary metabolites 

with mainly antifungal activity, 

also decreased gall formation, egg mass 

count and juvenile counts of M. incognita 

extracted from roots of tomatoes. Koumoutsi 

et al. (2004) reported that mutant 

analyses in B. subtilis sub sp. amyloliquefaciens 

strain FZB42 with fengycin and 

bacillomycinD act synergistically and inhibited 

the growth of F. oxysporum in 

vitro. Cellular leakage was also observed 

when bacillomycin L was used at higher 

concentration. 


Bacillus strains do have the capability 

to produce a variety of lipopeptides 

with outstanding surface active properties, 

disease reduction and biological actions. 

Among the three lipopeptides, iturin 

and fengycin family separately exhibited 

antifungal, antimicrobial and 

antinematicial activity, whereas surfactin 

retained the antifungal effect of iturin A 

as a synergistic factor. The lipopeptides 

are less toxic and helps in disease reduction 

with control of phytopathogens and 

pathogenic nematodes than agrochemicals. 

B. subtilis seems to be a good biocontrol 

agent and a flourishing antagonist 

with lipopeptide antibiotic production. 

Further research is needed to know the 

stability of lipopeptide antibiotics subject 

to field conditions. The biosurfactant 

lipopeptides are used in food industry, 

chemical industry, clinics, cosmetics and 

used for cleaning oil spills by bioremediation 

approach. These lipopeptides may be 

useful in various industries and agriculture 

as biosurfactants and biopesticides in 

plant protection, respectively. However, 

further research is needed to explore the 

full potential of lipopeptide biosurfactants. 

References 

Arima, K., Kakinuma, A. and Tamura, 

G. (1968). Surfactin, a crystalline 

peptidelipid surfactant produced by 

Bacillus subtilis: isolation, characterization 

and its inhibition of fibrin 

clot formation. Biochemical and Biophysical 

Research Communications 

31, 488–494. 

Asaka, O. and Shoda, M. (1996). Biocontrol 

of Rhizoctonia solani damping-off 

of tomato with Bacillus subtilis 

RB14. Applied Environmental 

Microbiololgy 62, 4081–4085. 

Bais, H. P., Fall, R. and Vivanco, J. M. 

(2004). Biocontrol of Bacillus subtilis 

against infection of Arabidopsis 

roots by Pseudomonas syringae is 

facilitated by biofilm formation and 

surfactin production. Plant Physiology 

134, 307–319. 

Bonmatin, J. M., Laprevote, O. and 

Peypoux, F. (2003). Diversity 

among microbial cyclic lipopeptides: 

iturins and surfactins. Activity-structure 

relationships to design 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 291




new bioactive agents. Combinatorial 

Chemistry & High Throughput 

Screening 6, 541–556. 

Cadena, M. B., Burelle, N. K., Kathy S. 

L., Santen, E. V. and Kloepper J. 

W. (2008). Suppressiveness of rootknot 

nematodes mediated by rhizobacteria. 

Biological Control 47, 55– 

59. 

Carrillo, C., Teruel, J. A., Aranda, F. J. 

and Ortiz, A. (2003). Molecular 

mechanism of membrane permeabilization 

by the peptide antibiotic 

surfactin. Biochimica et Biophysica 

Acta – Biomembranes 161, 91–97. 

Chung, S., Kong, H., Buyer, J. S., Lakshman, 

D. K., Lydon, J., Dal Kim, 

S. and Roberts, D. P. (2008). Isolation 

and partial characterization of 

Bacillus subtilis ME488 for suppression 

of soilborne pathogens of 

cucumber and pepper. Applied Microbiology 

and Biotechnology 80, 

115-123. 

Deleu, M., Paquot, M. and Nylander, T. 

(2005). Fengycin interaction with 

lipid monolayers at the air-aqueous 

interface – implications for the effect 

of fengycin on biological membranes. 

Journal of Colloid and Interface 

Science 283, 358–365. 

Desai, J. D. and Banat, I. M. (1997). 

Microbial production of surfactants 

and their commercial potential. Microbiology 

and Molecular Biology 

Reviews 61, 47–64. 

Georgiou, G., Lin, S. C. and Sharma, 

M. M. (1992). Surface-active compounds 

from microorganisms. Biotechnology 

10, 60–65. 

Gonzalez, S. M. A., Perez-Jimenez, R. 

M., Pliego, C., Ramos, C., de Vicente, 

A. and Cazorla, F. M. 

(2010). Biocontrol bacteria selected 

by a direct plant protection strategy 

against avocado white root rot show 

antagonism as a prevalent trait. 

Journal of Applied Microbiology 

109, 65-78. 

Hiraoka, H., Ano, T. and Shoda, M. 

(1992). Molecular cloning of a gene 

responsible for the biosynthesis of 

the lipopeptide antibiotics iturin and 

surfactin. Journal of Fermentation 

and Bioengineering 74, 323–326. 

Huang, C. C., Ano, T. and Shoda, M. 

(1993). Nucleotide sequence and 

characteristics of the gene, lpa-14, 

responsible for biosynthesis of the 

lipopeptide antibiotics iturin A and 

surfactin from Bacillus subtilis 

RB14. Journal of Fermentation and 

Bioengineering 76, 445–450. 

Jacques, P., Hbid, C. and Destain, J. 

(1999). Optimization of biosurfactant 

lipopeptide production from 

Bacillus subtilis S499 by Plackett- 

Burman design. Applied Biochemistry 

and Biotechnology 77, 223–233. 

Kinsella, K., Schulthess, C. P., Morris, 

T. F. and Stuart, J. D. (2009). Rapid 

quantification of Bacillus subtilis 

antibiotics in the rhizosphere. Soil 

Biology and Biochemistry 41, 374- 

379. 

Koumoutsi, A., Chen, X.-H., Henne, A., 

Liesegang, H., Hitzeroth, G., 

Franke, P., Vater, J. and Borriss, 

R. (2004). Structural and functional 

characterization of gene clusters directing 

nonribosomal synthesis of 

bioactive cyclic lipopeptides in Bacillus 

amyloliquefaciens strain 

FZB42. Journal of Bacteriology 

186, 1084-1096. 

Koumoutsi, A. (2006). Functional genome 

analysis of the plant growth 

promoting bacterium Bacillus amyloliquefaciens 

strain FZB42; characterizing 

its production and regulation 

of non-ribosomal peptide synthetases. 

Dissertation, Humboldt- 

Universitat, Berlin. 

Kunst, F., Ogasawara, N. and Moszer, 

I. (1997). The complete genome sequence 

of the Gram-positive bacterium 

Bacillus subtilis. Nature 390, 

249–256. 

Leclere, V., Marti, R., Bechet, M., 

Fickers, P. and Jacques, P. (2006). 

The lipopeptides mycosubtilin and 

surfactin enhance spreading of Ba- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 292




cillus subtilis strains by their surface-active 

properties. Archives of 

Microbiology 186, 475–483. 

Luo, A. H., Pouya, T. F., Roy, A. W., 

Carl, R. L. and Gary, A. J. (2011). 

Linking Context with Reward: A 

Functional Circuit from Hippocampal 

CA3 to Ventral Tegmental Area. 

Science 333, 353-357. 

Mora, I., Jordi, C. and Emilio, M. 

(2011). Antimicrobial peptide genes 

in Bacillus strains from plant environments. 

International Microbiology 

14, 213-223. 

Moyne, A. L., Shelby, R., Cleveland, T. 

E. and Tuzun, S. (2001). Bacillomycin 

D: an iturin with antifungal 

activity against Aspergillus flavus. 

Jounal of Applied Microbiology 90, 

622–629. 

Nagorska, K., Bikowski, M. and 

Obuchowski, M. (2007). Multicellular 

behavior and production of a 

wide variety of toxic substances 

support usage of Bacillus subtilis as 

a powerful biocontrol agent. Acta 

Biochimica Polonica 54, 495–508. 

Ohno, A., Ano, T. and Shoda, M. 

(1995). Effect of temperature on 

production of lipopeptide antibiotics, 

iturin A and surfactin, by a dual 

producer, Bacillus subtilis RB14, in 

solid-state fermentation. Journal of 

Fermentation and Bioengineering 

80, 517–519. 

Ongena, M. and Jacques, P. (2008). Bacillus 

lipopeptides: versatile weapons 

for plant disease biocontrol. 

Trends in Microbiology 16, 115– 

125. 

Peypoux, F., Pommier, M. T., Das, B. 

C., Besson, F., Delcambe, L. and 

Michel, G. (1984). Structures of bacillomycin 

D and bacillomycin L 

peptidolipid antibiotics from Bacillus 

subtilis. Journal of Antibiotics 

37, 1600-1604. 

Phae, C. G., Shida, M. and Kubota, H. 

(1990). Suppressive effect of Bacillus 

subtilis and its products on phytopathogenic 

microorganisms. Journal 

of Fermentation and Bioengineering 

69, 1-7. 

Ramarathnam, R., Bo, S., Chen, Y., 

Fernando, W. G. D., Xuewen, G. 

and de Kievit, T. (2007). Molecular 

and biochemical detection of 

fengycin- and bacillomycin D- 

producing Bacillus spp., antagonistic 

to fungal pathogens of canola 

and wheat. Canadian Journal of 

Microbiology 53, 901–911. 

Ramyabharathi, SA and Raguchander, 

T. (2014). Characterization of antifungal 

antibiotic synthesis genes 

from different strains of Bacillus 

subtilis. Journal of Pure and Applied 

Microbiology 8, 2337-2344. 

Ramyabharathi. SA and Raguchander 

T. (2014a). Efficacy of Secondary 

Metabolites Produced by Bacillus 

subtilis EPCO16 against Tomato 

Wilt Pathogen Fusarium oxysporum 

f.sp. lycopersici. Journal of Mycology 

and Plant Pathology, 44(2): 

148-153. 

Ramyabharathi, SA. (2015). Management 

of Fusarium wilt- root knot 

nematode disease complex in gerbera 

using lipopeptides producing 

PGPR under protected cultivation. 

PhD. Thesis, Tamil Nadu Agricultural 

University, Coimbatore, Tamil 

Nadu, India. 

Ramyabharathi, SA., Rajendran, L., 

Karthikeyan, G. and Raguchander, 

T. (2016). Liquid formulation 

of endophytic bacillus and its standardization 

for the management of 

Fusarium wilt in tomato. Bangladesh 

journal of botany 45(2), 283- 

290 

Rudrappa, T., Biedrzycki, M. L. and 

Bais, H. P. (2008). Causes and consequences 

of plant associated biofilms. 

FEMS Microbiology Ecology 

64, 153–166. 

Sandrin, C., Peypoux, F. and Michel, 

G. (1990). Coproduction of surfactin 

and iturin A, lipopeptides with 

surfactant and antifungal properties, 

by Bacillus subtilis. Biotechnology 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 293




and Applied Biochemistry 12, 370– 

375. 

Sankari Meena, K., Ramyabharathi, 

S.A. and Raguchander, T. (2016). 

Biomanagement of nematodefungus 

disease complex in tuberose 

using plant growth promoting rhizobacteria. 

International journal of 

science and nature. 7 (3), 1-9. 

Schippers, B., Bakker, A. W. and Bakker, 

P. A. H. M. (1987). Interactions 

of deleterious and beneficial 

rhizosphere microorganisms and the 

effect of cropping practices. Annual 

Review of Phytopathology 25, 339– 

358. 

Tsuge, K., Akiyama, T. and Shoda, M. 

(2001). Cloning, sequencing, and 

characterization of the iturin A operon. 

Journal of Bacteriology 183, 

6265-6273. 

Vanittanakom, N., Loeffler, W., Koch, 

U. and Jung, G. (1986). Fengycin - 

a novel antifungal lipopeptide antibiotic 

produced by Bacillus subtilis 

F-29-3. Journal of Antibiotics 39, 

888–901. 

Vater, J. (1986). Lipopeptides, an attractive 

class of microbial surfactants. 

Progress in Colloid & Polymer Science 

72, 12–18. 

Yao, S., Gao, X., Fuchsbauer, N., Hillen, 

W., Vater, J. and Wang, J. 

(2003). Cloning, sequencing, and 

characterization of the genetic region 

relevant to biosynthesis of the 

lipopeptides iturin A and surfactin 

in Bacillus subtilis. Current Microbiology 

47, 272–277. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 294



Biotechnology as a Tool for Conservation and Sustainable 

Utilization of Plant and Seaweed Genetic Resources 

of Tropical Bay Islands, India 

Pooja Bohra 1, *, Ajit Arun Waman 2 and Anuraj Anirudhan 3 

1 Division of Horticulture and Forestry, ICAR- Central Island Agricultural Research Institute, 

Port Blair- 744105, Andaman and Nicobar Islands, India; 2 Division of Horticulture 

and Forestry, ICAR- Central Island Agricultural Research Institute, Port Blair- 744105, 

Andaman and Nicobar Islands, India; 3 Division of Fisheries Sciences, ICAR- Central Island 

Agricultural Research Institute, Port Blair- 744105, Andaman and Nicobar Islands, 

India;*Correspondence: poojabohra24@gmail.com; Tel.: +91-3192-250436 

Abstract: Andaman and Nicobar Islands are known to harbor large diversity of plant and 

seaweed species, a number of them being endemic. Micropropagation could be used as an 

effective tool for large scale multiplication of economically important plants and seaweeds. 

The technique could also help in multiplying threatened species to conserve them. In vitro 

production of pharmaceutical macromolecules could be a viable option for avoiding destructive 

harvesting of plant species. Somaclonal variation, in vitro mutagenesis and transgenic 

could be useful in some cases. Molecular markers could help in assessment of genetic 

diversity, DNA barcoding, marker assisted selection etc. The article highlights the importance 

and relevance of various biotechnological tools in the management of biodiversity 

of the fragile ecosystem of Andaman and Nicobar Islands. Various research activities undertaken 

to conserve species of these islands are also highlighted. 

Keywords: Andaman and Nicobar Islands; Bay of Bengal; biodiversity; endemism; sustainable 

development 


Natural resources including flora 

and fauna have been the major associates 

of humankind since evolution. We are 

largely dependent on these resources for 

our existence and leading a normal day to 

day life. However, the increasing pressures 

of manmade and natural disasters 

have jeopardized these resources in such a 

way that every year the conservation status 

of a large number of species is pushed 

further in the red list. On the other hand, 

our dependence on a few species for 

meeting most of our requirements has 

worsened the situation by eliminating the 

so called non-useful types, which could 

be carrying potent genes for mitigating 

the stresses posed by climate change. The 

processes of industrialization, commercial 

synthetic farming, pollution, deforestation, 

urbanization etc. are the direct or 

indirect consequences of population explosion, 

which have largely contributed in 

misbalancing the resource utilization in a 

sustainable way. Considering the sensitivity 

of these issues, concerted efforts are 

required to protect our valuable resources 

so that they are available to the future 

generations too. 

The tropical rainforests are known 

to harbor wide array of unique floral diversity 

and a number of mega biodiversity 

hotspots are located in these regions. The 

Andaman and Nicobar Islands in the Bay 

of Bengal (a Union Territory of the India) 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 295


Genetic Resources Sustainability 

Bohra et al. 

are strategically placed between two such 

biodiversity hotspots viz. Arakan Yoma 

ranges of Myanmar and the Sumatra. This 

has resulted in unique confluence of flora 

of both these regions in ANI (Pandey and 

Diwakar, 2008). The islands are characterized 

by lush green forests occupying 

about 81.8% of the total geographical area. 

There are more than 2,314 species of 

flowering plants reported from these islands 

so far (Murugan et al., 2016) and 

the number may still increase considering 

the larger unexplored areas. Furthermore, 

these islands are known to harbor a large 

number of endemic species in a relatively 

smaller geographical area of about 8,249 

km 2 . So far, about 300 species of endemic 

plants have been reported from ANI 

(Murugan et al., 2016). Majority of the 

diversity is still unexplored and considerable 

scope exists for utilizing these species 

for the betterment of humankind. 

Since, horticulture has been the major 

source of livelihood and nutritional security 

for the island dwellers (Singh et al., 

2016), the present article focuses on the 

management of genetic resources of horticultural 

crops including their wild relatives. 

Similarly, seaweeds are important 

component of the diversity of these islands. 

The macro algae mainly belonging 

to Chlorophtya, Phaeophtya and Rhodophyta 

are found attached to the substratum 

in benthic zone. They are nonflowering 

plants with true roots, stem and 

leaves, and are known to contribute substantially 

to the primary production in the 

marine environment. Seaweeds have been 

used for centuries as food either in raw or 

processed form in many of the South East 

Asian countries and the trend is picking 

up in the western countries as well. Seaweeds 

are the only known natural sources 

of hydrocolloids viz. agar, algin and carrageenan. 

These multipurpose products 

find application in industrial, pharmaceutical 

and medicinal fields. Besides, seaweeds 

are used as animal feed and biofertilizers 

in crop production. The current 

research on seaweeds is centering on bioactives 

which are of great interest in the 

medical field. Andaman and Nicobar Islands, 

with 1/3 rd of India’s coastal line, 

supports good diversity of seaweeds and 

so far about 206 species including commercially 

important agarophytes and alginophytes 

have been reported from here. 

Interestingly, different parts of 

these islands are inhabited by six native 

tribes since centuries. Two Mongoloid 

tribes, Shompen and Nicobarese, reside in 

the Nicobar groups of islands, while the 

tribes of Negrito origin i.e. Jarawa, Great 

Andamanese, Onge and Sentinelese, are 

residing in the Andaman islands. These 

tribes differ in most of their cultures and 

habits. Some of the local species are presently 

being used by the native tribes for 

food, medicine, fodder, fuel and other 

purposes. Similarly, the settler population 

migrated from different parts of mainland 

India are utilizing these plants for variety 

of purposes. Underutilized fruits, indigenous 

leafy vegetables and tuber crops 

have immensely contributed in the livelihood 

and nutritional security of the island 

dwellers. Further, a large number of wild 

relatives of cultivated crop plants have 

been reported to occur in these islands. 

This diversity needs to be assessed and 

utilized sustainably to strengthen our resource 

base, while striking the fine balance 

between development and ecological 

soundness (Waman and Bohra, 2016). 

Biotechnology could be an effective 

tool for achieving this target through 

the application of techniques namely micropropagation, 

in vitro production of 

secondary metabolites, in vitro mutation, 

in vitro conservation, marker assisted selection, 

genetic diversity assessment, development 

of trait specific markers etc. 

(Waman et al., 2015; Waman and Bohra, 

2016). Present chapter concerned exploring 

the possibility of utilizing various biotechnological 

tools for management of 

biodiversity of the tropical Bay Islands of 

India. 

2. Relevance of biotechnological approaches 

and tools for island ecosystem 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 296



2.1. Micropropagation and in vitro conservation 

Andaman and Nicobar Islands are 

home to many endemic species belonging 

to different botanical families, which are 

of potential economic/ ecological significance 

and require timely attention for 

their conservation. A number of species 

belonging to rare, endangered and threatened 

(RET) category are also found distributed 

in these islands. Some of these 

species have problems in natural regeneration 

owing to the damage caused by 

birds/animals, poor seed viability, anthropogenic 

pressure etc. For example, Myristica 

andamanica is a vulnerable wild 

nutmeg species endemic to the islands 

and micropropagation could help to multiply 

it in large number. Similarly, natural 

populations of an underutilized fruit 

species – blood fruit (Haematocarpus 

validus) are dwindling (Bohra et al., 

2016a) and micropropagation could help 

in saving the species from extinction from 

the region. Experiments are in progress to 

standardize micropropagation protocol for 

this species. 

Secondly, banana is a major crop 

of the islands covering more than half of 

the area under fruit crops cultivation. 

However, the islands are largely dependent 

on the planting material supplies from 

mainland India. This has probably resulted 

in inadvertent introduction of dreadful 

banana bunchy top virus in the pristine 

islands. Developing protocols for in vitro 

multiplication of locally suitable varieties 

would help in production of their quality 

planting material. The importance of tissue 

culture technology for the island 

farmers has been emphasized earlier 

(Bohra et al., 2016b). Considering this, 

experiments have been initiated at authors’ 

institute for optimization of protocols 

for locally popular banana varieties 

of the islands. Other commercializable 

crops of the islands include variety of orchids 

and ornamental plants, which need 

further attention. Use of low cost options 

including concurrent ex vitro rooting cum 

Bohra et al. 

hardening has been emphasized in low 

price-high value crops, especially medicinal 

plants (Waman and Bohra, 2016). 

In vitro conservation is a technique, 

wherein plant tissues are cultured 

in vitro under sub-optimal growth conditions 

in order to reduce the frequency of 

sub-culturing. The technique has proven 

to be very efficient for short to medium 

term storage of a number of species. Considering 

vulnerability of the islands to 

natural disasters, the endemic species 

could be conserved under in vitro conditions 

and copies of the same could be 

maintained at other laboratories in mainland 

India. Some horticulturally important 

endemic species of islands have been 

listed in Table 1. 

2.2. In vitro production of secondary metabolites 

A large number of species are valued 

for their medicinal properties. However, 

the yield of bioactive molecules is 

very low in most of the cases. At times, 

complete plants are destroyed for obtaining 

the desired active ingredients. Such 

practice of destructive harvesting has 

been a cause of concern as it tends to 

threaten the natural populations to a great 

extent (Waman and Bohra, 2013). Biotechnological 

tools could help in large 

scale quality production of secondary metabolites 

under in vitro conditions. Induction 

of callus and extraction of active ingredients 

from them has been suggested 

as an important alternative for obtaining 

the desired molecules without disturbing 

the wild populations (Waman et al., 

2015). 

2.3. Creation of variability 

There are a few species e.g. mangosteen 

(Garcinia mangostana), which 

are economically important for the islands 

but have narrow genetic base. For improvement 

of such crops, creation of variability 

is possible through the induction of 

somaclonal variations in tissue culture or 

using the technique of in vitro mutation 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 297



Bohra et al. 

Table 1: List of selected endemic species of horticultural importance reported from ANI 

Family 

Species 

Anacardiaceae Mangifera andamanica, M. nicobarica, Semecarpus kurzii 

Apocyanaceae Carissa andamanensis 

Arecaceae Phoenix andamanensis 

Clusiaceae Garcinia andamanica, G. cadelliana, G. calycina, G. dhanikhariensis, 

G. kingii, G. kurzii, G. microstigma 

Dilleniaceae Dillenia andamanica 

Dioscoreaceae Dioscorea vexans 

Musaceae Musa bulbisiana var. andamanica, M. indandamanensis, M. sabuana, 

M. paramjitiana 

Myristicaceae Myristica andamanica, Knema andamanica 

Myrtaceae Syzygium andamanicum, S. manii 

Pandanaceae Pandanus lerum var. lerum 

Tiliaceae Grewia indandamanica 

Orchidaceae Vanilla andamanica, Eulophia nicobarica 

Zingiberaceae Kaempfaria siphonantha 

breeding. Being cornerstone of breeding 

activity, variability created will also be 

useful in development of island suitable 

varieties. 

2.4. Estimation of genetic diversity 

The ANI is considered as a centre 

of origin or diversity for a number of species 

e.g. wild populations of Piper betle 

are present in these islands. Both inter and 

intra specific diversity occurs for a number 

of species of ecological and economic 

importance (Singh et al., 2016). This diversity 

needs to be tapped in such a way 

that commercially viable types are identified 

for the benefit of island farmers. Selection 

of such elite types needs systematic 

characterization. Molecular characterization 

is one of the most reliable methods 

of estimating such diversity. Further, 

available diversity could also be compared 

with their counterparts occurring in 

mainland India or other parts of the 

world. Through this information, the studied 

population could be categorized in a 

way to pave the way for future breeding 

programmes. A few attempts were initiated 

at the authors’ Institute or other organizations 

in country in this direction, which 

have been summarized in Table 2. The 

similar technique could also be employed 

for diversity estimation and further studies 

in non-traditional and underutilized 

species, which play pivotal role in the 

lives of island dwellers. The generated 

information from such studies could be 

useful for selection of parents for carrying 

out conventional crop improvement programmes. 

2.5. Evolutionary studies 

From time to time, a number of 

new species have been reported by various 

research workers in the ANI, however, 

absence of requisite scientific information 

about their genetic relationship 

with their existing commercial counterparts 

could delay the process of their 

commercial utilization. Molecular phylogenetic 

studies could help in this regard. 

For example the presence of Indo- 

Myanmarese as well as Indonesian forms 

of Erianthus arundinaceus (Retz.) 

Jeswiet, a wild relative of sugarcane 

(Saccharum officinarum) was confirmed 

through the use of molecular markers 

(Nair and Mary, 2006). They concluded 

that collections from North Andaman 

were more similar to Indo-Myanmarese 

form, while that from Nicobar were of 

Indonesian form. This report supports the 

fact that ANI harbor confluence of flora 

of two different regions. Similarly, genetic 

diversity between the collections of 

Musa balbisiana from mainland India and 

the islands suggested that ANI is one of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 298



Bohra et al. 

Table 2: Selected examples of application of molecular markers for diversity assessment in 

various horticultural crops of ANI 

Species 

Molecular 

Salient findings 

Reference 

marker 

used 

Morinda citrifolia RAPD, 

ISSR 

Distinct clustering of collections from 

ANI islands 

Singh et al., 

2012 

Morus laevigata RAPD, Significant genetic divergence between 

Naik et al., 

ISSR 

collection from mainland India 2015 

and Andaman Is. 

Carica papaya ISSR Geographical clustering of collections Sudha et al., 

from various islands 

Bouea oppositifolia, 

SSR Genetic similarity of 43% between 

Mangifera 

both the species of Anacardiaceae 

andamanica 

family 

Cocos nucifera RAPD Two distinct clusters based on morphological 

and nut parameters 

SSR High genetic diversity among the collections, 

separate clustering of tall and 

dwarf types 

Syzygium cuminii 

escu- 

Colocasia 

lenta 

RAPD, 

ISSR 

RAPD, 

ISSR 

Genotype collected from Car Nicobar 

was distinct amongst 21 island genotypes 

and 2 mainland genotypes 

Island collections were distinctly different 

from 3 released varieties used 

as reference genotypes 

Mangifera indica SSR Separate clustering of monoembryonic, 

polyembryonic and wild mango 

species 

Orchid species RAPD Distinct clustering of green orchid 

species 

Costus speciosus RAPD Intra-specific variations to the extent 

of 35% 

the centres of diversity of the species 

(Uma et al., 2005). This new piece of information 

would go a long way in devising 

conservation strategy of Musa spp. 

from ANI. 

2013 

Damodaran et 

al., 2013 

Sankaran et 

al., 2012 

Rajesh et al., 

2008 

Ahmad et al., 

2012 

Singh et al., 

2012 

Damodaran et 

al., 2012 

Singh and Srivastava, 

2010 

Mandal et al., 

2007 

2.6. Development of trait linked markers 

As previously mentioned, a number 

of perennial dioecious species are 

known to occur in the islands. Important 

being Myristica andamanica, Knema andamanica, 

Horsfieldia glabra, Piper 

betle, Carica papaya, Garcinia spp., 

Momordica spp. etc. Development of sex 

linked markers would be a boon for the 

conservation and utilization of these species 

as the desired plants could be identified 

during early stages of development. 

In case of medicinal plants, identification 

of markers linked to the presence of their 

active ingredients would be very useful. 

Flowering behavior related markers in 

mango have been identified, which could 

be of practical utility (Damodaran et al., 

2006). 

3. Applications of biotechnological interventions 

in seaweeds 

In the wake of increasing demand 

for seaweeds for various applications, collections 

from the natural habitats are not 

sufficient to meet the requirement. Artificial 

culture of commercially exploited 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 299



seaweeds still remains the major means of 

raw material supply to the industries. 

However, on farm culture of seaweeds 

using vegetative fragments generally results 

in reduced growth rate and productivity 

over time. In order to overcome these 

problems and to develop strains with 

improved growth and yield parameters, 

biotechnological techniques such as micropropagation, 

transgenics and molecular 

markers have been tried in seaweed 

breeding and genetic studies. Micropropagation 

is a tool to produce large number 

of seeding material from explants of seaweed 

possessing desirable traits. Among 

the three cellular organization types in 

seaweeds, most of the work on micropropagation 

has been reported on parenchymatous 

and pseudo-parenchymatous 

types (Aguirre-Lipperheide et al., 1995). 

Although Gibor attempted to cultivate 

seaweeds axenically in as early as 1950, 

the first successful attempt is considered 

to be made during 1978 by Chen and Taylor 

in Chondrus crispus (Yokoya and 

Valentin, 2011). Seaweed micropropagation 

protocols have been developed in 

similar lines with that of higher plants and 

so far micropropagation protocols have 

been developed in about 85 species (Reddy 

et al, 2008). Considering the diversity 

present and diversified applications of 

seaweeds, the technique is considered to 

Bohra et al. 

be still in nascent stage. In vitro derived 

calli of economically important seaweeds 

have been commonly used for maintenance 

and clonal propagation of seed 

stock for mariculture (Dawes and Koch, 

1991; Reddy et al., 2003; Rajakrishna et 

al., 2004; Reddy et al, 2008). Studies 

suggested that growth and quality of carrageenan 

obtained from tissue cultured 

Kappaphycus alvarezii were superior 

when compared with conventional vegetative 

fragments (Rajakrishna et al., 

2007). Non-availability of standardized 

protocols for obtaining viable axenic cultures 

from wild, lack of knowledge about 

the role of culture incubation conditions, 

media supplements, explanting season 

etc. on callus induction are the major limiting 

factors in the development of seaweed 

micropropagation. Table 3 represents 

list of selected seaweed species reported 

from ANI in which micropropagation 

has been attempted elsewhere. 

Molecular marker assisted breeding 

based on quantitative trait loci (QTLs) 

offers several advantages over traditional 

phenotypic based breeding. Development 

of markers linked with genes of desirable 

traits could increase accuracy and efficiency 

of the breeding process. Several 

molecular markers such as Random Amplified 

Polymorphic DNA (RAPD), Inter 

Simple Sequence Repeat (ISSR), Se- 

Table 3: List of seaweed species reported from ANI in which cell and tissue culture has 

been accomplished elsewhere 

Seaweed 

Reference 

Chlorophyta Boergesenia forbessi Enomoto and Hirose, 1972 

Bryopsis plumosa Tatewaki and Nagata, 1970 

E. intestinalis Polne-Fuller and Gibor, 1987; Russig and Cosson, 

2001 

Rhodophyta Gracilaria corticata Subbaraju et al., 1981; Rajakrishna et al., 2007 

G. verrucosa Gusevet al., 1987; Kaczyna and Megnet, 1993 

Gelidiella acerosa Rajakrishna et al., 2004 

Phaeophyta Grateloupia filipina Huang and Fujita, 1997; Baweja and Sahoo, 2009 

Hypnea musciformis Rajakrishna et al., 2007 

Sargassum tenerrimum 

Rajakrishna et al., 2007 

Turbinaria conoides Rajakrishna et al., 2007 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 300



Bohra et al. 

quence Characterized Amplified Region 

(SCAR), Amplified Fragment Length 

Polymorphism (AFLP), Sequence Tagged 

Site (STS), Microsatellites etc. have been 

developed in seaweeds (Lin et al., 2012). 

Molecular markers have also been used 

for DNA barcoding, which could be used 

for assessing genetic diversity and taxonomic 

identification of seaweeds species, 

which lack reliable morphological characters 

for identification. For this purpose, 

RAPD, Restriction Fragment Length Polymorphism 

(RFLP), AFLP, Microsatellites, 

Single Nucleotide Polymorphism 

(SNP) etc. have been used (Liu and 

Cordes, 2004). Further, molecular markers 

could also be used for identification of 

invasive seaweed species, which could 

pose threats to the pristine biodiversity of 

these Islands. Transgenic technology has 

been mainly focused on genetic engineering 

of economically important seaweeds 

(Lin et al., 2012) like Gracilaria, Kappaphycus, 

Porphyra and Ulva. As with 

any other transgenic technology, the safety 

issues associated with culture of genetically 

modified seaweeds should be of 

prime consideration. Transgenic technology 

has been attempted in Chlorophyta 

(Huang et al., 1996), Rhodophyta (Wang 

et al., 2010) and Phaeophyta (Zhang et 

al., 2008). Production of valuable products 

in an indoor cultivation system with 

proper biosafety from transgenic kelp 

sporophyte has been successfully demonstrated 

(Qin et al., 2005). 

The general perception that compounds 

and chemicals produced from 

natural resources are safe and pose less 

risk to health has brought seaweeds in the 

limelight for extracting environmental 

friendly natural compounds and chemicals. 

The development of species-specific 

in vitro cell and tissue culture technology 

is essential for use of other biotechnological 

tools for genetic improvement and 

production of high value compounds from 

seaweeds. With plethora of problems 

faced in seaweed cultivation, the intervention 

of biotechnological tools is essential 

for overall growth of this sector. 


The tropical islands in the Bay of 

Bengal are known to harbor considerable 

diversity of flowering plants as well as 

seaweed species. Most of these species 

are ecologically important, while a large 

number of species could be utilized for 

their commercial potential. The diversified 

applications of biotechnology could 

be helpful in carrying out the major activities 

related to management of this unique 

diversity including conservation, regeneration, 

characterization, utilization etc. 

Though some efforts have been initiated 

in this direction, there is vast scope for 

employing biotechnological tools for sustainable 

development of such fragile ecosystem 

in near future. 


Authors are thankful to the Director, 

ICAR-CIARI, Port Blair for providing 

necessary facilities for conduct of various 

studies and extending support and 

guidance. 

References 

Aguirre-Lipperheide, M., Estrada- 

Rodríguez, F. J. and Evans, L. V. 

(1995). Facts, problems and need in 

seaweed tissue culture – an appraisal. 

Journal of Phycology 31, 677- 

688. 

Ahmad, I., Bhagat, S., Simachalam, P. 

and Srivastava, R. C. (2012). Molecular 

characterization of Syzygium 

cuminii from A&N Islands. Indian 

Journal of Horticulture 69, 306- 

311. 

Baweja, P. and Sahoo, D. (2009). Regeneration 

studies in Grateloupia 

filicina (J.V. Lamouroux) C. Agardh 

– an important Carrageenophyte and 

edible seaweed. Algae 24, 163-168. 

Bohra, P., Waman, A. A., Sakthivel, K., 

Gautam, R. K. and Dam Roy, S. 

(2016a). Plant Tissue Culture: A viable 

technique for augmenting the 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 301



Bohra et al. 

productivity of banana in Andaman 

and Nicobar Islands. Technical Bulletin, 

ICAR-CIARI, Port Blair. pp. 

1-12. 

Bohra, P., Waman, A.A. and Dam Roy, 

S. (2016b). Khoon phal (Haematocarpus 

validus (Miers.) Bakh. ex F. 

Forman). Technical Bulletin, ICAR- 

CIARI, Port Blair. pp. 1-8. 

Damodaran, T., Ahmad, I. and Nagarajan, 

B. (2013). Bouea oppositifolia 

- A fast disappearing native 

mango genetic resource 

from Andamans: morphological and 

molecular evidences. Indian Journal 

of Horticulture 70, 161-164. 

Damodaran, T., Kannan, R., Ahmad, 

I., Srivastava, R.C., Rai, R. B. and 

Umamaheshwari, S. (2012). Assessing 

genetic relationships among 

mango (Mangifera indica L.) accessions 

of Andaman Islands using inter 

simple sequence repeat markers. 

New Zealand Journal of Crop and 

Horticultural Sciences 40, 229-240. 

Damodaran, T., Medhi, R.P., Kapil 

Dev, G., Damodaran, V., Rai, R. 

B. and Kavino, M. (2006). Identification 

of molecular markers linked 

with differential flowering behavior 

of mangoes in Andaman and Nicobar 

Islands. Current Science 92, 

1054-1056. 

Dawes, C. J. and Koch, E. W. (1991). 

Branch, micropropagules and tissue 

culture of the red algae Eucheuma 

denticulatum and Kappaphycus alvarezii 

farmed in the Philippines. 


21–24. 

Enomoto, S. and Hirose, H. (1972). Culture 

studies on artificially induced 

aplano spores and their development 

in the marine alga Boergesenia 

forbesii (Harvey) Feldmann (Chlorophyceae, 

Siphonocladales). Phycologia 

11, 119–122. 

Gusev, M. V., Tambiev, A. H., Kirikora, 

N. N., Shelyastina, N. N. and 

Aslanyan, R. R. (1987). Callus 

formation in seven species of agarophyte 

marine algae. Marine Biology 

95, 593–597. 

Huang, W. and Fujita, Y. (1997). Callus 

induction and thallus regeneration 

insome species of red algae. Phycologia 

Research 45, 105–111. 

Huang, X., Weber, J. C., Hinson, T. K., 

Matheison, A. C. and Minocha, S. 

C. (1996). Transient expression of 

the GUS reporter gene in the protoplasts 

and partially digested cells of 

Ulva lactuca L. (Chlorophyta). Botanica 

Marina 39, 467-474. 

Kaczyna, F. and Megnet, R. (1993). The 

effects of glycerol and plant growth 

regulators on Gracilaria verrucosa 

(Gigartinales, Rhodophyceae). Hydrobiologia 

268, 57–64. 

Hanzhi L., Song Q. and Peng J. (2012). 

Biotechnology of seaweeds: Facing 

the coming decade. In: Se-Kwon 

Kim (Eds), Handbook of marine 

macroalgae: Biotechnology and Applied 

Phycology pp. 424-430. 

Liu, Z. J. and Cordes, J. F. (2004). 

DNA marker technologies and their 

applications in aquaculture genetics. 

Aquaculture 238, 1-37. 

Mandal, A.B., Thomas, V.A., 

Elanchezhian, R. (2007). RAPD 

Pattern of Costus speciosus Koen 

Ex. Retz., An Important Medicinal 

Plant from the Andaman and Nicobar 

Islands. Current Science 93, 

369-373. 

Murugan, C., Prabhu, S., Sathiyaseelan, 

R. and Pandey, R.P. (2016). A 

checklist of plants ofAndaman and 

Nicobar islands (Eds. Paramjit 

Singh and Arisdason, W.). ENVIS 

Centre on Floral Diversity, Botanical 

Survey of India, Kolkata, India. 

Naik, G. V., Dandin, S. B., Tikader, A., 

Pinto, M. V. (2015). Molecular Diversity 

of Wild Mulberry (Morus 

spp.) of Indian Subcontinent. 

Indian Journal of Biotechnology 14, 

334-343. 

Nair, N.V. and Mary, S. (2006). RAPD 

analysis reveals the presence of 

mainland Indian and Indonesian 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 302



Bohra et al. 

forms of Erianthus arundinaceus 

(Retz.) Jeswiet in the Andaman– 

Nicobar Islands.Current Science 90, 

1118-1122. 

Pandey, R.P. and Diwakar, P.G. (2008). 

An integrated check-list flora of 

Andaman & Nicobar Islands, India. 

Journal of Economic and Taxonomic 

Botany 32, 403–500. 

Polne-Fuller, M. and Gibor, A (1987) 

Callus and callus likegrowth in seaweeds: 

induction and culture. Proceedings 

of International Seaweed 

Symposium 12, 131–138. 

Qin, S. and Tseng, C. (2005). Transforming 

kelp into a marine bioreactor. 

Trends in Biotechnology 23, 

264-268. 

Rajakrishna Kumar, G., Reddy, C. R. 

K. and Jha, B. (2007). Callus induction 

and thallus regeneration 

from the callus of phycocolloid 

yielding seaweeds from the Indian 

coast. Journal of Applied Phycology 

19, 15–25. 

Rajakrishna Kumar, G., Reddy, C. R. 

K., Ganesan, M., Tiruppathi, S., 

Dipakkore, S., Eswaran, K., Subba 

Rao, P. V. and Jha, B. (2004). 

Tissue culture and regeneration of 

thallus from callus of Gelidiella acerosa 

(Gelidiales, Rhodophyta). 

Phycologia 43, 596–602. 

Rajesh, M. K., Nagarajan, P., Jerard, 

B. A., Arunachalam, V. and 

Dhanapal. R. (2008). Microsatellite 

variability of coconut accessions 

(Cocos nucifera L.) 

from Andaman and Nicobar Islands. 

Current Science 94, 1627-1631. 

Reddy, C. R. K., Jha, B., Fujita, Y. and 

Ohno, M. (2008). Seaweed micropropagation 

techniques and their potentials: 

an overview. Journal of 


Reddy, C. R. K., Raja Krishna Kumar, 

G., Eswaran, K., Siddhanta, A. K. 

and Tewari, A. (2003). In vitro somatic 

embryogenesis and regeneration 

of somatic embryos from pigmented 

callus of Kappaphycus alvarezii 

(Gigartinales, Rhodophyta). 

Journal of Phycology 39, 610–616. 

Russig A M and Cosson J (2001) Plant 

regeneration from protoplasts of Enteromorpha 

intestinalis (Chlorophyta, 

Ulvophyceae) as seedstock 

for macroalgal culture. Journal of 


Sankaran, M., Damodaran, V., Singh, 

D. R., Jaisankar, I. and Jerard, B. 

A. (2012). Characterization and Diversity 

Assessment in Coconut Collections 

of Pacific Ocean Islands 

and Nicobar Islands. African Journal 

of Biotechnology 11, 16320- 

16329. 

Singh, D. R. and Srivastava, R. C. 

(2010). Genetic Diversity Analysis 

among the Indigenous Orchids of 

Bay of Islands Using RAPD Markers. 

Indian Journal of Horticulture 

13, 142-145. 

Singh, D. R., Singh, S., Minj, D., Anbananthan, 

V., Salim, K. M., Kumari, 

C. and Varghese, A. (2012). 

Diversity of Morinda citrifolia L. 

in Andaman and Nicobar Islands 

(India) assessed through morphological 

and DNA markers. African 

Journal of Biotechnology 11, 

15214-15225. 

Singh, S., Singh, D. R., Faseela, F., 

Kumar, N., Damodaran, V. and 

Srivastava, R. C. (2012). Diversity 

of 21 taro (Colocasia esculenta (L.) 

Schott) 

accessions 

of Andaman Islands. Genetic Resources 

and Crop Evolution 59, 

821-829. 

Singh, S., Waman, A. A., Bohra, P., 

Gautam, R. K. and Dam Roy, S. 

(2016). Conservation and sustainable 

utilization of horticultural biodiversity 

in tropical Andaman and 

Nicobar Islands, India. Genetic Resources 

and Crop Evolution 63, 

1431–1445. 

Subbaraju, D. P., Ramakrishna, T., 

Sreedhara, S. and Murthy, M. 

(1981). Effects of some growth regulators 

on Gracilaria corticata, an 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 303



agarophyte. Aquatic Botany 10, 75– 

80. 

Sudha, R., Singh, D. R., Sankaran, M., 

Singh, S., Damodaran, V. and 

Simachalam, P. (2013). Genetic diversity 

analysis of papaya (Carica 

papaya L.) genotypes 

in Andaman Islands using morphological 

and molecular markers. African 

Journal of Agricultural Research 

8, 5187-5192. 

Tatewaki, M. and Nagata, K. (1970). 

Surviving protoplasts in vitro and 

their development in Bryopsis. 

Journal of Phycology 6, 401–403. 

Uma, S., Siva, S. A., Saraswathi, M. S., 

Durai, P., Sharma, T. V. R. S., 

Singh, D. B., Selvarajan, R., Sathiamoorthy, 

S. (2005). Studies on 

the origin and diversification of Indian 

wild banana (Musa balbisiana) 

using arbitrarily amplified DNA 

markers. Journal of Horticultural 

Science and Biotechnology 80, 575- 

580. 

Waman, A. A., Bohra, P., Sathyanarayana, 

B. N. and Hanumantharaya, 

B. G. (2015). In vitro approaches 

in medicinal plants- a viable 

strategy to strengthen the resource 

base of plant based systems 

of medicines. In: Biotechnological 

approaches for sustainable development. 

Pullaiah, T. (ed.). Regency 

Bohra et al. 

Publications, New Delhi, India. pp. 

141-156. 

Waman, A. A. and Bohra P. (2016). 

Sustainable development of medicinal 

and aromatic Plants sector in India: 

an overview. Science and Culture 

82, 245-250. 

Waman, A. A. and Bohra, P. (2013). 

Choice of explants- a determining 

factor in tissue culture of Ashoka 

(Saraca indica L.). International 

Journal of Forest Usufructs Management 

14, 10-17. 

Wang, J., Jiang, P., Cui, Y., Deng, X., 

Li, F., Liu, J. and Qin, S. (2010). 

Genetic transformation in Kappaphycus 

alvarezii using microparticle 

bombardment: a potential strategy 

for germplasm improvement. 

Aquaculture International 18, 

1027–1034. 

Yokoya, N. S. and Yoneshigue- 

Valentin, Y. (2011). Micropropagation 

as a tool for sustainable utilization 

and conservation of populations 

of Rhodophyta. Brazilian Journal of 

Pharmacognosy 21, 334-339. 

Zhang, Y. C., Jiang, P., Gao, J. T., 

Liao, J. M., Sun, S. L. and Shen, 

Z. L. (2008). Recombinant expression 

of rt-Pa gene (encoding Reteplase) 

in gametophytes of the seaweed 

Laminaria japonica (Laminariales, 

Phaeophyta). Science China 

Life Sciences 51, 1116-1120. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 304



Plantibodies for Global Health: Challenges and 

Perspectives 

Prasad Minakshi 1, *, Basanti Brar 1 , Manimegalai Jyothi 1 , Ikbal 1 , Koushlesh Ranjan 1 , 

Upendra Pradeep Lambe 1 and Gaya Prasad 2 

1 Department of Animal Biotechnology, LLR University of Veterinary and Animal Sciences, 

Hisar 125004, Haryana, India; 2 Sardar Vallabhbhai Patel University of Agriculture and 

Technology, Meerut 250110, Uttar Pradesh, India; *Correspondence: 

minakshi.abt@gmail.com; Tel: 09992923330 

Abstract: Antibodies are the important part of adaptive immune system. Plants do not naturally 

make the antibodies; but, they can be produced in plants by introducing antibodycoding 

genes from humans and animals. Plant derived antibodies are called as plantibodies 

and known to work in the same way as mammalian antibodies. The plantibodies bioproduction 

offers several advantages over the production of antibodies using mammals. Plants 

are more economic than all other forms of creating antibodies and the technology for obtaining 

and maintaining them is already present. Plantibodies are safer in use because, 

plants reduce the chance of coming in contact with pathogens. Plantibodies can be made at 

an affordable cost using plants as the genetic engineering methods are well established for 

agricultural crops such as tobacco, tomato, potato, soyabean, alfalfa, rice, and wheat. Plantibodies 

production is cost effective and safe. This review highlights the methods of production 

and purification of plantibodies as well as the various types of pharmaceutical antibodies 

produced in transgenic plants. 

Keywords: Antibody production; human and animal health; plants 


Plantibody is an antibody that is 

produced by plants that have been genetically 

engineered with animal DNA. These 

plant produced antibodies, namely 

plantibodies were first demonstrated by 

Hiatt et al. (1989) and Duering et al. 

(1990). They demonstrated that plants can 

express and assemble functionally active 

antibodies. Plants are used in this technology 

as antibody factories, to produce 

large amount of clinically viable proteins 

by using the endomemebrane and secretory 

systems of plants and later it will be 

derived from those plant tissue (Jain et 

al., 2011). For more than a decade, various 

kinds of antibody formats have been 

produced and studied in different species 

of plants and today it became the frontrunners 

among plant-derived pharmaceutical 

proteins. Plants offer many advantages 

and potential benefits for the 

production of recombinant proteins in 

terms of cost, safety and scalability (Stoger 

et al., 2014). For large-scale needs, 

production of recombinant protein using 

transgenic plants as bioreactors is more 

economical than alternative systems such 

as cell culture based antibody production. 

The main anticipated advantage is cost 

saving, low cast biomass production using 

agriculture in a short time without any 

specialized equipment or expensive media. 

Moreover, scale up process can be 

achieved quickly and inexpensively by 

cultivating more land (Stoger et al., 

2014). Other important advantage is the 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 305



Minakshi et al. 

ability of plant cells to correctly fold and 

assemble antibody fragments, single chain 

peptides and also full-length multimeric 

proteins and heterologous proteins such as 

antibodies accumulate in large amount to 

plant cells. Protein synthesis, secretion 

and folding as well as posttranslational 

modifications like signal peptide cleavage, 

di-sulphide bond formation and the 

initial stages of glycosylation are very 

similar in plants and animals. There is 

very low risk of product contamination by 

mammalian viruses, bacterial toxins, 

blood-borne pathogens and oncogenes 

(Jain et al., 2011). Use of these plantibodies 

avoids the ethical issue of producing 

transgenic animals, elimination of purification 

requirement when the plant containing 

recombinant protein is edible and 

production of disease resistant plants by 

raising antibodies in them. The plantibodies 

bioproduction process offers several 

advantages over the conventional method 

of antibody production in mammalian 

cells with the cost of plantibody production 

in plants are substantially lesser 

(Oluwayelu et al., 2016). Different types 

of plantibodies are produced from plants 

(Figure 1). 

Figure 1: Different types of plantibodies 

are produced in plants. 

2. Plants used as expression host for 

plantibody production 

A range of different plant systems 

have been developed for antibody production 

(Table 1). The choice of expression 

Table 1: Pharmaceutical antibodies produced in transgenic plants 

Antigen Plant Antibody form Application Reference 

Human chorionic 

Gonadotrophin 

Tobacco scFv, diabody, 

chimeric, IgG1 

Diagnostic/contraceptive 

Glycophorin 

Streptococcus 

surface antigen 

SAI/II 

Barley, potato, 

tobacco 

Tobacco 

Kathuria et al., 

2002 

ScFv-fusion Diagnostic (HIV) Schunmann et 

al., 2002 

SigA/G 

(CaroRx) 

Therapeutic (topical Ma et al., 2003 

Sperm Corn IgG Contraceptive (topical) 

Cone and 

Whaley, 2002 

Rhesus D Arabidopsis 

IgG Diagnostic Bouquin et al., 

2002 

Human IgG Alfalfa IgG Diagnostic Khoudi et al., 

1999 

Rabies Tobacco IgG Therapeutic Ko et al., 2003 

Herpes simples 

virus 

CD40 

Herpes simplex 

virus 

Soyabean, 

rice 

Tobacco 

cell culture 

Algae 

chlamydomonas 

chloroplast 

IgG 

Therapeutic (topical) 

Therapeutic 

ScFvimmunotoxin 

fusion 

One-chain antibody 

Therapeutic 

Zeitlin, 1998 

Francisco et al., 

1997 

Mayfield et al., 

2003 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 306





Antigen Plant Antibody form Application Reference 

Colon cancer 

antigen 

Tobacco IgG Therapeutic 

/Diagnostic 

Verch et al., 

1998 

Carcinoembryonic 

antigen 

Tobacco, 

rice, wheat, 

scFv, diabody, 

chimeric, IgG1 

Therapeutic/Diagnostic 

Stoger et al., 

2002 

(CEA) pea, tomato 

Herpes simplex Corn sIgA Therapeutic Hood et al., 

virus 

Non-Hodgkins 

lymphoma idiotypes 

Clostridium difficile 

Hepatitis B virus 

New castle disease 

virus 

Tobacco 

Virus vector 

scFv 

Personalized vaccines 

2002 

McCormick et 

al., 2003 

Corn IgG Therapeutic (oral) www.epicyte.co 

m 

Lettuce IgG Vaccine Kapusta et al., 

1999 

Corn Surface glycoprotein 

Vaccine 

Guerreroal, 

F 

Andrade et 

Cholera Tomato Cholera toxin B 

subunit (ctb 

gene) 

Oral vaccine 

2006 

Jiang et al., 

2007 

Enterovirus Tomato Serum IgG VP1 Oral vaccine Chen et al., 

2006 

Porcine reproductive 

and respiratory 

syndrome 

virus 

Banana IgG and IgA Oral immunization Chan et al., 

2013 

system depends upon many factors such 

as suitability for scale-up, storage and 

downstream processes. Other considerations 

attributed to host choice are anticipated 

production scale, the value and use 

of the product, geographical area of production, 

proximity of processing facility, 

biosafety, intellectual property right and 

economical aspects (Twyman et al., 

2003). Several works have shown that 

tobacco, potatoes, soya beans, corn, alfalfa 

and similar kind of crops are the alternative 

ways for production of recombinant 

proteins (Hiatt et al., 1989; Mason 

and Amtzen, 1995). Figure 2 showed that 

many benefits of plants used for plantibody 

production. 

2.1. Tobacco 

Among leafy crops tobacco have 

the greatest biomass yield per hectare and 

allow rapid scale up because they can 

crop several times in a year (Fischer et 

al., 2003). Tobacco grows quickly and 

has been shown to produce comparatively 

large quantity of antibodies. Additionally, 

tobacco is a non-food/non-feed crop if 

grown separately; there is less chance of 

cross-contamination food chain by pharmaceuticals 

(Schillberg et al., 2002). 

Chloroplast engineering in tobacco, an 

alternative to nuclear transgenics, transplastomic 

plants are produced by introducing 

DNA into the chloroplast genome 

instead of nuclear genome, and this will 

be achieved by particle bombardment 

(Daniell et al., 2002). Chloroplast transformation 

is more advantageous, includes 

high transgene copy number, absence of 

position effects and transgene silencing. 

Combination of these properties leads to 

extraordinary levels of expression, exceeding 

25 percent of the total soluble 

protein (Tregoning et al., 2003). Other 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 307



benefit of chloroplast engineering includes 

the ability to express several genes 

as operons and the accumulation of recombinant 

protein in the chloroplast, 

which reduces the toxicity of host plant. 

Biologically active and structurally accurate 

Human growth hormone and serum 

albumin produced at high levels in tobacco 

chloroplasts (Staub et al., 2000; Fernandez 

et al., 2003). Recently tetanus toxin 

fragment (Tregoning et al., 2003) and 

cholera toxin B subunit (Daniell et al., 

2001) has been expressed in tobacco chloroplast 

and was shown that tetanus toxin 

fragment induce protective levels of antitetanus 

antibodies and cholera B subunit 

shows that plastids can fold and assemble 

oligomeric proteins perfectly. Fischer et 

al. (1999) reported that recombinant proteins 

can also be produced from tobacco 

cell culture and several recombinant proteins 

including antibody derivatives were 

derived from a suspension cell line of tobacco 

strain BY-2. The expression of 

classical swine fever virus E2 protein was 

expressed in tobacco chloroplast elicited 

protective immune response in mice 


which was immunized with leaf extracts 

conferred specific antibodies (Shao et al., 

2008). 

2.2. Alfalfa and other legumes 

Another leafy crop used to produce 

recombinant antibodies are alfalfa 

and soya bean. Alfalfa is a perennial crop 

can propagated easily and also have good 

biomass yield. The Biotechnology Company 

Medicago selected it as a platform 

technology. The strong advantage of this 

crop is its tendency to synthesize homogenous 

N-glycans, which improves the 

consistency of recombinant proteins batch 

to batch (Bardor et al., 2003). One of the 

potential advantages of alfalfa is that recombinant 

antibodies produced as a single 

glycoform rather than heterogeneous collection 

of different glycoforms that is 

found in other plant systems. Pea, a grain 

legume also a useful production crop, reason 

that of its high protein content of 

seed. Although at present only low yield 

is possible with this species (Perrin et al., 

2000). 

Figure 2: Benefits of using plants for plantibodies production. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 308




2.3. Cereals 

The disadvantage of tobacco in 

recombinant protein production is its instability. 

The leafy tissue needs preservation 

such as freezing and drying for transportation 

(Ma et al., 2003). Cereals and 

legumes produce less biomass when compared 

to leafy crops but the accumulation 

of recombinant antibodies in seeds allows 

long-term storage in ambient temperature 

because the desiccated environment of the 

mature seeds reduces the exposure of 

stored proteins to non-enzymatic hydrolysis 

and protease degradation. It has been 

shown that antibodies expressed in rice 

seeds remains stable in room temperature 

for years with no detectable loss of activity 

(Stoger et al., 2000). Seeds of cereals 

lack phenolic substances, which are present 

in tobacco leaves, so it increases the 

efficiency of downstream process (Ma et 

al., 2003). Several crops have been investigated 

for antibody production includes, 

rice, wheat, barley, maize, legume pea 

and soya bean. Since the said seed crops 

are used as food, so the downstream processing 

steps may benefit from the food 

processing facilities. Maize is now the 

main commercial crop used for the production 

of two technical proteins Avidin 

and β-glucoronidase by a commercial molecular 

farming venture called Prodigene, 

and Maize also reflects the advantageous 

factors such as high biomass yield, ease 

of scale-up, ease of transformation and in 

vitro manipulation (Hood et al., 1997; 

Witcher et al., 1998). Maize is also being 

used for the production of recombinant 

antibodies, technical/pharmaceutical enzymes 

such as laccase, trypsin, and aprotinin 

(Hood et al., 2002a and 2002b). The 

expression of antibodies in other cereal 

crops has been explored and experiments 

have been carried out in wheat and rice 

(Stoger et al., 2000). The antibody level 

of 150 µg g -1 was expressed in transgenic 

barley was one of the most encouraging 

result have far come from the expression 

of a diagnostic scFv fusion protein called 

SimpliRED (Schunmann et al., 2002). 

2.4. Fruits and vegetables 

The benefit of fruits, vegetables, 

and other leafy crops is that they can be 

consumed raw or partially processed, 

which make them particularly suitable for 

the production and expression of recombinant 

antibodies for passive oral immunotherapy. 

Storage organs of plants such 

as tubers combine this advantage with a 

prolonged shelf-life when compared to 

leafy crops. Potato have been used widely 

for the production of plant-derived vaccines 

and been administered to humans in 

most of the clinical trials so far. Artseanko 

et al., 1995 first demonstrated the 

potential of potato tubers for antibody 

production and recently this crop has been 

investigated as a bulk-production system 

for antibodies (Wilde et al., 2002). Potatoes 

were also used for the production of 

diagnostic antibody-fusion proteins 

(Schunmann et al., 2002) and human milk 

proteins (Chong and Langridge, 2000). 

Tomatoes have outstanding properties for 

pharmaceutical protein production, such 

as high biomass yield and advantage of 

contained growth in greenhouse, by considering 

these potentials; tomatoes were 

used to produce the first plant-derived 

rabies vaccine (Garvey et al., 1995; stoger 

et al., 2000). Lettuce was used as a production 

host to produce edible recombinant 

vaccine against Hepatitis B virus. 

Kapusta et al., (1999) shown that mice 

fed with transgenic lupin tissue were developed 

significant levels of Hepatitis B 

virus specific antibodies and also human 

volunteers who fed with transgenic lettuce 

plants expressing HBV surface antigen 

developed specific serum IgG response to 

plant produced protein. Transgenic tomato 

based developed edible vaccine expressed 

cholera toxin B against cholera in 

the ripening tomato fruit under the control 

of tomato fruit-specific E8 promoter using 

Agrobacterium-mediated transformation 

(Jiang et al., 2007). The immunogenicity 

of the CTB protein expressed in tomato 

fruit was evaluated through determination 

of the serum and mucosal anti-CTB antibody 

levels in experimental mice. A study 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 309




conducted by Chen et al (2006), proved 

that VP1 protein of enterovirus in transgenic 

tomato plant provide both cellular 

and humoral immunity in orally immunized 

mice, those fed with tomato fruit expressing 

VP1 protein. Additionally, serum 

from mice fed with transgenic tomato 

could neutralize the EV71 infection to 

rhabdomyosarcoma cells, indicating that 

tomato fruit expressing VP1 was successful 

in orally immunizing mice. Pigs were 

immunized with recombinant GP5 protein 

expressed in transgenic Banana leaves 

using Agrobacterium-mediated transformation 

with ORF5 gene of porcine reproductive 

and respiratory syndrome virus 

envelope glycoprotein GP5. Pigs immunized 

orally with GP5 protein showed a 

gradual dependent increase in the elicitation 

of serum and saliva anti-PRRSV IgG 

and IgA was observed and significantly 

lower viraemia and tissue viral load were 

recorded when compared to the pigs 

which are fed with untransformed banana 

leaves (Chan et al., 2013). 

3. Methods for plantibody production 

Various techniques have been developed 

to exploit plants as bioreactors 

for the production of plantibodies. Some 

of the techniques are described below: 

3.1. Conventional method 

Once the desired DNA from the 

transformed host cell is isolated and purified, 

it can be injected into the embryo of 

a maturing plant, which we want to use 

for plantibodies production. After injecting 

the desired gene, followed by propagation 

of plant in open field allow large 

scale production of plantibodies. Plant 

tissue culture is most economic and time 

saving method for antibody production 

from plants. In this method, plant cells in 

differentiated states are grown in bioreactors 

with foreign proteins harvested either 

from biomass or culture liquid with less 

contaminant (Moffat, 1989). 

3.2. In vitro cell and tissue cultures 

This is an economically important 

method for producing plantibodies where 

in plant cells in differentiated stages are 

grown under controlled conditions having 

desired genes/ proteins and are harvested 

either in the form of biomass and 

culture liquid or combination of both. 

This method offers large amounts of recombinant 

proteins production in a shorter 

time (Doran, 1999). This method offers 

many advantages over conventional 

method of plantibodies production by 

overcoming the problem of extracting and 

purifying proteins but this method has not 

used for edible vaccines production. In 

this method no sexual reproduction is 

needed, so transgenic stability is increased 

because of absence of crossing 

over, segregation and recombination and 

provides more chances in plantibodies 

production (Ferrante and David, 2001). 

3.3. Breeding and sexual crossing 

An experiment on tobacco plant 

was established for its breeding and sexual 

crossing as a method for the production 

of plantibodies. In this experiment, transformation 

was used to introduce kappa 

chains of either light or heavy regions into 

tobacco plants. Same way was done with 

gamma heavy chains. Upon crossing one 

plant with kappa-chains and another with 

gamma-chains, an antibody was produced 

that expressed both chains (Hiatt et al., 

1989; Whitelam et al., 1994). This method 

provides an easy way to produce plantibodies 

without the need for double fertilization 

(Ferrante and David, 2001). 

3.4. Transgenic seeds 

Above mentioned methods have 

certain limitations. Further restrictions are 

found when plants used as storage system 

because plants cannot store antibodies for 

a longer time. This is due to certain proteases 

degrade the protein piece by piece. 

So, some researchers suggest that, use of 

transgenic seeds in place of green leafy 

plants, because seeds can store antibodies 

for an extended period without degradation. 

Seeds contain low level of proteases 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 310




that allows proteins to be stored for longer 

period when compared to green plants 

(Larrick et al., 1998; Fieldler and cornad, 

1995). So, seeds can be used as bioreactors 

and used as natural storage organs 

(Ferrante and David, 2001). 

3.5. Targeting and compartmentalization 

Antibodies can be targeted to 

some compartments by tagging with a 

small peptide sequence and this allows 

antibodies to be protected from proteases 

that present in the cytoplasm (Kusnadi et 

al., 1997). Compartmentalization to be 

easily isolated organelles and makes easy 

purification procedure (Kusnadi et al., 

1997). Targeting, however, has to be specifically 

controlled and this involves 

proper cleavage of targeted sequences. 

4. Purification techniques 

The main reason for raising antibodies 

in plants is its easy purification 

and low downstream processing. Easy 

purification of plantibodies makes biopharmaceutical 

production more economic 

(Arntzen, 1998). Transgenic seeds assure 

excellent storage properties and thus 

added flexibility in processing management 

and batch production. Separation of 

plantibody in seeds is less complicated 

because of limited range of endogenous 

proteins (Kusandi et al., 1997). Absence 

of human pathogens in plants eliminates 

expensive validation of virus removal 

steps during purification. But the probability 

of presence of a diverse burden on 

plants grown outdoors in non-sterile conditions 

is high. So the process for elimination 

or minimization of contamination 

with endotoxin and mycotoxins will be 

necessary in all commercial process to 

purify antibodies (Gegenheimer, 1990). 

Phenolics can interact with proteins in 

ways that can irreversibly alter the properties 

of proteins but most of the phenolics 

released during extraction are small in 

size, water soluble, and removable by ultrafilteration 

steps. Main techniques used 

for the purification of plantibodies are 

filteration, immunofluorescence, chromatography, 

diafilteration, polymer fusion 

and protein A-sepharose chromatography. 

Some other techniques such as RIA (Radioimmunoassay), 

northern blot technique, 

ELISA (Enzyme linked immune 

sorbent assay), western blot analysis and 

immunofluorescence southern blot analysis 

have been used of evaluation of plantibody. 

5. Applications of plantibodies 

5.1. Bioreactors 

Antibodies produced in plants 

have applications such as production of 

vaccine antigens, protein for clinical diagnosis, 

pharmaceutical and industrial 

proteins, carbohydrates, vitamins, minerals, 

biopolymer and food (Sharma and 

Sharma, 2009). These applications were 

proved in basic agronomy research (Jaeger 

et al., 2000). In recent years, many 

plant systems has been developed in order 

to using plants as bioreactors for the production 

of recombinant antibodies for 

many purposes (Stoger et al., 2002).Using 

plants as a bioreactor, or as a factories or 

using them as antibody replacement for 

microorganisms like bacteria to produce 

human antibodies to communicate with 

human health mainly due to two reasons. 

(i) When compared to prokaryotes, plants 

are better for the production of antibodies 

due to large scale production efficiency 

and low cost of production. 

(ii) Process of post-translational modification 

such as glycosylation that they are 

the kind of post-translational modifications 

of proteins, in plants can be done 

more carefully than bacteria (Ma and 

Hein, 1995). 

5.2. Therapeutic applications 

CaroRx, the first plant derived antibody 

created from tobacco (Fischer et 

al., 2006), is a Sig A secretory antibody. 

It is a clinically advanced anti Streptococcus 

mutans secretory immunoglobulin, a 

plantibody that binds specifically to the 

bacterium, thus protecting humans from 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 311



dental carries (Larrick et al., 2001). 

CaroRx is intended for regular topical 

preventive administration by both dental 

hygienists and patients allowing a thorough 

cleaning and intervention for any 

existing decay. Plantibodies have been 

investigated for inflammatory disease and 

to induce tolerance (Jain et al., 2011). A 

humanised antibody, another plantibody 

with human medical application was expressed 

in soya bean against herpes simplex 

virus (Zietlin et al., 1998). Antitumour 

antibodies against Burkitt`s lymphoma 

was expressed in rice and wheat 

(Ghasempour et al., 2014). Antibodies 

engineered to bind to Bacillus anthracis 

was extracted from transgenic strains of 

tobacco and tested in mice in a study conducted 

by Hull et al in 2005. The result of 

this study showed that the antibodies were 

effective in fighting B. anthracis strain 

and bodes well for the future in any anthrax 

epidemic will be a cheap and effective 

prevention against the disease. 


Treatment or cure for rabies through plantibodies 

has been investigated by Ko et al. 

2003. A plantibody based rabies vaccine 

produced in tobacco was experimentally 

administered in hamsters to check whether 

it could effectively target rabies. The 

plantibody proved to be safe and economically 

feasible alternative method compared 

to the current antibody production 

in animal systems. Another study, tobacco-derived 

plantibodies were experimentally 

administered in mice against the 

Lewis Y antigen found on tumour cells in 

mice and also in lung, breast, ovarian and 

colorectal cancer. According to Brodzick 

et al (2006), the plantibodies showed a 

definite positive effect on the cancerstriken 

mice by preventing tumour formation 

in them (Figure 3). 

5.2.1. Immunization 

One of the most interesting applications 

of this technology is production of 

edible vaccines or oral vaccines. Produc- 

Figure 3: Use of plantibodies for cancer treatment. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 312




Potential proteins such as cytokines, hormones, 

enzymes, epidermal growth factors, 

interferons, human protein C, and 

pharmaceutical foodstuff are produced, 

which are considered for oral immunization 

(Mason and Amtzen, 1995). Transgenic 

plants that express antigens might 

be used as an inexpensive oral vaccine 

production and delivery system, so immunization 

might be possible through 

consumption of an edible vaccine to provide 

immunization. Due to all these reasons 

transgenic plants are considered as 

better alternative for oral vaccines. This 

offers convenient immunization strategies 

for implementing universal vaccination 

programmes throughout the world (Tacket 

et al., 1998). In human host the pathogens 

usually attack mucosal sites in the 

respiratory tract, gastro-intestinal tract or 

genital tract. Stimulation of immune responses 

in these sites through mucosal 

vaccines to protect against illness is desirable 

and this can be achieved by applying 

vaccine to the mucosal surface directly, 

inducing systemic and cellular immune 

responses as well as local immune responses 

at the initial site of interaction 

between the pathogen and host directly 

(Kusnadi et al., 1997). Oral vaccines 

must be protected during passage through 

the hostile environment of the stomach 

and intestine to the sites of immune 

stimulation. 

5.2.2. Immunomodulation 

Immunomodulation is a technique 

that allows interference with cellular metabolism, 

signal transduction or pathogen 

infectivity by the ectopic expression of 

gene encoding antibodies or antibody 

fragments (Jaeger et al., 2000). Applications 

that are relaying on modulating antigen 

levels in vivo are dependent on expression 

and accumulation in specific sub 

cellular compartments and specific tissues. 

Development of crop resistance and 

passive immunization of plants by expression 

of pathogen-specific antibodies reduces 

infection and symptoms caused by 

viruses and mollicutes, and significant 

progress has been made towards engineering 

residence against insects (Schillberg 

et al., 2001; Jaeger et al., 2000). Immunomodulation 

is a dynamic tool for 

altering the function of an antigen in vivo. 

When an artificial abscisic acid sink was 

created by the production of an anti- abscisic 

acid specific scFv in the endoplasmic 

reticulum of potato and tobacco 

plants, both physiological and morphological 

changes were noticed (Conrad and 

Manteuffel, 2001). Moreover, agrofiltration 

of tobaco was used to produce a 

diabody against carcinoembryonic antigen 

(Vaquero et al., 2002). In addition, plantibodies 

may also prove useful as feed 

additives or for phytoremediation in human 

health care (Mason and Arntzen, 

1995). 

6. Pathogen resistance in plants 

Antibody mediated pathogen resistance 

in plants is a novel strategy for 

generating transgenic plants resistant to 

pathogens have been developed in many 

cases for therapeutic applications, for 

Immunomodulation. Peschen et al., 

(2004) demonstrated antibody-mediated 

resistance against fungal pathogens and 

protecting plants against fungal diseases. 

Schillberg et al (2000), targeted anti- 

TMV antibodies to the plasma membrane 

in vivo (in planta) results in evolvement 

of transgenic plants resistant to TMV infection. 

A study conducted by Boonrod et 

al (2004), paved the way for engineering 

broad-range virus resistance by expression 

of scFv antibodies in vivo that are 

specific and highly conserved motifs in 

viral replicase or polymerases. Boonrad 

and his colleagues demonstrated that 

transgenic tobacco plants expressing scFv 

antibodies against a conserved domain in 

plant viral RNA dependent RNA polymerase 

either in the cytosol or endoplasmic 

reticulum, showed high levels of resistance 

to four plant viruses from different 

genera. Malembic-Maher et al. (2005) 

studied the scFv 2A10 protein expression 

in engineered tobacco plants and for their 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 313



resistance to stobular disease. It was observed 

that tobacco plants producing secreted 

scFvs shown a short delay in symptom 

appearance, reduction to pathogen 

susceptibility and phyloplastoma multiplication. 

Isolated antipectinase scFv antibodies 

directed against extracellular proteins 

from Rhizoctonia solani secluded 

from a page display library. Soluble scFv 

antibodies shown to inhibit polygalacturonase 

in the culture supernatants of a 

range of fungal pathogens such as ascomycetes, 

basidiomycetes and oomycetes. 

This soluble antibody also inhibited maceration 

in potatoes (Manatunga et al., 

2005). First time antibody mediated fungal 

resistance was demonstrated by Wu et 

al in wheat and cereal grains. This fungal 

resistance in transgenic plants is mediated 

by generating specific antibodies against 

Fusarium graminearum, a predominant 

fungal species infecting wheat and small 

cereal grains in china (Wu et al., 2005). 

7. Treatment of Ebola patients 


Recently, antibodies against Ebola 

virus have been explored in plants. A 

high yielding geminivirus-based expression 

system in the tobacco plant, Nicotiana 

benthamiana, was used for the production 

of mAb (6D8) that protected animals 

from Ebola virus infection (Chen et al., 

2002). An Ebola immune complex (EIC) 

was produced by fusing Ebola glycoprotein 

GP1 to the C-terminus of the heavy 

chain of humanized 6D8 mAb that binds 

specifically to a linear epitope on GP1 

using the geminivirus-based expression 

system and Nicotiana benthamiana (Bhoo 

et al., 2011). The recombinant immunoglobulins 

were produced in leaves of Nicotiana 

benthamiana, which was purified 

by protein G affinity chromatography. 

The resultant recombinant antibody 

bound the the complement facto C1q, indicating 

immune complex formation. 

Therefore subcutaneous immunization of 

mice with purified EIC showed high level 

of production of anti-Ebola virus antibodies 

that provides protection against Ebola 

virus (Figure 4). This was the first published 

report of an Ebola virus candidate 

vaccine to be produced in plants. 

The Ebola virus disease outbreak 

in West Africa has provided a great opportunity 

for the use of plantibodies in 

resolving global human health challenges 

as 

Figure 4: Use of plantibodies for Ebola virus treatment. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 314




as two American medical aid workers 

who contracted the disease in Liberia 

were successfully treated with an experimental 

drug called ZMapp produced in 

the tobacco plant. The drug ZMapp contains 

combination of three humanized anti-Ebola 

virus mAbs and developed by 

Mapp biopharmaceutical incorporated, 

San Diego (Langreth et al., 2014). Although 

the drug ZMapp holds great promise 

for the future, the major limitation is 

producing large quantity of anti-Ebola 

virus antibody to meet the demand of 

widespread outbreak requirement, multiple 

doses, direct delivery of highly pure 

antibody into the blood stream and moreover 

the drug is intended originally for 

expression levels sufficient for animal 

trial (Powell, 2014). ZMapp is yet to receive 

the approval by the US food and 

Drug Administration who have to certify 

that the plant extraction process has not 

led to the contamination of the resulting 

drug (Begley, 2014). 

8. Advantages and challenges of plantibodies 

production 

Plantibodies production from 

plants has many potential advantages for 

creating biopharmaceuticals related to the 

medicine. First, the plant systems used for 

plantbodies production are more economical 

than industrial equipments using fermentation 

system. Second, this system 

provides large amount of plant products. 

Third, the system can be omitted the purification 

step when the aim is to produce 

edible vaccines. Fourth, plants can be 

directed to the desired proteins into compartments/organelles 

such as chloroplasts. 

Fifth, the amount of proteins produced in 

such an amount it can be suitable for industrial 

levels. Last, one of the important 

advantage, health risks from contamination 

with potential human pathogens are 

minimized (Sala et al., 2003; Daniell et 

al., 2001). 

After lots of advantages, there are 

some remaining challenges are associated 

with the plantibodies production. During 

down streaming processess of plantbodies 

production such as extraction and purification 

of plantibodies is an important 

step, covers more than half of the total 

cost. Thus purification system during 

plantibodies production is very expensive. 

Currently purification system in plant systems, 

an affinity purification protocol requires 

protein A-based matrices is mainly 

used (Valdes et al., 2003). So it is necessary 

to use alternative economic methods 

that use oleosin or polymer fusions for the 

purification of plantibodies (Daniell et al., 

2001). Till date, researches have had difficulty 

in achieving the high level of chloroplast 

gene expression. Each type of 

plants poses its own challenges and dosage 

of vaccines may be variable. Plantibodies 

are not suitable for infants (Doshi 

et al., 2013). 

9. Future perspectives 

Plant-derived systems for production 

of biopharmaceutics should meet the 

same standards of safety and performance 

as other production systems (Daniell et 

al., 2001). Plantibodies production system 

have many advantages over animal systems, 

such as well-established cultivation, 

quick scale-up, simple distribution by 

seeds, oral delivery, can be used as raw 

food or dry powder, desperate of cold 

chain requirement, mucosal and serum 

immune responses, cost efficiency, ease 

of genetic manipulation, ease of production 

and scale-up, safer than conventional 

vaccines, ideal to face bio-weapons and 

ideal for veterinary use as feed additive 

(Yoshida et al., 2004; Sala et al., 2003). 


Substantial progress has been 

made in recent years towards the production 

of a wide range of antibodies in 

plants and now it is a feasible and economical 

system of producing antibodies. 

These transgenic plants have been shown 

to be the most productive system in 

providing therapeutics and edible vac- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 315




cines, which are cheap and can be easily 

administered. Plants can be easily explored 

by pharmaceutical industries and 

Indian climate helps for the production of 

diversified crops. Engineering of increased 

pathogen resistance and alteration 

of phenotypes by immunomodulation 

provide a great platform to develop the 

prevention strategies. Various other strategies 

have been developed to exploit 

plants as bioreactors for the production of 

pharmaceutical antibodies and many plant 

produced antibodies are proved increased 

disease resistance. Recent developments 

focus on the detailed characterisation of 

recombinant products. Recent indications 

that tissue specific and physiological factors 

may have an impact on the quality 

and glycosylation pattern of a plantibodies 

will perhaps lead to new insights and 

production strategies. Although some 

plant-derived antibody products have successfully 

completed early phase clinical 

trials, several issues including regulatory 

guidelines and public acceptance must 

still be resolved. Currently, more than 200 

novel antibody-based potential products 

are in clinical trials worldwide, and market 

demand will certainly strain the capabilities 

of existing production systems. 

Moreover, adoption of plants as bioreactors 

on a larger scale would reduce the 

cost of antibody therapy and simultaneously 

increase the number of patients who 

access to these treatments. 

References 

Arntzen, C.J. (1998). Pharmaceutical 

food stuffs oral immunization with 

transgenic plants. Nature Medicine 4, 

502-503. 

Artsaenko, O., Peisker, M., Zurneieden, 

U., Fiedler, U., Weiler, 

E.W. and Muntz, K. (1995). Expression 

of a single-chain Fv antibody 

against abscisic acid creates a 

wilty phenotype in transgenic tobacco. 

The plant Journal 8, 745-750. 

Bardor, M., Loutelier-Bourhis, C., Paccalet, 

T., Cosette, P., Fitchette, A. 

and Vezina, L.P. (2003). Monoclonal 

C5-1 antibody produced in transgenic 

alfalfa plants exhibits a N- 

glycosylation that is homogenous 

and suitable for glyco-engineering 

into human compatible structures. 

Plant Biotechnology Journal 1, 451- 

462. 

Begley, S. (2014). Tobacco-derived 'plantibodies' 

enter the fight against Ebola. 

http://www.reuters.com/article/2014/ 

08/06/healthebolatobaccoidUSL2N0 

QB24N20140806. 

Bhoo, S.H., Lai, H., Ma, J., Arntzen, 

C.J., Chen, Q. and Mason, H.S. 

(2011). Expression of an immunogenic 

Ebola immune complex in Nicotiana 

benthamiana. Plant Biotechnology 

Journal 9, 807 -816. 

Boonrod, K., Galetzka, D., Nagy, P.D., 

Conrad, U. and Andkrczal, G. 

(2004). Single-chain antibodies 

against a plant viral RNA-dependent 

RNA polymerase confers virus resistance. 

Nature Biotechnology 22, 

856-862. 

Bouquin, T., Thomsen, M., Nielsen, 

L.K., Green, T.H., Mundy, J. and 

Hanefeld, D. (2002). Human antirhesus 

D IgG1 antibody produced in 

transgenic plants. Transgenic Research 

11, 115-122. 

Brodzick, R., Glogowska, M., Bandurska, 

K., Okulicz, M., Deka, D. 

and Ko, K. (2006). Plant-derived 

Anti-Lewis Y mAbexhibits biological 

activities for efficient immunotherapy 

against human cancer cells. 

Proceedings of the National Academy 

of Sciences USA 103, 8804 – 

8809. 

Chan, H.T., Chia, M.Y., Pang, V.F., 

Jeng, C.R., Do, Y.Y. and Huang 

P.L. (2013). Oral immunogenicity of 

porcine reproductive and respiratory 

syndrome virus antigen expressed in 

transgenic banana. Journal of Plant 

Biotechnology 11, 315–324. 

Chen, H.F., Chang, M.H., Chiang, 

B.L. and Jeng, S.T. (2006). Oral 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 316




immunization of mice using transgenic 

tomato fruit expressing VP1 

protein from enterovirus 

71.Vaccine 24, 2944-2951. 

Chen, Q., He, J., Phoolcharoen, W. and 

Mason, H.S. (2002). Geminiviral 

vectors based on bean yellow dwarf 

virus for production of vaccine antigens 

and monoclonal antibodies in 

plants. Human Vaccines 7, 331-338. 

Chong, D.K.X. and Langridge, W.H.R. 

(2000). Expression of full-length bioactive 

antimicrobial human lactoferrin 

in potato plants. Transgenic Research 

9, 71–78. 

Cone, R.A. and Whaley, K.J. (2002). 

Topical application of antibodies for 

contraception and for prophylaxis 

against sexually transmitted diseases. 

US patent 6, 355-235. 

Conrad, U. and Manteuffel, R. (2001). 

Immunomodulation of phytohormones 

and functional proteins in 

plant cells. Trends in Plant Science 

6, 399-402. 

Daniell, H., Khan, M.S. and Allison, L. 

(2002). Milestones in chloroplast genetic 

engineering: an environmentally 

friendly era in biotechnology. 

Trends in Plant Science 7, 84–91. 

Daniell, H., Lee, S.B., Panchal, T. and 

Wiebe, P.O. (2001). Expression of 

the native cholera B toxin subunit 

gene and assembly as functional oligomers 

in transgenic tobacco chloroplasts. 

Journal of Molecular Biology 

311, 1001-1009. 

Daniell, H., Streatfield, S.J. and 

Wycoff, K. (2001). Medical molecular 

farming: production of antibodies, 

biopharmaceuticals and edible vaccines 

in plants. Trends in Plant Science 

6, 219-226. 

Doran, P.M. (1999). Foreign protein production 

in plant tissue cultures. Current 

Opinon in Biotechnology 11, 

199-204. 

Doshi, V. Rawat, H. And Mukhergee, 

S. (2013). Edible vaccines from GM 

crops: current status and future 

scope. Journal of Pharmaceutical 

and scientific innovation 2, 1-6. 

Duering, K., Hippe, S., Kreuzaler, F. 

and Schell, J. (1990). Synthesis and 

self-assembly of a functional monoclonal 

antibody in transgenic Nicotiana 

tabacum. Plant Molecular Biology 

15, 281-293. 

Fernandez, S., Millan, A., Mingo- 

Castel, A., Miller, M. and Daniell, 

H. (2003). A chloroplast transgenic 

approach to hyperexpress and purify 

human serum albumin, a protein 

highly susceptible to proteolytic degradation. 

Plant Biotechnology 1, 77– 

79. 

Ferrante, E and David S. (2001). A review 

of the progression of transgenic 

plants used to produce plantibodies 

for human usage. Biological & Biomedical 


Fieldler, U. and Conrad, U. (1995). 

High-level production and long-term 

storage of engineered antibodies in 

transgenic tobacco seeds. Biotechnology 

13, 1090-1094. 

Fischer, R., Emans, N., Schuster, F., 

Hellwig, S. and Drossard, J. (1999). 

Towards molecular farming in the future: 

using plant-cell-suspension cultures 

as bioreactors. Biotechnology 

and Applied Biochemistry 30, 109– 

112. 

Fischer, R., Richard, M.T. and Schillberg, 

S. (2003). Production of antibodies 

in plants and their use for 

global health. Vaccines 21, 820-825. 

Fischer, R., Twyman, M.R., Hellwig, S., 

Drossard, J. and Schillberg, S. 

(2006). Facing the Future with 

Pharmaceuticals from Plants. Biotechnology 

and Sustainable Agriculture 

and Beyond. Xu, Z., Li, J., Xue, 

Y., Yang, W. (Eds.). Proceedings of 

the 11th IAPTC&B Congress. Beijing, 

China. pp. 13-32 

Francisco, J.A., Gawlak, S.L., Miller, 

M., Bathe, J., Russell, D. and Chace, 

D. (1997). Expression and characterization 

of bryodin 1 and a bryodin 

1- based single-chain immuno- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 317




toxin from tobacco cell culture. Bioconjugate 

Chemistry 8, 708-713. 

Garvey, P.B., Hammond, J., Dienelt, 

M.M., Hooper, D.C., Fu, Z.F., Dietzschold, 

B., Koprowski, H. and 

Michaels, F.H. (1995). Expression 

of the rabies virus glycoprotein in 

transgenic tomatoes. Biotechnology 

New York 13, 1484-1487. 

Gegenheimer, P. (1990). Preparation of 

extracts from plants. Methods in Enzymlogy 

182, 174-193. 

Ghasempour, H.R., Kahrizi, D. and 

Mehdiah, N. (2014). Application of 

transgenic plants as factories for producing 

biopharmaceuticals. Journal 

of Biodiversity and Environmental 


Guerrero-Andrade, O., Loza-Rubio, E., 

Olivera-Flores, T., Fehervari- 

Bone, T. and Gomez-Lim, M.A. 

(2006). Expression of the Newcastle 

disease virus fusion protein in transgenic 

maize and immunological studies. 

Transgenic Research 15, 455– 

463. 

Hiatt, A., Cafferkey, R. and Bowdish, 

K. (1989). Production of antibodies 

in transgenic plants. Nature 342, 76- 

78. 

Hood, E.E. (2002b). From green plants to 

industrial enzymes. Enzyme Microbial 


Hood, E.E., Witcher, D.R., Maddock, 

S., Meyer, T., Baszczynski, C. and 

Bailey, M. (1997). Commercial production 

of avidin from transgenic 

maize: characterization of transformant, 

production, processing, extraction 

and purification. Molecular 

Breeding 3, 291-306. 

Hood, E.E., Woodard, S.L. and Horn, 

M.E. (2002). Monoclonal antibody 

manufacturing in transgenic plants– 

myths and realities. Current Opinion 

in Biotechnology 13, 630-5. 

Hood, E.E., Woodard, S.L. and Horn, 

M.E. (2002a). Monoclonal antibody 

manufacturing in transgenic plantsmyths 

and realities. Current Opinion 

in Biotechnology 13, 630–635. 

Hull, A.K., Criscuolo, C.J., Mett, V., 

Groen, H., Steeman, W. and Westra, 

H. (2005). Human-derived, 

plant-produced monoclonal antibody 

for the treatment of anthrax. Vaccine 

23, 2082-2086. 

Jaeger, D.G., De Wilde, C., Eeckhout, 

D., Fiers, E. and Depicker, A. 

(2000). The plantibody approach: 

expression of antibody genes in 

plants to modulate plant metabolism 

or to obtain pathogen resistance. 

Plant Molecular Biology 43, 419- 

428. 

Jain, P., Pandey, P., Jain, D. and 

Dwivedi, P. (2011). Plantibody: an 

overview. Asian Journal of Pharmacy 

and Life Science 1, 87-94. 

Jiang, X.L., He, Z.M., Peng, Z.Q., Qi, 

Y., Chen, Q. and Su, S.Y. (2007). 

Cholera toxin B protein in transgenic 

tomato fruit induces systemic response 

in mice. Transgenic research 

16, 169-175. 

Kapusta, J., Modelska, A., Figlerowicz, 

M., Pniewski, T., Letellier, M., 

Lisowa, O., Yusibov, V., 

Koprowski, H., Plucienniczak, A. 

and Legocki, A.B. (1999). A plantderived 

edible vaccine against hepatitis 

B virus. Federation of American 

Societies for Experimental Biology 

Journal 13, 1796-1799. 

Kathuria, S., Sriraman, R., Nath, R., 

Sack, M., Pal, R. and Artsaenko, 

O. (2002). Efficacy of plant produced 

recombinant antibodies against 

HCG. Human Reproduction 17, 

2054-61. 

Khoudi, H., Laberge, S., Ferullo, J.M., 

Bazin, R., Darveau, A. and 

Castonguay, Y. (1999). Production 

of a diagnostic monoclonal antibody 

in perennial alfalfa plants. Biotechnology 

and Bioengineering 64, 135- 

43. 

Ko, K., Tekoah,Y., Rudd, P.M., Harvey, 

D.J., Dwek, R.A. and Spitsin, 

S. (2003). Function and glycosylation 

of plant-derived antiviral monoclonal 

antibody. Proceedings of the 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 318



National Academy of Sciences USA 

100, 8013-8018. 

Kusnadi, A.R., Nikolov, Z.L. and Howard, 

J.A. (1997). Production of recombinant 

proteins in transgenic 

plants: practical considerations. Biotechnology 

and Bioengineering 56, 

473-484. 

Langreth, R., Chen, C., Nash, J. and 

Lauerman, J. (2014). Ebola drug 

made from tobacco plant saves U.S. 

aid workers. Available at: 

http://www.bloomberg.com/news/20 

14-08-05/ebola-drug-made-fromtobacco-plant-saves-u-saidworkers.html 

Accessed January 10, 

2015. 

Larrick, J.W., Yu, L. and Chen, J. 

(1998). Production of antibodies in 

transgenic plants. Research in Immunology 

149, 603-608. 

Larrick, J.W., Yu, L., Naftzger, C., 

Jaiswal, S. and Wycoff, K. (2001). 

Production of secretory IgA antibodies 

in plants. Biomolecular Engineering 

18, 87–94. 

Ma, J.K.C. and Hein, M. (1995). Immunotherapeutic 

potential of antibodies 

produced in plants. Trends in Biotechnology 

13, 522–527. 

Ma, J.K.C., Drake, P.M.W. and Christou, 

P. (2003). The production of recombinant 

pharmaceutical proteins in 

plants. Nature Review Genetics 4, 

794-805. 

Malembic-Maher, S., Le Gall, F., 

Danet, J.L., Borne, F.D., Bove, 

J.M. and Garniersemancik, M. 

(2005). Transformation of tobacco 

plants for single-chain antibody expression 

via apoplastic and symplasmic 

routes, and analysis of their 

susceptibility to stolbur phytoplasma 

infection. Plant Science 168, 349- 

358. 

Manatunga, V., Sanati, H., Tan, P. and 

Andobrien, P.A. (2005). Maceration 

of plant tissue by fungi is inhibited 

by recombinant anti pectinase antibodies. 

European Journal of Plant 

Pathology 112, 211-220. 


Mason, H.S. and Arntzen, C.J. (1995). 

Transgenic plants as vaccine production 

systems. Trends Biotechnology 

13, 388-392. 

Mayfield, S.P., Franklin, S.E. and Lerner, 

R.A. (2003). Expression and assembly 

of a fully active antibody in 

algae. Proceedings of the National 


442. 

McCormick, A.A., Reinl, S., Cameron, 

T.I., Vojdani, F., Fronefield, M. 

and Levy, R. (2003). Individualized 

human scFv vaccines produced in 

plants: humoral anti-idiotype responses 

in vaccinated mice confirm 

relevance to the tumor Ig. Journal of 

Immunological Methods 278, 95-104. 

Moffat, A.S. (1989). Exploring transgenic 

plants as a new vaccine source. 

Science 268, 658-660. 

Oluwayelu, D.O. and Adebiyi, A.I. 

(2016). Plantibodies in human and 

animal health: a review. African 

Health Sciences 16, 640-645. 

Perrin, Y., Vaquero, C., Gerrard, I., 

Sack, M., Drossard, J., Stoger, E., 

Christou, P. and Fischer, R. (2000). 

Transgenic pea seeds as bioreactors 

for the production of a single-chain 

Fv fragment (scFV) antibody used in 

cancer diagnosis and therapy. Molecular 

Breeding 6, 345–352. 

Peschen, D., Li, H.P., Fischer, R., 

Kreuzaler, F. and Liao, Y.C. 

(2004). Fusion proteins comprising a 

Fusty-nun-specific antibody linked to 

anti-fungal peptides protect plants 

against fungal pathogens. Nature Biotechnology 

22,732-738. 

Powell, J.D. (2015). From Pandemic Preparedness 

to Biofuel Production: Tobacco 

Finds Its Biotechnology Niche 

in North America. Agriculture 5, 

901-917. 

Sala, F.M., Rigano, M., Barbante, A., 

Basso, B., Amanda M. and 

Walmsley, S. Castiglione. (2003). 

Vaccine antigen production in transgenic 

plants: strategies, gene con- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 319




structs and perspectives. Vaccine 21, 

803–808. 

Schillberg, S., Emans, N. and Fischer, 

R. (2002). Antibody molecular farming 

in plants and plant cells. Phytochemistry 

Reviews 1, 45-54. 

Schillberg, S., Zimmermann, S., 

Findlay, K. and Fischer, R. (2000). 

Plasma membrane display of antiviral 

single chain Fv fragments confers 

resistance to tobacco mosaic virus. 

Molecular Breeding 6, 317-326. 

Schillberg, S., Zimmermann, S., Zhang, 

M.Y. and Fischer, R. (2001). Antibody-based 

resistance to plant pathogens. 

Transgenic Research 10, 1-12. 

Schunmann, P.H.D., Coia, G. and Waterhouse, 

P.M. (2002). Biopharming 

the SimpliRED TM HIV diagnostic 

reagent in barley, potato and tobacco. 


Shao, H.B., He, D.M., Qian, K.X., Shen, 

G.F. and Su, Z.L. (2008). The expression 

of classical swine fever virus 

structural protein E2 gene in tobacco 

chloroplasts for applying chloroplasts 

as bioreactors. Comptes 

Rendus Biologies 331, 179–184. 

Sharma, A. and Sharma, K.M.K. 

(2009). Plants as bioreactors, Recent 

developments and emerging opportunities. 

Biotechnology Advances 27, 

811-832. 

Staub, J.M., Garcia, B., Graves, 

J., Hajdukiewicz, P.T., Hunter, 

P., Nehra, N., Paradkar, 

V., Schlittler, M., Carroll, 

J.A., Spatola, L., Ward, D., Ye, G. 

and Russell, D.A. (2000). Highyield 

production of a human therapeutic 

protein in tobacco chloroplasts. 

Nature Biotechnology 18, 

333-338. 

Stoger, E., Fischer, R., Moloney, M., 

Ma, J.K.C. (2014). Plant molecular 

pharming for the treatment of chronic 

and infectious diseases. Annual Review 

Plant Biology 65,743–768. 

Stoger, E., Sack, M., Fischer, R. and 

Christou, P. (2002). Plantibodies: 

applications, advantages and bottlenecks. 


13, 161-166. 

Stoger, E., Sack, M., Perrin, Y., 

Vaquero, C., Torres, E. and Twyman, 

R.M. (2000). Practical considerations 

for pharmaceutical antibody 

production in different crop systems. 


Stoger, E., Vaquero, C., Torres, E., 

Sack, M., Nicholson, L. and 

Drossard, J. (2000). Cereal crops as 

viable production and storage systems 

for pharmaceutical scFv antibodies. 

Plant Molecular Biology 42, 

583-590. 

Tacket, C.O., Mason, H.S., Losonsky, 

G., Clements, J.D., Levine, M.M. 

and Arntzen, C.J. (1998). Immunogenicity 

in humans of a recombinant 

bacterial antigen delivered in a transgenic 

potato. Nature Medicine 4, 

607-610. 

Tregoning, J.S., Nixon, P., Kuroda, H., 

Svab, Z., Clare, S., Bowe, F., Fairweather, 

N., Ytterberg, J., Wijk, 

K.J., Dougan, G. and Maliga, P. 

(2003). Expression of tetanus toxin 

Fragment C in tobacco chloroplasts. 

Nucleic Acids Research 31, 1174- 

1179. 

Twyman, R.M., Stoger, E., Schillberg, 

S., Christou, P. and Fischer, R. 

(2003). Molecular farming in plants: 

Host systems and expression technology. 

Trends Biotechnology 21, 

570-578. 

Valdes, R., Gomez, L., Padilla, S., 

Britom J., Reyes, B., Alvarez, T., 

Mendoza, O., Herrera, O., Ferro, 

W,, Pujol, M., Leal, V., Linares, 

M., Hevia, Y., Garcia, C., Mila, L., 

Garcia, O., Sanchez, R., Acosta, A., 

Geada, D., Paez, R., Luis Vega, J. 

& Borroto, C. (2003). Large-scale 

purification of an antibody directed 

against hepatitis B surface antigen 

from transgenic tobacco plants. Biochemistry 

and Biophysics Research 

Community 308, 94–100. 

Vaquero, C., Sack, M., Schuster, F., 

Finnern, R., Drossard, J. and 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 320



Schumann, D. (2002). A carcinoembryonic 

antigen-specific diabody 

produced in tobacco. Federation of 

American Societies for Experimental 

Biology 16, 408-410. 

Verch, T., Yusibov, V. and Koprowski, 

H. (1998). Expression and assembly 

of a full-length monoclonal antibody 

in plants using a plant virus vector. 

Journal of Immunological Methods 

220, 69-75. 

Whitelam, G.C., Cockburn, W. and 

Owen, M.R.L. (1994). Antibody 

production in transgenic plants. Biochemical 

Society Transactions 22, 

940-943. 

Wilde, D.C., Peeters, K., Jacobs, A., 

Peck, I. and Depicker, A. (2002). 

Expression of antibodies and Fab 

fragments in transgenic potato plants: 

a case study for bulk production in 

crop plants. Molecular Breeding 9, 

2871–282. 

Witcher, D., Hood, E.E., Peterson, D., 

Bailey, M., Bond, D. and Kusnadi, 

A. (1998). Commercial production of 


β-glucuronidase (GUS): a model system 

for the production of proteins in 

plants. Molecular Breeding 4, 301- 

312. 

Wu, A.B., Lt, H.P., Zhao, C.S. and 

Liao, Y.C. (2005). Comparative 

pathogenicity of Fusarium graminearum 

from China revealed by wheat 

coleoptile and floret inoculations. 

Mycopathologia 164, 75-83. 

www.epicyte.com 

Yoshida, K., Matsui, T. & Shinmyo, A. 

(2004). The plant vesicular transport 

engineering for production of useful 

recombinant proteins. Journal of Molecular 

Catalysis B: Enzymatic 28, 

167-171. 

Zeitlin, L., Olmsted, S.S., Moench, 

T.R., Co, M.S., Martinell, B.J. and 

Paradkar, V.M. (1998). A humanized 

monoclonal antibody produced 

in transgenic plants for immunoprotection 

of the vagina against genital 

herpes. Nature Biotechnology 16, 

1361-1364. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 321



Renewable Energy from Agro-industrial Processing 

Wastes: An Overview 

Sudhanshu S. Behera 1 , Ramesh C. Ray 2, * and S. Ramachandran 3 

1 Department of Fisheries and Animal Resource Development, Government of Odisha, India; 

2 ICAR- Central Tuber Crops Research Institute (Regional Centre), Bhubaneswar 

751019, India; 3 Department of Biotechnology, Birla Institute of Technology and Science, 

Pilani, Dubai campus, Dubai International Academic City , P. O. Box No: 345055, Dubai, 

UAE; *Correspondence: rc.ray6@gmail.com; Tel: +91- 674- 2470528 

Abstract: The continued use of fossil-derived fuels has resulted in progressive depletion of 

non-renewable energy resources and environmental deterioration that led to the development 

of an alternative renewable source of energy for sustainability. Agricultural and horticultural 

crops waste processing offer an option among alternatives for generating renewable 

energy (bio-refinery) due to its potential sustainable supply and abundance. The conversion 

of agro-industrial processing wastes typically contains mixed hexoses and pentoses with 

significant proportions of polymeric sugars (lingo-celluloses and cellulose) which are difficult 

to degrade and require advances in the technology. This article is highlighting the major 

developments in various biomass-based agro-industrial processing waste-based fuelgenerating 

processes and suggests a possible major solution to provide energy (bio-energy) 

in an eco-friendly way for the reduction of greenhouse gases and pollution. 

Keywords: Agro-industry; bio-ethanol; environment; processing; renewable energy 


The world is faced with a chronic 

energy crisis that has resulted in the crippling 

of most sectors of the economy. It is 

estimated that over 80% (about 450-500 

EJ/year) of the world’s energy demands is 

met by the combustion of fossil fuels 

(Ioelovich, 2015). The main energy 

sources of fossil fuels are coal, petroleum 

and natural gas. Coal does provide about 

28% of world’s consumed energy. Crude 

oil- petroleum, provides about 32% of 

world’s energy while natural gas provides 

about 20% of the world’s energy consumption 

(Ioelovich, 2015). However, 

fossil-based fuels are limited and excessive 

exploitation of such fossil fuel increases 

carbon footprints. The combustion 

of these fuels also caused the emission 

of harmful gases, including CO, 

CO 2 , NO x and sulfur-containing compounds 

which are main sources of air pollution 

and global warming (Crutzen et al., 

2016). Moreover, fossil-fuel exposes the 

earth to changes in price of petroleum resources 

and political instability from the 

oil producing region of the world (Behera 

and Ray, 2014; Aliyu et al., 2015). 

The renewable sources of energy, 

such as solar, wind, and biofuel (bioethanol, 

bio-diesel, bio-hydrogen) have 

gained huge attention from governments 

of many countries across the world. Recently, 

government policies have planned 

to replace the petroleum based fuels with 

renewable biomass fuels, which are derived 

from agricultural residues such as 

sugarcane, corn, switchgrass, algae, etc 

(Sarkar et al., 2012). These renewable 

resources are indigenous, non-polluting 

and virtually inexhaustible. Globally, 

more than 30% of the loss occurs from 

agricultural/horticultural substrates/ wast- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 322


Renewable Energy from Agro-industrial Processing Wastes 

es (rice husk, coffee wastes, sugar cane 

biogases, maize harvesting wastes, and 

bamboo cellulose pulp, etc.,) at the retail 

and consumer levels, of which the postharvest 

and processing level wastages account 

for the major share (Singh et al., 

2012).The different residues/biomass resulting 

from the production of agricultural 

crops might contribute to the achievements 

of the renewable energy target as 

proposed for further uses (Scarlat et al., 

2010). The use of biomass for transport 

fuel, heat and electricity production will 

have to increase substantially to meet the 

proposed binding target of renewable energy 

in the EU energy mix of 20% by 

2020 (Scarlat et al., 2010). The chapter 

discusses the agricultural/horticultural 

resources and food processing wastes for 

potential production of bioenergy. 

2. Agricultural and food processing 

wastes 

There are various forms of agricultural 

and horticultural (agro-industrial) resources 

in the world. Among the agroindustrial 

wastes, horticulture processing 

industries form a major share throughout 

the world. Horticultural wastes mainly 

generate from fruits and vegetable processing, 

starchy roots and tubers, coconut, 

olive and palm oil mills and fruit-based 

fermentation industries (Panda and Ray, 

2015; Panda et al., 2017). The fermentation 

industries of solid waste include 

items removed from fruits and vegetables 

during cleaning, processing, cooking, 

and/or packaging. These items may include 

leaves, peels, pomace, skins, rinds, 

cores, pits, pulp, stems, seeds, twigs, and 

spoiled fruits and vegetables (Bouallagui 

et al., 2005; Panda et al., 2016). Globally, 

more than billion tons of agroindustrial 

and food processing solid wastes are 

available. The losses in industrial countries 

are as high as in developing countries, 

but in developing countries more 

than 40% of losses occur at post-harvest 

and processing levels, while in industrialized 

countries, more than 40% of the 

Behera et al. 

losses occur at retail and consumer levels. 

Among them, lignocellulosic wastes derived 

from cereals, oil seeds, pulses and 

plantation crops, account for 222 million 

tons in industrialized countries. Fruits, 

vegetables and root crop processing 

wastes alone are available to the extent of 

12 million tons (Auer et al., 2017). However, 

over 86 x 10 6 t of fruits and 162 x 

10 6 t vegetables are produced annually in 

India, contributing 12.6% and 14.0% of 

the total world production of fruits and 

vegetables respectively (Source FAO 

website- February 2014-15; Horticulture 

Database, India-2015). Out of the total 

production, nearly 76% is consumed in 

fresh form, while wastage, and loss account 

for 20-22 percent. Of which only 2- 

4% is processed in the fruits and vegetables 

processing industries. This is in sharp 

contrast to the extent of processing of 

fruits and vegetables in several other developing 

countries such as Brazil (70%), 

Malaysia (83%), Philippines (78%), and 

Thailand (30%) (Horticulture Database, 

India- 2015). Disposal of these putrescible 

fruits and vegetables processing 

wastes (organic refuse) leads to environmental 

and economic problems (Viswanath 

et al., 1992; Scano et al., 2014). The 

nutrients of food waste may be re-used in 

agriculture by composting or by biotransformation 

of food waste into animal feed 

and/or converted to biofuels (Table 1). 

These wastes contain a high amount cellulose, 

hemi-cellulose, and pectin, which 

provide a suitable substrate for fermentation 

process (Khalid et al., 2011). These 

polymers can be hydrolyzed enzymatically 

by cellulose, β-glucosidase and patience 

to their corresponding soluble carbohydrates 

and subsequently to biofuels 

(Ariunbaatar et al., 2014; Behera and Ray 

2016). 

2.1.Bio-ethanol production 

Bio-ethanol has been proved to be 

a most promising alternate energy source 

with various added advantages (Manzano- 

Agugliaro et al., 2013). The worldwide 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 323



Behera et al. 

Table 1: Current status of biofuel technologies utilizing agricultural and horticultural substrates 

and wastes. (Lynd et al., 2015, modified) 

No Crop 

category 

Example Industry status 

1. Starch-rich Maize, wheat, 

sorghum 

About 50 billion L ethanol in the US based on 

maize 

2. Sugar -rich Sugar cane, sugar 

beet 

About 23 billion L ethanol in Brazil based on sugar 

cane 

3. Oil-rich Rapeseed, soy, 

sunflower, palm 

oil 

About 23 billion L produced worldwide, mostly in 

the EU, US and Brazil 

4. Cellulosic Grass trees, various 

horticultural 

wastes 

Liquid fuel capacity about 175 million L worldwide 

bio-ethanol production is increasing constantly. 

The world bio-ethanol production 

in 2006 was 39 billion liters and has 

grown to 100 billion liters in 2015 (Sarkar 

et al., 2012) and is expected to reach 180 

billion liters in 2020 (Ioelovich, 2015). 

Brazil and USA are the two major ethanol 

producers accounting for 62% of the 

world production (Sarkar et al., 2012). In 

Brazil ethanol is completely produced 

from sugar cane. In USA, the production 

of ethanol relies on corn starch (Ioelovich, 

2015). Conventional indigenous raw 

materials for bio-ethanol production include 

sugarcane, molasses/starch and 

corn-based material, although the amount 

of bio-ethanol produced can hardly meet 

the current global demand (Sarkar et al., 

2012; Saggi and Dey, 2016). Hence, cellulosic 

materials such as agro-residues are 

attractive feedstock for bio-ethanol production. 

The wastes from agro-residues 

being rich in polysaccharides (cellulose, 

hemi-cellulose and lignin) have been subjected 

to solid state fermentation to produce 

bioethanol (Singh et al., 2012). 

2.1.1. Waste pre-treatment 

The biomass from agro-industries 

are the most abundant on the earth. These 

biomass/substrates include corn (maize), 

wheat, oats, rice, potato, and cassava. On 

a dry basis, corn, wheat, sorghum (milo), 

and other grains contain around 60-70% 

(wt/wt) of starch, which are hydrolyzed to 

hexose and offered a good resource in 

fermentation processes (Lin and Tanaka, 

2006; Behera and Ray, 2016). However, 

bio-ethanol from agro-residues could be a 

promising technology that involves four 

processes of pre-treatment, enzymatic hydrolysis, 

fermentation and distillation 

(Gupta and Verma, 2015). These processes 

have several challenges and limitations, 

such as biomass transport and handling, 

and efficient pre-treatment process 

for removing the lining from the lignocellulosic 

agro-residues. Proper pretreatment 

process may increase the concentrations 

of fermentable sugars after 

enzymatic hydrolysis, thereby improving 

the efficiency of the whole process. Conversion 

of cellulose to ethanol requires 

some new pre-treatment, enzymatic and 

fermentation technologies, to make the 

whole process cost effective (Gupta and 

Verma, 2015). However, many agricultural 

and horticultural wastes have low 

lignin content and lesser amounts of cellulose 

with more easily degradable polysaccharides. 

This results in lower enzyme 

pretreatment or physio-chemical costs and 

increased yields of fermentable sugars 

(Sindhu et al., 2016). 

2.1.2. Microflora 

Most agricultural biomass/vegetable 

and fruit wastes (VFWs) 

have been used as a potential substrate for 

the ethanol fermentation by microbial 

processes. VFWs contain mainly starch, 

cellulose, soluble sugars and organic ac- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 324



ids (Table 2). Microbes are well-suited 

natural agents for recycling of organic 

wastes including VFWs (Zupancic and 

Grilc, 2012; Panda et al., 2016). The microorganisms 

such as Bacillus, Pseudomonas, 

Rhizopus and Trichoderma, well 

known for production of hydrolytic enzymes, 

are employed for biodegradation 

of VFWs (organic matter) to reduce the 

biological oxygen demand and chemical 

oxygen demand of the liquid wastes 

(Panda and Ray, 2015). More specifically, 

microorganisms, such as fungi (Mucor 

indicus), bacteria (Zymomonas mobilis), 

and yeasts (Candida utilis, Kluyueromyces 

marxianus) have been used for ethanol 

production from VFWs. However, 

yeast is most common microorganisms 

grown on VFWs as substrates (Stabnikova 

et al., 2005).Among the yeast, Candida 

utilis is selected for cultivation of 

concentrated effluents of the food industry 

after its anaerobic acidogenic treatment 

(Stabnikova et al., 2005). In contrast, 

Saccharomyces cerevisiae is the 

most commonly used yeast in industrial 

ethanol production from red beet (juice 

and bagasse) (Jimenez-Islas et al., 2014). 

Moreover, biotransformation of celluloseto-ethanol 

can be conducted by various 

anaerobic thermophilic bacteria, such as 

Clostridium thermocellum, and some filamentous 

fungi, including Monilia sp., 

Neurosprora sp., Zygosaccharomyces 

rouxii, Aspergillus sp., Paecilomyces sp., 

and Trichoderma viride (Lin and Tanaka, 

2006). Others efficient microbes and genetically 

modified microbes may also enhance 

the enzymatic hydrolysis. The approach 

of using depolymerizing enzymes 

producing co-culture or construction of 

engineered microorganisms are attractive 

for low capital cost and enhanced yield of 

fermentable sugars (Arora et al., 2015). 

2.2. Fermentation process 

The varied raw materials used in 

the fermentation are classified into three 

categories: sugars, starches, and cellulose 

materials (Mohapatra et al. 2017). The 

main sources of sugars (sugar cane, sugar 

Behera et al. 

beet, molasses and fruits) converted into 

ethanol directly. The starches (corn, cassava, 

potatoes, and root crops) are initially 

hydrolyzed to fermentable sugars by 

the action of enzymes (Ray and Naskar, 

2007; Ray et al., 2008). Celluloses (agricultural 

residues, wood, waste sulfite liquor 

from pulp and paper mills) are converted 

into sugars by the action of mineral 

acids. The simple sugar so formed, can 

readily fermented to ethanol by the action 

of microbial enzymes (Lin and Tanaka, 

2006; Monlauet al., 2013). 

2.2.1. Anaerobic digestion and methanogenesis 

Anaerobic digestion is a biochemical 

degradation process that is widely 

used for the treatment and energy recovery 

from many kinds of biomass, especially 

agricultural products and agroindustrial 

wastes (Scano et al., 2014). 

Hydrolysis and acidification are the advantages 

in two-phase in anaerobic digestion 

processes. FVWs are characterized 

by a high percentage of moisture (>80%), 

high organic content (volatile solids>95% 

of total solids), are readily biodegraded 

and are therefore suited to energy recovery 

through anaerobic digestion (Jiang et 

al., 2012). Scano et al. (2014) studied the 

biogas production through an anaerobic 

digestion pilot plant by using VFWs as 

single substrate. The experimental study 

was carried out using most suitable operating 

parameters and optimum organic 

loading rate was reported at 2.5-3.0 kg 

volatile solids/m 3 d with maximum biogas 

production of 0.78 Nm 3 /kg volatile solids 

(CH 4 content of 55%). 

2.3. Factors affecting biogas production 

Various aspects/factors influence 

the biogas production from the feed 

stocks. The C: N ratio, pH, temperature, 

total solid contents, organic loading rates, 

hydraulic retention time, design of digester, 

inoculum quality and volatile fatty acid 

content also regulate and impact biogas 

production. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 325



Behera et al. 

Table 2: Bio-energy production from lignocellulosic material by various microorganisms 

Microorganisms Waste substrate 

Bio-energy 

production 

References 

Bacteria 

Pichia stipites 

BCC15191 

Pichia stipitis DSM 

3651 

Clostridium acetobutylicum 

Sugar cane bagasse 

Wheat straw 

hydrolysates 

Rice straw 

C. beijerinckii Wheat straw 

hydrolysates 

Kluyveromyces Sunflower biomass 

marxianusATCC 

36907 

Yeasts 

Saccharomyces 

cerevisiae 

IBB10B05 

Saccharomyces 

cerevisiae 

Co-culture strains 

Saccharomyces sp. 

W0 + Pichia pastoris 

X-33/pPICZaA- 

INU1 

Candida shehatae + 

Saccharomyces 

cerevisiae 

Bacillus subtilis and 

Pseudomonas aeruginosa 

Trichoderma 

reesei + Saccharomyces 

cerevisiae 

Filamentous fungi 

Fusarium oxysporum 

Trichoderma sp. + 

Penicillium sp. 

+ Aspergillus sp. 

Recombinant (micro) 

organisms 

Saccharomyces 

cerevisiae 424A 

Spent sulfite 

liquor 

Banana & orange 

peels 

Tuber meal of 

Jerusalem artichoke 

Sugarcane bagasse 

Orange peel 

Oil palm EFB 

Glucose, birchwoodxylan, 

corn 

cob or wheat 

bran 

Sugarcane bagasse 

and corncob 

Corn stover 

Feature of the 

employed microorganism 

Ferment both 

glucose and xylose 

Bio-ethanol; 

0.92g/g 

- Bio-ethanol; 

0.41 g/g 

ABE; 10.5 g/L 

Ferment mono-, 

polysaccharides 

Ferment hexose 

and pentose 

Cellulase, & β- 

glucosidase activity 



Improved cellulase 

activity 

Recombinant 

inulinase 


activity 


activity 


activity 

Overexpression 

of 

xylanase 

ABE; 11.44 g/L 

Bio-ethanol; 

27.88 g/L 

Bio-ethanol; 

0.31-0.44g/g 

Bio-ethanol; 

28.6-40.7 g/L 

Bio-ethanol; 

0.319g/g 

Bio-ethanol; 

3.2g/L 

Bio-ethanol; 

82.7-92.2 g/L 

Bio-ethanol; 

4.6 mg/mL 

Bio-ethanol; 

2.68-2.85 g/L 

Over-produced Bio-ethanol; 58 

cellulases * g/L 

Ferment glucose 

and xylose 

Bio-ethanol; 40 

g/L 

Buaban et 

al. (2010) 

Bellido et 

al. (2013) 

Amiri et al. 

(2014) 

Bellido et 

al. (2014) 

Camargo et 

al. (2014) 

Novy et al. 

(2013) 

Singh et al. 

(2014) 

Zhang et al. 

(2010) 

Chandel et 

al. (2013) 

Gomaa, 

(2013) 

Karim et al. 

(2014) 

Anasontziset 

al. (2011) 

El-Bondkly 

and El- 

Gendy, 

(2012) 

Lau and 

Dale, (2009) 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 326



Behera et al. 

Table 2: Continued.. 

(LNH-ST) 

Escherichia coli 

FBR5 

Escherichia coli 

FBR5 

Immobilized (micro) 

organisms 

Zymomonas mobilis 

MTCC 92 

Saccharomyces 

cerevisiae MTCC 

3090 

Wheat straw 

hydrolysate 

Lignocellulosic 

biomass 

Sugarcane molasses 

Molasses 





Entrapped in 

luffa sponge 

disc & Caalginate 

beads 

Immobilized 

sodium-alginate 

Bio-ethanol; 

8.9-17.3 g/L 

Bio-ethanol; 50 

g/L 

Bio-ethanol; 

58.7-59.1 g/L 

Bio-ethanol; 

7.6 g/L 

Saha and 

Cotta, 

(2011) 

Cotta, 

(2012) 

Behera et al. 

(2012) 

Agnihotry et 

al. (2015) 

ABE: Acetone-butanol-ethanol; EFB: Empty fruit bunches; Luffa: Luffa cylindrica L.;Over 

produced cellulase * : exo-β-1,4-glucanase, endo-β-1,4-glucanase and β-1,4-glucosidase 

2.3.1. Carbon to nitrogen (C: N) ratio 

A C/N ration between 22 and 25 

seemed to be better for anaerobic codigestion 

of FVWs with its co-substrates. 

The most significant factor for enhanced 

FVWs digestion performance is the improved 

organic nitrogen content provided 

by the additional wastes (Bouallagui et 

al., 2009). 

2.3.2. PH 

The optimum pH and pH range 

differs with substrate and bio methanation 

technique. The optimum pH values for 

the acidogenesis and methanogenesis 

stages are different. During acidogenesis 

stage, lactic, propanoic, and acetic acids 

are formed and thus, the pH falls. The low 

pH (about 6.4) can be toxic for methaneforming 

bacteria (optimum between 6.6- 

7.0). Most of digested carbohydrate substrates 

are acidic and developing pH of 

6.2 or less and thus becoming toxic 

(Chanakya and Malayil, 2012). A suitable 

amount of lime is added to neutralize the 

acid accumulated in the carbohydrate residues 

of processing digesters. 

2.3.3. Temperature 

The temperature is an important 

parameter for bio methanation. Temperature 

has significant effect on the microbial 

community, process kinetics and stability 

and methane yield (Patil and Deshmukh, 

2015). The high temperature cooking 

(140-180 o C) is very effective for fermentation 

of ethanol from starchy materialin 

industrial-scale. The high temperature 

enhances the efficiency of starch saccharification 

and achieves high levels of ethanol 

under complete sterilization of harmful 

microorganisms (Krishania et al., 

2013). However, to resolve the difficulties 

of high production costs and requirement 

of additional amounts of enzymes 

(amylase), non-cooking and lowtemperature 

cooking fermentation system 

have been recently developed (Ghimire et 

al., 2015). 

2.3.4. Total solid content 

Total solid content influences biogas 

production from fruits and vegetable 

wastes. Abbassi-Guendouz et al. (2012) 

investigated the role of total solid content 

in anaerobic digestion in batch reactors. 

2.3.5.Organic loading rate /Volatile solids 

The organic loading rate determines 

the input of organic matter per unit 

volume of digester capacity per day 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 327



measured of the biological conversion 

capacity of the anaerobic digestion system 

(Patil and Deshmukh, 2015). There is 

an optimum feed rate for a size of digester 

is essential for optimum yield of biogas 

(Patil and Deshmukh, 2015). Shen et al. 

(2013) performed the anaerobic codigestion 

of FVWs and food waste in single-phase 

and two-phase digesters at various 

organic loading rate (3.5-5.0kg. Volatile 

solids. m -3 . d -1 ) to investigate biomethane 

production (0.328-0.544m 3 . kg -1 . 

Volatile solids). 

2.3.6. Hydraulic retention time 

The amount of time the feedstock 

stays in the digester is known as hydraulic 

retention time the retention time must be 

sufficient to carry out the necessary degree 

of biodegradation (Patil and 

Deshmukh, 2015). Bio methanation of 

banana peel and pineapple wastes studied 

at various hydraulic retention times 

showed a higher rate of gas production at 

lower retention time (Velmurugan and 

Ramanujam, 2011). The lowest possible 

hydraulic retention time for banana peel 

was 25 days, resulting maximum rate of 

gas production of 0.76 vol/vol/day with 

36% substrate utilization, while pineapple-processing 

waste digesters was operated 

at 10 days’ hydraulic retention time, 

with a maximum rate of gas production of 

0.93 vol/vol/day and 58% substrate utilization 

(Hosseini and Abdul Wahid, 

2014). To maximize the yield of biogas 

and to improve its quality (high CH 4 content 

and low H 2 S content) different strategies 

can be followed: 1) daily organic 

loading rate must be kept constant 2) use 

of well balanced mix of feeding substrate/wastes 

3) two stages process to separate 

the hydrolysis and acidogenesis 

phases from methanogenesis phase (Scano 

et al., 2014). 

Behera et al. 

2.3.7. Digester design 

Various kinds of digesters are 

used for anaerobic process such as onestage 

or two-stage digester, wet or dry 

digesters, batch or continuous process digesters, 

high rate digesters or digesters 

with combination of different approaches 

for bioenergy (Ganesh et al., 2014). 

However, most commonly used techniques 

of bio-hydrogen production, including 

direct bio-photolysis, indirect biophotolysis, 

photo-fermentation and darkfermentation 

and conventional or modern 

techniques (Mudroom et al., 2011). 

3. Anaerobic digestion process from 

fruit and vegetable wastes 

Normally, biogas is composed of 

45-70% methane, 30-45% carbon dioxide, 

0.5-1.0% hydrogen sulfide, 1-5% water 

vapor, and a small amount of other gases 

(hydrogen, ammonia, nitrogen, etc.). 

However, the composition varies with the 

sources of biodegradable biomass. Biomethane, 

obtained during anaerobic digestion 

by the microbial community of 

biodegradable agricultural and horticultural 

substrates/wastes (Singh et al., 

2012). 

3.1. Ensiling and methane generation 

Ensiling techniques is the process of bio 

methanation using the storing of forage 

crops and various other agricultural 

commodities such as mango peel, orange, 

lemon and lime peels, pineapple and tomato 

processing wastes for a prolong period 

(Kreuger et al., 2011; Panda et al., 

2017). Effects of ensiling process, storage 

of biological/agricultural silage additives 

are attributed to increases in organic acids 

and alcohols contents and showed positive 

effects on methane yield (Herrmann 

et al., 2011). Several processes have been 

developed for high rate bio-methanation. 

The processes include: 1) up-flow anaerobic 

sludge blanket, 2) expanded granular 

sludge bed, 3) fixed film, 4) fluidized bed 

and 5) plug flow. Fang et al. (2011) operated 

the up-flow anaerobic sludge 

blanketreactor using the potato juice for 

biogas production. The methane potential 

was determined at the highest organic 

loading rates of 5.1 g COD. (L-reactor. d) 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 328



with the methane yield of 240 mL-CH 4 /g 

volatile solids-added. 

3.2. Acetone-butanol-ethanol production 

There is also renewed interest in 

reviving the acetone-butanolethanolprocess 

through application of the 

recombinant strains (Lütke-Eversloh and 

Bahl, 2011) and process development and 

using cheaper agricultural 

wastes/substrates (Green, 2011). The bioconversion 

of lignocellulosic substrate/wastes 

to monomeric sugars and its 

consequent fermentation has been suggested 

for economic production of acetone-butanol-ethanol 

(Amiriet al., 2014). 

A variety of bacterial strains, such as 

Clostridium aurantibutyricum, C. beijerinckii 

and C. butyricumparticipatein 

acetone-butanol-ethanol production and 

utilize a variety of substrates including 

pentose, hexose, starch, and xylan but not 

cellulose (Bellido et al., 2014). Further, 

development can be directed by manipulating 

and controlling the fermentation 

conditions by reducing the toxic effect of 

products (repression) on cell physiology 

and promoting one dominant solvent 

product during production of acetonebutanol-ethanol. 

Behera et al. 

3.3. Microbial hydrogen production 

Hydrogen is produced by several 

processes, such as electrolysis of water, 

thermocatalytic reformation of hydrogenrich 

organic compounds, and biological 

processes. Currently, biological production 

of hydrogen (bio-hydrogen) from 

horticultural residues, using microorganisms, 

is an exciting new area of technology 

development (Levin et al., 2004). 

Asian countries possess significant potential 

for producing bio-hydrogen from crop 

residues. Bio-hydrogen production by 

culture of bacteria is highly attractive for 

larger-scale applications (Kumar et al., 

2015). Microbes, including strict anaerobes 

(clostridia, ruminococci and archaea) 

and facultative anaerobes, including 

Escherichia coli and Enterobacter 

aerogenes and aerobes, including Alcaligenes 

eutrophus and Bacillus licheniformis 

when held under anoxic conditions, can 

produce hydrogen from organic substrates/wastes 

(Sivagurunathan et al., 

2016). 

3.4. Biodiesel 

The technology implemented for 

production of liquid biofuels is based on 

transformation of food-grade biomass 

(carbohydrates) into bioethanol and vegetable 

oils into biodiesel fuel. The main 

sources of juices of sugar cane, sugar 

beet, and sweet sorghum, as well as 

starches of corn, wheat, potato, and some 

other agricultural plants (Ioelovich, 

2015). Oil-seed crops are the largest 

sources of exploitable biomass to produce 

liquid fuel, bio-diesel (i.e., fatty esters). 

Bio-diesel offers enhanced safety characteristics 

as compared to diesel fuel, having 

no emission of explosive air/fuel vapors 

(Bhuiya et al.,2014; Kumar and 

Sharma, 2015). Considerable research has 

been progressed on the use of vegetable 

oils as diesel fuel. Vegetable oils such as 

soybean oil, sunflower oil, coconut oil, 

rapeseed oil, Tung oil, and palm oil are 

the best choice (Carlsson, 2009). The 

most common way to produce bio-diesel 

is by transesterification, which refers to a 

catalyzed chemical reaction of vegetable 

oil and an alcohol to yield fatty acid alkyl 

esters (i.e., biodiesel) and glycerol (Shahid 

and Jamal, 2011). Indigenous to central-south 

America, Jatropha was introduced 

to Africa a few centuries ago. It is 

currently widely distributed throughout 

these areas where rural inhabitants generally 

make extensive use of it. Oil from the 

seeds of jatropa is used as a bio-diesel 

substitute (Osseweijeret al., 2015). 

4. Challenges and further prospective 

The production of bioenergy and 

food production is interrelated and is affected 

by global change of atmospheric 

(rising CO 2 and tropospheric 

ozone),climate (temperature and soil 

moisture), and land degradation (saliniza- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 329



tion, desertification, fertility loss) (Osseweijer 

et al., 2015).Recently, global energy 

crisis needs optimum yield of bioenergy 

from advanced fermentation technology 

converting residues/substrates from 

agro-industries into ethanol, enzyme 

technology for hydrolysis of lignocellulosic 

materials, immobilization of microorganisms 

in pilot-scale for production of 

bio-energy. Furthermore, C4-type crops 

possess the features of high photosynthetic 

yield, high rate of CO 2 fixation, produce 

more biomass, and resistance to 

aridity when compared with C3 crops. 

Therefore, C4 type of crops are to be 

more investigated and need to be focused 

for further bio-energy production (Koçar 

and Civaş, 2013). 


To date, bio-fuel has been evolved 

from first to fourth generation and they 

are mainly differed in feedstock and production 

technologies. The agricultural and 

horticultural residues based energy crops 

are critical and needs to be investigated as 

raw materials for bio-fuels for today and 

for the future demand. To attain the 

highest sustainability in bio-fuel production, 

continuous research and development 

on all sustainability-aspects is essential. 

References 

Abbassi-Guendouz, A. Brockmann, 

D.Trably, E. Dumas, C. Delgenès, 

J. P. Steyer, J. P.and Escudié, 

R.(2012). Total solids content drives 

high solid anaerobic digestion via 

mass transfer limitation. Bioresource 


Agnihotry, H. Sharma, S. Kaur, R.and 

Singh, H.(2015). Ethanol fermentation 

from molasses using free and 

immobilized cells of Saccharomyces 

cerevisiae [MTCC3090]–a comparative 

study.ACADEMICIA: An Inter- 

National Multidisciplinary Research 

Journal 5, 215-223. 

Behera et al. 

Aliyu, A. S. Dada, J. O. and Adam, I. 

K. (2015). Current status and future 

prospects of renewable energy in Nigeria. 

Renewable and Sustainable 

Energy Reviews 48, 336-346. 

Amiri, H. Karimi, K. and Zilouei, H. 

(2014).Organosolv pretreatment of 

rice straw for efficient acetone, butanol, 

and ethanol production. 

Bioresource Technology 152, 

450-456. 

Anasontzis, G. E. Zerva, A. Stathopoulou, 

P. M. Haralampidis, K. Diallinas, 

G. Karagouni, A. D. and 

Hatzinikolaou, D. G. (2011). Homologous 

overexpression of xylanase 

in Fusarium oxysporum increases 

ethanol productivity during 

consolidated bioprocessing (CBP) of 

lignocellulosics. Journal of Biotechnology 

152, 16-23. 

Ariunbaatar, J. Panico, A. Esposito, G. 

Pirozzi, F. and Lens, P. N. (2014). 

Pretreatment methods to enhance anaerobic 

digestion of organic solid 

waste. Applied Energy 123, 143-156. 

Arora, R. Behera, S. and Kumar, S. 

(2015). Bioprospecting thermophilic/thermotolerant 

microbes for 

production of lignocellulosic ethanol: 

A future perspective. Renewable and 

Sustainable Energy Reviews 51, 699- 

717. 

Auer, A. VandeBurgt, N.H. Abram, F. 

Barry, G. Fenton, O. Markey, B.K. 

Nolan, S. Richards, K. Bolton, D. 

De Waal, T. and Gordon, S.V. 

2017. Agricultural anaerobic digestion 

power plants in Ireland and 

Germany: policy and practice. 

Journal of the Science of Food 

and Agriculture 97, 719-723. 

Behera, S. and Ray, R.C. (2014). Batch 

ethanol production from cassava 

(ManihotesculentaCrantz) flour by 

Saccharomyces cerevisiae cells immobilized 

in calcium alginate. Annals 

of Microbiology (DOI: 

10.1007/s13213-014-0918-8) 

Behera, S.S. and Ray, R.C. (2016). Solid 

state fermentation for production 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 330



of microbial cellulases: Recent advances 

and improvement strategies. 

International Journal of Biological 

Macromolecule 86, 656–669. 

Behera, S. Mohanty, R. C. and Ray, R. 

C. (2012). Ethanol fermentation of 

sugarcane molasses by ZymomonasmobilisMTCC 

92 immobilized 

in Luffa cylindrica L. sponge discs 

and Ca-alginate matrices. Brazilian 

Journal of Microbiology 43, 1499- 

1507. 

Bellido, C. González-Benito, G. Coca, 

M. Lucas, S. and García-Cubero, 

M. T. (2013). Influence of aeration 

on bioethanol production from ozonized 

wheat straw hydrolysates using 

Pichia stipitis. Bioresource Technology 

133, 51-58. 

Bellido, C. Pinto, M. L. Coca, M. González-Benito, 

G. and García- 

Cubero, M. T. (2014). Acetone– 

butanol–ethanol (ABE) production 

by Clostridium beijerinckii from 

wheat straw hydrolysates: Efficient 

use of penta and hexa carbohydrates. 


198-205. 

Bhuiya, M. M. K. Rasul, M. G. Khan, 

M. M. K. Ashwath, N. Azad, A. 

K. and Hazrat, M. A. (2014). Second 

generation biodiesel: potential 

alternative to-edible oil-derived biodiesel. 

Energy Procedia 61, 1969- 

1972. 

Bouallagui, H. Lahdheb, H. Romdan, 

E. B.Rachdi, B. and Hamdi, M. 

(2009). Improvement of fruit and 

vegetable waste anaerobic digestion 

performance and stability with cosubstrates 

addition. Journal of Environmental 

Management 90, 1844- 

1849. 

Bouallagui, H. Touhami, Y. Cheikh, R. 

B. and Hamdi, M. (2005). Bioreactor 

performance in anaerobic digestion 

of fruit and vegetable 

wastes. Process Biochemistry 40, 

989-995. 

Buaban, B. Inoue, H. Yano, S. Tanapongpipat, 

S. Ruanglek, V. Cham- 

Behera et al. 

preda, V. Pichyangkura, R. Rengpipat, 

S. and Eurwilaichitr, 

L.(2010). Bioethanol production 

from ball milled bagasse using an onsite 

produced fungal enzyme cocktail 

and xylose-fermenting Pichia stipitis. 

Journal of Bioscience and Bioengineering 

110, 18-25. 

Camargo, D. Gomes, S. D. and Sene, L. 

(2014). Ethanol production from sunflower 

meal biomass by simultaneous 

saccharification and fermentation 

(SSF) with Kluyveromyces marxianus 

ATCC 36907. Bioprocess and 

Biosystems Engineering 37, 2235- 

2242. 

Carlsson, A. S. (2009). Plant oils as feedstock 

alternatives to petroleum–A 

short survey of potential oil crop 

platforms. Biochimie 91, 665-670. 

Chanakya, H. N. and Malayil, S. 

(2012). Anaerobic digestion for bioenergy 

from agro-residues and other 

solid wastes—an overview of science, 

technology and sustainability. 

Journal of the Indian Institute of 

Science 92, 111-144. 

Chandel, A.K. Antunes, F.F. Anjos, V. 

Bell, M.J. Rodrigues, L.N. Singh, 

O.V. Rosa, C.A., Pagnocca, F.C. 

and Da Silva, S.S.(2013). Ultrastructural 

mapping of sugarcane bagasse 

after oxalic acid fiber expansion 

(OAFEX) and ethanol production 

by Candida shehatae and Saccharomyces 

cerevisiae. 

Biotechnology for Biofuels 6, 

4-12. 

Cotta, M. A. (2012). Ethanol production 

from lignocellulosic biomass by recombinant 

Escherichia coli strain 

FBR5. Bioengineered 3, 197-202. 

Crutzen, P. J. Mosier, A. R. Smith, K. 

A. and Winiwarter, W. (2016). 

N2O release from agro-biofuel production 

negates global warming reduction 

by replacing fossil fuels. 

In: Paul J. Crutzen: A pioneer on 

atmospheric chemistry and climate 

change in the anthropo- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 331



cene. Springer International Publishing. 

pp. 227-238. 

El-Bondkly, A. M.and El-Gendy, M. M. 

(2012). Cellulase production from 

agricultural residues by recombinant 

fusant strain of a fungal endophyte of 

the marine sponge latrunculiacorticata 

for production of ethanol. Antonie 

Van Leeuwenhoek 101, 331-346. 

Fang, C., Boe, K. and Angelidaki, I. 

(2011). Biogas production from potato-juice, 

a by-product from potatostarch 

processing, in upflow anaerobic 

sludge blanket (UASB) and expanded 

granular sludge bed (EGSB) 

reactors. Bioresource Technology 

102, 5734-5741. 

Ganesh, R. Torrijos, M. Sousbie, P. 

Lugardon, A. Steyer, J. P. and 

Delgenes, J. P. (2014). Single-phase 

and two-phase anaerobic digestion of 

fruit and vegetable waste: comparison 

of start-up, reactor stability and 

process performance. Waste Management 

34, 875-885. 

Ghimire, A. Frunzo, L. Pirozzi, F. Trably, 

E. Escudie, R. Lens, P. N. and 

Esposito, G. (2015). A review on 

dark fermentative biohydrogen production 

from organic biomass: process 

parameters and use of byproducts. 

Applied Energy144, 73-95. 

Gomaa, E. Z. (2013). Bioconversion of 

orange peels for ethanol production 

using Bacillus subtilis and Pseudomonas 

aeruginosa. African Journal 

of Microbiology Research 7, 1266- 

1277. 

Green, E. M. (2011). Fermentative production 

of butanol—the industrial 

perspective. Current Opinion in Biotechnology 

22, 337-343. 

Gupta, A. and Verma, J. P. (2015). Sustainable 

bio-ethanol production from 

agro-residues: a review. Renewable 

and Sustainable Energy Reviews 41, 

550-567. 

Herrmann, C Heiermann, M. and 

Idler, C. (2011). Effects of ensiling, 

silage additives and storage period 

on methane formation of biogas 

Behera et al. 

crops. Bioresource Technology 102, 

5153-5161. 

Hosseini, S. E. and Abdul Wahid, M. 

(2014). The role of renewable and 

sustainable energy in the energy mix 

of Malaysia: a review. International 

Journal of Energy Research 38, 

1769-1792. 

Ioelovich, M. (2015). Recent findings 

and the energetic potential of plant 

biomass as a renewable source of 

biofuels–A review. BioResources 10, 

1879-1914. 

Jiang, Y. Heaven, S. and Banks, C. J. 

(2012). Strategies for stable anaerobic 

digestion of vegetable 

waste. Renewable Energy 44, 206- 

214. 

Jiménez-Islas, D.Páez-Lerma, J. Soto- 

Cruz, N. O. and Gracida, J. (2014). 

Modelling of ethanol production 

from red beet juice by Saccharomyces 

cerevisiae under thermal and acid 

stress conditions. Food Technology 

and Biotechnology 52, 93-99. 

Karim, R. A. Hussain, A. S. and Zain, 

A. M. (2014). Production of bioethanol 

from empty fruit bunches cellulosic 

biomass and Avicel PH-101 

cellulose. Biomass Conversion and 

Biorefinery 4, 333-340. 

Khalid, A. Arshad, M. Anjum, M. 

Mahmood, T. and Dawson, L. 

(2011). The anaerobic digestion of 

solid organic waste. Waste Management 

31, 1737-1744. 

Koçar, G. and Civaş, N. (2013). An 

overview of biofuels from energy 

crops: Current status and future prospects. 

Renewable and Sustainable 


Kreuger, E. Nges, I. A. and Björnsson, 

L. (2011). Ensiling of crops for biogas 

production: effects on methane 

yield and total solids determination. 

Biotechnology for Biofuels 4, 44-50. 

Krishania, M. Kumar, V. Vijay, V. K. 

and Malik, A. (2013). Analysis of 

different techniques used for improvement 

of biomethanation process: 

A review. Fuel 106, 1-9. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 332



Kumar, G. Bakonyi, P. Periyasamy, S. 

Kim, S. H. Nemestóthy, N. and Bélafi-Bakó, 

K. (2015). Lignocellulose 

biohydrogen: practical challenges 

and recent progress. Renewable and 


737. 

Kumar, M. and Sharma, M. P. (2015). 

Assessment of potential of oils for 

biodiesel production. Renewable and 


823. 

Lau, M. W. and Dale, B. E. (2009). Cellulosic 

ethanol production from 

AFEX-treated corn stover using Saccharomyces 

cerevisiae 424A (LNH- 

ST). Proceedings of the National 

Academy of Sciences 106, 1368- 

1373. 

Levin, D. B. Pitt, L. and Love, M. 

(2004). Biohydrogen production: 

prospects and limitations to practical 

application. International Journal of 

Hydrogen Energy 29, 173-185. 

Lin, Y. and Tanaka, S. (2006). Ethanol 

fermentation from biomass resources: 

current state and prospects. 

Applied Microbiology and 


Lütke-Eversloh, T. and Bahl, H. (2011). 

Metabolic engineering of Clostridium 

acetobutylicum: recent advances 

to improve butanol production. 


22, 634-647. 

Lynd, L.R. Sow, M. Chimphango, A.F. 

Cortez, L.A. Cruz, C.H.B. Elmissiry, 

M. Laser, M. Mayaki, I.A. 

Moraes, M.A.Nogueira, L.A. and 

Wolfaardt, G.M.(2015). Bioenergy 

and African transformation. 

Biotechnology for Biofuels 8, 

18-28. 

Manzano-Agugliaro, F. Alcayde, A. 

Montoya, F. G. Zapata-Sierra, A. 

and Gil, C. (2013). Scientific production 

of renewable energies 

worldwide: an overview. Renewable 

and Sustainable Energy Reviews 18, 

134-143. 

Behera et al. 

Mohapatra, S. Mishra, C. Behera, S. S. 

and Thatoi, H. N. (2017). Application 

of pretreatment, fermentation 

and molecular techniques for enhancing 

bioethanol production from grass 

biomass – A review. Renewable and 

Sustainable Energy Reviews 78, 

1007–1032. 

Monlau, F. Barakat, A. Trably, E. Dumas, 

C. Steyer, J. P. and Carrère, 

H. (2013). Lignocellulosic materials 

into biohydrogen and biomethane: 

impact of structural features and pretreatment. 

Critical Reviews in Environmental 


43, 260-322. 

Mudhoo, A. Forster-Carneiro, T. and 

Sánchez, A. (2011). Biohydrogen 

production and bioprocess enhancement: 

a review. Critical Reviews in 


Novy, V. Krahulec, S. Longus, 

K.Klimacek, M. and Nidetzky, B. 

(2013). Co-fermentation of hexose 

and pentose sugars in a spent sulfite 

liquor matrix with genetically modified 

Saccharomyces cerevisiae. 


439-448. 

Osseweijer, P. Watson, H. K. Johnson, 

F. X. and Batistella, M. (2015). Bioenergy 

and food security. Bioenergy 

& Sustainability: Bridging the Gaps 

(eds Souza GM, Victoria R, Joly C, 

Verdade L)(Chapter 4) 72, 779. 

Panda, S. K. and Ray, R. C. (2015). Microbial 

processing for valorization of 

horticultural 

wastes. 

In: Environmental microbial biotechnology. 

Springer International 

Publishing, Heidelberg.pp. 203-221. 

Panda, S.K. Mishra, S.S.Kayitesi, E 

and Ray, R.C. (2016).Microbialprocessing 

of fruit and vegetable 

wastes for production of vital enzymes 

and organic acids: Biotechnology 

and scopes. Environmental 

Research 146, 161- 172. 

Panda, S.K. Ray, R.C.Mshra, S. S. and 

Kayitesi1, K. (2017). Microbial processing 

of fruit and vegetable wastes 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 333



into potential biocommodities: a review. 

Critical Review in Biotechnology, 

http://dx.doi.org/10.1080/07388551.20 

17.1311295 

Patil, V. S.and Deshmukh, H. V. (2015). 

A review on optimization of parameters 

for vegetable waste biomethanation. 


Microbiology and Applied Sciences 

4, 488-493. 

Ray, R.C. and Naskar, S.K. (2008). Bioethanol 

production from sweet potato 

(Ipomoea batatas L.) by enzymatic 

liquefaction and simultaneous saccharification 

and fermentation. Dynamics 

Biotechnology, Process Biochemistry 

and Molecular Biology 2, 

47- 49. 

Ray, R. C. Mohapatra, S. Panda, S. 

and Kar, S. (2008). Solid substrate 

fermentation of cassava fibrous residue 

for production of α- amylase, 

lactic acid and ethanol. Journal of. 

Environmental Biology 29, 111-115. 

Saggi, S. K. and Dey, P. (2016). An 

overview of simultaneous saccharification 

and fermentation of starchy 

and lignocellulosic biomass for bioethanol 

production. Biofuels 4, 1-13. 

Saha, B. C. and Cotta, M. A. (2011). 

Continuous ethanol production from 

wheat straw hydrolysate by recombinant 

ethanologenic Escherichia coli 

strain FBR5. Applied Microbiology 

and Biotechnology 90, 477-487. 

Sarkar, N. Ghosh, S. K Bannerjee, S. 

and Aikat, K. (2012). Bioethanol 

production from agricultural wastes: 

an overview. Renewable Energy 37, 

19-27. 

Scano, E. A. Asquer, C. Pistis, A. Ortu, 

L.Demontis, V. and Cocco, D. 

(2014). Biogas from anaerobic digestion 

of fruit and vegetable wastes: 

Experimental results on pilot-scale 

and preliminary performance evaluation 

of a full-scale power 

plant. Energy Conversion and Management 

77, 22-30. 

Scarlat, N.Martinov, M. and Dallemand, 

J. F. (2010). Assessment of 

Behera et al. 

the availability of agricultural crop 

residues in the European Union: Potential 

and limitations for bioenergy 

use. Waste Management 30, 1889- 

1897. 

Shahid, E. M. and Jamal, Y. (2011). 

Production of biodiesel: a technical 

review. Renewable and Sustainable 


Shen, F. Yuan, H. Pang, Y.Chen, S., 

Zhu, B. Zou, D. and Li, X. (2013). 

Performances of anaerobic codigestion 

of fruit & vegetable waste 

(FVW) and food waste (FW): Singlephase 

vs. two-phase. Bioresource 

Technology144, 80-85. 

Sindhu, R. Binod, P. and Pandey, A. 

(2016). Biological pretreatment of 

lignocellulosic biomass–An overview. 


76-82. 

Singh, A. R. P. Singh, A. K. Charan, A. 

I.and Charan, A. A. (2014). Production 

of bioethanol by banana and orange 

peel using Saccharomyces 

cerevisiae. Trends in Biosciences 7, 

18-21. 

Singh, A. Kuila, A. Adak, S. Bishai, M. 

and Banerjee, R. (2012). Utilization 

of vegetable wastes for bioenergy 

generation. Agricultural Research 

1, 213-222. 

Sivagurunathan, P. Kumar, 

G.Bakonyi, P. Kim, S.H. Kobayashi, 

T. Xu, K.Q. Lakner, G., 

Tóth, G.Nemestóthy, N. and Bélafi- 

Bakó, K.(2016). A critical review on 

issues and overcoming strategies for 

the enhancement of dark fermentative 

hydrogen production in continuous 

systems. International Journal of 

Hydrogen Energy 41, 3820-3836. 

Stabnikova, O. Wang, J. Y. and Ding, 

H. B. (2005). Biotransformation of 

vegetable and fruit processing wastes 

into yeast biomass enriched with selenium. 


747-751. 

Velmurugan, B. and Ramanujam, R. 

A. (2011). Anaerobic digestion of 

vegetable wastes for biogas produc- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 334



tion in a fed-batch reactor. 

International Journal of Emerging 

Science 1, 478-486. 

Viswanath, P. Devi, S. S. and Nand, K. 

(1992). Anaerobic digestion of fruit 

and vegetable processing wastes for 

biogas production. Bioresource 


Zhang, T. Chi, Z. Zhao, C. H. Chi, Z. 

M. and Gong, F. (2010). Bioethanol 

Behera et al. 

production from hydrolysates of inulin 

and the tuber meal of Jerusalem 

artichoke by osp. W0. Bioresource 

Technology 101, 8166-8170. 

Zupančič, G. D. and Grilc, V. (2012). 

Anaerobic treatment and biogas production 

from organic 

waste. Management of Organic 

Waste 3, 1-28. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 335




Mohan Mani 1, *, Manohar Murugan 2 , Ganesh Punamalai 3 and 

Vijayalakshmi Ganesan Singaravelu 4 

1 Mahendra Engineering College(Autonomous), Namakkal, Tamil Nadu, India; 

2 Vivekananda College of Arts and Science (Autonomous), Elayampalayam, Namakkal Dt., 

Tamil Nadu, India; 3 Department of Microbiology, Faculty of Science, Annamalai University, 

Chithamparam, Tamil Nadu, India; 4 Former Professor of Environmental Biotechnology, 

Manonmaniam Sundaranar University, Tirunelveli, Tamil Nadu, India; 

*Correspondence: mohanrtt@gmail.com; Tel: +91-9486069246 

Abstract: Agricultural inputs and farming systems are playing vital role in the consumption 

of fossil fuels and climate change. Organic and non-organic production in terms of energy 

use is essential for understanding the energy inefficiencies of different agro systems and 

their potential for minimizing energy consumption and mitigating environmental impacts 

particularly climate change. Organic agriculture can provide a more energy efficient approach 

which leads to sustainable agricultural productions. Organic agro productions is 

contributing less greenhouse gas emissions and have a superior potential to sequester carbon 

available in biomass than conventional agro production. The energy efficiency of organic 

agriculture in terms of bioenergy production and thereby renewable fuel source is to 

reduce dependency of fossil fuel energy and mitigate environmental pollution caused by 

emissions. The industrialized production of agro products is responsible for a heavy impact 

on the environment and playing a major role in increasing global Green House Gas (GHG) 

emissions in respect to the usage of synthetic fertilizers and pesticides. More than 480 million 

tons of GHG is released to the atmosphere each year by the synthetic fertilizers and 

pesticides factories. Climate change is a serious environmental issue and has broad impacts 

on sustainable development and the future of our economy, health, and agricultural production 

sector. Integrated farm management system is reducing and sequestering GHG emissions. 

Organic farming is essential to ensure the future of our environment and food production 

in a sustainable manner. This article highlights the importance of organic agriculture 

and its role in mitigating climatic change. 

Keywords: Carbon sequestration; climatic change; greenhouse gases; organic farming; sustainable 

production 


Agriculture in India is the promising 

sector for millions of livelihood around 

two thirds of the work force in the country. 

Most of the population is dependent 

on agriculture and allied sectors which 

contribute nearly 24 per cent of gross 

domestic product (GDP) of India (Mahadevan, 

2003). Crop production has 

changed significantly in the past decades 

and agricultural practices highly dependent 

on synthetic pesticides and fertilizers. 

Equipment and machinery for cultivation 

is mainly dependent on consumption of 

fossil fuels. The modern agricultural 

practices and industrialization of agro 

system has created adverse effects on the 

environment and thereby emitting more 

Green House Gases (GHG).The main factor 

in anthropogenic climate change is the 

increase in the concentration of carbon in 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 336



the atmosphere over time. This increased 

concentration has been caused by the 

emission of GHGs as a result of economic 

activities, including energy, industry, 

transport, and land use, many of which 

rely upon fossil fuels (Banuri and Opschoor, 

2007). Climatic change is influencing 

the food caching behavior of a 

wide range of animals to store food for 

future use. (Sutton et al, 2016). Migrating 

animals are vulnerable to climate 

change and it affects the migratory bird 

movement. (Seebacher and Post, 2015). 

Mollusks, and coral reefs are observed 

negative impacts of climatic change 

(Tschakert 2015). Prolonged exposures to 

elevated CO 2 have affected structural proteins 

like actin (Ertl et al., 2016). The 

core objective of the this chapter is to elucidate 

the importance of organic farming 

as the alternative to modern farming system 

and leads to the sustainable agricultural 

system to overcome the recent 

changes in the environment and energy 

crisis faced by the present generation. 

2. Greenhouse gas emissions from agriculture 

Agricultural activities are responsible 

for the emission and sinks for 

greenhouse gases. The industrial production 

of nitrogen based fertilizers, the 

combustion of fossil fuels are the primary 

sources of greenhouse gases released 

from agriculture. Enteric fermentation or 

Mani et al. 

the fermentation that takes place in the 

digestive tract of ruminants observed in 

livestock resulted the emission of 40% of 

CO 2 eq (Figure 1). Photosynthesis is the 

natural process in which the CO 2 is converted 

to organic carbon and it is converted 

to CO 2 by respiration and decomposition. 

Carbon restoration to the soil is happened 

by many agricultural practices include, 

conservation tillage, recycling agricultural 

residues, cover cropping and 

crop rotations.Biological carbon sequestration 

is a process by which the greenhouse 

gases generated during agricultural 

activities have been removed from the 

atmosphere. Agriculture recorded from 

10 to 12 percent of total global human 

caused emissions of greenhouse gases 

during 2005, according to the report by 

Intergovernmental Panel on Climate 

Change (IPCC, 2007). India is recorded 

the second largest emitter of CO 2 from 

total agricultural practices and by the application 

of the synthetic fertilizers followed 

by China (Figure 2 and 3). 

3. Organic agriculture and climatic 

change 

Organic farming is the time immemorial 

practice in India and it was a part of the 

traditional farming systems. The ancient 

history of knowledge like the Vrikshayurveda, 

Agnipurana, Brihat Samhita 

and Arthasasthra is exemplifying the traditional 

Indian agriculture. Traditional 

Figure 1: Average CO 2 emission by sector observed during the year 2000 and 2014 

(Source: FAOSTAT 2014). 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 337



Mani et al. 

Figure 2: Top 10 CO 2 emitters during agricultural production (Source: FAOSTAT 2014). 

Figure 3: Top 10 CO 2 emitters by the application of synthetic fertilizers (Source: FAO- 

STAT 2014). 

farmers who are doing organic agriculture 

were efficiently utilized the land resource 

and appropriately utilizing the SOIL 

(Soul Of Infinite Life). 

Organic agriculture is a holistic production 

management system that avoids 

use of synthetic fertilizers, pesticides and 

genetically modified organisms, minimizes 

the pollution of air, soil and water and 

optimizes the health and productivity of 

interdependent communities of plants, 

animals and people (Codex Alimentarius 

Commission, 2001). 

Organic agriculture is playing a significant 

role in climate change and food security 

to the world’s population.Climate 

change mitigation and food security are 

vital management mediated by organic 

farming practices. The demand for food 

and renewable energy sources are increased 

with the increase of population. 

The impacts of climate change by the usage 

of fossil fuel supported chemical fertilizers, 

herbicides and pesticides will create 

deleterious issue on agricultural production. 

Organic agriculture is proved to be a 

remedy for climate change and the global 

food insecurity and potentially solve the 

issues related to vulnerability, unsustainability 

and social inequity of agricultural 

production. Climate change has its most 

significant impacts on agriculture because 

of its broad geographic dispersion and 

highly dependent on climate and environmental 

factors. Developing countries 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 338



are the least responsible for climate 

change, yet the most at risk from its effects 

(UNESCO, 2010).There is a need of 

a policies and practices to protect the ever 

changing environmental conditions and 

ecosystems that ensure the sustainable 

development. Organic agriculture is effectively 

retaining soil organic matter, 

soil carbon and there by balancing of ecosystem. 

Organic agriculture is the traditional 

practice that mitigates climate 

change, generate evergreen farming systems, 

eradicate poverty and augment the 

level of food security. Organic agriculture 

discharges very minimum levels of 

greenhouse gases (GHG) and efficiently 

sequesters carbon in the soil. Organic agriculture 

makes faming system and people 

dependent on this sector more resilient to 

climate change due to its water use efficiency, 

tolerance to extreme weather conditions 

and lower or no risk of total crop 

failure. Organic agriculture is extensively 

practiced agro-ecological farming system 

that accomplishes the main objectives of 

enabling people to thrive while improving 

our eco-systems and their natural cycling 

process. 

4. Soils for food security and climate - 

4 per 1000 initiative 

Mani et al. 

The 21 st Conference of the Parties 

(COP21) of the United Nations Framework 

Convention on Climate Change 

(UNFCCC) held in Paris in December 

2015 focused to lower the greenhouse gas 

(GHG) by launching a voluntary action 

plan named 4 per 1000 initiative - Soils 

for food security and climate (4o/oo Initiative). 

It initiated to increase soil carbon 

stock annually by 0.04 % through carbon 

sequestration. The annual growth rate 4 

parts per thousand of the soil carbon stock 

would make it possible to stop the present 

increase in atmospheric CO 2 . Food security 

is confirmed by providing proven technologies 

to the farmers in developing 

countries to repair and sustain the soils. 

To feed 9.5 billion in 2050, it is essential 

to keep our soils alive. Figure 4 shows 

various steps to ensure soil organic matter 

by providing various ecosystem services 

like capable of resisting the soil from erosion, 

retaining water, increasing soil fertility 

and proliferating soil biodiversity as 

per the initiation suggested by 4 per 1000. 

It can meet three fold challenges including 

food security, adaptation to food system 

and people to climate change and 

mitigation of anthropogenic emission. 

Soils under organic management will often 

increase the level of soil organic carbon 

more than conventional management 

(FAO 2011 and 2011a). 

Figure 4: Ecosystem services by soil organic matter and its effects and challenges. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 339



Mani et al. 

5. Climatic change – Indian scenario 

India is holding 2.4% world surface 

area, 17.5% of world’s population 

and is the fastest growing major economy 

in the world. It is the fourth largest 

greenhouse gas emitter, accounting for 

5.8 % of global emissions. India’s emissions 

increased by 67.1 % between 1990 

and 2012, and are projected to increase up 

to 85 % by 2030(MoEF, 2014). Increase 

in temperature beyond critical limits by 

increased industrialization resulted to reductions 

in rice, sorghum and maize yield 

(Saravanakumar, 2015). The climatic 

change includes changes in the intensity 

and distribution of rainfall and elevation 

of the level of oceans and a growing increase 

in the frequency and intensity of 

extreme climatic phenomena (Escobar, 

2009). 

India has abundance source of solar, 

wind, hydro power and more potential for 

bioenergy production. Sustainable development 

can be achieved by the integration 

of bioenergy sector with agricultural practices. 

Various regulatory frameworks are 

formed by India for the developmental 

strategies for encouraging bioenergy and 

sustainable agriculture. Biomass potential 

in integration with agriculture for 

power generation and climate change mitigation 

in Indian scenario is essential for 

the current situation (Kothari et.al, 2015). 

The contributions of our country 

will take in to account the imperatives for 

addressing the challenges of poverty eradication, 

food security and nutrition, universal 

access to education and health, 

gender equality and women empowerment, 

water and sanitation, energy, employment, 

sustainable cities and human 

settlement and last but not the least, the 

means of implementation for the following 

enhanced action for achieving among 

others sustainable development goals. 

• Jawaharlal Nehru National Solar Mission 

• National Mission for Enhanced Energy 

Efficiency 

• National Mission on Sustainable Habitat 

• National Water Mission 

• National Mission on Sustainable Agriculture 

• National Mission for Sustaining the 

Himalayan Ecosystem 

• National Mission for Green India 

• National Mission on Strategic 

Knowledge for Climate Change 

• State Action Plan on Climate Change 

• Auto Fuel Vision and Policy 2025 

• Indian Network for Climate Change 

Assessment 

An Expert Committee was constituted 

by the Government of India and 

have recommended a roadmap for improving 

auto fuel quality in India till 2025 

and provided vehicular emission norms 

for various categories of vehicles. As a 

result, a roadmap for rolling out Bharat 

Stage-IV (BS-IV), equivalent of Euro-IV, 

by 2017 and BS-V (Euro-V) auto fuels by 

2020 in the entire country was recommended 

for implementation (MoEF, 

2014). 

6. Sustainable development and climatic 

change 

Sustainable development, defined 

as “development that meets the needs of 

the present without compromising the 

ability of the future generations to meet 

their own needs” (WCED, 1987). It involves 

a harmonious incorporation of a 

viable economy, responsible governance, 

people’s empowerment, social consistency 

and ecological balance. Sustainable 

development means economic development 

with strong correlation with the 

improvement of environmental quality. 

Economic development and maintaining 

environmental quality are essential for 

sustainable development. 

Sustainable development is an effective 

tool for mitigation and adaptation 

from climate change, a major constraint 

faced by recent years. Eriksen et al. 

(2011) have charted out four principles 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 340



Mani et al. 

that can guide adaptation responses in a 

manner that supports sustainability. Sustainable 

adaptation should (1) recognize 

the context of vulnerability, including 

multiple stressors, (2) acknowledge that 

different values and interests affect adaptation 

outcomes, (3) integrate local 

knowledge into adaptation responses and 

(4) consider potential feedbacks between 

local and global processes. 


This chapter highlights that the 

organic farming system is an efficient tool 

for tackling the climatic change mitigation 

and energy efficient pathway for fulfil 

the goal of food and energy for all. 

Both food security and energy efficiency 

are consistently managed by organic agricultural 

practices. It is essential to mitigate 

the climate change and shift from 

modern agricultural practice to organic 

agriculture is an alternative that can conserve 

energy with environment protection 

without compromising the requirements 

of the human beings. It is essential to 

consider that organic agriculture will be 

the solution for the problem faced by the 

present agricultural practices. The global 

solution is necessary at this juncture to 

overcome the impact caused by climatic 

change. Adopting organic agricultural 

practices globally for the sustainability 

will boost the chances of achieving 2 o C 

target and minimize the temperature below 

1.5 o C. 


The authors are glad to express their 

deep sense of gratitude to Mr. R. Gopal 

Sharma, Leading farmer at Kallidaikurichi 

for providing valuable suggestions 

and views when writing this chapter. 

References 

Banuri, T. and Opschoor,H. (2007). 

Climate Change and Sustainable 

Development United Nations Department 

of Economic and Social 

Affairs Working Paper No. 

56.ST/ESA/2007/DWP/56. pp. 1 - 

26. 

Codex Alimentarius Commission. 

(2001). Guidelines for the production, 

processing, labelling and marketing 

of organically produced 

foods. First Revision. Joint Food 

and Agriculture Organization 

(FAO) and World Health Organization 

(WHO) Food Standards Program, 

Rome, Italy pp. 3. 

Eriksen,S., Paulina, A., Chandrasekar, 

B., Rafael, D. M., John I. M., 

Charles, N. K., O’Brien, Olorunfemi, 

F., Park, J., Sygna, L. and 

Ulsrud, K. (2011).When not every 

response to climate change is a 

good one: Identifying principles for 

sustainable adaptation. Climate and 

Development 3, 7–20. 

Ertl, N.G., O’Connor, W.A., Wiegand, 

A.N and Elizur, A. (2016). Molecular 

analysis of the Sydney rock 

oyster (Saccostrea glomerata) CO 2 

stress response. Climate Change 

Responses 3, 6. 1-19. 

Escobar, J. C., Lora, E. S., Venturini, 

O. J., Ya´n˜ez b, E. E., Castillo, 

E. F. and Almazan, O. (2009). 

Biofuels: Environment, technology 

and food security. Renewable and 

Sustainable Energy Reviews 13, 

1275–1287. 

FAO (2011). Energy Smart Food for 

People and Climate. Food and Agriculture 

Organization of the United 

Nations, Rome. pp. 1-78. 

FAO (2011a). Food and Agriculture Organization 

of the United Nations 

Natural Resources Management and 

Environment Department Rome, 

December 2011 - Organic Agriculture 

and Climate Change Mitigation 

- A Report of the Round Table on 

Organic Agriculture and Climate 

Change. pp. 1-82. 

FAO (2014). Asia and the Pacific Food 

and Agriculture Statistical Year 

book. pp. 152-168. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 341



Mani et al. 

Gliessman, S. (2017). Confronting climate 

change with agroecology in 

Mozambique. .Agroecology and 

Sustainable Food Systems 41, 99- 

100. 

Heather, E. D., Robert, H. G., Jonathan, 

C. W., William, K. P., Benjamin, 

Z., Guillermo, G., María, 

A. and William, C. (2014). El 

Niño adversely affected childhood 

stature and lean mass in northern 

Peru. Climate Change Responses 1, 

1-10. 

IEA (International Energy Agency) 

(2016). World Energy Outlook 

OECD/IEA, Paris. pp. 1-13. 

IPCC (2007). Climate Change: Working 

Group II: Impacts, Adaptation and 

Vulnerability. pp. 1-987. 

IPCC (2014). Climate Change: Impacts, 

Adaptation and Vulnerability. Fifth 

Assessment Report, Intergovernmental 

Panel on Climate Change, 

Working Group II. pp. 1-207. 

Julia, I. B. Chelsea, L. P. Andrew, 

J. F. and Ross, A. V. (2015). 

Temperature sensitivity of mineral 

soil carbon decomposition in shrub 

and Graminoid Tundra, West 

Greenland. Climate Change Responses 

3, 1-15. 

Kothari, R. V., Pathak, A. K., Chopra, 

S. A., Tanu, A. and Yadav, B. C. 

(2015). Developments in Bioenergy 

and Sustainable Agriculture Sectors 

for Climate Change Mitigation in 

Indian Context: A State of Art. Climate 

Change and Environmental 

Sustainability 3, 93-103. 

Lima Paris Agenda for Action (LPAA) 

The 4o/ooInitiative (2015). Ministry 

of Agriculture agrifood and forestry 

COP21 Paris. pp. 1-8. 

Mahadevan, R. (2003) Productivity 

growth in Indian Agriculture: The 

role of globalization and economic 

reform. Asia-Pacific Development 

Journal 10, 57-72. 

Ministry of Environment, Forests and 

Climate Change. Government of 

India, (2014). India’s Progress in 

Combating Climate Change. Briefing 

Paper for UNFCCC COP 20 

Lima, Peru. pp. 1-28. 

Nicholls, R. J., Hanson, S., Herweijer, 

C., Patmore, N., Hallegatte, S., 

Jan C. M., Jean, C. and Muir, W. 

R. (2007). Ranking the World’s Cities 

Most Exposed to Coastal Flooding 

Today and in the Future. Executive 

Summary, Environment Working 

Paper no. 1. Paris: Organization 

of Economic Cooperation and Development. 

pp. 1-10. 

Ortiz, R., Jarvis, A., Fox, P., Aggarwal, 

P. K. and Campbell, B. 

M. (2014). Plant genetic engineering, 

climate change and food security. 

CCAFS Working Paper no. 72. 

CGIAR Research Program on Climate 

Change, Agriculture and Food 

Security (CCAFS). Copenhagen, 

Denmark. pp.1-27. 

Osman, E. B. (2009). Climate change 

impacts, adaptation and links to sustainable 

development in Africa. 

Unasylva 231/232, 12-16. 

Ravenscroft, C. H., Raj, W. and Jason 

D. F. (2015). Rapid genetic divergence 

in response to 15 years of 

simulated climate change. Global 

Change Biology 21, 4165–4176. 

Robinson, J. B. and Herbert, D. (2001). 

Integrating climate change and sustainable 

development. International 

Journal of Global Environmental 

Issues, 1, 130 -149. 

Saravanakumar, V. (2015). Impact of 

Climate Change on Yield of Major 

Food Crops in Tamil Nadu, India - 

South Asian Network for Development 

and Environmental Economics 

(SANDEE) Working Paper No. 91- 

15, pp.1-33 

Seebacher, F. and Post, E. (2015). Climate 

change impacts on animal migration. 

Climate Change Responses 

2, 1-2. 

Sudhakar, K., Rajesh, M. and Premalatha, 

M. (2012). A Mathematical 

Model to Assess the Potential of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 342



Algal Bio-fuels in India. Energy 

Sources, Part A, 34, 1114–1120. 

Sutton, A.O., Strickland, D. and D. 

Norris, D.R. (2016). Food storage 

in a changing world: implications of 

climate change for food-caching 

species. Climate Change Responses. 

3, 12. 1-25. 

Trevor, D. (2014). Climate Change and 

the Food Chain. Royal Irish Academy. 

Climate Change Sciences 

Committee. Issue 12. pp. 1-6. 

Tschakert. (2015). 1.5°C or 2°C: a conduit’s 

view from the science-policy 

interface at COP20 in Lima, Peru. 

Climate Change Responses 2, 1-11. 

UNICEF (2009). Child Friendly Schools 

Manual, Climate Change and Environmental 

Education, United Na- 

Mani et al. 

tions Children’s Fund 3 United Nations 

Plaza New York, NY 10017, 

USA pp.1-6. 

UNESCO (2010). Climate Change Initiative 

- Climate Change Education for 

Sustainable Development.7, Place 

De Fontenoy 75253 Paris 07 SP 

France. pp. 1-20. 

WCED. (1987). Our common future: Oxford 

University Press. 

Wyss, W. S. and Yim. (2008). Carbon 

Trading, Climate Change, Environmental 

Sustainability and Saving 

Planet Earth. Paper no. PA21A- 

1292, 2007-2008. 

Yusuf Chisti. (2007). Biodiesel from microalgae. 

Biotechnology Advances 

25,294–306. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 343



Application of Anti-vibrio and Anti-quorum Sensing 

Technology for Sustainable Development in Shrimp 

Aquaculture 

Ramesh Kandasamy 1, *, Amutha Raju 2 and Manohar Murugan 1 

1 Department of Microbiology, Vivekanandha College of Arts and Sciences for Women (Autonomous), 

Elayampalayam - 637 205, Tiruchengode, Namakkal Dt., Tamil Nadu, India; 

2 Department of Biotechnology, Periyar University PG Extension Centre, Dharmapuri, 

Tamil Nadu, India; *Correspondence: ksrames@gmail.com; Tel:+91 9943112125 

Abstract: Farming of various marine shrimp species has been developed and commercialized 

in the last three decades. In the beginning, marine shrimp were cultivated in South-east 

Asia by farmers who raised them as incidental crops in tidal fish ponds. Over the years, 

enormous progress in developing shrimp culture techniques has been made. Shrimp culture 

evolved from an extensive farm using tidal zones to a super-intensive one in the 2000’s using 

ponds more inland. Outbreak of disease such as Vibriosis is considered as one of the 

important constraint and challenge in aquaculture industry of the world.Conventional approaches 

such as the use of antibiotics, disinfectants and other antimicrobial drugs have 

shown limited success in the disease prevention. In this context, there are sound reasons for 

studying the beneficial eco-friendly actions of probiotics and phyto-medicine in boosting the 

shrimp aquaculture production without or minimal adverse impacts on environment. This 

chapter highlights the anti-vibrio and anti-QS nature of probionts and plants against the Vibrio 

pathogen of luminous Vibriosis, a major bacterial disease in shrimp aquaculture. 

Keywords: Anti-vibrio; Penaeus monodon; probiotcs; quorum sensing; Vibrio harveyi 


Aquaculture is one which comprises 

all forms of culturing aquatic animals and 

plants in fresh, brackish and marine environments. 

According to FAO statistics, 

aquaculture is one of the fastest growing 

food producing industries. It has increased 

at an average rate of 8.9% per year since 

1970. In developing countries, in addition 

to agriculture the exploitation of aquatic 

resources can provide additional animal 

protein. Aquaculture can be an excellent 

complement to meet the food requirement 

of growing population. Further, it is estimated 

that half of the world’s seafood 

demand will be met by aquaculture in 

2020. Shrimp aquaculture is widespread 

throughout the tropical world. Currently, 

there are about 68 countries having shrimp 

farm operations. Among them, 22 countries 

reported producing Litopenaeus vannamei 

(white shrimp), while 23 countries 

are producing P. monodon (black tiger 

shrimp). In 2002, the global shrimp farming 

industry produced an estimate of 1.6 

million metric tons of shrimp and production 

is projected to increase at a rate of 12- 

15% per year over the next several years 

(Rosenberry, 2003). World shrimp aquaculture 

production has grown tremendously 

from a production of 200,000 tons in 

1985 to approximately 3.8 million metric 

tons in 2012 (GOAL 2011 shrimp aquaculture 

survey). In 2002, the global shrimp 

farming industry produced an estimate of 

1.6 million metric tons of shrimp and production 

is projected to increase at a rate of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 344


Sensing Technology for Shrimp Aquaculture Sustainability 

12-15% per year over the next several 

years (Rosenberry, 2003). The black tiger 

shrimp, Penaeus monodon and the Pacific 

white shrimp, Litopenaeus vannamei are 

the most widely cultured species everywhere. 

Currently, there are about 68 countries 

having shrimp farm operations. 

Among them, 22 countries reported producing 

Litopenaeus vannamei (white 

shrimp), while 23 countries are producing 

P. monodon (black tiger shrimp). But as 

with many other industries, rapid growth 

of this sector has brought with it the problem 

of environmental pollution. Though 

the shrimp hatchery technology has advanced 

over the decades, thehatchery production 

is frequently affected by viral and 

bacterial disease inflicting huge loss. 

2. Life cycle of penaeid shrimp 

Penaeid shrimp (Penaeus monodon) is 

otherwise called Giant black tiger shrimp 

because of its huge size and tiger-striped 

band appearance in the tail. Penaeid 

shrimp belong to the largest phylum in the 

animal kingdom, the Arthropoda. This 

group of animal is characterized by the 

presence of paired appendages and a protective 

cuticle or exoskeleton that covers 

the whole animal. Presence of cephalothorax 

with stiff rostrum and segmented abdomen 

on the external of the animal is 

unique characters which distinguished 

from other species. Organs like heart, gills 

and digestive tract are to be found in 

Cephalothorax. In the head region, antennules 

and antennae perform sensory functions. 

The mandibles and the two pairs of 

maxillae form the jaw-like structures that 

are involved in food uptake. In the thorax 

region, the maxillipeds are the first three 

pairs of appendages, modified for food 

handling and the remaining five pairs are 

the walking legs (pereiopods). Five pairs 

of swimming legs (pleopods) are found on 

the abdomen. 

Life cycle of a typical penaeid species 

includes several stages in different 

habitats. Mangrove estuaries and brackish 

water provide suitable environment for the 

Kandasamy et al. 

growth of Juveniles.Adults prefer high 

salinity for reproduction; hence they migrate 

into deep shore where the mating 

takes place. About 50,000 - 1,000,000 

eggs are laid by female per spawning 

(Rosenberry, 1997). The eggs hatched out 

and release the first stage of larvae called 

nauplius. After a few days they develop 

into the protozoeae and metamorphose 

into myses. The myses develop in to 

postlarvae (PLs), a stage of megalopas and 

share most of the adult characters. As they 

become larvae, migrate in to offshore 

plankton-rich surface water.PL reaches 

330 mm or above in length and 25-30 g in 

weight within 3-4 months after stocking in 

culture ponds with wide range of salinity 

(Lee and Wickins, 1992; Rosenberry, 

1997). This rapid growth of P. monodon 

made to initiate many culturing industries 

near the coastal area. Ultimately it leads to 

generate crowding and environmental 

degradation that make the rearing animal 

more susceptible to various diseases 

(Johnson, 1989). 

3. Factors influencing shrimp health 

Many factors influence shrimp 

health status such as the age of shrimp, 

management conditions, biotic and abiotic 

stress and pathogens (Figure 1). Infectious 

disease is one of the major limiting factors 

in shrimp farming. Shrimp can be threatened 

by protozoan, fungal, bacterial and 

viral pathogens but viral and bacterial diseases 

cause major troubles in shrimp 

farming (Lightner, 1996). Shrimp farming 

itself has got a significant effect on the 

environment such as loss of mangrove 

ecosystems, nutrient enrichment and eutrophication 

of coastal waters, development 

of antibiotic resistance in marine 

bacteria and accumulation of chemicals 

and toxicity to non-target species 

(Menasveta, 1997). 

4. Bacterial disease, vibriosis 

The reason of low level of aquaculture 

production has been due to a 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 345




Figure 1: Factors influencing the susceptibility of shrimp to pathogens. 

combination of both disease and pollution. 

While disease played the prominent role 

in declining aquaculture stocks, both factors 

caused massive mortality in the leading 

aquaculture countries. Among the 

groups of microorganisms that cause serious 

losses, the best known are bacteria 

because of the devastating economic effects 

they have on affected farms. Bacterial 

diseases mainly due to Vibriosis have 

been reported in Penaeid shrimp culture 

systems implicating several species of 

Vibrios which includes Vibrio harveyi, 

V.splendidus, V. parahaemolyticus, V. alginolyticus, 

V. anguillarum, V. vulnificus, 

V. campbelli, V. fischeri, V. damsella, V. 

pelagicus, V. orientalis, V. ordalii, V. 

mediterrani, V. logei etc. From the all 

above species V. harveyiis the main causative 

agent causing luminous vibrosis to 

larva in hatchery and pond cultivation 

(Won and Park, 2008). Being an important 

etiological agent, it causes mass mortalities 

in penaeid shrimp culture and leads to 

huge economic losses.They continue to 

cause chronic mortalities of up to 30% 

among P. monodon larvae, post larvae and 

adult under stressful conditions (Le 

Groumellec et al., 1996). In production 

unit it causes 100% losses at a time due to 

various virulence factors (Chythanya et 

al., 2002). 

The genus Vibrio is a gram negative 

motile rod shaped gamma proteobacter. 

The disease produced by Vibrio in shrimp 

culture is commonly called as vibriosis. 

The other names of bacterial vibriosis are 

luminescent vibriosis, penaeid bacterial 

septicaemia and red-leg disease (Aguirre- 

Guzmán et al., 2004). With the rapid developments 

in aquaculture particularly in 

Asia and South America, V. harveyi and 

related bacteria have become recognized 

as a serious cause of disease (Austin and 

Zhang, 2006). In many cases, Vibrios are 

opportunists causing disease when the 

host organism is immune-suppressed or 

otherwise physiologically stressed (Peddie 

and Wardle, 2005). Even though all crustaceans 

are suffered by this bacterium, 

most serious struggle was reported in Penaeid 

shrimp culture (Austin and Zhang, 

2006). 

The ill effects of adult animal due to 

vibriosis includes reddening body with red 

to brown gills, swimming lethargy and 

reduced feeding behaviours (Nash et al., 

1992). Similarly infected PL showed empty 

gut and reduced motility and phototaxis. 

Based on the syndrome,they are expressed 

as localised and systemic vibriosis, 

oral and enteric vibriosis, septic 

hepatopancreatitis and appendage and cuticular 

vibriosis (Lightner, 1996). Luminescent 

V. harveyi appears to release exotoxins 

and may cause 80-100% mortality 

in P. monodon hatcheries (Harris, 1995). 

Vibrio species also cause red-leg disease 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 346



characterised by red colouration of the 

pleopods, periopods and gills in juvenile 

to adult shrimps. Shrimps suffering vibriosis 

may display localised lesions of the 

cuticle typical of bacterial shell disease, 

localised infections from puncture 

wounds, loss of limbs, cloudy musculature, 

localised infection of the gut or 

hepatopancreas and general septicemia 

(Lightner, 1993). Virulence factors associated 

with V. harveyi pathogenicity is due 

to the production of bacteriocin-like substance, 

phage induced haemolytic activity, 

extracellular products such as chitinases, 

cysteine protease, haemolysin, luciferase, 

metalloprotease, proteases, phospholipases, 

siderophores and proteinaceous exotoxins. 

High loads of either V. parahaemolyticus 

or V. harveyi induced the rounding 

up and detachment of epithelial cells 

from the basal lamina of the mid gut trunk 

(MGT) and can cause high mortality in 

shrimp by eliminating two layers that protect 

the shrimp from infections: the epithelium 

and the peritrophic membrane. In 

addition, loss of the epithelium may affect 

the regulation of water and ion uptake into 

the body. It is well-known that there are 

significant differences between different 

V. harveyi isolates in terms of pathogenicity 

with some strains being highly virulent 

and others being non-pathogenic (Austin 

and Zhang, 2006). Even if many virulence 

factors have been already reported in V. 

hrarveyi, complete pathogenic mechanisms 

should be elucidated. 

5. Quorum sensing in V. harveyi 

The term ‘Quorum Sensing’ refers 

to the process of bacterial cell-to-cell 

communication. It is a population dependent 

phenomenon first characterized in the 

1970’s in luminescent marine species of 

Vibrio (Nealson et al., 1970). Through 

this mechanism bacteria coordinate gene 

expression in a density-dependent manner. 

It is solely depends on the production, release 

and detection of chemical signal 

molecules called autoinducers (Miller and 


Bassler, 2001). These molecules are constantly 

produced and received at a basal 

level by bacterial cells. With high population 

density, there is a surplus of signalling 

molecules in the environment. These 

signals diffuse back into the cell where 

they facilitate the regulation of gene expression 

(Hastings and Greenberg, 1999). 

The quorum sensing signal molecules are 

found to be involved in the regulation of 

various physiological processes such as 

the bioluminescence, biofilm formation, 

pigment production, toxin production, exopolysaccharide 

production, motility and 

virulence factor production in many 

Gram-negative bacteria including fish and 

shrimp pathogens like V. harveyi (Bruhn 

et al., 2005; Kennedy et al., 2006). 

In each QS system, the autoinducer 

attaches to a gene that is known as 

the transcriptional activator and this attachment 

can lead to alterations in DNA 

and activation of virulence factors (Waters 

and Bassler, 2005). Gram-negative bacteria 

use N-acyl homoserine lactones 

(AHLs) as autoinducers, while Grampositive 

bacteria use oligopeptides to 

communicate (Miller and Bassler, 2001). 

The most extensively investigated intercellular 

signaling molecules are the AHLs. 

Quorum sensing in V. harveyi is regulated 

via a multichannel phosphorylation / 

dephosphorylation cascade. This bacterium 

produces and responds to two autoinducers 

namely (i) harveyi autoinducer 1 

(HAI-1) and (ii) autoinducer 2 (AI-2), 

which regulate the expression of bioluminescence 

and other virulence factors.Miller 

and Bassler (2001) have thoroughly 

studied the mechanism of QS in V. 

harveyi. HAI-1 is an AHL and its biosynthesis 

is catalysed by the luxM enzyme 

(Figure 2). AI-2 is a furanosyl borate 

diester and its biosynthesis is mediated by 

the luxS enzyme. Both HAI-1 and AI-2 

are detected at the cell surface by the 

LuxN and LuxP-LuxQ receptor proteins 

respectively. In the absence of the signals, 

LuxN and LuxQ autophosphorylate and 

transfer phosphate to LuxO via LuxU. The 

phosphorylated luxO is an active repressor 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 347




Figure 2: Mechanism of QS in V. harveyi (Miller and Bassler, 2001). 

for the target genes. In the presence of the 

signal molecules, LuxN and LuxQ interact 

with their autoinducers and change from 

kinases to phosphatases that drain phosphate 

away from LuxO via LuxU. The 

dephosphorylated LuxO is inactive. Subsequently, 

transcription of the target genes 

is activated by LuxR. Recently, a third QS 

component, a Vibrio cholerae-like autoinducer 

CAI-1 was discovered and identified 

as (S)-3-hydroxytridecan-4-one in V. 

Harveyi (Henke and Bassler, 2004). Several 

investigations have been made to find 

the effect of V. harveyi quorum sensing on 

the production of the virulence factors like 

caseinase, gelatinase, lipase, hemolysin 

and phospholipase by determining their 

expression levels both in vitro and invivo 

during infection of gnotobiotic brine 

shrimp (Natrah et al., 2011; Darshanee et 

al., 2011). 

6. Antibiotics vs anti-vibrio probiotics 

Traditionally antibiotics have 

been used in attempts to control bacterial 

disease in aquaculture. When antibiotics 

are used, the resistant strains can multiply 

rapidly because their (sensitive) competitors 

get removed. Moreover, horizontal 

exchange of resistant determinants can 

occur between resistant bacteria and virulent 

pathogens that re-enter the culture 

facilities after the treatment. Thus, antibiotic-resistant 

strains of pathogenic V. harveyi 

can evolve rapidly. In view of the indiscriminate 

use of antibiotics in aquaculture, 

it cannot be surprising that many reports 

have mentioned multiple resistance 

of V. harveyi strains to several antibiotics 

(Table 1).From all this, it might be clear 

that the efficacy of antibiotics to treat luminescent 

vibriosis is very poor. The 

presence of residual antibiotics in commercialized 

aquaculture products constitutes 

another problem with respect to human 

health as this can lead to an alteration 

of the normal human gut microbiota and 

can also generate problems of allergy and 

toxicity (Cabello, 2006). Anbiotics residues 

in animals are also regarded as hazardous 

for exporting quality. In order to 

limit the use of antibiotics, many workers 

have been exploring the use of new bioactive 

compounds for controlling bacterial 

diseases of shrimp particularly that of 

caused by V. harveyi. Various solutions 

have been proposed such as the use of 

probiotics, immunostimulation, vaccination, 

specific pathogen-free (SPF) and 

specific pathogen-resistant (SPR) shrimp. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 348




Table 1: Multiple antibiotic resistances in V. harveyi isolated from aquaculture facilities 

Multiple 

Location Antibiotic(s) 

Reference 

resistance 

India 

Cotrimoxazole, chloramphenicol, 

+ Karunasagar et al.,1994 

erythromycinand streptomycin 

Java Tetracyclin, ampicillin and other β- 

lactams 

Mexico Ampicillin, amikacin, carbenicillin, 

cephalotin and oxytetracycline 

Philippines Oxytetracycline, furazolidone, oxolinic 

acid and chloramphenicol 

Philippines 

Taiwan 

Thailand 

Kanamycin, gentamycin, carbenicillin 

and ampicillin 

Nitrofurantoin, novobiocin and sulphonamide 

+ Teo et al., 2002 

+ Molina et al.,2002 

+ 

Tendencia and De La Peña, 

2001 

+ Nakayama et al., 2006 

+ Liu et al., 1997 

Kanamycin and carbenicillin + Nakayama et al., 2006 

The addition of beneficial bacteria 

to exclude potential pathogens from 

shrimp larviculture has been suggested as 

early as 1991. Beneficial bacteria may enhance 

larval nutrition by supplying essential 

nutrients, improving digestion through 

essential enzymes, mediating direct uptake 

of dissolved organic material and producing 

substances which may inhibit the 

growth of opportunistic pathogens (Browdy, 

1998). The potential negative consequences 

of using antibiotics in aquaculture 

have led to the use of non-pathogenic bacteria 

as probiotic control agents (Vaseeharan 

and Ramasamy, 2003). Probiotics 

are defined as ‘‘live microbial feed supplement 

which when consumed in adequate 

amounts confer a health benefit for 

the host’’. As antibiotics become less 

popular for controlling the aquatic microflora 

in hatcheries the use of probiotics in 

qauaculture has become increasingly popular. 

The probionts commonly used for 

aquaculture are isolated from healthy larvae 

and adults. However, some probionts 

used for humans and terrestrial animals 

have also shown promise in aquaculture 

(Vine et al., 2006). Probiotic use in aquaculture 

is practiced throughout the world 

and the results showed improved management 

of shrimp culture practices 

(Vaseeharan et al., 2004). Probiotics in 

contrast to antibiotics can be a safer ecological 

alternative tool for sustainable aquaculture. 

In order to be considered as 

biological control agents in aquaculture, 

probiotics should be non-pathogenic and 

biochemically and physiologically well 

characterized. It should be genetically stable. 

They should be normal inhabitants of 

the host and able to survive and grow at 

the site of application while exerting their 

beneficial effect. Finally, they should 

maintain their viability and activity 

throughout the product manufacturing and 

storage. 

Balcázar et al., (2006) found that 

V. alginolyticus UTM102, Bacillus subtilis 

UTM126, Roseobacter gallaeciensis 

SLV03 and Pseudomonas aestumarina 

SLV22 are effective probiotics in preventing 

V. parahaemolyticus infection in 

shrimp culture. Feed conversion ratio, 

specific growth rate and final production 

were higher in shrimp receiving a probiotic 

mixture of five Bacillus species (B. 

subtilis, B. licheniformis, B. polymyxa, 

B. laterosporus and B. circulans) than in 

control shrimp which had received no 

probiotic (Ziaei et al., 2006). Bacillus S11 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 349



bacterium provided protection in P. monodon 

shrimp when challenged with V. 

harveyi. After a 100 days feeding trial, 

shrimp in the treatment groups displayed 

100% survival after challenge with V. 

harveyi whereas high mortality was observed 

in the control group (Rengpipat et 

al., 1998). Continuous addition of Bacillus 

sp. as probiotic to tanks containing black 

tiger shrimp for over 160 days decreased 

shrimp mortality caused by luminescentpathogenic 

Vibrio (Moriarty, 1998). 

Cell-free extract of Bacillus subtilis 

BT23 showed greater inhibitory effects 

against the growth of V. harveyi. 

Their probiotic effect was tested by exposing 

P. monodon larvae to B. subtilis BT23 

before a challenge with V. harveyi. The 

results showed 90% decrease in accumulated 

mortality (Vaseeharan and Ramasamy, 

2003). Decamp et al.(2008) reported 

some field data of the use of a. The addition 

of the commercial mixture of Bacillus 

strains in Thai and Brazilian hatchery 

water significantly improved the survival 

of P. monodon and Litopenaeus vannamei 

larvae. There have been reports that Pseudomonas 

species produce bioactive compounds 

with the ability to control vibrios 

such as V. harveyi and V. parahaemolyticus 

and that have no effect on the shrimp 

(Vijayan et al., 2006). The culture supernatant 

or culture filtrate of Pseudomonas 

sp. W3 contain secreted secondary metabolites 

(anti-vibrio) that inhibited the pathogenic 

bacteria responsible for shrimp 

luminous vibriosis disease The active antivibrio 

compound produced by Pseudomonas 

sp. W3 is a small molecule with heat 

stable, pH resistant and mostly tolerant to 

a variety of enzymes such as lysozyme, 

protease, lipase and amylase (Rattanachuay 

et al., 2010). A bioactive compound 

produced by Pseudomonas MCCB 102 

and 103 that inhibited V. harveyi was 

identified as N-methyl-1- 

hydroxyphenazine, a phenazine antibiotic 

(Preetha et al., 2009). Cell free extracts of 

four Bacillus species isolated from Penaeid 

shrimp gut have shown the inhibitory 


activity against luminous strain of V. harveyi 

(Ramesh and Umamaheswari, 2011). 

Possible modes of action that 

have been mentioned in literature for probiotics 

include: (1) Production of inhibitory 

compounds, (2) Competition for nutrients, 

(3) Competition for adhesion sites in 

the gastrointestinal tract, (4) Enhancement 

of the immune response, (5) Production of 

essential nutrients such as vitamins and 

fatty acids and (6) Enzymatic contribution 

to digestion. A protocol for the development 

of probiotics as biocontrol agents in 

aquaculture was proposed by Verschuere 

et al. (2000) and later by Vine et 

al.(2006). It involves the following major 

steps: (1) In vitro screening, (2) Identification, 

(3) Pathogenicity or toxicity test, (4) 

In vivo validation and (5) Cost-benefit 

analysis. In addition to this, bacteria that 

are able to improve the water quality by 

removing toxic inorganic nitrogen or by 

mineralizing organic matter are also considered 

as probiotics. 

7. Plant based anti-vibrio 

Antibacterial compounds from 

natural resources would be the alternative 

to overcome the resistance problem in 

most of the pathogens. Screening of antibacterial 

activity of medicinal plants is 

very important since vast number of medicinal 

plants have been used for centuries 

as remedies for human and animal diseases. 

Much interest is now directed towards 

the vast untapped source of plant-based 

antimicrobials, many of which reduce the 

side effects of synthetic antimicrobials. 

Medicinal plant extracts have been used 

for centuries as remedies for animal diseases 

because they contain components of 

therapeutic value. Various chemotherapeutic 

agents isolated from plants have 

proved effective against drug-resistant 

bacteria. The role of plants in the discovery 

of drugs has increased notably in recent 

years due to a substantial improvement 

in biological screening methods. Unfortunately, 

the chemical nature of phytocompounds 

present in many plants are still 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 350



has to be elucidated. Though numerous 

studies have reported the basic bioactive 

nature of various herbals, only a small 

percentage of that have been examined 

and explored thoroughly for their bioactive 

potential. Many terrestrial and coastal 

plants and marine seaweeds were reported 

to have promising antibacterial (antivibrio) 

activity against aquaculture pathogens 

including V. harveyi. Three mangrove 

species (Avicennia marina,Bruguiers 

cylindrical and Acanthus 

ilicifolius) collected from the coast was 

extracted in methanol and tested for different 

range of biological activities including 

antimicrobial activity against five species 

of fish and shrimp Vibrio pathogens 

(Manilal et al., 2009). 

8. Anti-quorum sensing (quorum 

quenching) 


Strategies to interfere with quorum 

sensing provide new avenues to combat 

bacterial diseases in humans, animals and 

plants. These strategies are termed as 

quorum quenching which targets different 

components of bacterial quorum-sensing 

communication systems and disintegrates 

quorum-sensing-dependent bacterial attacks. 

Due to the increase in antibiotic resistance, 

disruption of QS could lead to 

new pharmaceuticals and it can significantly 

decrease the virulence factor production 

in bacteria without interfering 

their growth. Hence, the disruption of 

quorum sensing (quorum quenching) has 

been suggested as a new anti-infective 

strategy in aquaculture and several techniques 

that could be used to disrupt quorum 

sensing have been investigated (Dong 

and Zhang, 2005). This can be done by 

one of the three methods: degradation of 

the enzyme that produces autoinducers, 

degradation of the autoinducers or degradation 

of the gene that the autoinducer attaches 

to and by autoinducers analog 

(Roche et al., 2004). Acylases and lactonases 

are two kinds of AHL-degrading 

enzymes also known as quorum sensing 

inhibitors (QSI) which have been identified 

(Figure 3). Lactonases block signal 

reception by removing the lactone ring 

from the AHL molecule resulting in just 

the acylhomoserine. Acylases detach the 

nitrogen bond to form a fatty acid chain 

and homoserine lactone. By changing the 

structure of the AHL molecules, lactonases 

and aclyases keep AHLs from attaching 

to the transcriptional activator (Czajkowski 

and Jafra, 2008). Several AHLdegrading 

enzymes identified in various 

bacteria have the potential to be used as 

quorum quenchers. The first application of 

autoinducer quenching for the purpose of 

disease control involves aiiA [autoinducer 

inactivation (aiiA)], a Bacillus gene encoding 

AHL lactonase, which inactivates 

AHL by hydrolyzing its lactone bond 

(Dong et al., 2001). AHL degradation 

protects aquatic animals from infection, 

hence AHL-degrading Bacillus sp. might 

be interesting novel biocontrol strains for 

use in aquaculture. Similarly one of the 

first AHL acylases identified was AiiD 

from Ralstonia eutropha (Lin et al., 

2003). The homologs of aiiA were later 

found in the closely related Bacillus species 

including B. subtilis, B. cereus, B. 

mycoides and many subspecies of B. thuringiensis 

(Dong et al., 2002; Lee et al., 

2002; Pan et al., 2008; Uroz et al., 2003). 

Medicinal plants contain large 

varieties of chemical substances with important 

therapeutic properties that can be 

effectively utilized in the treatment of animal 

diseases like Vibriosis caused by luminous 

Vibrio pathogens. Because of their 

history of medicinal properties, many folk 

medicinal plants have been screened for 

anti-quorum sensing activities. Among all 

the possibilities to inhibit QS activity, the 

use of anti-QS (AHL analogue) compounds 

may be of great interest to avoid 

bacterial infections. Therefore, screening 

of anti-QS compounds or QS inhibitors 

(QSI) from natural resources have been 

used for centuries as remedies for various 

diseases. QSI compounds have been identified 

from a wide range of natural resources 

particularly medicinal plants, edible 

vegetables and fruits, marine sponges 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 351




Figure 3: Mode of action of AHL lactonase and AHL acylase. 

and seaweeds (Kim et al., 2007; Skindersoe 

et al., 2008; Daglia et al., 2010; 

Musthafa et al., 2010). 

The QS interference from novel 

sources may also be an important as the 

antibacterial effects. Anti-QS agents were 

first characterized in the red marine alga 

D. pulchura (Manefield et al., 1999). 

This alga was investigated for its antifouling 

properties and was found to contain 

halogenated furanones which block 

AHLs via competitive inhibition and destabilization 

of LuxR (Manefield et al., 

2002). Delisea furanones have been 

shown to reduce light emission in Vibrio 

species (Givskov et al., 1996), inhibit 

pigment production in C. violaceum 

(Martinelli et al., 2004) and attenuate exoenzyme 

production and swarming motility 

in Serratia liquefaciens (Rasmussen et al., 

2000). The quorum sensing-disrupting 

natural furanone, (5Z)-4-bromo-5- 

(bromomethylene)-3-butyl-2(5H)- 

furanone was found to block autoinducer 

2 quorum-sensing in V. harveyi in a concentration-dependent 

way (Defoirdt et al., 

2006). Persson et al., (2005) reported that 

toluene extracts of garlic contained several 

compounds with varying levels of QSI 

against Gram-negative transcriptional regulators 

Lux R or Lux R. Sergey et al., 

(2011) have evaluated 78 natural products 

from chemical libraries containing compounds 

from marine organisms (Sponges, 

algae, fungi and cyanobacteria) and terrestrial 

plants were screened for the inhibition 

of bacterial QS using a reporter strain 

C. violaceum CV017. Various fruits and 

herbs were shown to possess anti-QS activity 

in a C. violaceum biomonitor strain 

and on the swarming motility of E. coli 

and P. aeruginosa (Vattem et al., 2007). 

The subsequent discovery of compounds 

that inhibit cell-to-cell communication, 

dubbed anti-quorum sensing (anti- 

QS) agents could provide a novel method 

of combating infection. It is possible that 

several terrestrial plants also produce 

quorum signal mimics capable of controlling 

bacterial quorum sensing (Gao et al., 

2003). Even bacteria themselves produce 

QSI substances (Nithya et al., 2010). 

Spices such as garlic, ginger and turmeric 

have been reported for their QSI potential 

(Vattem et al., 2007). Similarly, the essential 

oils of cinnamon (Niu et al., 2006) and 

clove (Khan et al., 2009) are also known 

to possess QSI potentials. Acyl-homo serine 

lactone analogs and other quorum 

sensing inhibitors (QSI) have been investigated 

to determine their ability to prevent 

expression of quorum sensing controlled 

genes. The complex of signalling 

molecules (AHLs) and receptor proteins 

trigger the expression of specific genes 

responsible for bioluminescence in V. 

harveyi (LuxM/N). Hence the disintegration 

of signals with receptor by plant derived 

QSI prevents the bioluminescence 

and other virulence factors in V. harveyi. 

Several compounds have been identified 

that have the ability to interfere with QSmediated 

gene expression through com- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 352



petitive inhibition thus reducing biofilm 

thickness (Hentzer et al., 2002). 


The oceans cover more than 70% of 

the earth’s surface and they are a promising 

source of novel pharmacologically 

active compounds. Although macroorganisms 

of the ocean have proved to be good 

sources of novel bioactive metabolites, 

large-scale productions of these bioactive 

metabolites have been difficult. Microorganisms 

isolated from marine sources 

have been reported to produce antibacterial, 

anti-fungal, anti-viral and antitumor 

substances. Several studies have 

suggested that such marine bacteria can be 

used as bio-control to combat epizootics 

in aquaculture systems.During the past 

two decades, the use of probiotics as an 

alternative to antibiotics has shown to be 

promising in aquaculture. Data about the 

impact of quorum sensing on virulence of 

aquatic pathogens are still lacking. Few 

reports that deal with disruption of quorum 

sensing of aquatic pathogens indicate 

that this new approach has potential in 

fighting infections in aquaculture. The 

furanones reported earlier as quorum 

quenchers (QS inhibitors) are toxic and 

chemically synthetic (non-degradable). 

Consequently the invention of non-toxic, 

broad spectrum QS inhibitors is needed 

for its successful exploitation against bacterial 

infections due to their drug resistance. 

References 

Aguirre-Guzmán, G., Meija Ruíz, H. and 

Ascencio, F. (2004). A review of extracellular 

virulence product of Vibrio sp. 

important in disease of cultivated 

shrimp. Aquaculture Research 35, 1395- 

1404. 

Austin, B. and Zhang, X. H. (2006). Vibrio 

harveyi: a significant pathogen of marine 

vertebrates and invertebrates. Letter 

of Applied Microbiology 43, 119-124. 


Balcàzar, J. L., de Blas, I., Ruiz-Zarzuela, 

I., Cunningham, D., Vendrell, D. and 

Muzquiz, J.L. (2006). The role of probiotics 

in Aquaculture. Veterinary Microbiology 

114, 173-186. 

Browdy, C. L. (1998). Recent developments 

in penaeid broodstock and seed production 

technologies: improving the outlook 

for superior captive stocks. Aquaculture 

164, 3-21. 

Bruhn, J. B., Dalsgaard, I., Nielsen, K. F., 

Buchholtz, C., Larsen, J. L. and 

Gram, L. (2005). Quorum sensing signal 

molecules (acylated homoserine lactones) 

in Gram-negative fish pathogenic 

bacteria. Disease of Aquatic Organism 

65, 43-52. 

Cabello, F. C. (2006). Heavy use of prophylactic 

antibiotics in aquaculture: a growing 

problem for human and animal 

health and for the environment. Environmental 


Chythanya, R., Karunasagar, I. and 

Karunasagar, I. (2002). Inhibition of 

shrimp pathogenic vibrios by a marine 

Pseudomonas I-2 strain. Aquaculture208, 

1-10. 

Czajkowski, R. and Jafra, S. (2008). 

Quenching of acyl-homoserine lactonedependent 

quorum sensing by enzymatic 

disruption of signal molecules. Journal 

of Acta Biochimica Polonica 56, 1-16. 

Daglia, M., Stauder, M., Papetti, A., Signoretto, 

C., Giusto, G.,Canepari, P., 

Pruzzo, C. and Gazzani, G. (2010). 

Isolation of red wine components with 

anti-adhesion and anti-biofilm activity 

against Streptococcus mutans. Food 

Chemistry 119, 1182-1188. 

Darshanee, R. H. A., Bhowmick, P. P., 

Karunasagar, I., Bossier, P. and 

Defoirdt, T. (2011). Quorum sensing 

regulation of virulence gene expression 

in Vibrio harveyiin vitro and in vivo during 

infection of gonotobiotic brine 

shrimp larvae. Environmental Microbiology 

Report 3, 597-602. 

Decamp, O., Moriarty, D. J. W. and Lavens, 

P. (2008). Probiotics for shrimp 

larviculture: review of field data from 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 353



Asia and Latin America. Aquaculture 

Research 39,334-338. 

Defoirdt, T., Crab, R., Wood, T.K., 

Sorgeloos, P. and Verstraete, W. 

(2006). Quorum sensing-disrupting 

brominated furanones protect the gnotobiotic 

brine shrimp Artemia franciscana 

from pathogenic Vibrio harveyi, Vibrio 

campbellii and Vibrio parahaemolyticus 

isolates. Appllied Environmental Microbiology 

72, 6419-6423. 

Dong, Y. H. and Zhang, L. H. (2005). 

Quorum sensing and quorum-quenching 

enzymes. Journal of Microbiology 

43,101-109. 

Dong, Y. H., Gusti, A. R., Zhang, Q., Xu, 

J. L. and Zhang, L. H. (2002). Identification 

of quorum-quenching N- 

acylhomoserine lactonases from Bacillus 

species. Appllied Environmental Microbiology 

68, 1754-1759. 

Dong, Y. H., Wang, L. H., Xu, J. L., 

Zhang, H. B., Zhang, X. F. and Zhang 

L. H. (2001). Quenching quorumsensing-dependent 

bacterial infection by 

an N-acyl homoserine lactonase. Nature 

411, 813-817. 

Gao, M., Teplitski, M., Robinson, J. B. and 

Bauer, W. D. (2003). Production of 

substances by Medicago truncatula that 

affect bacterial quorum sensing. Molecular 

Plant Microbe Interaction 16, 827- 

834. 

Givskov, M., de Nys, R., Manefield, M., 

Gram, L., Maximilien, R., Eberl, L., 

Molin, S.,Steinberg, P. D. and 

Kjellerberg, S. (1996). Eukaryotic interference 

with homoserine lactone mediated 

prokaryotic signalling. Journal of 

Bacteriology 178, 6618-6622. 

Harris, L. (1995). The involvement of toxins 

in the virulence of Vibrio harveyi strains 

pathogenic to the black tiger shrimp Penaeus 

monodon and the use of commercial 

probiotics to reduce shrimp hatchery 

disease outbreaks caused by Vibrio harveyi 

strains. CRC for aquaculture, Scientific 

Conference abstract, Bribie Island, 

Australia. 

Hastings, J. W. and Greenberg, E. P. 

(1999). Quorum sensing: the explanation 


of a curious phenomenon reveals a 

common characteristic of bacteria. Journal 

of Bacteriolology 181, 2667-2668. 

Henke, J. M. and Bassler, B. L. (2004). 

Three parallel quorum-sensing systems 

regulate gene expression in Vibrio harveyi. 

Journal of Bacteriology 186,6902– 

6914. 

Hentzer, M., Riedel, K., Rasmussen, T.B., 

Heydorn, A., Andersen, J.B.,Parsek, 

M. R., Rice, S. A., Eberl, L., Molin, S., 

Hoiby, N., Kjelleberg, S. and Givskov, 

M. (2002). Inhibition of quorum sensing 

in Pseudomonas aeruginosa biofilm 

bacteria by a halogenated furanone 

compound. Microbiology 148, 87-102. 

Johnson, S. K. (1989). Handbook of shrimp 

diseases. Texas A and M University 

Press Sea Grant, Texas. pp. 27. 

Karunasagar, I., Pai, R., Malahti, G. R. 

and Karunasagar, I. (1994). Mass mortality 

of Penaeus monodon larvae due to 

antibiotic-resistant Vibrio harveyi infection. 


Kennedy, B., Venugopal, M. N., 

Karunasagar, I. and Karunasagar, I. 

(2006). Bacterial flora aassociated with 

the giant fresh water prawn Macrobrachium 

rosenbergii in the hatchery system. 

Aquaculture 261, 1156 -1167. 

Khan, M. S. A., Zahin, M., Hasan, S., Husain, 

F.M. and Ahmad, I. (2009). Inhibition 

of quorum sensing regulated bacterial 

functions by plant essential oils 

with special reference to clove oil. Letters 

in Applied Microbiology 49, 354- 

359. 

Kim, J.S., Kim, Y.H., Seo, Y.W. and Park, 

S. (2007). Quorum sensing inhibitors 

from the red alga, Ahnfeltiopsis flabelliformis. 

Biotechnology and Bioprocess 

Engineering 12, 308-311. 

Le Groumellec, M., Goarant, C., Haffner, 

P., Berthe, F. and Costa, R. (1996). 

Syndrome 93 in New Caledonia: Investigation 

of the bacterial hypothesis by 

experimental infections with reference to 

stress induced mortality. SICCPPS book 

of abstracts, SEAFDEC, Iloilo City, 

Phillippines. pp. 46. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 354



Lee, C.F., Han, C. K. and Tsau, J. L. 

(2004). In vitro inhibitory activity of 

Chinese leech extract against Campylobacter 

species. International Journal of 

Food Microbiology 94, 169-174. 

Lee, D. O. C. and Wickins, J. F. (1992). 

Crustacean farming. Blackwell Scientific 

Publications, The University Press, 

Cambridge, pp. 392 

Lee, S. J., Park, S. Y., Lee, J. J., Yum, 

D.Y., Koo, B.T. and Lee, J. K. (2002). 

Genes encoding the N-acyl homoserine 

lactone-degrading enzyme are widespread 

in many subspecies of Bacillus 

thuringiensis. Appllied Environmental 


Lightner, D. V. (1993). Diseases of cultured 

penaeid shrimp. In: Handbook of Mariculture, 

Crustacean Aquaculture. 

McVey, J.P. (ed.). CRC Press Inc., Boca 

Raton, FL. pp. 393-486. 

Lightner, D. V. (1996). A Handbook of pathology 

and diagnostic procedures for 

diseases of Penaeid shrimp. World Aquaculture 

Society, Baton Rouge, Louisiana, 

USA, pp. 304. 

Lin, Y. H., Xu, J. L., Hu, J., Wang, L. H., 

Ong, S. L., Leadbetter, J. R. and 

Zhang, L. H. (2003). Acyl-homoserine 

lactone acylase for Ralstonia strain 

XJ12B represents a novel and potent 

class of quorum-quenching enzymes. 

Molecular Microbiology 47, 849-860 

Liu, P. C., Lee, K. K. and Chen, S. N. 

(1997). Susceptibility of different isolates 

of Vibrio harveyi to antibiotics. 

Microbiololgy91, 175-180. 

Manefield, M., de Nys, R., Kumar, N., 

Read, R., Givskov, M., Steinberg, P. 

and Kjelleberg, S. (1999). Evidence 

that halogenated furanones from Delisea 

pulchra inhibit acylated homoserine lactone 

(AHL)-mediated gene expression 

by displacing the AHL signal from its 

receptor protein. Microbiololgy 145, 

283-291. 

Manefield, M., Rasmussen, T. B., Henzter, 

M., Andersen, J. B., Steinberg, P., 

Kjelleberg, S. and Givskov M. (2002). 

Halogenated furanones inhibit quorum 


sensing through accelerated LuxR turnover. 

Microbiology148, 1119-1127. 

Manilal, A., Sujith, S., Kiran, G. S., Selvin, 

J. and Shakir, C. (2009). Biopotentials 

of mangroves collected from the Southwest 

coast of India. Global Journal of 

Biotechnology and Biochemistry 4, 59- 

65. 

Martinelli, D., Grossmann, G., Séquin, U., 

Brandl, H. and Bachofen, R. 

(2004).Effects of natural and chemically 

synthesized furanones on quorum sensing 

in Chromobacterium violaceum. 

BMC Microbiology 4, 1-10. 

Menasveta, P. (1997). Mangrove destruction 

and shrimp culture systems. World aquaculture 

Society 28, 36-42. 

Miller, M. B. and Bassler, B. L. (2001). 

Quorum sensing in bacteria. Annual Review 

of Microbiology 55, 165-199. 

Molina, A., Alejandra, G., Alberto, A. G., 

Carmen, B., Ana, R. and Gomez, G. 

B. (2002). Plasmid profiling and antibiotic 

resistance of Vibrio strains isolated 

from cultured Penaeid shrimp. FEMS 

Microbiology Letter 213,7-12. 

Moriarty, D. J. W. (1998). Control of luminous 

Vibrio species in penaeid aquaculture 

ponds. Aquaculture 164, 351-358. 

Musthafa, K. S., Ravi, A.V., Annapoorani, 

A., Packiavathy, I. S and Pandian, S. 

K. (2010). Evaluation of anti-quorumsensing 

activity of edible plants and 

fruits through inhibition of the N-acylhomoserine 

lactone system in Chromobacterium 

violaceum and Pseudomonas 

aeruginosa. Chemotherapy 56, 333-339. 

Nakayama, T., Ito, E. and Nomura, N. 

(2006). Comparison of Vibrio harveyi 

strains isolated from shrimp farms and 

from culture collection in terms of toxicity 

and antibiotic resistance. FEMS 

Microbiology Letter 258, 194-199. 

Nash, G., Nithimathachoke, C., Tungmandi, 

C., Arkarjarmorin, A., Prathampipat, 

P. and Ruamthaveesub, P. 

(1992). Vibriosis and its control in pondreared 

Penaeus monodon in Thailand. 

In: Diseases in Asian Aquaculture. Shariff, 

I.M., Subasinghe, R.P. and Arthur, 

J.R. (eds.).International Fish Health Sec- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 355




tion, Asian Fisheries Society, Manila, 

pp. 143-155. 

Natrah, F. M. I.,Kenmegne, M. M., Wiyoto, 

W., Sorgeloos, P., Bossier, P. and 

Defoirdt, T. (2011). Effects of microalgae 

commonly used in aquaculture on 

acyl-homoserine lactone quorum sensing. 


Nealson, K. H., Platt, T. and Hastings, J. 

W. (1970). Cellular control of the synthesis 

and activity of the bacterial luminescent 

system. Journal of Bacteriology 

104, 313-322. 

Nithya, C., Aravindraja, C. and Pandian, 

S. K. (2010).Bacillus pumilus of Palk 

Bay origin inhibits quorum-sensingmediated 

virulence factors in Gramnegative 

bacteria. Research in Microbiology 

161, 293-304. 

Niu, C., Afre, S. and Gilbert, E.S. (2006). 

Subinhibitory concentrations of cinnamaldehyde 

interfere with quorum 

sensing. Letter in Applied Microbiology 

43, 489-494. 

Pan, J., Huang, T., Yao, F., Huang, Z., 

Powell, C. A., Qiu, S. and Guan, X. 

(2008). Expression and characterization 

of aiiA gene from Bacillus subtilis BS-1. 

Microbiological Research 163, 711-716. 

Peddie, S. and Wardle, R. (2005). Crustaceans: 

the impact and control of vibriosis 

in shrimp culture worldwide. Aquaculture 

Health International 2, 4-5. 

Persson, T., Hansen, T. H., Rasmussen, T. 

B., Skindersoe, M. E., Givskov, M. 

and Nielsen, J. (2005). Rational design 

and synthesis of new quorum-sensing 

inhibitors derived from cylated homoserine 

lactones and natural products from 

garlic. Organic and Biomolecular 

Chemistry 3, 253-262. 

Preetha, R., Jose, S., Prathapan, S., Vijayan, 

K.K., Jayaprakash, N. S., Philip, 

R. and Singh, I.S. B. (2009). An inhibitory 

compound produced by Pseudomonas 

with effectiveness on Vibrio 

harveyi. Aquaculture Research 41, 

1452-1461. 

Ramesh, K. and Umamaheswari, S. (2011). 

Inhibitory activity of antibiotics and anti-vibrio 

Probiotics against Vibrio harveyi 

isolated from Penaeid shrimp 

hatcheries. Asian Fishery Science 24, 

186-196. 

Rasmussen, T. B., Manefield, M., Andersen, 

J. B., Eberl, L., Anthoni, 

U.,Christophersen, C., Steinberg, P., 

Kjelleberg, S. and Givskov, M (2000). 

How Delisea pulchra furanones affect 

quorum sensing and swarming motility 

in Serratia liquefaciens MG1. Microbiolology 

146, 3237-3244. 

Rattanachuay, P., Kantachote, D., Tantirungkij, 

M., Nitoda, T. and Kanzaki, 

H. (2010). Inhibition of shrimp pathogenic 

vibrios by extracellular compounds 

from a proteolytic bacterium 

Pseudomonas sp. W3. Electronic Journal 

of Biotechnology 13, 1-11. 

Rengpipat, S., Phianphak, W., Piyatiratitivorakul, 

S. and Menasaveta, P. 

(1998). Effects of a probiotic bacterium 

in black tiger shrimp Penaeus monodon 

survival and growth. Aquaculture 167, 

301-313. 

Roche, D. M., Byers, J. T., Smith, D.S., 

Glansdorp, F.G., Spring, D.R. and 

Welch, M. (2004). Communications 

blackout? Do N-acylhomoserinelactone-degrading 

enzymes have any 

role in quorum sensing?.Micobiolology 

150, 2023-2028. 

Rosenberry, B. (1997). World Shrimp farming. 

In:Shrimp News International. San 

Diego, California, USA, pp. 284. 

Rosenberry, B. (2003). World Shrimp farming. 

In: Shrimp News International.San 

Diego, California, USA. 

Sergey, D., Teplitski, M., Bayer, M., Gunasekera, 

S., Proksch, P. and Paul, V. 

J. (2011). Inhibition of marine biofouling 

by bacterial quorum sensing inhibitors. 

Biofouling 27, 893-905. 

Skindersoe, M. E., Ettinger-Epstein, P., 

Rasmussen, T. B., Bjarnsholt, T., de 

Nys, R. and Givskov, M. (2008). Quorum 

sensing antagonism from marine organisms. 

Marine Biotechnology 10, 56- 

63. 

Tendencia, E. and De La Peña, L.D. 

(2001). Antibiotic resistance of bacteria 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 356



from shrimp ponds. Aquaculture 195, 

193-204. 

Teo, J. W., Tan, T. M. and Chit, L. P. 

(2002). Genetic determinants of tetracycline 

resistance in Vibrio harveyi. Antimicrobial 

Agents and Chemotherapy 46, 

1038-1045. 

Uroz, S., Angelo-Picard, D. C., Carlier, A., 

Elasri, M., Sicot, C., Petit, A., Oger, 

P., Faure, D. and Dessaux, Y. (2003). 

Novel bacteria degrading N- 

acylhomoserine lactones and their use as 

quenchers of quorum-sensing-regulated 

functions of plant-pathogenic bacteria. 


Vaseeharan, B. and Ramasamy, P. (2003). 

Control of pathogenic Vibrio sp. by Bacillus 

subtilis BT23, a possible probiotic 

treatment for black tiger shrimp Penaeus 

monodon. Letters in Appllied Microbiology 

36, 83-87. 

Vaseeharan, B., Lin, J. and Ramasamy, P. 

(2004). Effect of probiotics, antibiotic 

sensitivity, pathogenicity, and plasmid 

profiles of Listonella anguillarum like 

bacteria isolated from Penaeus monodon 

culture systems. Aquaculture 241, 77– 

91. 

Vattem, D. A., Mihalik, K., Crixell, S. H. 

and McClean, R. J. C. (2007). Dietary 

phytochemicals as quorum sensing inhibitors. 

Fitoterapia 78, 302-310. 


Verschuere, L., Rombaut, G., Sorgeloos, P. 

and Verstraete, W. (2000). Probiotic 

bacteria as biological control agents in 

Aquaculture. Microbiology Molecular 

Biology Review 64, 655-671. 

Vijayan, K. K., Singh, I. S. B., Jayaprakash, 

N. S., Alavandi, S.V.,Pai, S. S., 

Preetha, R.,Rajan, J. J. S. and Santiago, 

T. C. (2006). A brackishwater isolate 

of Pseudomonas PS-102, a potential 

antagonistic bacterium against pathogenic 

vibrios in penaeid and non-penaeid 

rearing systems. Aquaculture 251, 192- 

200. 

Vine, N.G., Leukes, W. D. and Kaiser, H. 

(2006). Probiotics in marine larviculture. 

FEMS Microbiology Review 30, 404- 

427. 

Waters, C. M. and Bassler, B. L. (2005). 

Quorum sensing: Cell-to-cell communication 

in bacteria. Annual Review of Cell 

and Development Biology 21, 319-346. 

Won, K. M. and Park, S. (2008). Pathogenicity 

of Vibrio harveyi to cultured 

marine fishes in Korea. Aquaculture 

285, 8-13. 

Ziaei, N. S., Rezaei, M. H., Takami, G. A., 

Lovett, D. L. and Mirvaghefi, A. R. 

(2006). The effect of Bacillus sp. bacteria 

used as probiotics on digestive enzyme 

activity, survival and growth in the 

Indian white shrimp Fenneropenaeus 

indicus. Aquaculture 252, 516-524. 

© 2017 by the authors. Licensee,Editors and AIMST University, Malaysia. 




ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 357



Promiscuous Rhizobia: A Potential Tool to Enhance 

Agricultural Crops Productivity 

Ikbal 1 , Prasad Minakshi 1, *, Basanti Brar 1 , Upendra Pradeep Lambe 1 , Manimegalai 

Jyothi 1 , Koushlesh Ranjan 2 , Deepika 3 , Virendra Sikka 4 and Gaya Prasad 5 

1 Department of Animal Biotechnology, LUVAS, Hisar, Haryana, 125004, India; 

2 Department of Veterinary Physiology and Biochemistry, SVPUAT, Meerut, 250110, Uttar 

Pradesh, India; 3 Department of Botany and Plant physiology, CCSHAU Hisar, 125004, 

Haryana, India; Department of Molecular Biology, Biotechnology & Bioinformatics , 

CCSHAU Hisar, 125004, Haryana, India; 5 SVPUAT, Meerut, 250110, Uttar Pradesh, India; 

*Correspondence: minakshi.abt@gmail.com / minakshi.abt@luvas.edu.in; Tel: +91 

9992923330 

Abstract: Rhizobium-legume symbiosis is a complex and regulated association between 

plant and bacteria. This symbiosis is under the coordinated and tight regulation of several 

species specific (symbiosis related) genes of bacterium and respective host plant. Thus, rhizobia 

require action of several classes of specific genes for the formation of an effective 

symbiosis and dictate the host range. Other nod genes mediate the „decoration‟ of the core 

signaling compounds with various substituents and make them hostspecific. But, there are 

some reports that highlight that the rhizobia can infect non-legume plants. The signaling 

compounds are responsible for the effective symbiosis; however, there are several other 

factors which influence symbiosis and needs to be discovered. Certain modifications in the 

signaling molecules can cause changes in legume host range. Genetic exchange and rearrangement 

among heterologous Rhizobium spp. leading to broadening of host range and 

become promiscuous. Such type of rhizobia having broad host range and could be beneficial 

for the agricultural practices; because, choosing the correct inoculant group for a particular 

legume host is difficult for effective nodulation. Most of the commercially available 

strains are known to have a very narrow host range. Promiscuous Rhizobium strains for 

greater symbiotic association and ability to infect across strict host specificity would be of 

greater importance for the farming community. Farmers can enhance Biological Nitrogen 

Fixation by inoculating such correct rhizobia to their legume crops. The potential of this 

system is appealing because the whole world is seeking to adopt the organic farming. This 

could provide an alternate method to improve the soil fertility and could boost the agricultural 

sustainability. 

Keywords: Biofertilizer; nitrogen fixation; promiscous; Rhizobium 


Biological nitrogen fixation occurs 

mainly through symbiotic association of 

plants with N 2 -fixing microorganisms 

(Shiferaw et al., 2004). BNF supply nitrogen 

more than 2x10 13 g/year to the 

world agriculture system (Falkowski, 

1997). It is one of the most economically 

system ever studied, involves bacteria 

(Rhizobium) and legume plants. The establishment 

of symbiosis involves several 

signaling molecules exchange between 

bacteria and host plant. These molecules 

are regulated by several nod genes and 

work in coordinated manner (Cohn et al., 

1998; Long, 1996). In a successful symbiosis 

rhizobia colonize on roots of host 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 358


Promiscuous Rhizobia and its Potential to Enhance Crops Productivity 

Ikbal et al. 

plants and elicit the nodule formation 

where they colonize and differentiate into 

non-dividing endocellular symbionts. 

These symbionts convert atmospheric dinitrogen 

into NH 3 through the induction 

of the nitrogenase complex (Patriarca et 

al., 2002). Rhizobium species have been 

successfully used worldwide as a bioinoculant 

leading to effective establishment 

of nitrogen fixing symbiosis with 

leguminous crop plants (Miller et al., 

2007). Nitrogen applied as fertilizers usually 

provides high yields to plants. Therefore 

efficient monitoring of biological nitrogen 

fixation and status of chemical fertilizers 

are essential to balance the yield 

of crops and need to minimize environmental 

pollution, especially water and soil 

quality (Jaynes et al., 2001). The role of 

BNF, especially in legumes, is well established 

and documented but Legume- 

Rhizobium symbiosis is not so extensively 

studied the system of nitrogen-fixation. 

Soil containing adequate and diverse 

communities (Figure 1) of rhizobia and 

become less effective at nitrogen fixing. 

The application of sufficiently high 

numbers of improved inoculant strains 

can successfully compete with established 

soil rhizobia and replace them (Figure 2). 

The aim is to increase, the percentage of 

crops that are inoculated in terms of biomass 

yield and extra nitrogen in the soil. 

Improved Rhizobium strains for greater 

symbiotic association and ability to infect 

across strict host specificity would be of 

greater importance for the farming community. 

Farmers can enhance BNF by inoculating 

such correct rhizobia to their 

legume crops. Such promiscuous Rhizobium 

strains with improved efficiency to 

fix nitrogen would acts as a single inoculum 

for all the legumes and may add 

higher amount of nitrogen per unit area. 

2. Classification of biofertilizers as per 

host specificity 

Biofertiliser are the low cost source of 

plant nutrients, eco-friendly and have 

supplementary role with chemical fertilizers. 

The Bio-fertilizers are bacteria, algae 

and fungi and may broadly be classified 

into two categories viz. Nitrogen fixing 

like Rhizobium, Azotobactor, Azospirilum, 

Acetobacter, Blue Green Algae and 

Azola and Phosphorous solubilisers/mobilisers 

like PSM and Mycorrizae 

(Figure 3). Rhizobia and legumes establish 

a mutualistic symbiosis. Host specificity 

is an important characteristic of 

symbiosis, where specific species of rhizobia 

forms nodules on defined legumes 

(Ampomah et al., 2008). Rhizobia currently 

consist of 61 species belonging to 

13 different genera, namely Rhizobium, 

Bradyrhizobium, Mesorhizobium, Azorhizobium, 

Allorhizobium, Sinorhizobium, 

Methylobacterium, Cupriavidus, 

Burkholdera, Devosia, Ochrobactrum, 

Herbaspirullum and Phyllobacterium. 

Some Rhizobia have a narrow host range 

and form nodules with specific legume. 

For example Azorhizobium caulinodans, 

Sinorhizobium saheli and sesbaniae 

biovar of Sinorhizobium terange nodulate 

only Sesbania rostrata (Boivin et al., 

1997) and Rhizobium galegae is the only 

symbiont of Galega offcinalis and Galega 

orientalis (Lindstrom, 1989). In contrast 

some rhizobia are capable to infect a 

spectrum of legumes as they have broad 

host range (various degree of promiscuity). 

For example, Sinorhizobium sp. 

NGR234 and closely related Sinorhizobium 

fredii USDA257 nodulate at least 112 

and 77 legumes from two different tribes, 

respectively (Pueppke and Broughton. 

1999). 

3. Symbiotic infection is a regulated 

pathway 

Symbiosis is a developmental process 

driven by bacteria but ultimately under 

the control of host plant (Murray, 2011). 

The successful establishment of infection 

requires several factors such as nod factors 

and plant exudates (flavonoids). These 

flavonoids activate different kinds of 

nitrogen fixing genes and the interaction 

takes place between bacteria and 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 359



Ikbal et al. 

Figure 1: Diverse communities of microorganisms found in soil. 

Figure 2: Atmospheric nitrogen fixation by microorganisms. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 360



Ikbal et al. 

Figure 3: Classification of biofertilizers based on microorganisms. 

plants. In positive interaction rhizobia 

moves towards the localized sites of plant 

root and followed by formation of infection 

pocket (Barbour et al., 1991; Brewin, 

2004). A bacterial colony established in 

this infection pocket and become a new 

organ called nodule (Fournier et al., 2008 

and Oldroyd and Downie, 2004). 

These pre infection responses ready the 

plant for infection by rhizobia. However 

some β-rhizobia use an alternative pathway 

to initiate symbioses in some legumes, 

where a purine derivative plays a 

key role in triggering nodule formation. 

The universality of the nod factor paradigm 

was recently overturned by some 

bacteria that elicit root and stem nodules 

on a particular group of plants lack the 

canonical nodABC genes required for the 

synthesis of the Nod factor (Giraud et al., 

2007). This indicates that a group of rhizobia 

uses a NF-independent strategy to 

enter into symbiosis. Madsen et al., 

(2010) found that snf1/nfr1/nfr5 triple 

mutants allowed rhizobia to invade 

through an “intercellular” route (crack 

entry) but in this case rhizobial infection 

was not accompanied by the formation of 

infection threads within root hairs. This 

finding suggests that alternative pathway 

may facilitate the entry of the bacterium 

in to the roots of diverse legumes. 

4. Specificity of symbiotic infection 

between legume plant species and 

rhizobia 

Work on molecular basis of host 

specificity began at the end of the last 

century. Experimental evidence suggests 

that the progression of invasive rhizobia 

towards nodule primordial is challenged 

at various steps. The host range is determined 

at early stages of the plantbacterium 

interaction. During initial phases 

of nodulation (bacterial entry), molecular 

signals are given by flavonoids and 

Nod factors (Martinez et al., 1988). In 

this process, NodD proteins are the chief 

interlocutors of molecular traffic in the 

rhizosphere (Perret et al., 2000). NodD 

shows specificity to certain flavonoid secreted 

by plants (Figure 4). Therefore, 

NodD takes part in determining host specificity 

(Miller et al., 2007). Although 

some host plants and rhizobia have the 

ability to enter into symbiosis with more 

than one companion, only certain combination 

of symbionts results in the formation 

of nitrogen fixing nodules. Several 

other studies have also shown that the 

length of the oligosaccheride backbone of 

LCOs determine the host specificity of 

nodulation (Bec-Ferte et al., 1994; Felle 

et al., 1995; Heidstra et al., 1994; Stokkermans 

et al., 1995). These results 

demonstrate that nodC contributes to the 

host specificity of rhizobium. 

The amount of Nod factors released 

by rhizobia also play important role in 

determining the host range. For instance, 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 361



Ikbal et al. 

Figure 4: Symbiotic interaction between legume plant species and rhizobia. 

introduction of strain NGR234 nodD1 

into R. Meliloti increases Nod factor production 

by about two-fold and permits the 

nodulation of V. Unguiculata, a non-host 

(Relic et al., 1994). In S. Meliloti these 

NodD proteins respond to different 

groups of flavonoids, suggesting that 

NodD redundancy allows the bacterium to 

infect multiple hosts secreting a wide 

range of flavonoids (Maillet et al., 1990). 

To demonstrate that nod is a key determinant 

of host specificity, Melicent et al. 

(2006) expressed nod genes from different 

species of rhizobia in a strain of S. 

Meliloti which was lacking endogenous 

nodD activity. They observed that nod 

gene expression was initiated in response 

to distinct set of flavonoid inducers. Furthermore, 

data from several researches 

suggest that nodD controls the response 

of rhizobia to flavonoids in speciesspecific 

manner (Hovath et al., 1987) 

Herman et al. (1989) found that node 

product is the main factor that distinguish 

the host range for symbiosis. Hybrid nodE 

genes, which consist of a 5‟ part Rhizobium 

leguminosarum nodE genes and a 3‟ 

part of the Rhizobium trifolii gene, were 

constructed. From the properties of these 

hybrid genes it was concluded that a central 

region determine different host ranges. 

Louise et al. (2002) have mutagenised 

Rhizobium strains with transposon Tn5 to 

determine if additional negatively-acting 

traits exist that can alleviate cultivarspecific 

nodulation failure. They reported 

two new mutants, proficiently nodulate 

cv. Woogenellup. They suggested that 

simple gene to gene interaction is not sufficient 

for symbiosis but there are at least 

two independent mechanisms which mediated 

the cultivar-specificity. Although 

much information is available on the influence 

of Nod factors on the host range 

yet no strict correlation can be drawn the 

types of LCOs produced by rhizobia and 

host plants. 

5. Molecular factors control the symbiosis 

The nitrogen fixation and nodulation 

by Rhizobium strains is controlled at various 

levels by certain factors (Hooykaas et 

al., 1981). The importance of each individual 

step would depend on the specific 

legume–Rhizobium combination and Nod 

factors (NF) in the rhizosphere (Figure 5). 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 362



Ikbal et al. 

Figure 5: Molecular factors which are involved in symbiosis (Okazaki et al., 2013). 

Bacterial Nod factors functions as a key 

to opens the door of its host (Perret et al., 

2000), and there is a high degree of stringency 

for chemical structure of Nod factor 

that determine whether the host allows 

bacterial invasion to proceed. Nod factor 

elicits significant changes in the expression 

of host gene (D‟Haeze and Holster, 

2002; Oldroyd and Dowine, 2008). Nod 

factors are complex signaling molecules 

secreted from bacteria as a cocktail of β 

1-4-linked N acetyle D-glucosamine 

(GlcNAc) trimers, tetramers or pentamers 

(D‟Haeze and Holster, 2002). The hostrhizobia 

co-evolution involved modifications 

of Nod factor structure such as replacement 

of fucosyl, which made the interaction 

more specific and increased affinity 

between partners (Mario et al., 

2006). 

All the rhizobial species have 

common nod genes (nod A, B, and C), 

which are capable of cross species complementation 

and responsible for the synthesis 

of nod factor backbone. These 

genes confer specificity for nodulation of 

a particular host and are involved in various 

modification of the chitin backbone 

(Gibson et al., 2008). The nodulation 

gene expression is activated when bacteria 

perceive flavonoids that are secreted 

by plant roots (Perret et al., 2000). nodD, 

gene is central to the regulation of nod 

box which activates other nod gene expression 

(Loh and Stacey, 2003). It induces 

the transcription of nodulation 

genes involved in the synthesis of nod 

factors (Capela et al., 2005 and Peck et 

al., 2006). In response of isoflavone signals 

which are produced by plants 

NodVW, positively regulate nod genes, 

thus these are thought to activate transcription 

via a series of phosphorylation 

steps. Mutation in either of these two 

genes results in the complete loss of 

nodulation activity in certain plant hosts. 

The proposed role of nodV as a sensor of 

plants signals adds another point of complexity 

of nod gene regulation. In response 

of plant signal, phosphorylation of 

NodV takes place which subsequently 

activate NodW via the transfer of phos- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 363



Ikbal et al. 

phoryl group to an aspartate residue in its 

receiver domain. The nodW play a key 

role in the regulation of nod gene expression 

and in the ability of B. Japonicum to 

infect the host plant (Sanjuan et al., 

1994). The phosphorylation of NodV and 

NodW is essential not only for nod gene 

expression but also for the nodulation. 

Nod factors permit rhizobia to enter their 

hosts and certain additional factors probably 

act within plant (D‟Haeze et al., 

1998). Thus a number of physiological 

responses to nod factor are observed 

when rhizobium applied to the plant roots 

(Gibson et al., 2008). 

Said et al., (1998) have found that nolO 

factor is the principal host range determinant. 

They have mobilized large 

fragments of the symbiotic plasmid of 

Rhizobium spp. NGR234 into heterologous 

rhizobia. The trans-conjugants nodulate 

V. unguiculata at low frequency and 

extended the host range. They have confirmed 

that the conjugation of nolO into 

Rhizobium fredii extends the host range 

of the recipient to the non-hosts. Nod factors 

are essential to the nodulation and 

their modification contributes to host 

specificity, thus these signaling molecules 

probably one of the several elements 

specifying host range. It should also be 

noted that although nodFE mutants of 

Rhizobium melilotii secrete nod factors in 

which C16 unsaturated fatty acids are replaced 

by vaccenic acid, the mutant still 

form nodules on various Medicago cultivars 

(Ardourel et al., 1994). Several other 

examples contradict the dogma that Nod 

factors determine host specificity. Despite 

the fact that predominant nod factors secreted 

by R. Leguminosarum bv. trifolii 

and R. Leguminosarum bv. viciae are 

identical yet these two bacteria have distinct 

host ranges (Orgambide et al., 1995). 

In contrast, two rhizobia that secrete different 

nod factor may nodulate the same 

plant: R. Tropici and R. etli produce different 

nod factors but both effectively 

nodulate Phaseolus vulgaris (Poupot et 

al., 1995). It would thus seem that nod 

factors in absolute level are not only important 

for induction of different components 

of the nodulation pathway but also 

some bacterial cell surface components 

such as LPS, cyclic-β-glucans, EPS, capsular 

proteins and K antigens recognised 

by plants help to determine host specificity 

(Mathis et al., 2005). These additional 

factors must have role in symbiotic development 

between rhizobia and plant. 

6. Broadening of host range (promiscuity) 

of rhizobia 

The host specificity concept has 

now almost defunct, because many overlapping 

host ranges have observed so the 

concept of host specificity has been challenged. 

A single legume e.g. Acacia, Glycine 

max or Leucaena can be associated 

with genetically dissimilar symbionts. 

Closely related rhizobia infect legumes 

from different tribes and distantly related 

rhizobia infect closely related legumes 

(Quesada et al., 1997). The Rhizobium sp. 

strain NGR234 is good examples of this 

phenomenon. It has broad host range and 

nodulate legume species from 112 genera 

and the non-legume Parasponia (Pueppke 

and Broughton, 1999). Zhu et al., (2002) 

characterized the rhizobia that nodulate 

legume species of the genus Lespedeza by 

analysing whole cell proteins, and crossnodulation 

with selected legume species. 

They have observed that the strains isolated 

from Sesbania spp. and Lespedeza 

spp. represent a cross-nodulating group of 

bacteria. Hernandez-Lucas et al., (1995) 

also found two strains of R. etli and three 

stains of R. tropici and tested on 43 legume 

species. Out of these 22 of the tested 

legume species were nodulated by three 

or more of these strains. These strains 

have broad host range and nodulate 

woody species also such as Albizia 

lebbeck. 

Setiyo Hadiwaluyo (2011) characterized 

forty one cross inoculating rhizobial 

isolates from Java and Sumatra and 

these isolates were used to inoculate soybean 

and mungbean plants. He found 19 

isolates from Java and 15 isolates from 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 364



Ikbal et al. 

Sumatra were promiscuous. Beatrix et 

al., (1987) have studied nodD gene from 

wide host range rhizobium strain 

MPIK3030 to verify the nodD function, 

as well as the host-range extension ability 

of R. Meliloti. The 2.9-kb nodDl region 

was mobilized into R. Meliloti and transconjugants 

were tested for their nodulation 

phenotype on siratro and alfalfa. The 

double mutant R. meliloti nodDI12 PP659 

carrying the MPIK3030 nodDI-region 

(pBH264), showed a clear restoration of 

nodulation on alfalfa and simultaneously 

extended the host range of the R. meliloti 

trans-conjugants to siratro. Similarly 

Transfer of nodD1 of NGR234 into R. 

meliloti results in host range extension to 

M. atropurpureum and Vigna unguiculata, 

whereas R. meliloti nodD1is incapable 

of restoring the ability of an NGR234 

nodD1 mutant to nodulate M. atropurpureum 

(Relic et al., 1994). Conjugation 

of NGR234 nodD1 into R. leguminosarum 

bv. Trifolii extends its hostrange 

to the non-legume Parasponia andersonii 

(NGR234 host) whereas nodD1 

mutant of R. trifolii did not regain the 

ability to nodulate Trifolium repens when 

it was complimented with nodD1 of R. 

meliloti (Spaink et al., 1987). 

Promiscuity is not only the characteristics 

of the rhizobia, but some legumes also 

harbor diverse rhizobia (Perret et al., 

2000). Several plants such as Phaseoleae 

are nodulated by R. leguminosarum bv. 

Phaseoli as well as Bradyrhizobium and 

Sinorhizobium species (Gaultieri and Bisseling, 

2000). Arya K. Bal (1982) studied 

how a legume interacts with Rhizobium 

species of two different cross inoculation 

groups. They have compared physiology 

and morphology of root nodules induced 

by two Rhizobium species of different 

cross inoculation groups. They have 

found that Rhizobium sp. 127E15 promiscuously 

induce effective root nodules on 

pole bean. Similarly, Shantharam and Peter 

(1982) have shown that R. phaseoli 

127K14 is capable of forming effective 

nodules on different legume. They have 

showed that R. phaseoli 127K14 nodulates 

a host of different cross-inoculation 

group. Promiscuity is probably ancestral 

to restricted host range. In this support 

hypothesis comes from the observation 

that NGR234 and USDA257, both nodulate 

Parasponia andersonii (van Rhijn et 

al., 1996). There are some other reports of 

promiscuous strains that have broad host 

range and nodulate soybean as well as 

many other legumes, including cowpea, 

pigeon pea and mungbean (Scholla and 

Elkan 1984; Stowers and Eaglesham 

1984; Chamber and Iruthayathas 1988). 

In view of these reports it was concluded 

that promiscuity is widely dispersed in 

nature and not only associated with a particular 

bacterial or plant taxonomic group. 

In further studies, nodulation capacities of 

large collections of rhizobia have been 

evaluated by inoculation of numerous 

legumes. 

7. Hydrolytic-cell wall degrading enzymes 

in rhizobial infection 

In the development of the Rhizobiumlegume 

symbiosis localized erosion of 

cellulosic plant wall is the central event 

through which the bacterial symbiont enter 

into host plant and establish a nitrogen 

fixing, intracellular endosymbiotic state. 

Previous studies found that rhizobia produce 

hydrolytic enzymes capable of degrading 

the cell wall polymers, but little is 

known about their molecular mechanism 

(Angle 1986). In considering the process 

of active penetration of plant cell wall by 

Rhizobium sp., McCoy (1932) was the 

first to investigate the involvement of hydrolytic 

enzymes. Ljunggren and 

Fahraeus (1961) gave the “polyglacturonase 

hypothesis” which describes the 

involvement of pectolytic enzymes at the 

site of nodule formation. The hypothesis 

in essence proposes a physical penetration 

of the root hair cell wall. Callaham and 

Torrey in 1981 gave the strongest evidence 

for the involvement of wall hydrolysis 

by R. leguminosarum bv. trifolii in 

white clover infection process. Baker et 

al. (1989) also found that many cells of R. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 365



Ikbal et al. 

leguminosarum bv. trifolii attached to the 

root surface of white clover and produce 

pit erosions in epidermal wall that follow 

the penetration of the bacterium, suggesting 

that wall-degrading enzymes are involved 

in symbiosis. Vashishat et al., 

(1985b) observed that R. trifolii strains 

were capable of producing hydrolytic enzymes 

like pectinase, hemicellulase and 

cellulose and these enzymes play important 

role in symbiosis. Considering 

these evidence Angle in 1986 tried to determine 

whether differences exists between 

fast and slow growing soyabean 

rhizobia to produce pectinase and proteolytic 

enzymes. It was proposed that wide 

spread production of proteolytic enzymes 

indicates indirect evidences for their involvement 

in the invasion of host. Al- 

Mallah et al. (1990) pre-treated clover 

roots with an enzyme mixture of 1% 

(w/v) cellulase and 0.1% pectolase before 

inoculating clover with R. trifolii. Increase 

in nodulation indicated the role of 

cellulase and pectinase in nodulation process 

of clover. 

In 1992, Pedro et al., verify the production 

of R. leguminosarum bv. trifolii 

enzymes that deteriorate polygalacturonate 

and carboxymethyl cellulose 

(CMC) as model substrates of plant cell 

wall polymers. Similarly Mateos et al. 

(1992) reported the production of enzymes 

from R. leguminosarum bv. trifolii 

that degrade carboxymethyl cellulose and 

polypectate substrates. Their studies 

shows that R. leguminosarum bv. trifolii 

produces multiple enzymes that cleave 

glycosidic bonds in the plant cell wall. 

Mateos et al., (2001) also found that cellulase 

(Cel2) enzyme is important for 

symbiotic development because rhizobial 

symbionts require its activity to breach 

the host barrier to establish nitrogen fixing 

association with legumes. Robledo et 

al. (2008) have purified cell bound cellulase 

(Cel2) isozymes and analysed its 

symbiotic function by reverse genetics 

and plant microscopy approaches. These 

results provide compelling evidence that 

this enzyme could erode the tip of root 

hair wall of the host and making a localized 

hole of sufficient size to allow rhizobial 

cell penetration. This leads to develop 

more nodules for successful nitrogenfixation. 

Hussain et al. (1995) studied the involvement 

of hydrolytic enzymes in the 

nodulation of berseem (Trifolium alexandrinum). 

They selected a single mutant 

hrt20m7 in wild type strain hrt20 of R. 

trifolii by screening for reduction in activity 

of degradative enzymes, the relative 

activities shown by the mutant for pectinases 

and cellulase were 33 and 4 percent, 

respectively of wild type strain. It 

was observed that mutants unlike its parent 

failed to nodulate clover seedlings. 

Aggarwal et al. (2000) found rhizobia behaving 

as super nodulating rhizobia. They 

suggest cellulases over pectinases in the 

process of symbiotic infection of berseem 

by R. leguminosarum bv. trifolii. They 

derived rhizobia mutants showing better 

growth on CMC and /or pectin i.e. behaving 

as super-nodulating rhizobia. Emtiazi 

et al. (2007) also studied the cellulase activities 

in nitrogen fixing Paenibacillus 

isolated from nitrogen free media. The 

cellulase positive Paenibacillus were selected 

by reduction of congored color on 

CMC medium. They have observed that 

nitrogen fixing strains with cellulase activities 

grow well on nitrogen free media 

with sucrose or manitol as the only 

sources of carbon. They have concluded 

that most plant associated microorganism 

might have cellulase activities for adoption 

or establishment of a plant microbe 

interaction. Egamberdieva et al. (2010) 

determined the bacterial cellulase activity 

on media containing the substrate carboxy-methylcellulose. 

They have found 

that cellulase producing bacteria were 

significantly increase nodule numbers and 

nitrogen content of the plants. Fouts et al. 

(2008) studied the complete genome sequence 

of the nitrogen fixing broad host 

range endophyte Klebsiella pneumonia 

342. They have found the gene related to 

carbohydrates, including pectins and cellulosic 

compound degradation are essen- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 366



Ikbal et al. 

tial for Kp342 to form endophytic associations. 

So, it was concluded that hydrolytic 

enzyme activity in the form of cellulase 

and pectinase is an essential property of 

Rhizobium for infection of white clover 

during symbiosis. 

8. Improvement in symbiotic efficiency 

of rhizobia 

The rhizobium-legume symbiosis 

accounts for a significant proportion of 

nitrogen available to leguminous plants. 

Thus there is a need to improve rhizobia 

to increase their symbiotic efficiency and 

host range. The traditional method for obtaining 

Rhizobium strains with improved 

properties has been the selection of naturally 

occurring field isolates that best exhibit 

the trait desired (Figure 6). An alternative 

approach is to construct improved 

Rhizobium strains by genetic transfer of 

symbiotically favorable determinants. 

Genomic rearrangements have been reported 

to occur frequently in R. leguminosarum 

phaseoli (Flores et al., 1988, 

Garg et al., 1999). 

Better nitrogen fixation may be 

brought by manipulating both rhizobia 

and plant host by eventually creating an 

artificial rhizosphere. Schlaman et al., 

(1998) observed nodulation and the levels 

of nitrogen fixation can be significantly 

higher when plants are infected with rhizobia 

containing the hybrid gene 

nodD604, which activates the transcription 

of nod genes independent from flavonoids. 

For introduction of foreign DNA 

into bacterial species electroporation is a 

novel approach (Chassy et al., 1988). 

Garg et al. (1999) successfully carried out 

electro-transformation of R. leguminosarum 

with 15.1kb plasmid, pMP154 

(Cmr), containing a nodABC-lacZ fusion 

by electroporation. Chitchanok et al. 

(2011) derived mutants from wild type 

Rhizobium sp. 6-1C1 through 0.8 and 1.0 

kGy gamma radiation. They have observed 

that Rhizobium meliloti nodH gene 

mutants result in a change of host range. 

They infect vetch with this mutated strain 

but fail to nodulate their normal host, alfalfa. 

Figure 6: Improvement and large scale production of biofertilizes. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 367



Ikbal et al. 

The nodQ on the other hand, are 

able to infect both alfalfa and vetch. 

Mostly mutations results in the alteration 

or extension of the host range (Faucher et 

al., 1989 and Horvath et al., 1986). In R. 

leguminosarum bv. viciae and bv. trifolii, 

the nod product is the main factor that 

distinguishes the host range for nodulation. 

In contrast to wild type, R. leguminosarum 

bv. Trifolii nodEF mutants nodulate 

white and red clover poorly but have 

acquired the ability to infect peas. When 

these nodEF mutants harbour the node 

gene of R. leguminosarum bv. Viciae, 

they have an extended host range to vicia 

and lathyrus species (Spaink et al., 1989). 

Transfer of nodD1 gene of strain 

NGR234 to restricted host range rhizobia 

extend the nodulation capacity of the recipients 

to new hosts, including the nonlegume 

Parasponia andersonii (Horvath 

et al., 1987). Nonetheless, nodD gene represents 

a molecular interface between the 

bacterium and the plant. Plasmid transfer 

may increase nodulation or nitrogen fixation 

in R. Leguminosarum bv. viciae 

strains (DeJonj et al., 1982), and there is 

one report of a plasmid loss that improves 

symbiotic properties in Rhizobium loti 

(Pankhurst et al., 1986). In R. meliloti, a 

non-symbiotic plasmid enhances nodulation 

of the strains harboring it (Urban, J. 

1988). Esperanza and Monica (1990) genetically 

modified R. leguminosarum bv. 

phaseoli CFN42, through transfer of a 

225kb plasmid from typeII strain 

CFN299. They have observed that more 

nodules were obtained with the transconjugants 

on P. vulgaris. These strains also 

have a diminished competitive ability. 

Philippe et al., (1996) introduced an IncP 

plasmid, pGMI149, carrying the main R. 

meliloti nodulation region into R. tropici. 

The R. tropici (pGMI149) transconjugants 

poorly nodulate M. sativa. When a second 

plasmid, of the IncQ group (compatible 

with pGMI149), carrying the nodL gene 

(pGMI1962) was introduced into R. 

tropici (pGMI149), a better nodulation 

was observed. They have also prepared 

NFs from R. tropici, R. meliloti (with 

pMH682 for increasing NF production), 

and the R. tropici (pGMI149) 

(pGMI1962) hybrid strain and then tested 

the ability of these NFs to form nodules 

on alfalfa. NFs produced by the R. tropici 

hybrid strain were able to induce nodule 

formation. The presence of R. meliloti 

nodulation genes therefore enables R. 

tropici to produce new NFs that can induce 

nodule formation. Their results show 

that allelic variation of the common nod- 

ABC genes is a genetic mechanism that 

plays an important role in signaling variation 

and in the control of host range. They 

have also found that mutations in the regulatory 

genes nodD1 and nodD3 did not 

result in a detectable decrease in nodulation. 

Falguni et al., (2009) amplified 2.4 kb 

fegA gene (encoding ferrichrome receptor) 

along with its native promoter from 

Bradyrhizobium japonicum 61A152 and 

cloned in a broad host range plasmid vector 

pUCPM18. The plasmid construct pFJ 

was transferred by conjugation into Rhizobium 

sp. ST1 to give trans-conjugant 

ST1pFJ12. Inoculation of pigeon pea 

seedlings with trans-conjugant ST1pFJ12 

led to a marked increase in plant growth 

parameters as compared to plants inoculated 

with the parent strain ST1, Nodule 

occupancy on pigeon pea plant when inoculated 

with the trans-conjugant was increased. 

Gene fegA not only supports the 

growth of the trans-formants rhizobia under 

iron limited laboratory conditions, but 

also increases its survivability under natural 

soil conditions, which led to higher 

nodulation on peanut plant. Yoshitake et 

al., (2010) introduced vktA into R. leguminosarum 

cells and the strain with a remarkably 

high catalase activity was constructed. 

The vktA trans-formant was inoculated 

to the host plant P. vulgaris and 

the nodulation efficiency was evaluated. 

The nitrogen-fixing activity of nodules 

was increased 1.7 to 2.3 times as compared 

to the parent. Results show that the 

increase of catalase activity in rhizobial 

cells could be a valuable way to improve 

the nodulation and nitrogen-fixing ability 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 368



Ikbal et al. 

of nodules. Therefore, it was concluded 

that some genetic determinants of rhizobia 

involved in host range infectivity have 

been worked out leading to the extension 

of their infectivity but their effectivity in 

terms of nitrogenase expression in enlarged 

host is still not achieved. Rhizobium 

symbiosis with non-legume host clearly 

indicates the ability of this symbiont to 

nodulate legume as well as non-legume 

(Louise et al., 2002). 

9. Perspective 

Considerable research efforts have 

been made through development of promiscuous 

rhizobia for improving the efficiency 

of biological nitrogen fixation; because, 

this process has the potential to 

reduce our dependence on nitrogenous 

chemical fertilizers. 

References 

Aggarwl, M., Sikka, V. K. and Vashishat, 

R. K. (2000). Symbiotic 

properties of Rhizobium trifolii mutants 

altered for cell wall degradative 

ability. Tropical Agriculture. 

77, 109-111. 

Al-Mallah, M. K., Davey, M. R. and 

Cocking, E. C. (1990). Enzymatic 

treatment, PEG, biotin and mannitol 

stimulate nodulation of whiteclover 

by Rhizobium trifolii. J. 

Plant. Physio. 137: 15-19. 

Ampomah, O. Y., Ofori-Ayeh, E., Solheim, 

B. and Svenning, M. M. 

(2008). Host range, symbiotic effectiveness 

and nodulation competitiveness 

of some indigenous cowpea 

bradyrhizobia isolates from the 

transitional savanna zone of Ghana. 

African Journal of Biotechnology 7 

(8), 988-996. 

Angle, J. S. (1986). Pectic and pectinolytic 

enzymes produced by fast ad 

slow growing soyabean rhizobia. 

Soil Biocemistry 18, 115-116. 

Ardourel, M., Demont, N., Debellé, F., 

Maillet, F., de Billy, F., Promé, J. 

C., Dénarié, J. and Truchet, G. 

(1994). Rhizobium meliloti lipooligosaccharide 

nodulation factors: 

different structural requirements 

for bacterial entry into target root 

hair cells and induction of plant 

symbiotic developmental responses. 

Plant Cell 6, 1357-1374. 

Arya, K. B., Shantharam, S. and Peter, 

P. W. (1982). Nodulation of Pole 

Bean (Phaseolus vulgaris L.) by 

Rhizobium species of two crossinoculation 

groups. Applied and 

Environment Microbiology 44, 

965-971. 

Baker, D., Petersen, M., Robeles, M., 

Chen, J., Squartini, A., Dazzo, F. 

and Hubbell, D. (1989). Pit erosion 

of root epidermal cell walls in 

the Rhizobium-white clover symbiosis. 

12th North American Symbiotic 

Nitrogen Fixation Conference, 

Iowa State University Press, Ames. 

Barbour, W. M., Hatterman, D. R. and 

Stacey, G. (1991). Chemotaxis of 

Bradyrhizobium japonicum to soybean 

exudates. Applied and Environment 

Microbiology 57, 2635– 

2639. 

Beatrix, H., Christian, W. B., Bachem, 

J. S. and Adam, K. (1987). Hostspecific 

regulation of nodulation 

genes in Rhizobium is mediated by 

a plant-signal, interacting with the 

nodD gene product The EMBO 

Journal 6, 841-848. 

Bec-Ferte, M. P., Krishnan, H. B., 

Prome, D., Savagnac, A., 

Pueppke, S. G. and Prome, J. C. 

(1994). Structures of nodulation 

factors from the nitrogen fixing 

soybean symbiont Rhizobium fredii 

USDA257. Biochemistry 33 (39), 

11782–11788. 

Boivin, C., Ndoye, I., Lortet, G., 

Ndiaye, A., De Lajudie, P. and 

Dreyfus, B. (1997). The Sesbania 

root symbionts Sinorhizobium saheli 

and S. teranga bv. sesbaniae 

can form stem nodules on Sesbania 

rostrata, although they are less 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 369



Ikbal et al. 

adapted to stem nodulation than 

Azorhizobium caulinodans. Applied 

and Environment Microbiology 

63, 1040–1047. 

Brewin, N. J. (2004). Plant cell wall remodelling 

in the Rhizobium legume 

symbiosis. Critical. Rev. Plant Sci. 

23, 293–316. 

Callaham, D. and Torrey, J. (1981). The 

structural basis for infection of root 

hairs of Trifolium repens by Rhizobium. 

Canadian Journal of Botany 

59, 1647-1664. 

Capela, D., Carrere, S. and Batut, J. 

(2005). Transcriptome-based identification 

of the Sinorhizobium meliloti 

NodD1 regulon. Applied and 

Environment Microbiology 71, 

4910–13. 

Chamber, M. A. and Iruthayathas, E. 

E. (1988). Nodulation and nitrogen 

fixation by fast and slow growing 

strains of soybean on several temperate 

and tropical legumes. Plant 

Soil 112, 239 – 245. 

Chassy, B. M., Mercenier, A. and Flickinger, 

J. (1988). Transformation of 

bacteria by electroporation. Trends 

Biotechnology 6, 303–309. 

Chitchanok, A., Rattasaritt, P., 

Suthatip, S., Suphaporn, P., Nattayana, 

P. and Yanee, T. (2011). 

Improvement of vitamin B6 production 

from Rhizobium sp. 6-1C1 

by random mutation. KKU Research 

Journal 16(8), 911-918. 

Cohn, J., Day, R. B. and Stacey, G. 

(1998). Legume nodule organogenesis. 

Trends in plant science 3, 

105-110. 

D’Haeze, W. and Holsters, M. (2002). 

Nod factor structures, responses 

and perception during initiation of 

nodule development. Glycobiology 

12, 79–105. 

D’Haeze, W., Gao, M. S., De-Rycke, R., 

Van Montagu, M., Engler, G., 

and Holsters M. (1998). Roles for 

azorhizobial Nod factors and surface 

polysaccharides in intercellular 

invasion and nodule penetration, 

respectively. Molecular Plant 

Microbe Interaction 11, 999-1008. 

De Jonj, T. M., Brewin, N. J., Johnston, 

A. W. B. and Phillips, D. A. 

(1982). Improvement of symbiotic 

properties in Rhizobium leguminosarum 

by plasmid transfer. Journal 

of Genetics and Microbiology 

128, 1829-1838. 

Egamberdieva, D., Berg, G., 

Lindströmc, K. A. and Rasanen 

L. A. (2010). Co-inoculation of 

Pseudomonas spp. with Rhizobium 

improves growth and symbiotic 

performance of fodder galega 

(Galega orientalis L.) European 

Journal of Soil Biology 46, 269- 

272. 

Emtiazi, G., Pooyan, M. and Shamalnasab, 

M. (2007). Cellulase 

activities in Nitrogen fixing Paenibacillus 

isolated from soil in N-free 

media World Journal of Agricultural 

Science 3(5), 602-608. 

Esperanza, Martinez-Romero and 

Monica R. (1990). Increased Bean 

(Phaseolus vulgaris L.) nodulation 

competitiveness of genetically 

modified Rhizobium strains Applied 


56(8), 2384-2388. 

Falguni, R. J, Dhwani, K., Desai, G. A. 

and Anjana J. D. (2009). Enhanced 

Survival and Nodule Occupancy 

of pigeon pea nodulating 

Rhizobium sp. ST1 expressing 

fegA Gene of Bradyrhizobium japonicum 

61A152. Journal of Biological 

Science 9(2), 40-51 

Falkowski and Paul, G. (1997). Evolution 

of the nitrogen cycle and its influence 

on the biological sequestration 

of CO 2 in the ocean. Nature. 

387, 272–275. 

Faucher, C., Camut, H., Denarie, J. 

and Truchet, G. (1989). The nodH 

and nodQ host range genes of Rhizobium 

meliloti behave as avirulence 

genes in R. leguminosarum 

bv. viciae and determine changes in 

the production of plant-specific ex- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 370



Ikbal et al. 

tracellular signals. Molecular Plant 

Microbe Interaction 2, 291–300. 

Felle, H. H., Kondorosi, E., Kondorosi, 

A. and Schultze, M. (1995). Nod 

signal induced plasma membrane 

potential changes in alfalfa root 

hairs are differentially sensitive to 

structural modifications of the 

lipochito-oligosaccharide. Plant 

Journal 7, 939–947 

Flores, M., Gonzalez, V., Pardo, M. A., 

Leija, A. Martinez, E. Romero, 

D., Piniero, D., Davila, G. and 

Palacios R. (1988). Genomic instability 

in Rhizobium phaseoli. 

Journal of Bacteriology 170, 1191- 

1196. 

Fournier, J., Timmers, A. C., Sieberer, 

B. J., Jauneau, A., Chabaud, M., 

and Barker, D. G. (2008). Mechanism 

of infection threads elongation 

in root hairs of Medicago truncatula 

and dynamic interplay with 

associated rhizobial colonization. 


Fouts, D. E., Tyler, H. L., De Boy, R. 

T., Daugherty, S. and Ren, Q. 

(2008). Complete Genome Sequence 

of the N 2 -fixing broad host 

range endophyte Klebsiella pneumoniae 

342 and virulence predictions 

verified in Mice. Genetics 

4(7), e1000141. 

Garg, B., Dogra, R. C. and Sharma, P. 

K. (1999). High efficiency transformation 

of Rhizobium leguminosarum 

by electroporation. Applied 


65(6), 2802–2804. 

Gibson, K. E., Kobayashi, H. and 

Walker, G. C. (2008). Molecular 

determinants of a symbiotic chronic 

infection. Annual Review of Genetics 

42, 413–441. 

Giraud, E. (2007). Legumes symbioses: 

absence of Nod genes in photosynthetic 

bradyrhizobia. Science 316, 

1307–1312. 

Gualtieri, G. and Bisseling, T. (2000). 

The evolution of nodulation. Plant 

Molecular Biolology 42, 181–194. 

Heidstra, R., Geurts, R., Franssen, H., 

Spaink, H. P., van Kammen A. 

and Bisseling, T. (1994). Root hair 

deformation activity of nodulation 

factors and their fate on Vicia sativa. 

Plant Physiology 105, 787–797 

Herman, P. S., Jeremy, W., Michael, 

A., Djordjevic, C., A., Wijffelman, 

R., Okker, J. H. and Ben 

J. J. L. (1989). Genetic analysis 

and cellular localization of the Rhizobium 

host specificitydetermining 

NodE protein. The 

EMBO Journal 8(10), 2811-2818. 

Hernandez, L., Segovia, I. L., Martinez, 

R. and Steven, G. P. (1995). Phylogenetic 

relationships and host 

range of Rhizobium spp. that nodulate 

Phaseolus vulgaris L. Applied 


61(7), 2775–2779. 

Hooykaas, P., Van Brussel, J. J., Den, 

A. A. N., Dulk-Ras, H., Van 

Slogteren, G. M. G. and 

Schilperoort, R. A. (1981). Sym 

plasmid of Rhizobium trifolii expressed 

in different rhizobial species 

and Agrobacterium tumefaciens. 

Nature. 291, 351-353. 

Horvath, B., Bachem, C. W., Schell, J. 

and Kondorosi. A. (1987). Hostspecific 

regulation of nodulation 

genes in Rhizobium is mediated by 

a plant signal interacting with the 

nodD gene product. EMBO Journal 

6, 841–848. 

Horvath, B., Kondorosi, E., John, M., 

Schmidt, J., Torok, I., Gyorgypal, 

Z., Barabas, I., Wieneke, 

U., Schell, J. and Kondorosi, A. 

(1986). Organization, structure and 

symbiotic function of Rhizobium 

meliloti nodulation genes determining 

host specificity for alfalfa. Cell. 

46, 335–343. 

Hussain, Z., Sikka, V. K. and Vashishat, 

R. K. (1995). Symbiotic infection 

of clover by Rhizobium trifolii 

depends on degradative enzymes 

cellulase and pectinse. Annals 

of Biology 11, 285-289. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 371



Ikbal et al. 

Jaynes, D. B., Colvin, T. S., Karlen, D. 

L., Cambardella, C. A., and 

Meek, D.W. (2001). Nitrate losses 

in subsurface drainage as affected 

by nitrogen fertilizer rate. Jornal of 

Environment 30, 1305-1314. 

Lindstom, K., (1989). Rhizobium 

galegae, a new species of root nodule 

bacteria. International Journal 

of System Bacteriology 39, 365– 

367. 

Ljunggren, H., and G. Fahraeus. 

(1961). The role of polygalacturonase 

in root-hair invasion by 

nodule bacteria. J. Gen. Microbiol. 

26,521-528. 

Loh, J. and Stacey, G. (2003). Nodulation 

gene regulation in Bradyrhizobium 

japonicum: a unique integration 

of global regulatory circuits. 

Applied and Environment Microbiology 

69, 10-17. 

Long, S. R. (1996). Rhizobium symbiosis: 

Nod factors in perspective. 

Plant Cell 8, 1885-1898. 

Louise, F., Roddam, A., Wendy, R., 

Lewis, H. B. and Michael, A. D. 

(2002). Two novel chromosomal 

loci influence cultivar-specific 

nodulation failure in the interaction 

between strain ANU794 and subterranean 

clover cv. Woogenellup 

Funct. Plant Biololgy 29, 473–483. 

Madsen, L. H., Tirichine, L., Jurkiewicz, 

A., Sullivan, J. T., Heckmann, 

A. B., Bek, A. S. (2010). 

The molecular network governing 

nodule organogenesis and infection 

in the model legume Lotus japonicus. 

Nature Communication 1,10. 

Maillet, F., F. Debelle, and J. Denarie. 

(1990). Role of the nodD and syrM 

genes in the activation of the regulatory 

gene nodD3 and of the 

common and host-specific nod 

genes of Rhizobium meliloti. Mol. 

Microbiol. 4:1975–1984 . 

Mario, O., Aguilar, M., Veronica, L., 

Mariano, D., Belen, M., Eugenia, 

M., Soria, D., Clemente, M., Antonio, 

G. S., Carolina, S. and 

Manuel, M. (2006) Soil Biology 

and Biochemistry 38, 573–586. 

Martinez, E., Flores, M., Brom, S., 

Romero, D., Davila, G., and Palacios, 

R. (1988). Rhizobium 

phaseoli: a molecular genetics 

view. Plant Soil 108, 179-184. 

Mateos, P. F. (2001). Erosion of root epidermal 

cell walls by Rhizobium 

polysaccharide-degrading enzymes 

as related to primary host infection 

in the Rhizobium–legume symbiosis. 

Canadian Journal of Microbiology 

47,475–487. 

Mateos, P. F., Jimenez-zurdo, J. I., 

Chen, J., Squartini, A. S., Haack, 

S. K., Martinez-Molina, E., Hubbell, 

D. H. and Dazzo, F. B. 

(1992). Cell associated pectinolytic 

and cellulolytic enzymes in Rhizobium 

leguminosarum bv trifolii. 

Applied and Environment Microbiology 

58(6), 1816-1822. 

Mathis, R., De Rycke, R., D’Haeze W., 

Van Maelsaeke, E., Anthonio, E., 

Van Montagu, M., Holsters, M., 

Vreecke, D. and Van Gijsegem, 

F. (2005). Lipopolysaccharides as a 

communication signal for progression 

of legume endosymbiosis. 

Proceeding of Natural Academy of 

Science USA. 102(7), 2655-2660. 

McCoy, E. (1932). Infection by B. 

radicicola in relation to the microchemistry 

of the host's cell walls. 

Proceeding of Royal Society London 

110,514-533. 

Melicent, C. P., Robert, F. F. and Sharon, 

R. L. (2006) Diverse flavonoids 

stimulate NodD1 binding to 

nod gene promoters in Sinorhizobium 

meliloti. Journal of Bacteriology 

188(15), 5417–5427. 

Miller, S. H., Elliot, R. M., Sullivan, J. 

T. and Ronson, C. W. (2007). 

Host specific regulation of symbiotic 

nitrogen fixation in Rhizobium 

leguminosarum biovar trifolii. Microbial. 

153, 3184-3195. 

Murray, J. D. (2011). Invasion by Invitation: 

Rhizobial infection in leg- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 372



Ikbal et al. 

umes. Molecular Plant- Microbe 

Interactions 24(6), 631–639. 

Okazaki, S., Takakazu, K., Shusei, S. 

and Kazuhiko, S. (2013). Hijacking 

of leguminous nodulation signaling 

by the rhizobial type III secretion 

system. PNAS 110(42), 

17131-17136. 

Oldroyd, G. E. and Downie, J. A. 

(2008). Coordinating nodule morphogenesis 

with rhizobial infection 

in legumes. Annual Review of Plant 

Biology 59, 519–46. 

Oldroyd, G. E. D. and Downie, J. A. 

(2004). Calcium, kinases and nodulation 

signalling in legumes. Natural 

Cell Biology 5, 566–576. 

Orgambide, G. G., Lee, J. I., Hollingsworth, 

R. I., and Dazzo, F. 

B. (1995). Structurally diverse chitolipooligosaccharide 

Nod factors 

accumulate primarily in membranes 

of wild-type Rhizobium leguminosarum 

biovar trifolii. Biochemistry 

34, 3832-3840. 

Pankhurst, C. E., Macdonald, P. E. and 

Reeves, J. M. (1986). Enhanced 

nitrogen fixation and competitiveness 

for nodulation of Lotus pedunculatus 

by a plasmid-cured derivative 

of Rhizobium loti. Journal 

of Genetics and Microbiology 132, 

2321-2328. 

Patriarca, E. J., Tatè, R. and Iaccarino, 

M. (2002). Key role of bacterial 

NH + 

4 metabolism in Rhizobiumplant 

symbiosis. Microbiology and 

Molecular Biology Review 66(2), 

203-222. 

Peck, M. C., Fisher, R. F. and Long, S. 

R. (2006). Diverse flavonoids 

stimulate NodD1 binding to nod 

gene promoters in Sinorhizobium 

meliloti. Journal of Bacteriology 

188, 5417–27. 

Pedro, F., Mateos, J. I., Jimenez, Z., 

Jin, C., Andrea, S., Squartini, 

Sheridan, K., Haack, E., Martinez, 

M., David, H., Hubbell and 

Frank, B. D. (1992) Cell associated 

pectinolytic and cellulolytic enzymes 

in Rhizobium leguminosarum 

biovar Trifolii. Applied 


1816-1822. 

Perret, X., Staehelin, C. and Broughton, 

W. J. (2000). Molecular basis of 

symbiotic promiscuity. Microbiology 

and Molecular Biology Review 

64, 180–201. 

Philippe, R., Fabienne, M., Claire, P., 

Frede, R. D., Myriam, F., 

Georges, T., Jean-Claude, P. and 

Jean De, N. (1996). The common 

nodABC genes of Rhizobium meliloti 

are host-range determinants 

Proceeding in Natural Academy of 

Science USA 93, 15305–15310. 

Poupot, R., Martínez-Romero, E., Gautier, 

N. and Prome, J. C. (1995) 

Wild-type Rhizobium etli, a bean 

symbiont, produces acetylfucosylated, 

N-methylated and carbamoylated 

nodulation factors. 

Journal of Biological Chemistry 

270, 6050-6055. 

Pueppke, S. G. and Broughton, W. J. 

(1999). Rhizobium sp. strain 

NGR234 and R. fredii USDA257 

share exceptionally broad, nested 

host ranges. Molecular Plant Microbe 

Interction 12, 293-318. 

Quesada, Vincen, D., Fellay, R., Nassim, 

T., Viprey, V., Burger, U., 

Promé, J. C., Broughton, W. J., 

and Jabbouri, S. (1997). Rhizobium 

sp. NGR234 NodZ protein is a 

fucosyltransferase. Journal of Bacteriology 

179, 5087-5093. 

Relic, B., Perret, X., Estrada-Garcia, 

M. T., Kopcinska, J., Golinowski, 

W., Krishnan, H. B., Pueppke, S. 

G., and Broughton, W. J., (1994). 

Nod factors of Rhizobium are a key 

to the legume door. Molecular Microbiology 

13, 171-178. 

Relic, B., Staehelin, C., Fellay, R., Jabbouri, 

S., Boller, T., Broughton, 

W. J. (1994) Do Nod-factor levels 

play a role in host-specificity? In 

Proceedings of the first European 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 373



Ikbal et al. 

nitrogen fixation conference. 

Press: Szeged, Hungary 69-75. 

Robledo, M., Jimenez-Zurdo, J. I., Velazquez, 

Trujillo, E. M. E., Zurdo-Pineiro, 

J. L., Ramírez- 

Bahena, M. H., Ramos, B., Díaz- 

Mínguez, J. M., Dazzo, F., Martínez-Molina, 

E. and Mateos, P. 

F. (2008). Rhizobium cellulase 

CelC2 is essential for primary 

symbiotic infection of legume host 

roots. Proceeding in National 

Academy of Science USA. 105(19), 

7064–7069. 

Saı¨d, J., Biserka, R., Moez, H., 

Philippe, K., Ulrich, B., Danielle, 

Prome., Jean C. P. and William, 

J. B. (1998) nolO and noeI (HsnIII) 

of Rhizobium sp. NGR234 are involved 

in 3-O-carbamoylation and 

2-O-methylation of Nod factors. 


273(20), 12047–12055 

Sanjuan, J., Gro, P., Gottfert, M., 

Hennnecke, H. and Stacey, G. 

(1994). NodW is essential for the 

full expression of the common 

nodulation genes in Bradyrhizobium 

japonicum. Molecular Plant 

Microbe Interaction 7, 364-369. 

Schlaman, H. R., Horvath, B., 

Vijgenboom, E., Okker, R. J. and 

Lugtenberg, B. J. J. (1998). Suppression 

of nodulation gene expression 

in bacteroids of Rhizobium leguminosarum 

biovar viciae. Journal 

of Bacteriology 173, 4277- 

4287. 

Scholla, M. H., and Elkan, G. H. 

(1984). A fast-growing species that 

effectively nodulates soybeans. International 

Journal of Systematic 

Bacteriology 34, 484-486. 

Setiyo, H. (2011) Characterization and 

phylogenetic analysis of Soybean 

rhizobial strains from Java and 

Sumatra Microbiology Indonesia 

5(4), 170-181. 

Shantharam, S. and Peter, P. (1982). 

Recognition of leguminous hosts 

by a promiscuous Rhizobium Strain 

Applied and Environmental Microbiology 

43(3), 677-685. 

Shiferaw, B., Bantilan, M. C. S. and 

Serraj, R. (2004). Harnessing the 

potential of BNF for Poor Farmers: 

Technological Policy and institutional 

constraints and research 

need. Symbiotic Nitrogen Fixation; 

prospects for enhanced application 

in tropical agriculture. (ed.): R. 

Serraj. Oxford and IBH publishing 

Co. Pvt. Ltd. New Delhi. pp.3. 

Spaink, H. P., Weinman, J., Djordjevic, 

M. A., Wijfelman, C. A., Okker, 

J. H. and Lugtenberg, B. J. J. 

(1989). Genetic analysis and cellular 

localization of the Rhizobium 

host specificity-determining NodE 

protein. EMBO Journal 8, 2811– 

2818. 

Spaink, H. P., Wijffelman, C. A., Pees, 

E., Okker, R. J. H. and Lugtenberg, 

B. J. J. (1987). Rhizobium 

nodulation gene nodD as a determinant 

of host specificity. Nature 

328, 337-340. 

Stokkermans, T. J. W., Ikeshita, S., 

Cohn, J., Carlson, R. W., Stacey, 

G., Ogawa, and Peters, N. K. 

(1995). Structural requirements of 

synthetic and natural product lipochitin 

oligosaccharides for induction 

of nodule primordia on Glycine 

soja. Plant Physiology 108, 

1587–1595. 

Stowers, M. D., Eaglesham, A. R. J. 

(1984). Physiological and symbiotic 

characteristics of fast-growing. 

Plant Soil 77, 3-14. 

Urban, N. R. and Eisenreich, S. 

J. (1988). Nitrogen cycling in a 

forested Minnesota bog. Canadian 

Journal of Botany 66, 435-449. 

van Rhijn, P., Luyten E., Vlassak K. 

and Vanderleyden, J. (1996). Isolation 

and characterization of a 

pSym locus of Rhizobium sp. 

BR816 that extends nodulation 

ability of narrow host range 

Phaseolus vulgaris symbionts to 

Leucaena leucocephala. Molecular 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 374



Ikbal et al. 

Plant Microbe Interaction 9, 74– 

77. 

Vashishat, R. K., Yadav, A. S. and 

Chaudhary, K. (1985b). Production 

of hydrolytic enzymes in nonnodulating 

strains of Rhizobium trifolii. 

Haryana Agricutural University 

Journal Research 15, 403-405. 

Yoshitake, O.,Yoshinobu, N., Takuji, 

O., Hidetoshi, O., Nobutoshi, I., 

Zhu, Y. Y., Feng, L. K., En, T. W., Ge, 

H. W. and Wen, X. C. (2002). 

Characterization of rhizobia that 

nodulate legume species of the genus 

Lespedeza and description of 

Bradyrhizobium legumingense sp. 

nov. Inter. Journal of Systemic and 

Evolution of Microbiology 52, 

2219–2230. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 375



Organic Farming and Halalan Toyyiban Foods: An 

Attempt to Relate Them 

Quamrul Hasan 1, 2, * and Zakirah Othman 1 

1 Knowledge Science Research Lab., School of Technology Management and Logistics, 

College of Business, Universiti Utara Malaysia, 06010 Sintok, Kedah, Malaysia; 

2 Japan Halal Research Institute for Products and Services (JAHARI), Kobe, 

Japan;*Correspondence: quamrul@uum.edu.my 

Abstract: Everyone wants to consume safe and healthy food. Also, the producers want to 

position their products according to the customers‟ demands. In the context of safe and 

healthy foods, among others, there are two different terminologies, „Organic‟ and „Halalan 

Toyyiban‟. However, our understandings on these two terminologies are not clear enough 

especially when it comes to relate them. Therefore, this research work was undertaken to 

better understand the terminologies - organic and halalan toyyiban, and find out the relationship 

between them, if any. The research methodology involves both primary data by 

visiting an organic farm and face-to-face interviewing farmers, and secondary data. The 

findings might help the consumers in selecting the produce/product and business people in 

promoting their products. Research informants were farmer, volunteer, and intern at the Sri 

Lovely Farm, a government-certified organic farm at Sik, Kedah, Malaysia. The research 

reveals new insights on the relationship of characteristics of organic farming with halalan 

toyyiban. The three commonly found characteristics are: 1) quality; 2) healthy; 3) environmental 

friendly. Based on the findings, we are proposing a model on the relationship of organic 

farming with halalan toyyiban. This study is the first of its kind and undertaken as an 

exploratory research; therefore, further study should be conducted to obtain more understanding 

and knowledge on this subject. 

Keywords: Environment; halal food; halalan toyyiban; organic farming; organic food 


In the last two decades, globalization 

has significantly advanced leading to 

not only technological and economic advancement 

but also in agriculture, food 

production, food safety and security. 

While efforts to establish trade rules led 

by the World Trade Organization and further 

progress in global free trade are advantageous 

to create new and mutually 

benefitted opportunities, such developments 

have highlighted several risks associated 

with agriculture and food, which 

were originally characterized by an unclear 

food chain (Huynen et al., 2005). 

Consumers are mainly concerned 

about health issues, protection of the environment 

and animal welfare besides 

food safety in terms of food processing 

methods, innovative food technologies, 

and presence of chemical substances in 

foods such as pesticides, toxins and food 

additives (Borin et al., 2011; Hansen et 

al., 2011; Stanton et al., 2012). Literature 

suggests that cultural diversity is an important 

criterion to expedite more sustainable 

food consumption patterns among 

society (Nicolaou et al., 2009; Schösler et 

al., 2012). The role of religion in shaping 

consumers food choice is rather vague 

except where the impact of food consumption 

depends on the religion itself 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 376


Organic Farming and Halalan Toyyiban Foods… 

(Bonne et al., 2007). Religion can influence 

consumers‟ attitudes and behavior, 

including food purchasing decisions and 

eating habits (Pettinger et al., 2004). Eating 

‘halal’ food by the Muslim community 

strictly follows the Islamic values as a 

reflection of obedience and adherence to 

the religion‟s beliefs and teachings 

(Bonne et al., 2007). Muslim consumers‟ 

attitudes towards ‘halal’ food consumption 

are influenced by religious belief, 

mass media and people around them 

(Aiedah, 2014). Further, the appearance 

of a religious (halal) logo on product 

packaging helps Muslims to choose and 

justify their product purchases without 

hesitation guided by their religious beliefs 

and laws (Bakar et al., 2013). 

Though ‘halal’ concept applies specifically 

to the Muslim society (Alam and 

Nazura, 2011), there is a huge potential to 

tap this in to the non-Muslim community 

as well especially in case of food. The 

fact that food is a common need for all 

people, the market potential is even more 

promising though people from different 

cultural backgrounds and religious faith 

do not have same perceptions and experiences 

to food. In Muslim community, the 

increasing awareness and concern over 

health is the basis for acceptance of ‘halal’ 

food as it covers the whole understanding 

of consuming clean and hygienic 

food to promote better health. In general, 

consumers are more conscious of their 

health which influences their behavior 

while selecting their food. They search 

for food with the benefits to keep them 

healthy and improve their mental state 

leading to quality of life. The role of food 

in cultural practices and religious beliefs 

might be complex; but, it has a unified 

understanding among Muslims. For instance, 

the halal logo or label helps to 

convince Muslim consumers that the food 

product is suitable for their consumption. 

On the other hand, the non-Muslim consumers 

understand that food items carrying 

the halal logo are prepared in the most 

hygienic way. Furthermore, it has also 

been proven that non-Muslim consumers 

Hasan and Othman 

do respond positively to ‘halal’ food certification 

(Hasnahet al., 2009). 

Besides the religious value, the other 

motives behind the halalan toyyiban 

concept include: 1) preserve life, 2) safeguard 

future generations, and 3) maintain 

self-respect and integrity (Muhammad et. 

al., 2007). Today, the concept of halalan 

toyyiban is beyond the religious value. 

Now a day, the rising concern of food 

consumers is health which could be an 

untapped opportunity for the ‘halal’ food 

producers. This is because the concern of 

health due to food consumption basically 

shares the same value with the halalan 

toyyiban concept. Being healthy means, 

being watchful over food on the cleanliness, 

the source, and the method of handling 

and preparing it. The most important 

thing is to ensure and minimize 

any possible harmful effects to the body 

from the food. There could be several determinants 

for the market acceptance of 

the ‘halal’ food. It is believed that consumers 

accept a product when they have 

the true intention to use it, or have used 

the product earlier and want to continue 

in using it. Generally, consumers respond 

positively to the products with high quality. 

In the case of food, quality is defined 

mainly by its cleanliness and freshness. 

To achieve this, the food processing 

methods are the key in ensuring the cleanliness 

and freshness of the food, which 

can also affect the nutritional value and 

quality of the food. The food quality is 

also critical to determine food safety. 

Grunert et al. (1996) classified the food 

quality dimensions into: hedonic, healthrelated, 

and convenience related. They 

explained: “Hedonic quality is related to 

sensory pleasure and is therefore mainly 

linked to taste, smell, and appearance. 

Health-related quality is concerned with 

the ways in which consumption of the 

product will affect consumers‟ physical 

health. Convenience-related quality is related 

to the time and effort which has to 

be expended while buying, storing, preparing 

and consuming the product” 

(Grunert et al., 1996). These explana- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 377



tions, too, relate to the food quality and 

safety as well. Furthermore, Rezai et al. 

(2011) stressed that the benefits of ‘halal’ 

food could be explained from the context 

of food safety, which is also demanded by 

non-Muslims. 

Studies on the consumers‟ attitude 

towards the use of chemicals in agriculture 

were explored since 1960‟s (Bearler 

and Willits, 1968). It marked the beginning 

of the era when human beings started 

to be more concerned and aware about 

preserving the environment. The findings 

from earlier studies confirmed that consumers 

showed positive attitudes towards 

the products of organic farming where 

one of the most commonly found reasons 

for choosing these products was - the 

products of organic farming were perceived 

as healthier than the conventional 

counterparts (Chinnici et al., 2002; Harper 

and Makatouni, 2002). Consumers do 

not necessarily buy sustainable products 

due to environmental concern, giving 

benefit to the community, and personal 

beliefs; but, mainly to give priority to 

health (Vermeir and Verbeke, 2004). Researchers 

have shown that the consumers 

of organic food are less likely to pay attention 

to the price as compared to those 

who do not purchase organic product 

(Yiride et al., 2005). 

In the past two decades, the increased 

awareness about the environment 

has had an effect on consumers‟ behavior, 

resulting in to expansion of market of the 

green product at a remarkable rate (Aini 

et al., 2003). As a result, there is a huge 

increase in production and consumption 

of organic products. It is believed that organic 

products have lesser negative effect 

to the environment. The National Organic 

Standards Board of the U.S. Department 

of Agriculture (USDA), established a national 

standard for the term „organic‟ in 

December 2000. According to them, organic 

food is defined by how it cannot be 

made rather than how it can be made. The 

organic food must be produced without 

the use of sewer-sludge fertilizers, most 

synthetic fertilizers, pesticides, genetic 


engineering, growth hormones, irradiation 

and antibiotics. Many kinds of agricultural 

products can be produced organically. 

These include produce of grains, meat, 

dairy, egg and processed food products. 

The term „organic‟ does not mean „natural‟. 

There is no fixed definition as to 

what constitutes a „natural‟ food. Nevertheless, 

the food industry uses the term 

„natural‟ to indicate that a food has been 

minimally processed and is preservativefree. 

A natural food can be called as an 

organic food, but not all natural foods are 

organic foods. 

This exploratory study aims to understand 

about meaning of organic farming 

and halalan toyyiban foods and find 

out the relationships between them based 

on the common characteristics, if any. 

The key research questions to be addressed 

in this study were: i. What is our 

understanding about the organic farming 

and halalan toyyiban foods? ii. What are 

the common characteristics to relate them, 

if any? 

2. Literature review 

For the better understanding of the 

concept of halalan tayyiban, the discussion 

here starts with the two Arabic 

words, „halal’ and „haram’. The „halal’ 

means to set free, to let go, to dissolve 

and to allow, or to exit from something 

that is not allowed (haram) (Ibn Manzur, 

n. d). Alternatively, „halal’ can be defined 

as something that is allowed and the follower 

cannot be punished if it is conducted 

properly (Jayyib, 1998). In other 

words, „halal’ means anything which is 

not prohibited or lawful, especially for 

food and meat from permitted animal 

which is ritually slaughtered (Cyril, 

1989). The opposite of „halal’ is „haram’ 

(Ibn Manzur, n. d.). It means prohibited, 

forbidden, unlawful, restricted and or unpermitted 

(Mohammad, 1993). ‘Haram’ 

can be defined as something that must be 

avoided by the Muslims, and committing 

the act of „haram’ is sinful and immoral 

for them (Ibn Hazm, 1983). 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 378



Allah s.w.t. (God) commanded 

specifically on the intake of „halal’ food, 

referring to the term „al-tayyib’ or „altayyibat’ 

and urging to eat „halal’ and 

good quality food and avoiding filthy 

food. The word „al-tayyibat’ came from 

„taba’ which means good, tasty, delicious, 

sweet, pure, clean, and free from any materials 

which are makruh (detested) (Ibn 

Manzur, n.d; al-Ghazzali, n.d). Some Islamic 

scholars suggested to integrating 

tayyib and halal (al-Qurtubi, al-Suyuti, 

Ibn „Ashur and Ibn Kathir). This was 

supported by Sazelin and Ridzwan 

(2011). 

The halalan tayyiban concept covers 

all the necessary factors (physical 

and spiritual) of the food for the human 

being. In this connection, the halalan tayyiban 

can be translated as the foods which 

are permitted (halal) for human intake for 

providing benefits to the human body and 

mind as well. The food classified as the 

halalan tayyiban should fulfill two criteria: 

firstly, the food is ‘halal’ (and taken 

from a halal source), and secondly, it is a 

quality food as it provides benefit to human. 

If the food misses these two criteria, 

it cannot be called as the halalan tayyiban. 

Hence, it must be avoided by the 

followers of Islam. 

The halalan tayyiban also indicates 

that the determination of ‘halal’ 

food encompasses both the tangible and 

intangible aspects of the food: Before 

consumption, the food must be ensured as 

‘halal’, in good quality, hygienic and 

safe. These preconditions are applicable 

from the initial sourcing and handling to 

the final stage (preparation, manufacturing, 

storage, distribution and serving). 

The idea of tayyiban does not limit the 

food to be ‘halal’, good, delicious, tasty 

and pure only. It goes further with the requirement 

of beneficial and not causing 

any harm to the body. Al-Ghazali said 

that “what is beneficial for the body is 

also beneficial for the mind and soul”. In 

addition, Sazelin and Ridzwan (2011) 

stated that the good quality food bounded 

by Islam, also has a relationship in the 


development of good quality human capital. 

Halalan tayyiban food should be 

viewed from the aspect of its complete 

supply chain, beginning from the farm 

and reaching up to the dining table. This 

means, it is important to ensure that during 

the whole process, the food should not 

be contaminated by anything which may 

be harmful to the human health. 

As underlined by the Syariah law, 

the term „halalan toyyiban‟ refers to the 

products which are safe to be consumed 

(Omar et al., 2013). As Allah s.w.t. (God) 

says in the Quran, „O mankind! Eat of 

that which is lawful and good on the 

earth‟ (Surah Al Baqarah 2: 172). They 

ask you (O Muhammad SAW) what is 

lawful for them (as food) ... Lawful unto 

you are at Tayyibaat (all kind of ‘halal’ 

foods) (Surah Al Maidah 5: 4). As explained, 

Islam requires that Muslims find 

rizk (sustenance) and consume food that 

is halalan toyyiban because it ensures a 

healthy living that reflects good attitudes 

and behaviors as well (Yousef, 2010). It 

goes further by covering the concept of 

wholesomeness, which includes quality, 

cleanliness, and safety of the food (Omar 

et al., 2013). 

The results from an earlier study 

suggest that non-Muslim consumers are 

aware of the existence of ‘halal’ food in 

Malaysia. In general, socioenvironmental 

factors such as socially 

mixing (of non-Muslims) with Muslims 

and the presence of advertised ‘halal’ 

food significantly influence non- 

Muslims' understanding of the ‘halal’ 

principle. These findings also suggest 

that non-Muslims understand that ‘halal’ 

principle that addresses the issues of 

food safety and environmental friendlyness. 

In the study, at least 94 percent 

non-Muslims agree that the ‘halal’ 

principle is religious obligation, while 

90 percent and 71 percent agree that it 

is concerned with food safety and environmental 

friendliness, respectively 

(Rezai et al., 2012). Therefore, these 

suggest that a close relationship exists 

among halalan toyyiban food safety and 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 379



environmental friendliness, which are 

also the essential characteristics of the 

products from organic farming. In an 

earlier and related study, we have highlighted 

that sustainable agriculture can 

be achieved through organic farming 

(Othman and Hasan, 2016). 

3. Methodology 

This study employed a qualitative 

research using the face-to-face interviews 

and secondary data approaches. The interviews 

were conducted with six respondents 

who also allowed the interviews 

to be recorded. Later, phone calls 

were made to the selected respondents to 

obtain clarity of the information from 

them. The location of this field study was 

at Sri Lovely Farm, Sik, Kedah, Malaysia. 

The interviews were conducted with the 

managing director (Farmer 1), his two 

assistants (Farmer 2 and Farmer 3), a volunteer 

(Farmer 4), and two interns by visiting 

the farm on October 24, 2016. 

Traditional and computer-based qualitative 

methodologies were used to analyze 

the data and to compare and contrast 

the observation. The method suggested by 

Corbin and Strauss (1990) was used in the 

data analysis. All data were first reviewed 

and then categorized. 

4. Results 

4.1. Characteristics of halalan toyyiban 

The characteristics of halalan toyyiban 

can be divided into four: 1) Quality, 

2) Healthy, 3) Clean, and 4) Environmental 

friendly, which are further explained 

below. 


4.1.1. Quality 

The whole process of ‘halal’ accreditation 

is stringent; therefore, it has 

some beneficial characteristics which can 

also be enjoyed by non-Muslim consumers. 

Its requirements meet many of the 

conventional quality standards, like ISO, 

Codex Alimentarius, Hazard Analysis and 

Critical Control Point, and Good Hygienic 

Practice. Therefore, implementing the 

halalan toyyiban requirements (to obtain 

‘halal’ accreditation) should ensure to 

produce higher quality food products 

(Talib and Ali, 2009). Considering this, 

the ‘halal’ values may become popular 

among non-Muslim consumers, if the society 

at large is made to be more aware of 

issues concerning health, hygiene, safety, 

environment, and animal welfare which 

come along with the ‘halal’ ways of doing 

the things. 

4.1.2. Healthy 

Halalan toyyiban foods are those, 

which have been handled and prepared by 

following the strict hygiene, and the high 

standards of nutrition, cleanliness and 

safety. In other words, the food must be 

produced and handled by fulfilling the 

stringent requirements of the Islamic Dietary 

Law, which as a result guarantees 

that the food is healthy. Since more and 

more people are becoming healthconscious, 

the halalan toyyiban principles 

of preparing food may no longer remain 

confined to the strictly religious need but 

may become an alternative to non- 

Muslims for a healthy life. 

4.1.3. Clean 

Allah‟s s.w.t. (God) command to 

select halalan toyyiban food can be seen 

in the verses of the al-Quran, and among 

several one is surah al-A„raf (7) verse 

157. In this, the word „al-tayyibat‟ is interpreted 

as ‘halal’ (al-Qurtubi, n. d.; al- 

Tabari, n. d.; al-Suyuti, 1990); ‘halal’ and 

not repugnant (Ibn ‟Ashur, 1984). One 

more interpretation is: ‘halal’ is good, 

beneficial to the body and helpful in 

terms of habits and the law of Islam (Ibn 

Kathir, n. d.). Also, “tayyib” is mentioned 

in surah al-Baqarah (2) verse 168. Furthermore, 

al-Sharbini (n.d) explained that 

the “toyyiban” has four principal elements 

as listed below: 

i) Both the source and whole content of 

food is ‘halal’, no haram is included 

ii) It is clean, therefore, does not contain 

any impurities 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 380




Table 1: Key differences between conventional and organic farming-Sri Lovely Farm focusing 

on the seed used 

Key practices Conventional Sri Lovely Farm 

1. Seed preparation Seed not selected Seed selected: Seeds soaked for 24 

hours prior to sowing to eliminate 

non-viable ones 

2. Quality of seedling 

at transplant 

All kinds of seedlings Only healthy seedlings transplanted 

iii) It does not cause any negative effect 

upon intake 

iv) The food contents are nutritious; 

therefore, beneficial to human. 

4.1.4. Environmental friendly 

Taking the paradigm shift into account 

which emphasizes on the need of 

the green supply chain, the ‘halal’ principles 

are no more only for the Muslims of 

slaughtering permitted animals in the Islamic 

way. It also emphasizes on the sustainability, 

environmental friendliness, 

food safety and animal welfare. Hence, 

the ‘halal’ standard implies the Halalness 

of the products to Muslims and it stands 

for not only just and fair business transactions 

but also caring for the environment, 

sustainability, and animal welfare. 

4.2. Characteristics of organic farming 

The characteristics of organic 

farming can be divided in to four: 1) 

Healthy seed, 2) Natural fertilizer, 3) 

Quality soil, and 4) Natural insect control. 

These characteristics are further explained 

below. 

4.2.1. Healthy seed 

The seeds being used in Sri Lovely 

Farm are of a very good quality. As 

depicted in the Table 1, only viable seeds 

are selected after soaking in water for 24 

hours followed by sowing in the container 

for 4 days before planting on the ground. 

The seeds are free of GM seeds. The concept 

of the healthy seed selection can be 

related with the quality of produce (in this 

case rice). “Original seed instead of the 

modified seeds”. The original seeds are 

hereditary which, when we planted the 

seeds can later use to return to replanting”- 

(Farmer 4, personal communication, 

October 24, 2016) 

4.2.2. Natural fertilizer 

Fertilizers used in Sri Lovely 

Farm are natural, from 100% natural ingredients 

produced locally, and which are 

organic entirely. Fertilizers produced in 

Sri Lovely Farm are from rice straw, and 

fruit waste collected. 

4.2.3. Quality soil 

The quality of the soil in Sri Lovely 

Farm is monitored by the Department 

of Agriculture, Malaysia in order to maintain 

soil quality of the farm land. 

“Our soil was often taken to be 

used as a sample, enter the lab. 

From there, we could know the 

soil contains heavy metal or not” - 

(Farmer 1, personal communication, 

October 24, 2016) 

The soil/land of high quality can 

produce rice of high quality, and also help 

in balancing the ecosystem, which is good 

for the environment. Quality of soil is 

maintained by the nutrients - carbon (C), 

hydrogen (H), oxygen (O), Potassium 

(K), calcium (Ca), magnesium (Mg), sulfur 

(S), Phosphorus (P) and nitrogen (N). 

Carbon, hydrogen, oxygen and nitrogen 

can be obtained from the air whereas; potassium, 

calcium, magnesium and sulfur 

are usually obtained through fertilizers. 

Fertility of the soil is very important and 

it is one of determining factors of the crop 

yield. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 381



4.2.4. Natural insect control 

Insect control is being practiced 

by Sri Lovely Farm in an ecofriendly way 

to avoid the synthetic and dangerous 

chemicals. 

“We have own methods, so how 

we want to control of caterpillars, 

we use 'ubi gadung', so, we use the 

traditional concept of back to nature” 

- (Farmer 1, personal communication, 

October 24, 2016) 

According to the one of respondents 

from the Sri Lovely Farm, the insect 

control is being carried out by using „ubi 

gadung‟ (Dioscorea daemona). 

5. Discussion 


This study was exploratory with 

an aim to further understand about the 

food choices classified under “organic” 

and “halalan toyyiban”. To our surprise, 

it was found that the key issues of environmental 

friendliness and food safety 

were addressed by the ‘halal’ principles 

as revealed by the non-Muslims. Considering 

about our future generations, we 

must put in our best effort in promoting 

and maintaining a sustainable green environment, 

these issues are very critical in 

that sense. And the halalan toyyiban food 

helps by providing a choice to consumers 

to meeting the sustainability goal. We already 

know that some non-Muslim consumers 

are familiar with the ‘halal’ principles 

and food products available in the 

market. To make it further successful, 

more awareness promotion about the halalan 

toyyiban food is needed by emphasizing 

that it‟s not only about the religious 

point of view but also about the common 

benefits for all such as the food safety, 

wholesomeness, hygiene, caring for animal 

and environment. In this effort, the 

Muslim consumers have a key role to 

play by promoting and making their non- 

Muslim friends aware about the halalan 

toyyiban principles of producing the food. 

To the best of our knowledge, this 

study was the first attempt to relate the 

foods of halalan toyyiban with organic 

farming. Therefore, enough information 

was not available in the published form 

especially when it was about to establish 

the relationship between the halalan toyyiban 

and organic farming. However, it 

was possible to collect some materials 

relevant which were found separately under 

halalan toyyiban and organic farming. 

By combining our insights obtained from 

both primary and secondary data, we have 

been able to come up with three common 

characteristics to show the significant inter-relationships 

between halalan toyyiban 

and organic (farming) foods. These 

are: 1) quality; 2) healthy; and 3) environmental 

friendly (natural). This is further 

illustrated in the Figure 1. 

Characteristics of 

organic farming: 

Characteristics of 

halalan toyibban: -Healthy seed 

-Quality 

-Natural fertilizer 

-Healthy 

-Quality soil 

-Clean (safe) -Natural insect control 

-Environmental friendly 

Relationship between 

organic farming and 

halalan toyyiban foods: 

-Quality 

-Healthy 

-Environmental 

friendly/Natural 

Figure 1: A proposed model to show relationship between organic farming and halalan 

toyyiban foods 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 382



6. Conclusion 

We conclude that there is a significant 

relationship between the integral 

part of the halalan toyyiban principles 

and practices of organic farming to produce 

food. At least three characteristics 

namely, „quality‟, „healthy‟ and „environmental 

friendly (natural)‟ were identified 

in common between halalan toyyiban 

principles and practices of organic farming. 

These insights might help in the value 

proposition of the both kind of produce 

and products to all consumers regardless 

of their faith and religion. Furthermore, 

Malaysia, as the pioneer and promoter of 

the halalan toyyiban food, should be able 

to further promote and successfully enter 

in to the emerging global market with its 

own produce and products by utilizing 

insights reported in this article. 


The authors would like to extend 

their gratitude to Captain Zakaria Kaman 

Tasha at Sri Lovely Farm; Noor Azian 

Mohamad and Siti Noor Ashikin Abd 

Latif at UUM for their assistance and 

support to complete this study. 

References 

Aiedah, A.K. (2014). Young consumers‟ 

attitude towards halal food outlets 

and JAKIM‟s halal certification in 

Malaysia. Procedia-Social and Behavioural 

Sciences 121, 26-34. 

Aini, M.S., Fakhru’l-Razi., A. Laily, P., 

and Jariah, M. (2003). Environmental 

concerns, knowledge and 

practices gap among Malaysian 

teachers. International Journal of 

Sustainability in Higher Education. 

4(4), 305 – 313. 

Alam, S.S, and Sayuti, N. M. (2011). 

Applying the Theory of Planned Behavior 

in Halal Food Purchasing. International 

Journal of Commerce and 

Management 21(1), 8-20. 


Al-Ghazzali, Abu Hamid Muhammad 

ibn Muhammad ibn Muhammad. 

(n. d.). Ihya’ „Ulum al-Din (v. 2). 

Beirut: Dar al-Ma„rifah. 

Al-Qurtubi, Abi ‘Abd Allah Muhammad 

ibn Ahmad al-Ansari. (n. d.). 

Tafsir al-Qurtubi al-Jami„ li 

Ahkamal-Qur’an (v. 7). Beirut: Dar 

al-Sha„bi. 

Al-Sharbini, Shams al-Din Muhammad 

ibn al-Khatib. (n. d.). Mughni al- 

Muhtaj ila Ma‘rifat Ma„ani Alfazal- 

Minhaj (v. 4). Dimashq: Dar al-Fikr. 

Al-Suyuti, Jalal al-Din ‘Abd al- 

Rahman ibn Abu Bakr ibn Muhammad 

ibn Sabiq al-Din. (1990). 

al-Daral-Manthur, v. 3. Beirut: Dar 

al-Kutub al-„Ilmiyyah. 

Al-Tabari, Abu Ja‘far Muhammad ibn 

Jarir. (n. d.). Tafsir al-Tabari, v. 9. 

Mesir: Dar al-Ma„arif. 

Bakar, A., Lee, R. and Rungie, C. 

(2013). The effects of religious symbols 

in product packaging on Muslim 

consumer responses. Australasian 

Marketing Journal, 21, 198-204. 

Bearler, R.C., and Willits, F.K. (1968). 

Worries and non-worries among consumers 

about farmers use of pesticides. 

Journal of consumer Affairs, 2, 

189. 

Bonne, K., Vermeir, I., Bergeaud- 

Blackler, F. and Verbeke, W. 

(2007). Determinants of halal meat 

consumption in France. British Food 

Journal, 109 (5), 367-386. 

Borin, N., Cerf, D.C. and Krishnan, R. 

(2011). Consumer effects of environmental 

impact in product labelling. 

Journal of Consumer Marketing 

28 (1), 76-86. 

Chinnici, G., D’Amico, M., and Pecorino, 

B. (2002). A multivariate statistical 

analysis on the consumers of organic 

products. British Food Journal. 

104, 187-199. 

Corbin, J. M. and Strauss, A. (1990). 

Grounded theory research: Procedures, 

cannons, and evaluative criteria. 

Qualitative Sociology, 13 (1), 3- 

21. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 383



Grunert, K. G., Hartvig Larsen, H., 

Madsen, T. K., and Baadsgaard, 

A. (1996). Market orientation in food 

and agriculture. Kluwer, Boston, MA 

Hansen, T., Mukherjee, A. and Thyra, 

U.T. (2011). Anxiety and search during 

food choice: the moderating role 

of attitude towards nutritional claims. 

Journal of Consumer Marketing 28 

(3), 178-186. 

Harper, G.C., and Makatouni, A. 

(2002). Consumer perception of organic 

food productions and farm animal 

welfare. British Food Journal 

104, 287-299. 

Hasnah, S., H., Dann, S., Annuar, 

M.K., and De Run, E.C. (2009). Influence 

of the Halal Certification 

Mark in Food Product Advertisement 

in Malaysia. (Chapter 14: The New 

Culture of food. Marketing Opportunities 

from ethnic, religious and cultural 

diversity. Edited by Adam 

Lindgreen and Martin K. Hingleg), 

Gower Publishing Limited, England. 

Huynen, M.M.T.E., P. Martens and 

H.B.M. Hildetink (2005). The health 

impacts of globalisation: A conceptual 

framework. Globalization and 

Health 1, 1-12. 

Ibn ‘Ashur, M. al-T. (1984). Tafsir al- 

Tahrir wa al-Tanwir, v. 6. Tunisia: 

al-Dar al-Tunisi. 

Ibn Hazm, A. M. (1983). al-Ihkam fi 

Usul al-Ahkam, v. 3. Beirut: Dar al- 

Afaq al-Jadidah. 

Ibn Manzur, J. al-Din M. ibn M. al-A. 

(n. d.). Lisan al-‘Arab, v. 4. Mesir: 

Dar al-Misriyyah li al-Ta‟lif wa al- 

Tarjamah. 

Jayyib, S. A. (1998). al-Qamus al-Fiqhi 

Lughatan wa Istilahan. Beirut: Dar 

al-Fikr. 

Mohammad, M. H. (1993). Islamic Dietary 

Concepts and Practices. The Islamic 

Food and Nutrition Council of 

America (IFANCA), Chicago, IL. 

Mohani, A. , Hasanah, I., Haslina, H. 

and Juliana, J., (2009). SMEs and 

halal certification. China-USA Business 

Review 8 (4). 


Muhammad, N., Norhaziah, N., Nuradli, 

R., and Hartini, M. (2007). 

Halal Branding: An Exploratory Research 

among consumers in Malaysia. 

Available at nuradli.com 

Nicolaou, M., Doak, C.M., van Dam, 

R.M., Brug, J., Stronks, K. and 

Seidell, J.C. (2009). Cultural and social 

influences on food consumption 

in Dutch residents of Turkish and 

Moroccan origin: a qualitative study. 

Journal of Nutrition Education and 

Behaviour 41 (4), 232-241. 

Omar, E.N., H.S. Jaafar and M.R. Osman. 

(2013). Halalan toyyiban supply 

chain of the food supply industry. 

Journal of Emerging Economics and 

Islamic Research 1, 1-12. 

Othman, Z. and Hasan, Q. (2016). Sustainable 

agriculture through organic 

farming: A case in paddy farming in 

peninsular Malaysia. Focus on Environment. 

AIMST University, Malaysia. 

pp. 38-50. 

Pettinger, C., Holdsworth, M. and 

Gerber, M. (2004). Psycho-social influences 

on food choice in Southern 

France and Central England. Appetite, 

42 (3), 307-316. 

Rezai, G., Mohamed, Z., and 

Shamsudin, Nasir Mad (2012). 

Non-Muslim consumers‟ understanding 

of Halal principles in Malaysia. 

Journal of Islamic Marketing 3 (3), 

35-46. 

Sazelin, A., and Ridzwan, A. (2011). 

Food quality standards in developing 

quality human capital: An Islamic 

Perspective. African Journal of Business 

Management 5(31), 12242- 

12248. 

Schösler, H., de Boer, J. and Boersema, 

J.J. (2012). The organic food philosophy: 

a qualitative exploration of the 

practices, values, and beliefs of 

Dutch organic consumers within a 

cultural-historical frame. Journal of 

Agricultural and Environmental Ethics 

26 (2), 439-460. 

Stanton, J.L., Wiley, J.B. and Wirth, 

F.F. (2012). Who are the locavores? 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 384



Journal of Consumer Marketing 29 

(4), 248-261. 

Taib, H. A., and Ali, K.A. (2009). An 

overview of Malaysian food industry: 

the opportunity and quality aspects. 

Pakistan Journal of Nutrition 8 (5), 

507-17. 

Vermeir, I., and Verbeke, W. (2006). 

Sustainable food consumption: Exploring 

the consumer attitudebehavioral 

intention gap. Journal of 

Agricultural and Environmental Ethics 

19 (2), 169-194. 

Yiridoe, E.K., Bonti-Ankomah, S., and 

Martin, R.C. (2005). Comparison of 

consumers perceptions and preferences 

toward organic versus conventionally 

produced foods: A review 


and update of the literature. Renewable 

Agriculture and Food System 20, 

193-205. 

Yousef, D. K. (2010). UAE: Halal food 

numbers look tasty. Available at: 

http://tinyurl.com/jn9f8m7 

Yunus, A. M., Yusof, W. M., Chik, W., 

and Mahani, M. (2010), The Concept 

of Halalan Toyyiba and Application 

in Product Marketing: A Case 

Study at Sabasun HyperRuncit Kuala 

Terengganu, Malaysia. International 

Journal of Business and Social Science 

1(3), 239-248. 

Zeithaml, V., Parasuraman, A. and 

Berry, L. (1996). The behavioral 

consequences of service quality. 

Journal of Marketing 60, 31-46. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 385



Biotechnological Approaches: Sustaining Sugarcane 

Productivity and Yield 

Ashutosh Kumar Mall and Varucha Misra* 

ICAR-Indian Institute of Sugarcane Research, Lucknow- 226 002, Uttar Pradesh, India; 

*Correspondence: Ashutosh.Mall@icar.gov.in / misra.varucha@gmail.com; Tel: +91 522- 

2480726 

Abstract: Biotechnology is an important field of science which is playing a vital role in 

agriculture and other domains. The idea of creating new hybrid varieties is not new; however, 

earlier this process was possible only in close species associated with each other. With 

the use of biotechnological techniques, it is now possible even in species which are not 

closely associated. Sugarcane crop is also not left untouched by this field of science. It has 

paved a new way for improving the cane production and productivity. It even helps in enhancing 

the sucrose content of the crop. Sugarcane researchers have achieved success in 

several aspects with the use of these techniques like developing high yielding cane varieties; 

enhance accumulation of sucrose content in cane stalks, etc. Although there are still 

certain constrains which have yet not be solved in this crop but the way field of biotechnology 

is developing, it is not far that these constrains will also be overcome. In this article we 

are highlighting the usefulness and potential of biotechnology approaches to boost the sugarcane 

productivity and yield for the sustainability. 

Keywords: Abiotic stress; high yielding; productivity; sugarcane 


Sugarcane is a crop that imparts 

sweetness to human’s life. It is a major 

sugar producing crop that contributes to 

more than 70 per cent for production of 

sugar. It covers an area of around 3.8 million 

hectares with an annual cane production 

of around 270 mt. 2.8 per cent of the 

cultivated land area is occupied by this 

crop and in respect to agricultural production 

about 7.5 per cent is contributed by 

this crop to India. In India, 42.02 (%) and 

57.98 (%) is contributed to sugarcane area 

in tropical and sub-tropical zone, respectively, 

while in terms of production it is 

48.58 (%) and 51.42 (%), respectively 

(Shukla et al., 2016). It is well known that 

this crop is a major source of food as well 

as fuel production. The new field of biotechnology 

has the power of improving 

cane production as well as yield. Using 

this new field of science as a tool, researches 

specialized in plant breeding can 

able to produce better crops. The technologies 

used in this field have the capability 

to transfer and alleviate a single gene/or 

number of genes of desired trait rather 

than thousand of genes from one species 

to the other one (Nel, 2009). 

Biotechnological approaches in 

the plant kingdom has been playing significant 

role from past many decades. 

This field of science had encompassed the 

magnificent genetic engineering developments 

within itself in several folds. The 

crops obtained from such methods have 

been known to be the latest technological 

approaches that had helped in boosting up 

the production of food to a great extent. 

These transgenic crops had many beneficiary 

points like easier application of 

herbicide and that too in very low levels 

as per the normal practices which in turn 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 386


Biotech Approaches for Sustainable Sugarcane Productivity 

Mall and Misra 

helps in reducing the cost of production 

as well as in overcoming the environmental 

pollution (Baker and Pretson, 2003). In 

case of sugarcane crop, on worldwide basis 

there is high pressure to augment cane 

productivity for sustaining the profits of 

sugar mills (Halon et al., 2000). In this 

regards, various sugarcane researchers 

have been showing effort in developing 

new hybrid cane varieties that posses high 

yield and high sugar contents under conventional 

breeding programmes of sugarcane 

at different institutes. With the use 

of these approaches, new cultivars are 

being able to develop which possess high 

sugar content, better ability of ratooning 

as well as resistance towards various diseases. 

These newer techniques and methodology 

have paved new way in the field 

of breeding for improving varieties and 

also helped in rapid multiplication of these 

varieties. A common breeding constrain 

in developing new varieties is its 

slow multiplication rate as well as its rapid 

spread. This creates a problem in not 

fulfilling the seed requirement of the newly 

developed varieties, biotechnology in 

this aspect, had helped in faster multiplication 

of new varieties (Source access: 

http://shodhganga.inflibnet.ac.in/bitstrea 

m/10603/42274/7/07_chapter%202.pdf, 

3.05.2017). 

2. Biotechnological achievements 

2.1. In improving cane production 

Being a major food and fuel 

source all over the world, biotechnology 

in this regard has the power for improving 

the economically important traits in this 

crop. The key approach in improving 

sugarcane production lies in the classical 

plant breeding method but plant breeders 

always encounter difficulty in this regard 

as cane genome is highly complex and 

possess narrow genetic base (Roach, 

1989; Lima et al., 2002). The biotechnological 

approaches had successfully improved 

cane production especially the inter-specific 

Saccharum officinarium and 

S. spontaneous hybrids (Usman, 2015). 

Another most important constrain is the 

time required for a new variety to develop 

and commercialize that generally takes a 

long time of 12-15 years. As mentioned 

before that biotechnology helps in transferring 

a desired trait of gene from one 

plant to other so in case of sugarcane 

crop, there is certain desired traits which 

would not be able to introduced into it 

through the normal plant breeding methods. 

The victory of improving the crop 

production by biotechnological tools lies 

in the high levels of the trans-gene expression. 

In this aspect, promoters have 

been identified in driving the high levels 

of gene expression in transgenic sugarcane, 

particularly in stem and leaves. The 

first identified promoter was obtained 

from Cestrum yellow leaf curling virus 

which impels the elevated level of constitutive 

trans-gene expression significantly 

higher than the ones obtained by the 

maize polyubiquitin-1 (Zm-Ubi1) promoter 

(a well known benchmark). Another 

identified promoter was the 

maize phosphonenolpyruvate carboxylate 

promoter which facilitates the expression 

levels, particularly in the leaf 

region of sugarcane, compared to Zm- 

Ubi1. By the process of gene modification, 

the transgenic expression was enhanced 

by approximately 50-fold for better 

cane production (Kinkema et al., 

2014). Bower and Birch (1992) had 

achieved success in the sugarcane transformation 

trailing with the development 

of micro-projectile system. Some studies 

had showed improved resistance in developing 

a transgenic sugarcane crop towards 

micro-organisms acting as pathogens 

(Joyce et al., 1998a, b; Ingelbrecht et 

al., 1999; Zhang et al., 1999; Gilbert et 

al. 2005;), towards pests like stem borer 

(Arencibia et al., 1999; Braga et al., 

2003) and towards herbicide (Gallo- 

Meagher and Irvine, 1996; Enriquez- 

Obregon et al., 1998). 

The use of techniques of genetic 

engineering in the past two decades by 

plant breeders had caused transmission of 

noble gene into the plant for developing 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 387




better characteristics in them. The technique 

involves insertion of foreign genes 

into the parent plant, use of protoplasmic 

cells or tissues for the development of 

transgenic plant having normal physiological 

and biological functions. Several advances 

have been seen from past several 

years in field of molecular biology as well 

as genetic engineering pertaining to crops 

(Jenes et al., 1993). With the use of a 

technology based on recombinant DNA it 

is now even achievable to clone a gene, 

modify or mobilize it and even integrate it 

in any other without any discrimination 

from where the gene has been taken 

(Chakrabarty et al., 2002). About 50 % 

losses are being occurring by different 

types of borers but the use of Bacillus 

thurigenesis by these technologies have 

shown harmful effects towards these borers. 

In the present scenario, by the use of 

cry genes, greater than 30 species of different 

plants have been transformed 

(Schuler et al., 1998). 

2.2. In developing high yielding sucrose 

cane varieties 

Biotechnological approaches had 

made possible in increasing the frequency 

of capability of parental clones possess 

with very high sucrose content which in 

prevailing breeding programmes is at a 

very low level. In case of sugarcane crop, 

Sugarcane Breeding Institute had initiated 

programme in this aspect. The target of 

this programme is to develop genetic 

stocks that consist of high sucrose by the 

use of recurring cycles of intensive crossing 

and selection. In cycle I, about 5420 

seedlings obtained from 30 bi-parental 

crosses were considered. Out of all the 

crosses performed, crosses between CoC 

671 x CoT 8201, Co 86002 x Co 62198, 

Co 85002 x CoT 8201, CoC 671 x Co 

94019, PR 1080 x Co 94008 and PR 1080 

x CoT 8201 had shown high levels of sucrose 

contents. The approach of biotechnology 

in this aspect of developing high 

sugarcane varieties have opened up a new 

option which will reward to sugar millers 

in two ways, firstly productivity will 

raise and secondly cost of production will 

be reduced (Shanthi, 2016). 

2.3. Towards abiotic stress 

For tolerance to a particular stress 

or multiple stresses, biotechnology had 

showed a new way for developing transgenic 

plants. The best short-term approach 

for development of stress tolerant 

cane variety lies on the base of selection 

and breeding wherein wide crosses are 

being made. For the development of the 

stress tolerant variety certain steps have 

been outlined (Figure 1) (Epstein and 

Rains, 1987). Researchers had identified 

candidate genes in sugarcane for imparting 

tolerance to various abiotic and biotic 

stresses. In this view, some of the examples 

of candidate genes which have been 

identified in case of drought/water deficit 

condition are DREB (dehydration responsive 

transcription factor), HSP (heat 

shock proteins), LEA (late embryogenesis), 

RAB (responsive to abscisic acid), 

osmotin, choline oxidase and annexin 

(Nair, 2011), stress-related clusters showing 

differential expression (>2-fold) during 

biotic and abiotic stress conditions 

(Gupta et al., 2010), sugarcane ethyleneresponsive 

factor, SodERF3 (Trujillo et 

al., 2009), up-regulation of genes regulating 

intracellular redox status (Prabu et al., 

2011) and presence of LEA (late embryogenesis 

abundance)-related proteins and 

dehydrin (Iskandar, et al., 2011), accumulation 

of trehalose and proline (Molinari 

et al., 2007; Guimarães et al., 2008), other 

stress-inducible proteins (Jangpromma 

et al., 2010), early response to dehydration 

protein 4 (ERD4) (McQualter et al., 

2007). Wahid and Close (2007) had identified 

various expressions of genes or proteins 

in sugarcane grown under temperature 

and salinity induced stress. Some of 

the instances of such gene expression under 

stress are heat stress-induced DHNs 

(Wahid and Close, 2007), genes encoding 

for O - /OH - radicals and reduction of H 2 O 2 

by peroxidase/ catalase under heat stress 

(McQualter et al., 2007; Chagas et al., 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 388




Figure 1: A diagrammatic sketch depicting the steps involved in using molecular approaches 

for the development of stress tolerant sugarcane variety (Source: Data from 

Shrivastava et al., 2016). 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 389




2008; Shrivastava et al., 2012), coldinducible 

ESTs, PPDK and NADP-ME 

proteins and dehydrin-like proteins which 

protect membranes against chilling stress 

(Nogueira et al., 2003), reduced activity 

of sucrose phosphate synthase, NADP- 

MDH and pyruvate orthophosphate 

dikinase to maintain photosynthesis under 

chilling damage (Du et al., 1999), induction 

of Galactional synthase (GolS) and 

pyrroline-5-carboxylase synthetase 

(P5CS) (McQualter et al., 2007) and osmolytes 

like proline and glycine betaine 

(Patade et al., 2008) during salinityinduced 

stress. Shaik et al. (2007) had 

mediated transformation in sugarcane 

through a microorganism Agrobacterium 

tumifaciens with two plasmid LBA4404 

pB1 121 construct GLY1 that bestowed 

stress tolerance in crop. Another bacterial 

transformation of A. tumifaciens imparted 

tolerance to drought and salinity in sugarcane 

using Arabidopsis Vascular Pyrophosphatase 

(AVP1) gene (Kumar et al., 

2014). 

Some recent achievements in providing 

tolerance to abiotic stress in sugarcane 

through biotechnological tools are as follows: 

i. Identification of nearly 600 differentially 

expressed genes in cane grown 

under low temperature for activity of 

the trans-membrane transporter with 

an enhancement of ~2.5 fold in Ssp- 

NIP2 expression (Saccharum homolog 

of a NOD26-like major intrinsic 

protein gene (Park et al., 2015). 

ii. For enhancing tolerance power of 

cane towards drought and salinity, 

over expression of PDH45 (a DEADbox 

helicase gene- a pea isolated 

gene) in transgenic sugarcane. This 

gene also exhibited an up-regulation 

of DREB2-induced downstream 

stress-related genes (Augustine et al., 

2015). Another expression of genes in 

response to drought in sugarcane, expression 

of a set of genes majorly accountable 

for synthesis or expression 

of trehalose 5-PO 4 and sucrose-PO 4 in 

response to foliar application of salicylic 

acid (Almeida et al., 2013). 

iii. Identification and expression of genes 

related to defense/ signaling sequences 

in smut and eyespot disease inoculated 

cane plants- 62 differentially 

expressed genes having 19 transcript 

derived fragments (TDFs) and a chitinase 

gene ScChi which is concerned 

in interaction of host with pathogen 

(Borrás-Hidalgo et al., 2005; Que et 

al., 2014). 

iv. Identification and expression of EST 

clusters that are responsible in signaling 

of reactive oxygen species (ROS), 

defense response and sugarcane innate 

immunity against red rot infection 

(Sathyabhama et al., 2016). 

v. Development of drought tolerant 

transgenic sugarcane- PT Perkebunan 

Nusantara in Indonesia, University of 

Jember (East Java) and Ajinomoto 

Co., Inc., Japan had developed this 

transgenic plant by using bet A gene 

from the Rhizobium meliloti that produces 

glycine-betaine. This product is 

an osmo-protectant which imparts tolerance 

towards drought stress. This 

GM transgenic cane produced 20-30 

per cent more sugar in comparison to 

other cane varieties opted for drought 

conditions (Marshall, 2014; Waltz, 

2014). With the approval by the national 

genetically Modified Products 

Bio-safety Commission of Indonesia 

this has gained the first position of the 

world’s first commercialized GM 

sugarcane (Anon, 2013). 

3. Sugarcane production and productivity 

Biotechnology has paved a new 

way for improving the cane production 

and productivity. To increase the sucrose 

content in the crop, genetic manipulation 

are being used which requires a complete 

knowledge and command in the processes 

involved in sucrose accumulation within 

the cane stalks (the storage house of the 

plant). Researchers have been successful 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 390




in identifying the enzymes that gives a 

start to these processes, however, through 

the modern technique of genetic engineering 

these enzymes cane be hasten or 

slowed down to attain more efficient storage 

and accumulation of sucrose in cane 

stalks. The application of genetic manipulation 

in cane stalks are being conducted 

one step at a time. Towards first step for 

success South African scientists had 

knocked down a certain enzyme by genetic 

means which enhanced the sucrose 

content in young stalks of sugarcane 

(Groenewald and Botha, 2008). Another 

perspective is making cellulosic bio-fuel 

production easier. It is well known that 

the sucrose is an essential component for 

production of bio-ethanol, an alternative 

for fossil fuels, through the process of 

fermentation. Breeders are focusing now 

on enhancing the sucrose yield for increasing 

production of ethanol without 

compromising the sucrose content as food 

commodity. By the modern use of biotechnology 

tools the cellulose content in 

cane leaves as well as bagasse are being 

used for the production of ethanol thereby 

not utilizing the main cane product, sugar. 

The intricate structure of cellulose can be 

broken down into simpler molecules of 

carbohydrates by number of enzymes 

which later can be used for production of 

ethanol by fermentation nevertheless cellulosic 

structure is guarded by lignin. This 

hard guarding material requires a costly 

procedure for its removal. Brazilian scientists 

are taking initiatives through genetic 

engineering in modifying the cellulosic 

structure so that it could be separated 

from bagasse without any difficulty 

(http://agencia.fapesp.br/en/16756, accessed 

on 03.04.2017). Adding to it, Australian 

researchers developed transgenic 

canes by inserting genes capable of producing 

the cellulose degrading enzymes 

in leaves of mature plants (Harrison et al., 

2011). Besides production of small products 

through sugarcane bio-factory which 

involves the tweaking of genetic mechanism 

occurring in cells of sugarcane plant 

that instructs them for the production of 

these small products which in turn converts 

the complete cane plant into a biofactory. 

A number of high value products 

are being produced like therapeutic proteins 

(Wang et al., 2005) and biopolymers 

(Petrasovits et al., 2007; McQualter et al., 

2005). Another important production of 

small product is production of isomaltose 

which was possible by insertion of a bacterial 

gene into the cane plant for production 

of an enzyme responsible for conversion 

of sucrose into iso-maltose, an alternative 

sweetener (Wu and Birch, 2007). 

Biotechnological efforts gave positive 

results in stream of characterization of 

genome structure, specific traits mapping, 

marker assisted selection in resistance 

towards insect/disease, variability of 

pathogens on molecular basis, transformation, 

pathogen detection in precise 

manner in plants and many more. The 

sharpness in sensitivity of the assays 

made them more rapid for routine analysis 

of plant pathogen detection and identification. 

Besides, the assay has become 

even more economical. The better ability 

to detect the infection at early or latent 

stages help in improving the management 

of disease as well as restricting the 

movement of the various diseases and 

even assist in solving the phylogenetic 

relationship amongst the various pathogens. 

This also assists in developing newer 

strategies for enhancing the resistance 

activity of the host by genetic transformation 

methods. There is a need to improve 

the technologies for development 

of transgenic before making it a part of 

the varietal developmental programmes of 

sugarcane. There is still a challenge for 

the researchers in developing a pathogenic 

free transgenic as this requires complete 

understanding of interaction of plant 

and pathogen, however, gene cloning, to 

some extent, is making a new revolution 

in this aspect. There is still a need to identify 

and characterize the genes responsible 

for the antifungal activity in case of 

disease resistance. In grassy shoot disease 

of sugarcane and Sugarcane yellow leaf 

syndrome, a common occurring disease 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 391




development of PCR diagnostic kits are 

needed for Phytoplasma. 

For improving the crop quality as 

well as productivity and even providing 

resistivity to pathogen, tissue culture is 

playing an effective role. The use of cryotherapy 

has enhanced the likelihood of 

attaining healthy plants. In case of attaining 

virus resistant plants, micropropagation 

is the best tool which requires the use 

of shoot apical meristem (acting as explants). 

In minimizing the somaclonal 

variation in times to come, application of 

clonal propagation as well as research in 

transgenic are needed. There is a need to 

work on the structure of genomes so that 

identification of markers associated with 

important agronomic characters may be 

performed. Transgene constructs that help 

in lessening the hazardous effect of environment 

and even bio-safety jeopardize of 

these plants are need of the upcoming 

times. The way researchers are moving 

towards the biotechnological techniques 

many mysteries will unravel (Tiwari et 

al., 2010). 

4. Biotechnological challenges in enhancing 

the cane production and 

productivity 

The use of biotechnology in sugarcane 

crop has drawn researchers as well 

as entrepreneurs towards itself but its application 

over this crop on commercial 

basis had always be a regulatory challenge 

particularly in case of field cultivation. 

There is higher probability of transferring 

of genes along with unwanted 

genes from the source plant to other 

plants used as a food commodity. Therefore, 

the practicability of these techniques 

on commercial cane bio-factory will be 

dependent on how much amount of risk 

containment as compared to the non-food 

product plants, for example tobacco 

(http://isaaa.org/resources/publications/po 

cketk/45/default.asp, 27.3.2017). A commonly 

used technique now-a-days is tissue 

culture technique that helps in developing 

uniform disease free plantlets in 

short time, however, a major problem in 

this technique is its cost of production 

(Usman, 2015) . 

One of the most talked topics in 

biotechnology achievements is the development 

of transgenic varieties and the 

area under the transgenic obtained plants 

has increased to approximately greater 

than 81 million hectares but there are certain 

limitations too in developing it. Singh 

et al. (2013) showed that the drawbacks 

in developing cultivars are not even removed 

by the process of transformation; 

however, other methods for precise integration 

and control trans-gene expression 

are still to be performed in sugarcane 

crop. Furthermore, in studies related to 

sugarcane association, there is still need 

for developing high throughput markers 

as well as producing more markers and 

even ensure the proper availability of these 

markers. The newly developed markers 

will enhance the knowledge and mystery 

of complex structure of sugarcane genome. 

DNA-based molecular markers of 

progenitor plants have the potential to 

show the prevailing genetic polymorphism 

that may be helpful in case of this 

crop as parental genome is much less 

complex in comparison to the hybrids 

ones (Henry et al., 2012). 

5. Constrains in improving cane production 

using biotechnological tools 

Biotechnology is generating 

enormous information which had played 

an important role in transforming the 

world of science. Biotechnological interference 

in sugarcane crop provides a 

chance for sugar producers and cane 

farmers to enhance the production and 

sustainability. These approaches act as a 

proactive approach in alleviation of the 

troubles occurring in cane production 

(Usman, 2015). Though researchers are 

attaining much success in improving the 

cane production and yield yet there are 

certain constrain in this regard using biotechnological 

tools. Some of these constrains 

are: High-throughput sugarcane 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 392




transformation that involves efficiency of 

the low transformation, inactivation of 

trans-gene, soma-clonal variation, and the 

long time required for regeneration and its 

commercial release. In restricting the access 

in exploiting gene technology, transformation 

and tissue culture-induced soma-clonal 

variation are the two things that 

remain important for sugarcane improvement 

(Arencibia et al., 1997). The substantial 

alteration in the current transformation 

systems are required to make sure 

of the clonal fidelity of transgenic cultivars. 

Also as stated before the time lag of 

developing and releasing a cane variety is 

also a crucial challenge as well as constrain 

for cane breeders. At times the cane 

grown and developed by classical method 

yields similar/or enhancing gains as compared 

to the ones developed through biotechnological 

approaches. There are two 

major factors, viz., polyploidy and aneupolyploidy 

that cause molecular characterization 

of cane genome more difficult. 

Aitken et al. (2014) had reported that the 

recent available genetic maps and markers 

obtained from sugarcane provide partial 

information in aspect of genome organization. 

The reason behind is the low 

density of markers and its coverage. This 

causes difficulty in allocating the markers 

into linkage groups (Souza et al., 2011). 


This chapter concludes that various 

successful achievement have been 

attained by plant breeders by using the 

biotechnological techniques in this crop 

but the way, application of biotechnology 

is spreading its hands in agriculture especially 

in sugarcane crop, the left over 

mysteries could be unravelled much easier 

in the times to come. Progress in traditional 

breeding of sugarcane, a highly 

polyploid and frequently aneuploid plant, 

is impeded by its narrow gene pool, complex 

genome, poor fertility, and the long 

breeding/selection cycle. These constraints, 

however, make sugarcane a good 

candidate for molecular breeding. 

References 

Aitken, K. S., McNeil, M. D., Hermann 

S., Bundock, P. C., Kilian, A., Heller 

Uszynska, K., Henry, R. J. and 

Li, J. (2014). A comprehensive genetic 

map of sugarcane that provides 

enhanced map coverage and integrates 

high-throughput diversity array 

technology (DArT) markers. 

BMC Genomics 15, 152. 

Almeida, C. M. A., Donato, V.M.T.S., 

Amaral, D. O. J., Lima, G. S. A., 

Brito, G. G., Lima, M. M. A., Correia, 

M. T. S. and Silva, M. V. 

(2013). Differential gene expression 

in sugarcane induced by salicyclic 

acid under water deficit conditions. 

Agricultural Science Research J. 

3(1), 38-44. 

Anonymous (2013). Indonesia approves 

first GM sugarcane. Crop Biotech 

Update (May 22, issue), International 

Service for the Acquisition of Agri- 

Biotech 

Applications 

(http://www.isaaa.org/kc/cropbiotech 

update/ 

article/default.asp?ID=10989). 

Arencibia, A., Vazquez, R. I., Prieto, 

D., Tellez, P., Carmona, E. R., 

Coego, A., Hernandez, L., Dela Riva, 

G. A. and Selman, H. G. (1997). 

Transgenic sugarcane plants resistant 

to stem borer attack. Molecular 

Breeding 3, 247–255. 

Arencibia, A.D., Carmona, E.R., 

Cornide, M.T., Castiglione, S., 

O’Relly, J., Chinea, A., Oramas, P. 

and Sala, F. (1999) Somaclonal variation 

in insect-resistant transgenic 

sugarcane (Saccharum hybrid) plants 

produced by cell electroporation. 

Transgenic Res. 8, 349–360. 

Augustine, S. M., Narayan, J. A., Syamaladevi, 

D.P., Appunu, C., 

Chakravarthi, M., Ravichandran, 

V., Tuteja, N. and Subramonian N. 

(2015). Introduction of pea DNA helicase 

45 into sugarcane (Saccharum 

spp. hybrid) enhances cell membrane 

thermostability and upregulation of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 393




stress-responsive genes leads to abiotic 

stress tolerance. Molecular Biotechnology 

57, 475-488. 

Baker J., Preston C. (2003). Predicting 

the Spread of Herbicide Resistance 

in Australian Canola Fields. Transgenic 

Research 12, 731-737. 

Borrás-Hidalgo, O., Thomma, P.H., 

Bart, J., Carmona, E., Borroto, C. 

J., Pujol, M., Arencibia, A. and 

Lopez J. (2005). Identification of 

sugarcane genes induced in diseaseresistant 

somaclones upon inoculation 

with Ustilago scitaminea or Bipolaris 

sacchari. Plant Physiology 

and Biochemistry 43, 1115-1121. 

Bower, R. and Birch, R. G. (1992) 

Transgenic sugarcane plants via microprojectile 

bombardment. Plant J. 

2, 409–416. 

Braga, D. P. V., Arrigoni, E. D. B., Silva-Filho 

M. C., Ulian, E. C. (2003). 

Expression of the Cry1Ab protein in 

genetically modified sugarcane for 

the control of Diatraea saccharalis 

(Lepidoptera: Crambidae). J New 

Seeds, 5, 209–222. 

Chagas, R. M., Silveira, J. A. G., Ribeiro, 

R. V., Vitorello, V. A. and Carrer 

H. (2008). Photochemical damage 

and comparative performance of 

superoxide dismutase and ascorbate 

peroxidase in sugarcane leaves exposed 

to Paraquat-induced oxidative 

stress. Pesticide Biochemistry and 

Physiology 90, 181-188. 

Chakrabarty, R., Viswakarma, N., 

Bhat, S. R., Kirti, P. B., Singh, B. 

D. and Chopra, V. L. 

(2002). Agrobacterium-mediated 

transformation of cauliflower: optimization 

of protocol and development 

of Bt-transgenic cauliflower. 

Journal of Biosciences 27, 495-502. 

Du, Y. C., Nose, A. and Wasano, K. 

(1999). Thermal characteristics of C4 

photosynthetic enzymes from leaves 

of three sugarcane species differing 

in cold sensitivity. Plant Cell Physiology 

40,298-304. 

Enríquez-Obregón, G.A., Vázquezpadrón, 

R.I., Prieto-sansonov, 

D.L., de la Riva, G.A. and Selman- 

Housein,G. (1998). Herbicide resistant 

sugarcane (Saccharum officinarum 

L.) plants by Agrobacteriummediated 

transformation. Planta, 

206, 20-27 

Epstein, E. and Rains, D. W. (1987). 

Advances in salt tolerance. Plant & 

Soil 99, 17- 29. 

Gallo-Meagher, M. and Irvine J.E. 

(1996). Herbicide resistant transgenic 

sugarcane plants containing the bar 

gene. Crop Science, 36 (5), 1367- 

1374. 

Gilbert, R. A., Gallo-Meagher, M., 

Comstock, J.C., Miller, J.D., Jain, 

M. and Abouzid, A. (2005). Agronomic 

evaluation of sugarcane lines 

transformed for resistance to Sugarcane 

mosaic virus strain E. Crop Sci. 

45 , 2060–2067. 

Groenewald, J. H. and Botha, F. C. 

(2008). Down-regulation of pyrophosphate: 

fructose 6-phosphate 1- 

phosphotransferase (PFP) activity in 

sugarcane enhances sucrose accumulation 

in immature internodes. 

Transgenic Research 17, 85- 

92. 

Guimarães, E. R., Mutton, M. A., Mutton, 

M. J. R., Ferro, M. I. T., Ravaneli, 

G. C. and Silva, J. A. (2008). 

Free proline accumulation in sugarcane 

under water restriction and spittlebug 

infestation. Sci. Agric. 65, 

628-633. 

Gupta V., Raghuvanshi, S., Gupta, A., 

Saini N., Gaur, A., Khan, M. S., 

Gupta, R. S., Singh, J., Duttamajumder, 

S. K., Srivastava, S., Suman, 

A., Khurana, J. P., Kapur, R. 

and Tyagi, A. K. (2010). The waterdeficit 

stress- and red-rot-related 

genes in sugarcane. Functional Integrative 

Genomics 10 (2), 207-214. 

Hanlon, D., MacMahon, G. G., 

McGuire, P., Beattie, R. N. and 

Stringer, J. K. (2000). Managing 

low sugar prices on farms – short 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 394




term and long term strategies. In: D. 

M. Hogarth (ed.): Proceeding of 

Australian Society of Sugarcane 

Technology, 22, pp. 1–8. 

Harrison, M. D., Jason, Geijskes, 

Coleman, Heather, D., Shand, Kylie, 

Kinkema, Mark, Palupe, Anthony, 

Hassall, Rachael, Sainz, 

Manuel, Lloyd, Robyn, Miles, Stacy 

and Dale, L. James (2011). Accumulation 

of recombinant cellobiohydrolase 

and endoglucanase in the 

leaves of mature transgenic sugar 

cane. Plant Biotechnology Journal 9, 

884-896 

Henry, R. J., Edwards, M., Waters, D. 

L., Gopala Krishnan, S., Bundock, 

P., Sexton, T. R., Masouleh A. K, 

Nock, J. C. and Pattemore, J. 

(2012). Application of large-scale 

sequencing to marker discovery in 

plants. Journal of Bioscience 37, 

829–841. 

http://shodhganga.inflibnet.ac.in/bitstrea 

m/10603/42274/7/07_chapter%202.p 

df, 3.05.2017 

http://agencia.fapesp.br/en/16756, 

03.04.2017 

http://isaaa.org/resources/publications/poc 

ketk/45/default.asp, 27.03.2017 

http://isaaa.org/resources/publications/poc 

ketk/45/default.asp, 27.3.2017 

Ingelbrecht I.L., Irvine J.E. and 

Mirkov T.E. (1999). Post trancriptional 

gene silencing in transgenic 

sugarcane. Dissection of homologydependent 

virus resistance in a monocot 

that has a complez polyploid 

genome. Plant Physiol. 119, 1187- 

1198. 

Iskandar, H. M., Casu, R.E., Fletcher, 

A. T., Schimdt, S. Xu, J. Maclean. 

D.J., Manners, J. M. and Bonnett 

G.D. (2011). Identification of 

drought response genes and a study 

of their expression during sucrose 

accumulation and water deficient in 

sugarcane culms. BMC Plant Biol., 

11 , 12 

Jangpromma, N., Kitthaisong, S., 

Lomthaisong, K., Daduang, S., 

Jaisil, P. P. and Thammasirirak S. 

(2010). A proteomics analysis of 

drought stress-responsive proteins as 

biomarker for drought tolerant sugarcane 

cultivars. American Journal of 

Biochemistry & Biotechnology 6 (2), 

89-102. 

Jenes, B., Moore, H., Cao, J., Zhang, 

W. and Wu, R. (1993). Techniques 

for gene transfer in Transgenic 

Plants. Kung S, Wu R (eds), San Diego: 

Academic Press Inc 1, 125-146. 

Joyce P., McQualter R., Handley J., 

Dale J., Harding R., Smith G. 

(1998). Transgenic sugarcane resistant 

to sugarcane mosaic virus. 

Proceedings-Australian Society of 

Sugar Cane Technologists (Salisbury: 

Watson Ferguson and Company), 

204–210. 

Kinkema, M., Geijskes, J., Delucca, 

P., Palupe, 

A., Shand, 

K., Coleman, H. D., Brinin, 

A., Williams, B., Sainz, M., Dale, J. 

L. (2014). Improved molecular tools 

for sugar cane biotechnology. Plant 

Molecular Biology, 84(4-5), 497- 

508. 

Kumar, T., Uzma, Khan, M. U., Abbas, 

Z. and Ali, G. M. (2014). Genetic 

improvement of sugarcane for 

drought and salinity stress tolerance 

using Arabidopsis Vascular pyrophosphatase 

(AVPi) gene. Molecular 

Biotechnology 56(3), 199-209. 

Lima, M.L.A., Garcia, A.A. F., 

Oliveira, K.M., Matsuoka, S. Arizono, 

H. et al., (2002). Analysis of 

genetic similarity detected by AFLP 

and coefficient of parentage among 

genotypes of sugar cane (saccharum 

spp.) Theoretical and Applied Genetics, 

104, 30-38. 

Marshall, A. (2014). Drought tolerant 

varieties begin global march. Nature 

Biotechnology, 32, 308. 

McQualter, R. B. and Dookun- 

Saumtally, A. (2007). Expression 

profiling of abiotic-stress-inducible 

genes in sugarcane. Proceeding of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 395




Australian Society of Sugar Cane 

Technology 29, pp. 878-888. 

McQualter, R. B., Chong, B. F., Meyer, 

K., Van, Dyk D. E., Oshea, M. G., 

Walton, N. J., Vitanen, P. V. and 

Brumbley, S. M. (2005). Initial 

evaluation of sugarcane as a production 

platform for a p-hydroxybenzoic 

acid. Plant Biotech Journal 2, 1-13 

Molinari, H. B. C., Marur, C. J., Daros, 

E., Campos, M. K. F., Carvalho, J. 

F. R. P., Bespalhok, J. C. Filho, 

Pereira, L. F. P. and Vieira, L. G. 

E. (2007). Evaluation of the stressinducible 

production of proline in 

transgenic sugarcane (Saccharum 

spp.): Osmotic adjustment, chlorophyll 

fluorescence and oxidative 

stress. Physiology Plant 130, 218- 

229. 

Nair, N.V. (2011). Sugarcane varietal 

development programmes in India: 

An overview. Sugar Tech 13(4), 275- 

280. 

Nel, S. (2009). The potential of biotechnology 

in the sugarcane industry: are 

you ready for the next evolution? 

Proceeding of Sugar African Sugar 

Technology Association 82, 225– 

234. 

Nogueira, F. T. S., Vicente, V. E. D., 

Menossi, Jr. M., Ulian, E. C. and 

Arruda, P. (2003). RNA expression 

profiles and data mining of sugarcane 

response to low temperature. 


Park, J. W., Benatti, T. R., Marconi, T., 

Q.Yu, N. Solis-Gracia, V. Mora 

and Silva, J. A. da (2015). Cold responsive 

gene expression profiling of 

sugarcane and Saccharum spontaneum 

with functional analysis of a cold 

inducible Saccharum homolog of 

NOD26- like intrinsic protein to salt 

and water stress. PloS One 10(5), 

e0125810. doi:10.1371/journal. 

pone.0125810. 

Patade, V. Y., Suprasanna, P. and 

Bapat, V. A. (2008). Effects of salt 

stress in relation to osmotic adjustment 

on sugarcane (Saccharum officinarum 

L.) callus cultures. Plant 

Growth Regulation 55,169-173. 

Petrasovits, L., Purnell A., Matthew, 

P., Nielsen, K. Lars and Brumbley, 

M. Stevens (2007). Production of 

polyhydroxybutyrate in sugarcane. 

Plant Biotech Journal 5, 162- 

172. 

Prabu, G. R., Kawar, P. G., Pagariya, 

M. C. and Prasad, D. Theertha 

(2011). Identification of water deficit 

stress upregulated genes in sugarcane. 

Plant Molecular Biology Reporter 

29, 291-304. 

Que, Y., Su, Y., Guo, J., Wu, Q. and 

Xu, L. (2014). A global view of transcriptome 

dynamics during Sporisorium 

scitamineum challenge in sugarcane 

by RNAseq. PloS One 9(8), 

e106476. 

doi:10.1371/journal.pone.0106476. 

Roach, B. T. (1989). Origin and improvement 

of the genetic base of 

sugarcane. Proceedings of the Australian 

Society of Sugar Cane Technology, 

34–47. 

Shaik, M.M., Hossain, M.A., Khaton, 

M.M. and Nasiruddin, K.M. 

(2007). Efficient Transformation of 

Stress tolerance Glygene in Transgenic 

Tissue of Sugarcane (Saccharum 

officinarum L.) Mol. biol. 

biotechnol. J., 5(1&2): 37-40. 

Shanthi, R. M. (2016). Pre-breeding for 

developing high sucrose genetic 

stocks in sugarcane. Winter School 

on Biotechnological and conventional 

approaches for biotic and abiotic 

stress management in sugarcane, pp 

Sathyabhama, M., Viswanathan, R., 

Malathi, P. and Sundar, A. R. 

(2016). Identification of differentially 

expressed genes in sugarcane during 

pathogenesis of Colletotrichum 

falcatum by suppression subtractive 

hybridization (SSH). Sugar Tech 18, 

176. 

Schuler. T. H., Poppy, G. M., Kerry, B. 

R. and Denholm, I. (1998). Insect 

resistant transgenic plants. Trends 

Biotechnology 16,168-175. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 396



Shrivastava, A. K. and Srivastava, S. 

(2012). Sugarcane: Physiological and 

molecular approaches for improving 

abiotic stress tolerance and sustaining 

crop productivity. In Improving 

Crop Resistance to Abiotic Stress 

(Tuteja N., S S. Gill, A.F. Tiburcio 

and R. Tuteja, eds.) Vol. 2, Wiley- 

Blackwell, Germany, pp. 885-922. 

Shrivastava, A. K., Srivastava, T. K., 

Srivastava, K. Arun, Misra, Varucha, 

Shrivastava, Sangeeta, 

Singh, V. K. and Shukla, S. P. 

(2016). Climate change induced abiotic 

stresses affecting sugarcane and 

their mitigation. ICAR-Indian Institute 

of Sugarcane Research, Lucknow, 

Pp. 108 

Singh, R. K., Kumar, P., Tiwari, N. N., 

Rastogi, J., Singh, S. P. (2013). 

Current Status of Sugarcane Transgenic: 

an Overview. Advance Genetic 

Engeneering 2,112. doi: 

10.4172/2169-0111.1000112. 

Souza, G. M., Berges, H., Bocs, S., 

Casu, R., D’Hont, A., Ferreira, J. 

E., Henry, R., Ming, R., Potier B., 

Van Sluys M. S., Vincetz M. and 

Paterson A. H. (2011). The sugarcane 

genome challenge: strategies for 

sequencing a highly complex genome. 

Tropical Plant Biology 4, 

145–156. doi:10.1007/s12042-011- 

9079-0. 

Tiwari, A. K., Tripathi, Shivanand, 

Lal, Madan and Rao, G. P. (2010). 

Biotechnological approaches for improving 

sugarcane crop with special 

reference to disease resistance. Acta 

Phytopathologica et Entomologica 

Hungarica 45(2), 235-349. 


Trujillo, L. E., Menéndez, C., Ochogavía, 

M. E., Hernández, I., 

Borrás, B., Rodríguez, R., Coll, Y., 

Arrieta, J.G., Banguela, A., Ramírez, 

R. and Hernández, L. (2009). 

Engineering drought and salt tolerance 

in plants using SodERF3, a 

novel sugarcane ethylene responsive 

factor. Biotechnology Applicada 26 

(2),168-171. 

Usman Shehu Inuwa (2015). Biotechnology 

interventions for production 

of good quality seed canes. International 

Journal of Scientific Research 

and Innovative Technology 2(9), 96- 

104. 

Wahid, A and Close, T.J. (2005). Expression 

of dehydrins under heat 

stress and their relationship with water 

relations of sugarcane leaves. Biol. 

Plantarum. 51, 104-109. 

Waltz, E. (2014). Beating the heat. Nature 

Biotechnology 32(7), 610-613. 

Wang, M. L., Goldstein, C., Su W., 

Moore, H. Paul, and Henrik, H. 

Albert (2005). Production of biologically 

active GM-CSF in sugarcane: 

a secure biofactory. Transgenic Research 

14, 167-178. 

Wu, L. and Birch, R. G. (2007) Doubled 

sugar content in sugarcane plant 

modified to produce a sucrose isomer. 

Plant Biotech Journal 5,109– 

117. 

Zhang, L. Xu, J and Birch, R. G. 

(1999). Engineered detoxication confers 

resistance against a pathogenic 

bacterium. Nature Biotechnology, 17, 

1021-1024. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 397




Niharika Chandra, Ankita Srivastava, Swati Srivastava, Shailesh Kumar Mishra and 

Sunil Kumar* 

Faculty of Biotechnology, Institute of Biosciences and Technology, Shri Ramswaroop 

Memorial University, Barabanki, Uttar Pradesh, India;*Correspondence: 

sunil.bio@srmu.ac.in / sunilsbt@gmail.com 

Abstract: The term bioremediation refers to the use of natural biological agents such as 

microbes (bacteria, fungi, and yeast), plants or the enzymes released by them to return the 

polluted natural environment to its original uncontaminated state. Bioremediation is a 

novel, safe, cost effective, and ecologically feasible technology to detoxify accumulated 

pesticides, toxic chemicals, fertilizers, aromatic compounds, xenobiotics, hydrocarbons (oil 

spills), heavy metals in soil and water. Both in situ and ex situ bioremediation are being 

used at a large number of sites all around the world with varying level of success. Although 

bioremediation cannot degrade all the toxicity, particularly all the inorganic contaminants, 

but it is still more eco-friendly and less expensive as compared to other remediation 

methods like incineration, chemical treatment and thermal recovery of pollutants. 

Furthermore, advances in bioremediation are being achieved by coupling this biological 

method with molecular, genetic engineering, microbiology, pathway engineering, and 

enzyme design and immobilization tools. In this chapter we have discuss the general 

process for bioremediation, in situ and ex situ classes of bioremediation followed by the 

types of bioremediation techniques being used till date. The role of bioremediation to deal 

with different types of pollutions and comprehend the advantages, disadvantages and 

sustainable use of its approaches is also highlighted. 

Keywords: Ex situ bioremediation; GMOs; in situ bioremediation; microbes; pollutants 


A rapid increase in human 

population accompanied with 

technological advancements in fields 

related to agriculture, industries and 

health has led to accumulation of various 

toxic chemicals and xenobiotic 

compounds in our environment. 

Indiscriminate use of fertilizers and 

pesticides in agriculture, poor handling of 

wastewater and solid waste, untreated 

release of polluted discharge from 

industries has led to shortage of clean 

water sources and disturbances in soil 

content and quality (Kamaludeen et al. 

2003). Contamination of soil with heavy 

metals, xenobiotic compounds and 

municipal waste is responsible for loss of 

biodiversity and functional aspects such 

as cycling of nutrients (Su et al., 2014). 

Similarly, increased pollution in aquatic 

ecosystem is resulting in decreased purity 

and content of ground water as well as 

surface fresh water (Zhang et al., 2011). 

Hence, we are in urgent need to seek a 

feasible and efficient system to manage 

such pollutants. 

Bioremediation is the use of living 

organisms, primarily microorganisms, to 

degrade the environmental contaminants 

into less toxic forms. The process 

involves the degradation or detoxification 

of hazardous substances, which are 

harmful to human health and/or the 

environment, with the help of naturally 

occurring bacteria, fungi and plants. 

Microorganism which perform the 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 398



function of bioremediation are known as 

Bioremediators (Kumar et al., 2011). 

Bioremediation is a natural, effective and 

environment friendly alternative to 

previously used methods for degradation 

of harmful contaminants, such as physical 

removal, absorbents, catalytic destruction, 

incineration etc. which are high cost and 

nonspecific methods (Head and Swannell, 

1999; Gillespie and Philp, 2013) 

Although use of microbial 

consortia have proved their capability for 

remediation, application of biotechnology 

and genetic engineering tools is further 

improving the efficiency and decreasing 

the cost involved in treating toxic 

substances . Bacteria, fungi, yeast and 

algae along with several plants are being 

used for this purpose. 

We will discuss the general 

process for bioremediation, in situ and ex 

situ classes of bioremediation followed by 

the types of bioremediation techniques 

being used till date. The role of 

microorganisms in bioremediation as well 

as their genetic modification by the use of 

recombinant DNA technology will be 

understood. Further we will discuss the 

role of bioremediation to deal with 

different types of pollutions and 

comprehend the advantages, 

disadvantages and sustainable use of 

bioremediation approaches. 

2. Bioremediation 

Bioremediation is the branch of 

biotechnology that deals with the 

solutions of problems related to the 

environment. Bioremediation also 

involves the process of cleaning the 

environment from different types of 

pollutants as well as contaminants by 

using bacteria, fungi etc. Bacteria play a 

vital role in the process of bioremediation 

since it break down the dead materials 

into organic matter and nutrients. Several 

types of contaminants such as chlorinated 

pesticides etc. can be easily digested by 

Chandra et al. 

bacteria. Also oil spills can be treated by 

bacteria (Agarwal and Liu, 2015). 

Recently it has been noticed that 

the awareness of the dangers of many 

chemicals used in our society has led to 

research on formulation of products that 

are more easily degraded in the 

environment. The process of 

bioremediation involves the degradation 

of contaminants using microorganisms 

that have adverse impact on environment 

and humans. Bioremediation includes the 

actions of several microorganisms that are 

acting in parallel or sequence, in order to 

complete the process of degradation. In 

this process, both in situ as well as ex situ 

remediation are used. Hence, 

bioremediation is a technology applied in 

case of different environmental conditions 

where the numerous and versatile 

microbes degrades a vast array of 

pollutants (Majone et al., 2015). 

On the basis of ecological point of 

view, the term bioremediation involves 

the interactions between three factors that 

is substrate (pollutant), organisms, and 

environment, as shown in Figure 1. The 

interactions between these three factors 

primarily affect the biodegradability and 

bioavailability of pollutants, and 

physiological requirements of microbes, 

which plays a vital role in the assessment 

of the feasibility of bioremediation. 

Biodegradability defines whether any 

chemical can be degraded by microbes or 

not, whereas bioavailability refers to the 

availability of a pollutant to organisms 

that are capable of degrading it. For 

instance, the substrate has low 

bioavailability if it is tightly bound to soil 

organic matter or it is trapped inside its 

aggregates. The conditions that are 

required by microorganisms to carry out 

the process of bioremediation include 

different factors like nutrient availability, 

optimal pH, and availability of electron 

acceptors, such as oxygen and nitrate etc. 

are referred to as physiological 

requirements. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 399




Figure 1: Bioremediation from an environment perspective. 

Table 1: Summary of bioremediation approaches 

S.N. Approaches About Advantages Disadvantages Examples 

1. In situ At the 

site 

2. Ex situ Away 

from the 

site 

Non-invasive 

Most cost efficient 

Relatively passive 

Treats soil and 

water 

Can be done on 

site 

Low cost 

Time efficient 

3. Bioreactors Rapid degradation 

kinetic 

Enhances mass 

transfer 

Monitoring 

difficulties 

Extended 

treatment time 

Environmental 

constraints 

Space 

requirements 

Need to control 

abiotic loss 

Bioavailability 

limitation 

Mass transfer 

problem 

High cost capital 

High operating 

cost 

Bioventing 

Bioaugmentation 

Biosparging 

Landfarming 

Biopiles 

Composting 

Slurry reactors 

Aqueous 

reactors 

3. Classes of bioremediation 

3.1. In situ bioremediation (ISB) 

In situ bioremediation (ISB) is the class of 

bioremediation that is performed at the 

original site of contamination. There is no 

excavation or removal of polluted 

soil/ground water to any secondary 

location for conduction of remediation 

process. In situ remediation includes 

techniques such as bioventing, 

biosparging, bioslurping and 

phytoremediation along with physical, 

chemical, and thermal processes. This 

class of bioremediation technology is 

beneficial because of its low cost, more 

effective method as an alternative to the 

standard pump and treats methods that are 

used to clean up aquifers and soils 

contaminated with organic chemicals 

including fuel hydrocarbons, chlorinated 

solvents. ISB has the potential to 

provide advantages such as complete 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 400



destruction of the contaminants, lower 

risk to site workers, and lower equipment 

or operating costs (Koning et al., 2000; 

Vidali. 2001). 

3.2. Ex-situ bioremediation (EXB) 

Ex-situ bioremediation (EXB) is 

defined as a biological process which 

involves excavation of polluted soil or 

pumping of groundwater that is placed in 

a lined above-ground treatment area to 

facilitate microbial removal of 

contaminants. Ex situ remediation 

includes techniques such as Landfarming, 

biopiling, and processing by bioreactors 

along with thermal, chemical, and 

physical processes (Koning et al., 2000). 

Ex situ remediation is a more thorough 

remediation technique, but due to the 

costs associated not only with the 

remediation processes, but also with the 

excavation and transportation of the soil, 

many people are looking towards in situ 

remediation techniques (Vidali, 2001) as 

depicted in Table 1. 

4. Types of bioremediation 

For the better understanding of 

bioremediation and for convenience of the 

study it may be divided into following 

types according to biological agents used 

for the treatment of toxicants: 

4.1. Phytoremediation 

In this method plants are used to 

remove pollutants from the environment. 

Phytoremediation targets include 

contaminating metals, metalloids, 

petroleum hydrocarbons, pesticides etc. In 

comparison to conventional purification 

technologies, phytoremediation is a cost 

effective one. This technology is the main 

driving force of the researches done in 

this area resulting into commercialization 

of the technology. Commercial 

phytoremediation systems for clean-up of 

shallow aquifers and water borne 

contaminants are now in the market. 

Today, we know about the plants which 

purify air by absorbing toxic gases and 


metals by the application of 

biotechnology (Li and Li, 2017). Such 

types of transgenic plants are being 

developed which can remove more and 

more contaminants from the environment. 

Sometimes it is also referred to as „green 

clean‟ which means cleaning up of 

environment with the help of plants. Some 

plants, most notably, the Chinese brake 

fern (Pteris vittata) has been reported to 

be suitable for arsenic phytoremediation 

(Alkorta et al., 2004). An American 

patent registered in 1994 describes how 

genetically altered members of the family 

Brassicaceae (family of scavengers) like 

Brassica juncea have shown tremendous 

potential for clean-up of polluting metals 

through their roots. The plants accumulate 

metals to levels between 30 and 1000 

times higher than their concentration in 

the surrounding soil. The metals absorbed 

by various members of the Brassica plant 

family include antimony, arsenic, barium, 

beryllium, cadmium, cesium, chromium, 

cobalt, copper, gold, both stable and 

unstable form of lead, manganese, 

mercury, molybdenum, nickel, palladium, 

plutonium, selenium, silver, strontium, 

uranium, vanadium, zinc etc. 

Dust is a major air pollutant and 

around 40% of total air pollution in India 

is contributed by dust pollution. Based on 

extensive field surveys and experimental 

studies, the following species have been 

recommended by NBRI, Lucknow for 

raising green belts around industrial and 

urban areas to reduce the dust load: 

Ipomoea fisstulosa, Peltophorum 

pterocarpum, Tectona grandis, Ficus 

bengalensis, F. infectoria, Terminalia 

arjuna. 

Following are examples of some plants 

tolerant to gaseous pollutants: 

Plants tolerant to SO 2 : Polyalthia 

longifolia, Terminalia arjuna, Acer 

platanoides, Thuja orientalis. 

Plants tolerant to Ozone (Bowler and 

Fluhr, 2000): Zinnia elegans, Gladiolus 

spp., Pelargonium graveolens. 

Plants tolerant to NOx: Carrisa carandas, 

Codiaeum variegatum. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 401



Plants tolerant to PAN: Acer platanoides, 

Chrysanthemum spp. 

4.2. Rhizofiltration 

In this form of phytoremediation 

roots of plants act as biofilters i.e. these 

absorb toxic metals from the 

contaminated water and accumulate them. 

Therefore, the water which passes 

through root zone becomes free from 

pollutants (Macek et al., 2004). Later, the 

contaminants are removed from the 

system by harvesting root biomass. 

Members of family Brassicaceae (the 

family of scavengers) have shown 

tremendous potential for the cleanup of 

polluting metals. The plants roots 

accumulate metals from the surrounding 

soil giving a metal content as high as 30% 

of the dry weight of the plant roots. 

Alan Baker, a geobotanist from 

University of Sheffield, England, 

discovered a tree Sebertia acuminate (the 

nickel lover) which hyper-accumulates Ni 

so much that when slashed, it bleeds a 

jade green liquid. The tree is a native of 

New Caledonia and accumulates Ni as 

high as 20% of its dry body weight. 

Besides, Acedium plant absorbs 

Vanadium and some plants of family 

papilionaceae (e.g. Pea) are known to 

absorb higher amounts of Molybdenum 

from soil. 

4.3. Microbial bioremediation 

Microorganisms like bacteria are 

proving very important tools for the 

removal of pollutants from the 

environment and are thus continuously 

doing their job of detoxification, 

tirelessly, with or without coming into the 

attention of man, who is the only culprit 

of dumping more and more pollutants into 

the environment. In the recent past, 

microbes were first used to treat industrial 

wastewater as early as 1930s. There was a 

significant movement in the field of 

microbial bioremediation when Dr. 

Howard Worne (USA) first discovered 

phenol degrading microbes, when began 


his research in this field in the early 

1950s. 

In another breakthrough, Dr. 

Ananda Mohan Chakrabarty of the 

General Electric Company (USA) 

developed a genetically engineered oil 

eating bacterium (Pseudomonas putida). 

The patent was registered to him in 

1980s. It was welcomed by the scientists‟ 

community as an answer to pollution 

problem. The Environment Protection 

Agency (EPA) reported that 

bioremediation eliminated both soil and 

water borne oil contamination at about 

1/5 th cost of previous method. Since then, 

bioremediation has been increasingly 

used to clean up oil pollution in United 

States and in other countries. In 1989, oil 

spilled from the Exxon Valdez tanker off 

the coast of Alaska where these oil eaters 

helped in clean-up of oil. They degraded 

the oil efficiently trapped between rocks 

and under gravel beaches where all other 

means had failed. 

4.4. Zooremediation 

It is the process of 

decontamination of polluted environment 

by using animals as bioagents. Animals so 

far used for bioremediation purposes are 

fish, different arthropods and other filter 

feeders in aquatic systems and 

earthworms in solid organic waste 

management systems. Use of animals in 

bioremediation is not very encouraging 

except earthworms because there are so 

many limitations with them e.g. fish and 

arthropods may bioaccumulate the toxic 

compounds and metals which may be 

biomagnified in the food chain creating 

many problems to the environment 

(Gifford et al., 2007). 

Solid organic waste generation is a 

major problem of cities which are facing 

the threat of being overrun by garbage 

and the piled up waste and is adversely 

threatening our health, environment and 

wellbeing. In this context, 

vermicomposting - waste degradation 

through earthworms has proved to be very 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 402




Figure 2: Ex-situ bioremediation by composting. 

promising. The principles behind this are 

relatively simple and related to those 

involved in traditional composting 

(Figure 2 summarizes composting as a 

basic Ex-situ bioremediation process). In 

general, vermicomposting consists of 4 

major phases: Phase I: Collection of the 

waste, separation of metal, glass, 

ceramics etc. from the organic waste, and 

storage of the organic waste. 

Phase II: Earthworm beds are 

maintained and the earthworms are fed 

with the organic waste. 

Phase III: After the organic waste 

has been worked over by the earthworms, 

the vermicompost, cocoons, earthworms 

and the undigested material are separated. 

Phase IV: Packaging of the 

vermicompost and reintroduction of 

undigested material into the vermipits. 

Certain species of earthworms 

(Eisenia fetida, E. Andrei, Lumbricus 

rubellus, L. hortensis, L. terristris etc.) 

can consume organic residues very 

rapidly and fragment them into much 

finer particles by passing them through a 

grinding gizzard, an organ that all 

earthworms possess. The earthworms 

derive their nourishment from the 

microorganisms that grow upon the 

organic materials. At the same time they 

promote further microbial activity in the 

residues so that the faecal matter or casts 

that they produce are much more 

fragmented. During this process, the 

important plant nutrients in the organic 

material particularly nitrogen, 

phosphorus, potassium and calcium are 

released and converted into forms that are 

much more soluble and available to plants 

than those in the parent compounds. 

Worms can digest waste several times 

their own weight each day. 

4.5. Bioventing 

Bioventing is a process of 

stimulating the natural in situ 

biodegradation of contaminants in soil by 

providing air or oxygen to existing soil 

microorganisms. Bioventing uses low air 

flow rates to provide only enough oxygen 

to sustain microbial activity in the vadose 

zone (Hinchee, 1994). This is an on-site 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 403



or in-situ bioremediation option for 

reducing or eliminating contaminants in 

soil and water. With benefits that include 

minimal site disturbance and lower cost 

compared to other remediation 

technologies, in-situ bioremediation 

continues to be researched and applied 

with the goal of helping 'close out' 

specific sites, that is, reducing toxins to 

safe and/or legally acceptable levels 

(Agency, 1995, Gibbs et al., 1999). 

Among the most promising of 

these technologies is soil bioventing, the 

process of supplying oxygen to 

contaminated soil in hopes of stimulating 

microbial degradation of contaminants. A 

typical bioventing setup is appealingly 

simple: a blower or compressor connected 

to one or more air-supply wells and a 

series of soil-gas monitoring wells 

(Sellers, 1999). The technology of choice 

for remediating many petroleum wastes, 

bioventing may eventually be used to 

treat a wider variety of more recalcitrant 

toxins (McCauley, 1999a). Bioventing 

has noteworthy remediation relatives, 

with distinct principles, goals and 

applications. Air sparging forces 

compress air into saturated soil in hope of 

promoting biodegradation. Unlike 

sparging, bioventing uses low-pressure air 

and is generally focused on the vadose or 

unsaturated zone of soil (McCauley, 

1999b). Bioslurping combines bioventing 

and direct vacuum extraction of 

contaminants. Soil vapor extraction or 

soil vacuum extraction (SVE) maximizes 

volatilization of contaminants and sucks 

them out of the soil. Bioventing began to 

mature as a technology after 1988, when 

researchers on a SVE operation at Hill Air 

Force Base, Utah, concluded that a 

significant proportion of contaminant 

decrease was not due to volatilization, but 

biodegradation (Agency, 1995; Litchfield, 

1993). 

4.6. Bioleaching 

This principle of bioleaching 

involves the use of living organisms like 

microbes in the metal extraction from 


their ores. It is one of several applications 

within biohydrometallurgy and several 

methods are used to recover Cu, Zn, Pb, 

As, Ni, Mo, Au, Ag, and Co. 

Heterotrophic bacteria are widely used in 

the study of bacterial leaching of 

manganese from manganese dioxide ores 

and glucose or other organic compounds 

are used as a source of energy, rendering 

their commercial utilization uneconomic 

(Cornu et al. 2017). Coal, especially 

brown coal from certain coal mines may 

contain a certain amount of rare metals, 

such as Germanium (Ge) and Gallium 

(Ga). The conventional process to recover 

Ge from brown coal is a lengthy process 

involving many steps, i.e. burning of the 

brown coal, recovering of Ge from ashes 

by sulphuric acid leaching, precipitating 

of Ge with tannin, roasting of Gecontaining 

tannin to produce Ge 

concentrate with the grade of 11%. This 

process is complex and has a low 

recovery of 60%, which is sure to bring 

about a great waste of resource. Instead, a 

novel process to recover Ge from brown 

coal in the presence of microorganism has 

been developed where a germanium 

recovery of up to 85.33% has been 

achieved. 

4.7. Landfarming 

Landfarming is a process in which 

the soil is excavated and mechanically 

separated via sieving. In land farming, 

which is performed in the upper soil zone 

or in biotreatment cells, contaminated 

soils are mixed with soil amendments 

such as soil bulking agents and nutrients 

and then they are tilled into the earth. 

Contaminated soils, sediments, or sludges 

are incorporated into the soil surface and 

periodically turned over or tilled to aerate 

the mixture. The material is periodically 

tilled for aeration. Contaminants are 

degraded, transformed, and immobilized 

by microbiological processes and by 

oxidation. Soil conditions are controlled 

to optimize the rate of contaminant 

degradation (Datta et al., 2016). Moisture 

content, frequency of aeration, and pH are 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 404



all conditions that may be controlled. 

Land Farming differs from composting 

because it actually incorporates 

contaminated soil into soil that is 

uncontaminated. Landfarming is most 

successful in removing polycyclic 

aromatic hydrocarbons (PAH) and 

pentchlorphenol (PCP). 

4.8. Bioreactor 

Bioreactors treat contaminated 

soils in both solid and liquid (slurry) 

phases. The solid phase treatment process 

mechanically decomposes the soil by 

attrition and mixing in a closed container. 

The objective of the mixing is to 

guarantee that the pollutants, water, air, 

nutrients, and microorganisms are in 

permanent contact. An acid or alkalinity 

may also be added to control the pH (van 

Deuren and Lloyd, 2002). Infixed bed 

reactors, compost are added and 

significantly increase the degradation rate. 

In rotating drum reactors, the drum has a 

screw like mechanism in the middle of it 

that rotates to mix and transport the soil. 

The liquid phase treatment process uses 

suspension bioreactors and treats as 

slurry. The slurry feed enters the system 

and is rinsed through a vibrating screen to 

remove debris. Sand is then removed 

using a sieve or hydrocyclone. If a 

hydrocyclone is used to remove the sand, 

the sand falls to the bottom of the cyclone 

and the fines remain on top. The fines are 

then treated in a bioreactor. After the 

treatment, the slurry must be dewatered 

and the water is then treated with standard 

wastewater techniques (Kleijntjens and 

Luyben, 2000). 

4.9. Biopiling 

It is an in situ process that is also 

known as the heap technique. The first 

step in the biopiling process is to perform 

laboratory tests that will determine the 

biological degradation capabilities of the 

soil sample. The next step involves the 

mechanical separation of the soil, which 

will homogenize the sample and remove 

any disruptive material such as plastics, 


metals, and stones. The stones will then 

be crushed into smaller pieces and then 

depending on the degree of contamination 

will either be added to a pile or sent out 

for reuse. The soil is then homogenized, 

meaning that the pollution concentration 

is averaged out across the entire soil 

sample. Homogenization allows for 

biopiling to be more effective (Schulz– 

Berendt, 2000). Once the soil is piled, 

nutrients, microbes, oxygen, and substrate 

are added to start the biological 

degradation of the contaminants. The 

results of the initial laboratory tests 

indicate to the operators which substrates 

such as bark, lime, or composts needs to 

be added to the soil. Nutrients such as 

mineral fertilizers may also be added. 

Additionally, microorganisms such as 

fungi, bacteria, or enzymes could be 

added (Schulz-Berendt, 2000). 

4.10. Bio-stimulation 

Bio stimulation could be 

perceived as including the introduction of 

adequate amounts of water, nutrients, and 

oxygen into the soil, in order to enhance 

the activity of indigenous microbial 

degraders. The concept of bio stimulation 

is to boost the intrinsic degradation 

potential of a polluted matrix through the 

accumulation of amendments, nutrients, 

or other limiting factors and has been 

used for a wide variety of xenobiotics 

(Kadian et al., 2008). Microorganisms do 

extremely well in thriving on herbicide 

compounds in the soil by utilizing them as 

a supply of nutrients and energy. Many 

herbicides serve as good carbon and/or 

nitrogen sources for soil microorganism 

(Qiu et al., 2009). Evidence for their 

remarkable range of degradative abilities 

can be seen in the recycling rather than 

accumulation of vast quantities of 

biological materials that have been 

produced throughout the history of life on 

earth (Dua et al., 2002). 

4.11. Bio-augmentation 

Bio augmentation is the 

enhancement of biodegradation of waste 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 405



and contaminants in the media by the 

introduction of adapted competent 

microbes and nutrients. Microorganisms 

from Geobacteraceae family due to their 

physiological characteristics can play an 

important role in the bioremediation of 

subsurface environments contaminated 

with organic or metal contaminants 

(Lovley et al., 2004). In some instances, 

the rate of biological degradation can be 

increased through the addition of 

microorganisms that have been shown to 

degrade the contaminants of concern at 

high rates or are particularly well suited to 

remain active under prevailing site 

conditions. This process is referred to as 

bio augmentation. This can be useful if 

the contaminants are particularly 

recalcitrant to degradation or if site 

conditions are extreme (for example: high 

concentrations or toxicity of 

contaminants). To be effective, the 

introduced organism(s) must become 

distributed throughout the contaminated 

matrix and compete with the indigenous 

microorganisms for available nutrients. If 

they are not distributed throughout the 

matrix the positive effect will be 

localized. On the other hand if the 

introduced organisms compete poorly, 

they will not persist and the treatment 

effect will be short lived. The problems 

encountered using this approach include 

biofouling of equipment, injection wells 

and seepage beds. Adjustments to the 

system, such as the use of new discharge 

areas, may be required to prevent this 

from occurring. This approach to 

bioremediation must be evaluated on a 

site specific basis. 

4.12. Intrinsic bioremediation 

Often bioremediation can be 

accomplished without human intervention 

by microorganisms that are naturally 

found in the contaminated matrix. For 

this approach to be used, it is usually 

necessary for the rate of contaminant 

degradation to exceed the rate of 

contaminant migration. Knowledge of the 


following key site characteristics are 

required to evaluate the likely success of 

intrinsic remediation; the bioavailability 

of contaminants, levels of nutrients, the 

presence of minerals to buffer the pH of 

the matrix, adequate levels of electron 

acceptors (either oxygen, nitrate, ferric 

iron, or sulphate), and site specific 

contamination migration rates. This 

approach deals with stimulation of 

indigenous or naturally occurring 

microbial populations by feeding them 

nutrients and oxygen to increase their 

metabolic activity. 

5. Microbes involved in bioremediation 

Microorganisms are responsible 

for biodegradation in various diverse 

environmental conditions. These 

microorganisms include: Acinethobacter, 

Actinobacter, Acaligenes, Arthrobacter, 

Bacillins, Berijerinckia, Flavobacterium, 

Methylosinus, Mycrobacterium, 

Mycococcus, Nitrosomonas, Nocardia, 

Penicillium, 

Phanerochaete, 

Pseudomonas, Rhizoctomia, Serratio, 

Trametes and Xanthofacter. Individual 

microorganisms are not efficient in 

mineralization of harmful substances. 

Thorough mineralization results in a 

progressive degradation by a group of 

microorganisms (or microbial 

consortiums) and involves coaction and 

co-metabolism actions. Microorganisms 

in various habitats have remarkable 

physiological flexibility, so they are able 

to make use of and often mineralize an 

enormous number of organic molecules. 

Several other requirements for microbial 

growth in biodegradation process are 

listed in Table 2. Some microbes with 

specific biodegradation capabilities are 

discussed below. 

Pseudomonas putida: In context 

of bioremediation, it is a microorganism 

found in farmland soil involving high 

impact xenobiotics including 

organophosphate insecticides, petroleum 

hydrocarbons, and both monocyclic and 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 406




Table 2: Requirements for microbial growth in bioremediation process (Source: Vidali, 

2001) 

Requirement 

Description 

Nutrients The growth and activity of the microorganisms must be estimated by adequate 

maintenance and supply of nutrients. These nutrients are the basic building 

blocks of life and allow microbes to create the necessary enzymes to break down 

the contaminants. Bio-stimulation usually involves the addition of nutrients and 

oxygen to help native microorganisms. Nitrogen (ammonic, nitrate, or organic 

nitrogen) and phosphorous (orthophosphate or organic phosphorous) are 

commonly used as the limiting nutrients. In certain anaerobic systems, the use of 

trace metals (e.g. iron, nickel, cobalt, molybdenum and zinc) is generally 

preferred. 

Carbon source Carbon which is considered as the most basic element of living forms is required 

in larger quantities than other elements. Carbon contained in many organic 

contaminants may function as a carbon source for cell growth. If the organism 

involved is an autotroph CO 2 or HCO 3 in solution is required. In some cases, 

contaminant levels are too low to supply suitable levels of carbon to cell. In these 

cases the addition of carbon sources may be required. 

Electron All respiring bacteria require a terminal electron acceptor. In some cases, the 

acceptor organic contaminant may serve in this capacity. The most common electron 

acceptor in aerobic bioremediation processes is dissolved oxygen. Under 

anaerobic conditions, NO 3- , SO 3- 4 , Fe 3+ , and CO 2 may serve as terminal electron 

acceptors. Certain co-metabolic changes are carried out by fermentative and other 

anaerobic organisms, in which terminal electron acceptors are not necessary. 

Energy source In the case of primary metabolism, the organic contaminant supplies energy 

required for growth. This is not the case when the contaminant is metabolized 

via secondary metabolism or co-metabolism or as a terminal electron acceptor. If 

the contaminant does not serve as a source of energy, the addition of a primary 

substrate(s) is required. 

Soil moisture Microbial growth and activity is also affected by moisture content. The waterholding 

capacity suggested for bioremediation process may range from 25% – 

28%. 

Temperature Temperature regulates the rates of growth and metabolic activity. Surface soils 

are particularly susceptible to wide variations in temperature. Generally, 

mesophilic conditions are appropriate for most applications (with composting 

being a notable exception). 

pH 

A pH is another important factor that affects bioremediation process. If the soil is 

acidic, it is possible to raise pH by adding lime. A pH fluctuating between 6.5 

and 7.5 is generally considered optimal. The pH of most ground water (8.0–8.5) 

Absence of 

toxic metals 

Adequate 

contact 

between 

microorganisms 

and substrates 

Time 

is not considered inhibitory. 

Many contaminated sites contain a mixture of chemicals, organic and inorganic, 

which may be inhibitory or toxic to microorganisms. Heavy metals and phenolic 

compounds are of particular concerns. 

For contaminants to be available for microbial uptake it must be present in 

aqueous phase. Thus contaminants that exist as non-aqueous phase liquids or are 

sequestered within a solid phase may not be readily metabolized. For degradation 

it is necessary that bacteria and the contaminants be in contact. This is not easily 

achieved, as neither the microbes nor contaminants are uniformly spread in the 

soil. It is possible to develop the mobilization of the contaminant utilizing some 

surfactants such as sodium dodecyl sulphate (SDS). 

Time is an important factor in the start-up of bioremediation systems. Even the 

above mentioned parameters are met, lag phases are often observed prior to the 

onset of activity. In some cases, the intense bacterial population shifts that are 

required for bioremediation will increase periods of slow activity. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 407



polycyclic aromatics (Iyer and Damania, 

2016). 

Dechloromonas aromatic: This 

bacterium is involved in degradation of 

aromatic compounds like benzene in 

nitrate reducing conditions as well as 

physiological and molecular 

characterization in anaerobic mixed 

cultures (Ulrich and Edwards, 2003). 

Deinococcus radiodurans – In 

field of development of bioremediation 

strategies, this bacterium plays a role as a 

radiation resistant organism. It is used for 

the treatment of mixed radioactive wastes 

containing ionic mercury (Brim et al., 

2000). The radioactive waste sites can be 

treatedby this strategy of bioremediation. 

Methylibium petroleiphilum – 

Also known as PM1 strain that is 

involved in methyl tert butyl ether 

(MTBE) bioremediation. MTBE is 

degraded by this strain using the 

contaminants as source of carbon and 

energy (Hanson et al., 1999). 

Alcanivorax borkumensis is a rodshaped 

bacterium having capability of 

consuming hydrocarbons and produces 

carbon dioxide. Hence it can be used 

readily in oil damaged environment 

(Santisi et al., 2015). 

Phanerochaete chrysosporium – It 

is the first fungi involved in degradation 

of organic pollutants (Kadri et al., 2017). 

6. Genetically modified organisms 

Bioremediation by means of 

microorganisms is not significant for 

treatment of all types of pollutants. For 

example, heavy metals such as cadmium 

and lead are not freely absorbed or taken 

by organisms. The role of genetically 

modified organisms in the process of 

bioremediation has emerged as a new tool 

(Jafari et al., 2013). A genetically 

modified organism, or GMO, is an 

organism that has an altered DNA 

configuration made through genetic 

engineering. Most of the genetically 

modified organisms have been 

transformed with DNA from other 


organisms like bacteria, plant, virus or 

animal and thus these are also referred to 

as transgenic organisms (Ozcan et al., 

2012). 

6.1. Role of GMOs in environmental 

management 

Genetically modified organisms 

can be used to clean up the environment 

by bioremediation. Effects of some 

genetically modified microorganisms are 

unstable and vary according to species, 

changes in population structure and loss 

of some functions, to the formation of 

toxic metabolites. Presence of high and 

active microorganisms makes the process 

of bioremediation more operative and 

they must adapt with the changing 

environmental conditions. Deinococcus 

radiodurans that exhibit toluene 

dioxygenase to clear-out toxic elements 

that are found in radioactive waste sites 

was used by Lange (1998) as a 

recombinant. Deinococcus radiodurans is 

known to have two properties, first it is 

resistant to radiation and secondly it can 

degrade chlorobenzene in radioactive 

environments (Lange et al., 1998). Then 

again, it can only be produced in an 

environment at temperatures less than 

39°C and as radioactive sites generally 

have high temperatures, so a bacterium is 

required that can function at higher 

temperatures. Another well-known 

example for the application of GMOs in 

the management of environmental issues 

can be cited through certain bacteria that 

can yield biodegradable plastics and this 

quality of bacteria were transferred to 

microbes which were cultured in the 

laboratory and now a days they have 

enabled the wide scale greening of plastic 

industry. 

In the early 1990s, Zeneca, a 

British company, established a 

microbially manufactured biodegradable 

plastic called Biopol 

(polyhydroxyalkanoate, or PHA). The 

plastic was made using a GM bacterium, 

Ralstonia eutropha, to transform glucose 

and a variety of organic acids into a 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 408



flexible polymer (Perez-Pantoja et al., 

2008). GMOs which are able to 

metabolize oil and heavy metals through 

their bacterially encoded ability may 

prove effective for the bioremediation 

process. Simultaneously, genetically 

engineered microorganisms (GEMs) have 

shown possible uses for bioremediation in 

soil, groundwater, and activated sludge 

environments, due to the enriched 

degradative capabilities for extensive 

range of contaminants. Recent advances 

in molecular biology have unlocked new 

perceptions for the development of 

engineering microorganisms with the 

purpose of performing specific 

bioremediation. 

From the biological safety view it 

has also been reported that not all 

naturally occurring bacteria are ideal as 

bioremediation agents. For instance, 

Burkholderia cepacia would be both used 

as an agent for bioremediation and for 

biological regulator of phytopathogens. 

However, it causes cystic fibrosis in 

humans and it is also found to be resistant 

to many antibiotics (Holmes et al., 1998). 

For these reasons, the US Environmental 

Protection Agency (EPA) has led to its 

elimination to be used as an 

environmental agent (Davison, 2005). 

7. Types of pollution controlled by 

bioremediation 

The population explosion throughout the 

world has led to an increase in the 

polluted soil and water regions. As the 

number of people continues increasing 

day by day it also results in the overuse of 

natural resource like air, water and land 

resources. For these reasons, there occurs 

rapid expansion of industries, food, health 

care, vehicles, etc. but it is very 

challenging to retain the quality of life 

with all these new expansions, which are 

critical to the environment in which we 

live. Since the quality of life is very much 

linked to the overall quality of the 

environment, worldwide measures are 

taken to sustain and preserve the 


environment. Bioremediation is one of the 

emerging biological strategies which is 

applicable to the repair of damaged 

environment. 

The three main types of pollution 

(Soil, water and marine pollution) that are 

controlled by bioremediation using a 

variety of microorganisms which belong 

to different environments and are active 

members of microbial associations are 

discussed here. 

7.1. Marine pollution 

The derivatives of petroleum are 

the most important source of energy for 

industry and societies. The probable cause 

of oil spills in marine environment is 

mainly through the frequent transport of 

petroleum across the world. Moreover, it 

is broadly known that petroleum 

hydrocarbons pollution has obstructed, 

and spoiled the world oceans, seas and 

coastal zones and due to this, the Earth‟s 

health sustainability is at high risk. In 

marine environment too, bioremediation 

is considered as an economic and 

ecological biotechnology tool for the 

handling of polluted wastes (Paniagua- 

Michel and Rosales, 2015). The 

frequently applied bioremediation 

methods that can be used in marine 

environments facing disturbance due to 

oil spills are (i) using the process of bio 

augmentation by the addition of oil 

degrading bacteria so as to grow or 

improve the existing bacterial biota, and 

(ii) use of composts (nutrients), to 

encourage and stimulate the growth of 

native oil degraders, which is called biostimulation. 

In the case of oil spills, the 

processes make use of the catabolic skill 

of microorganism feeding on oil. Several 

workers (Odu, 1978; Sloan, 1987; Ijah 

and Antai, 1988; Okpokwasili and 

Okorie, 1988; Barnhart and Meyers, 

1989; Anon, 1990; Pritchard, 1991; 

Pritchard and Costa, 1991; Hoyle, 1992; 

Ijah, 2002; and Ijah, 2003) have 

pronounced numerous application of 

microorganism in the bioremediation of 

oil pollution with promising results. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 409



7.2. Water pollution 

Water pollution is a subject of 

great global concern, and it can be largely 

distributed into three main groups, that is, 

contamination by organic compounds, 

inorganic compounds (e.g., heavy 

metals), and microorganisms. It has 

caused an irreparable damage to aquatic 

ecosystems through environmental 

contamination by heavy metals from 

anthropogenic and industrial activities. 

Other sources of heavy metals comprise 

the mining and smelting of ores, run-off 

from storage batteries and automobile 

exhaust, and the manufacturing and 

inadequate use of fertilizers, pesticides, 

and many others. The bioremediation 

approach works on the high metal binding 

ability of biological agents, which can 

eliminate heavy metals from polluted sites 

with high efficacy. Specimens of 

microorganisms studied and 

advantageously used in bioremediation 

treatments for heavy metals include the 

following: (i) Bacteria: Arthrobacter spp., 

Pseudomonas veronii, Burkholderia spp., 

Kocuria flava, Bacillus cereus, and 

Sporosarcina ginsengisoli (Gautam et al., 

2011; Cycon et al., 2017); (ii) fungi: 

Penicillium canescens, Aspergillus 

versicolor, and Aspergillus fumigatus;(iii) 

yeast: Saccharomyces cerevisiae and 

Candida utilis; (iv) algae: Cladophora 

fascicularis, Spirogyra spp. and 

Cladophora spp., and Spirogyra spp. and 

Spirullina spp. 

7.3. Soil pollution 

Decontamination of soil can be 

processed through both ex situ and in situ 

remediation techniques. Ex situ thermal 

remediation processes are best for use for 

the following contaminants: petroleum 

hydrocarbons (TPH), polycyclic aromatic 

hydrocarbons (PAH), benzene, toluene, 

ethylbenzene, xylenes (BTEX), phenolic 

compounds, cyanides, and chlorinated 

compounds like polychlorinated 

biphenyls (PCB), pentchlorphenol (PCP), 

chlorinated hydrocarbons, chlorinated 

pesticides, 

polychlorinated 


dibenzodioxins (PCDD), and 

polychlorinated dibenzofurans (PCDF) 

(Koning et al., 2000). The biological 

processes of ex situ remediation involve: 

composting, landfarming, biopiling and 

the use of bioreactors. Alternatively, 

bioventing, biosparging, bioslurping and 

phytoremediation along with physical, 

chemical, and thermal processes are 

included in in situ remediation techniques 

for treating soil pollution. 

8. Advantages of bioremediation 

For successful bioremediation to 

occur, the bioremediation methods rest on 

having the right microbes in the right 

place with the right environmental factors 

for the process of degradation. 

Bioremediation is considered more 

advantageous over conventional 

techniques like land filling or 

incineration. Microbes capable of 

destroying the contaminants increase in 

number when the pollutant is present and 

when the pollutant is degraded, the 

biodegradative population declines. The 

remains for the treatment are generally 

harmless products and include carbon 

dioxide, water and cell biomass. 

Theoretically, bioremediation is useful for 

the thorough damage of a wide variety of 

contaminants. Many compounds that are 

officially considered to be unsafe can be 

transformed to nontoxic products. 

Bioremediation also reduces the chance of 

future problem associated with treatment 

and disposal of polluted waste (Rajwade 

et al., 2015). Bioremediation that is 

performed on site is often less expensive 

and site interruption is nominal, it 

removes waste permanently, reduces 

long-term problem, and has better public 

acceptance, with regulatory 

encouragement, and it can be tied with 

other physical or chemical treatment 

methods. 

9. Disadvantages of bioremediation 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 410



There are limitations to every 

process and so is bioremediation which is 

limited to those compounds that are 

biodegradable. It has been found that 

compounds such as heavy metals, 

radionuclides and some chlorinated 

compounds are not prone to rapid and 

complete degradation through 

bioremediation and there are cases where 

microbial breakdown of contaminants has 

resulted in toxic metabolites. Like most 

of the biological processes, 

bioremediation is also highly specific. 

The site factors that are significant and 

required for the success include the 

presence of metabolically proficient 

microbial populations, proper 

environmental growth conditions, and 

appropriate levels of nutrients and waste 

products (Tran et al., 2015). 

Bioremediation is logically a thorough 

procedure which can be designed for sitespecific 

conditions, i.e. before proceeding 

to cleaning of the sites, one has to do 

treatment studies on a minor scale 

(Ramrakhiani et al., 2016). The process of 

bioremediation often involves the time 

factor as it takes much more time than 

other treatment options, such as diggings 

and removal of soil or incineration. A 

second drawback to this technique in 

contrast to other remediation techniques is 

its relative sensitivity to environmental 

factors for example temperature, pH, and 

the presence of various other substances 

or organisms. There are many questions 

that should be answered before using 

bioremediation: Whether the contaminant 

is biodegradable? Is biodegradation 

occurring in the natural site? Are 

environmental conditions appropriate for 

biodegradation? Where the waste will be 

disposed if it is not degraded completely? 

These questions can be answered by 

doing site classification and also by 

treatability studies. 

10. A current update on bioremediation 

The existing bioremediation 

strategies are based upon either 


absorption or metabolism of the polluting 

compound. If the xenobiotic compound 

acts as a source of energy, carbon or any 

nutrients then it is absorbed by the 

bioremediators. Otherwise, it is cometabolised 

by a single organism or a 

group of different organisms‟ together 

acting as bioremediators. To enhance 

bioremediation by these strategies an 

adequate understanding of microbial 

behaviour is of prime importance 

(Alvarez et al., 2017). Furthermore, 

advanced engineering techniques have 

been developed to stimulate the 

microorganism involved in detoxification 

process. For instance, sparging of gaseous 

phase by enhanced mechanisms has led to 

complete utilization of bioremediation 

potential of several aerobic microbes. 

Research is also being carried out to 

promote the availability of pollutants to 

the microbes. Advance techniques such as 

use of surfactants, solubilisation of 

pollutants by exposure to steam, heat or 

heated water, and application of high 

pressure are being used for this purpose 

(Rittmann, 1993; Shukla et al., 2014). 

The approach of developing 

designer microbes (GMOs) with the help 

of recombinant DNA technology has 

already being discussed in detail. Several 

other innovative technologies such as 

transcriptome and proteome analysis, 

molecular profiling, pyro sequencing, 

metatranscriptomics and metaproteomics, 

mass spectrometry, microarrays, and 

numerous bioinformatics applications are 

helping in realization of complete 

potential of microbes for bioremediation 

(Kulshreshtha, 2012; Rittmann, 1993). 

In another very innovative 

approach the toxic industrial, domestic 

and agricultural waste can be converted 

into useful forms and products such as 

bioethanol, biogas, biofuels, single cell 

proteins etc., (Kulshreshtha, 2012). 

Recently, immobilization of microbial 

cells or the enzymes released by them 

through several mechanisms such as 

adsorption, electrostatic binding, covalent 

binding, aggregation, crosslinking, 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 411



entrapment, and encapsulation has been 

used extensively for bioremediation. Such 

immobilization of microbes improves the 

bioremediation process by facilitating 

reuse of microbes or the catalyst involved, 

and also reduce the costs of the process 

(Dzionek et al., 2016). 

11. Bioremediation and sustainability 

Sustainable remediation 

technology has been defined as the 

cautious use of natural resources by using 

a combination of remedies which 

maximizes the net benefit on human 

health and environment. Environmental 

modification through bioremediation is a 

dominant part of bio economy and 

sustainable development. In recent years, 

there has been an increase in the use of 

biodiversity as raw material for 

environmental decontamination. On the 

other hand volume and diversity of 

contaminated substrates (water, soil and 

air) are increasing due to anthropogenic 

and technogenic sources. Microorganisms 

have occupied some of the most lifethreatening 

environments on the earth and 

some of them are capable of degrading 

the pollutants that are produced through 

our industries. Ecological engineering has 

been suggested as a theoretical framework 

to project “sustainable ecosystems that 

incorporate human society with its natural 

environment for the profit of both”. 

Energy use is one of the most important 

sustainability concerns for conventional 

remediation projects. Ex-situ remediation 

is typically too energy intensive to be 

considered ecological engineering. In 

sustainable bioremediation external 

energy input is preferably used only in the 

initiation phase to start a process that is 

later driven by solar energy and the 

exemplified chemical energy of the 

pollutant itself. The engineer‟s role is to 

help provide the proper conditions in 

which such a process can take place. 

Since the objective of bioremediation 

projects is to eliminate pollution that 

employs stress on the ecosystem, it 


involves an ecosystem conservation 

approach. However, if large amounts of 

soil are physically removed from the site 

in ex-situ operations the remediation itself 

might be a threat to the ecosystem. In 

view of economic sustainability, a 

number of organic by-products are used 

which include lignocellulosic wastes such 

as sugarcane, bagasse and sawdust, crop 

residues such as coffee pulp and molasses 

and whey, a by-product of the dairy 

industry. These are also identified to 

increase the degradation of diverse toxic 

compounds. A good bioremediation 

methodology will include the planned use 

of all native microbes in an engineered 

way to accomplish the best possible 

purification levels. In summary, we can 

conclude that although bioremediation 

appears to be a promising alternative for 

the remediation of contaminants in 

different ecosystems and is also 

contributing to sustainability of the 

environment but it is still in the 

developmental phase. 

12. Future perspectives 

The application of 

microorganisms to increase the fertility of 

soil conditions and removing the soil 

contaminations through bioremediation 

technology is extensively used in Europe 

and USA. In Asia particularly in India as 

major agriculture dependent country, 

progress has been made in applying 

microorganisms to the restoration of 

polluted soil through bioremediation 

processes. However, the application of 

bioremediation technology in the 

restoration of ecosystem and soil 

management is used less compared to 

Europe and USA. Hence, extensive 

research programs are needed to increase 

the capabilities of bioremediation to deep, 

extensive, subsurface contamination due 

to chlorinated hydrocarbons and complex 

mixed wastes, including soils and 

groundwater. Besides that, The American 

Academy of Microbiology (AAM) has 

concluded that enough knowledge is now 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 412



available for field trials of bioremediation 

technology for organic compounds and 

further they emphasized that research is 

needed for the following classes of 

environmental pollutants: metals, 

metalloids, radionuclides and complex 

polycyclic hydrocarbons. The on-going 

microbial genomics studies will deliver 

more robust technologies for the 

bioremediation of metal – contaminated 

waters and land. Exciting developments 

in the use of microorganisms for the 

recycling of metal waste, with the 

formation of novel biominerals with 

unique properties are also predicted in the 

near future. Moreover, a wide diversity of 

microbes with detoxification abilities is 

waiting to be explored. The inadequate 

knowledge about microbes and their 

natural role in the environment could 

affect the acceptability of their uses. The 

understanding of the diversity of 

microbial community's in petroleum 

contaminated environment is essential to 

get a better insight into potential oil 

degraders and to understand their genetics 

and biochemistry that will result in 

developing appropriate bioremediation 

strategies, thus, preserving the long-term 

sustainability of natural terrestrial and 

marine ecosystems. 


Authors are thankful to Shri 

Ramswaroop Memorial University, 

Barabanki, Uttar Pradesh, India for 

providing facility and space for this work. 

References 

Agarwal, A. and Liu, Y. (2015). 

Remediation technologies for oilcontaminated 

sediments. Marine 

Pollution Bulletin 101, 483-90. 

Agency, U. S. E. P. (1995). Bioventing 

Principles and Practice. Bioventing 

Principles, Washington D.C, 1, 15- 

20. 

Alkorta, I., J. Hernández-Allica, J. M. 

Becerril, I. Amezaga, I. Albizu 


and C. Garbisu (2004). Recent 

findings on the phytoremediation of 

soils contaminated with 

environmentally toxic heavy metals 

and metalloids such as Zinc, 

Cadmium, Lead, and Arsenic. Rev. 

Reviews in Environmental Science 

and Bio/Technology, 3, 71 - 90. 

Alvarez, A., J. M. Saez, J. S. Davila 

Costa, V. L. Colin, M. S. Fuentes, 

S. A. Cuozzo, C. S. Benimeli, M. 

A. Polti & M. J. Amoroso (2017). 

Actinobacteria: Current research 

and perspectives for bioremediation 

of pesticides and heavy metals. 

Chemosphere, 166, 41-62. 

Bowler, C. and R. Fluhr (2000). The 

role of calcium and activated 

oxygens as signals for controlling 

cross-tolerance. Trends in Plant 

Science, 5, 241-6. 

Brim, H., S. C. McFarlan, J. K. 

Fredrickson, K. W. Minton, M. 

Zhai, L. P. Wackett and M. J. 

Daly (2000). Engineering 

Deinococcus radiodurans for metal 

remediation in radioactive mixed 

waste environments. Nature 

Biotechnology, 18, 85-90. 

Cornu, J. Y., D. Huguenot, K. Jezequel, 

M. Lollier and T. Lebeau (2017). 

Bioremediation of coppercontaminated 

soils by bacteria. 

World Journal of Microbiology and 


Cycon, M., A. Mrozik and Z. 

Piotrowska-Seget (2017). 

Bioaugmentation as a strategy for 

the remediation of pesticidepolluted 

soil: A review. 

Chemosphere, 172, 52-71. 

Datta, S., J. Singh and S. Singh (2016). 

Earthworms, pesticides and 

sustainable agriculture: a review. 

Environmental Science and 

Pollution Research (international), 

23, 8227-43. 

Davison, J. (2005). Risk mitigation of 

genetically modified bacteria and 

plants designed for bioremediation. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 413



Journal of Industrial Microbiology 

and Biotechnology, 32, 639-50. 

Dua, M., A. Singh, N. Sethunathan and 

A. K. Johri (2002). Biotechnology 

and bioremediation: successes and 

limitations. Applied Microbiology 

and Biotechnology, 59, 143-52. 

Dzionek, A., D. Wojcieszyńska and U. 

Guzik (2016). Natural carriers in 

bioremediation: A review. 

Electronic Journal of 

Biotechnology,19, 28 - 36. 

Gautam, V., L. Singhal and P. Ray 

(2011). Burkholderia cepacia 

complex: beyond pseudomonas and 

acinetobacter. Indian Journal of 

Medical Microbiology, 29, 4-12. 

Gibbs, J. T., B. C. Alleman, R. D. 

Gillespie, E. A. Foote, S. E. 

McCall, F. A. Snyder, J. E. Hicks, 

R. K. Crowe and J. Ginn (1999). 

Bioventing Nonpetroleum 

Hydrocarbons. IN Engineered 

Approaches for In Situ 

Bioremediation of Chlorinated 

Solvent Contamination. Battelle 

Press, Columbus. OH, USA. pp 7- 

14 

Gifford, S., R. H. Dunstan, W. 

O'Connor, C. E. Koller and G. R. 

MacFarlane (2007). Aquatic 

zooremediation: deploying animals 

to remediate contaminated aquatic 

environments. Trends in 


Gillespie, I. M. and J. C. Philp (2013). 

Bioremediation, an environmental 

remediation technology for the 

bioeconomy. Trends in 


Hanson, J. R., C. E. Ackerman and K. 

M. Scow (1999). Biodegradation of 

methyl tert-butyl ether by a bacterial 

pure culture. Applied and 

Environmental Microbiology, 65, 

4788-92. 

Head, I. M. and R. P. Swannell (1999). 

Bioremediation of petroleum 

hydrocarbon contaminants in marine 

habitats. Current Opinion in 



Hinchee, R. E. (1994). Air sparging for 

site remediation. CRC Lewis 

Publishers. 

Holmes, A., J. Govan and R. Goldstein 

(1998). Agricultural use of 

Burkholderia (Pseudomonas) 

cepacia: a threat to human health? 

Emerging Infectious Diseases, 4, 

221-7. 

Iyer, R. and A. Damania (2016). Draft 

Genome Sequence of Pseudomonas 

putida CBF10-2, a Soil Isolate with 

Bioremediation Potential in 

Agricultural and Industrial 

Environmental Settings. Genome 

Announcements 4,4, e00670-16 

Jafari, M., Y. R. Danesh, E. M. 

Goltapeh and A. Verma (2013). 

Bioremediation and Genetically 

Modified Organisms. Springer- 

Verlag Berlin Heidelberg.29, 

33811-3 

Kadian, N., A. Gupta, S. Satya, R. K. 

Mehta and A. Malik (2008). 

Biodegradation of herbicide 

(atrazine) in contaminated soil using 

various bioprocessed materials. 

Bioresource Technology, 99, 4642- 

7. 

Kadri, T., T. Rouissi, S. Kaur Brar, M. 

Cledon, S. Sarma and M. Verma 

(2017). Biodegradation of 

polycyclic aromatic hydrocarbons 

(PAHs) by fungal enzymes: A 

review. Journal of Environmental 

Sciences (China), 51, 52-74. 

Kamaludeen, S. P., K. R. Arunkumar, 

S. Avudainayagam and K. 

Ramasamy (2003). Bioremediation 

of chromium contaminated 

environments. Indian journal of 

experimental biology, 41, 972-85. 

Kleijntjens, R. H. and K. C. A. M. 

Luyben (2000). Bioremediation. 

Biotechnology, 11b, 329-347. 

Koning, M., K. Hupe and R. Stegmann 

(2000). Thermal Processes, 

Scrubbing/Extraction, 

Bioremediation and Disposal. 11b, 

306 - 317. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 414



Kulshreshtha, S. (2012). Current Trends 

in Bioremediation and 

Biodegradation. Journal of 

Bioremediation 


Biodegradation, 3:e114. 

Kumar, A., B. S. Bisht, V. D. Joshi and 

T. Dhewa (2011). Review on 

Bioremediation of Polluted 

Environment: A Management 

Tool. International Journal of 

Environmental Sciences,1, 1079- 

1093 

Lange, C. C., L. P. Wackett, K. W. 

Minton and M. J. Daly (1998). 

Engineering a recombinant 

Deinococcus radiodurans for 

organopollutant degradation in 

radioactive mixed waste 

environments. 

Nature 


Li, J. and R. Li (2017). Current research 

scenario for microcystins 

biodegradation - A review on 

fundamental knowledge, application 

prospects and challenges. Science of 

The Total Environment, 595, 615- 

632. 

Litchfield, C. D. (1993). In situ 

Bioremediation : Basis and Practice 

IN Biotreatment of Industrial and 

Hazardous Wastes, M.A. Levin and 

Michael A Gealt (eds.)McGraw- 

Hill, Inc.34, 167 -197 

Lovley, D. R., D. E. Holmes and K. P. 

Nevin (2004). Dissimilatory Fe(III) 

and Mn(IV) reduction. Advances in 

Microbial Physiology, 49, 219-86. 

Macek, T., K. Francova, L. 

Kochankova, P. Lovecka, E. 

Ryslava, J. Rezek, M. Sura, J. 

Triska, K. Demnerova and M. 

Mackova (2004). 

Phytoremediation: biological 

cleaning of a polluted environment. 

Reviews on Environmental Health, 

19, 63-82. 

Majone, M., R. Verdini, F. Aulenta, S. 

Rossetti, V. Tandoi, N. 

Kalogerakis, S. Agathos, S. Puig, 

G. Zanaroli and F. Fava (2015). In 

situ groundwater and sediment 


bioremediation: barriers and 

perspectives at European 

contaminated sites. New 


McCauley, P. (1999a). Land 

Remediation and Pollution Control 

Division (Treatment and 

Destruction branch), . Personal 

communication. Chemist, U.S. EPA 

National Risk Management and 

Research Laboratory. Web Site 



composites in biomedicine. Applied 

Microbiology and Biotechnology, 

99, 2491-511. 

Ramrakhiani, L., S. Ghosh and S. 

Majumdar (2016). Surface 

Modification of Naturally Available 

Biomass for Enhancement of Heavy 

Metal Removal Efficiency, 

Upscaling Prospects, and 

Management Aspects of Spent 

Biosorbents: A Review. Applied 

Biochemistry and Biotechnology, 

180, 41-78. 

Rittmann, B. E. (1993). In Situ 

Bioremediation: When does it 

work? National Academy Press 

Washington, D.C. 1st Ed. 

Santisi, S., S. Cappello, M. Catalfamo, 

G. Mancini, M. Hassanshahian, L. 

Genovese, L. Giuliano and M. M. 

Yakimov (2015). Biodegradation of 

crude oil by individual bacterial 

strains and a mixed bacterial 

consortium. Brazilian Journal of 

Microbiology, 46, 377-87. 

Schulz - Berendt, V. (2000). 

Bioremediation with heap 

technique, Biotechnology. 320-328. 

11b, 2nd Ed. 

Shukla, A. K., S. N. Upadhyay and S. 

K. Dubey (2014). Current trends in 

trichloroethylene biodegradation: a 

review. Critical Reviews in 



Su, C., L. Jiang and W. Zhang (2014). 

A review on heavy metal 

contamination in the soil worldwide: 

Situation, impact and remediation 

techniques. Environmental Skeptics 

and Critics, 3, 24 - 38. 

Tran, N. H., H. H. Ngo, T. Urase and 

K. Y. Gin (2015). A critical review 

on characterization strategies of 

organic matter for wastewater and 

water treatment processes. 

Bioresource Technology, 193, 523- 

33. 

Ulrich, A. C. and E. A. Edwards (2003). 

Physiological and molecular 

characterization of anaerobic 

benzene-degrading mixed cultures. 

Environmental Microbiology, 5, 92- 

102. 

Van Deuren, J. and T. Lloyd (2002). 

Remediation technologies screeing 

matrix and reference guide. Federal 

Remediation technologies 

Roundtable, 4th Ed. 

Vidali, M. (2001). Bioremediation: An 

overview. Journal of Applied 

Chemistry, 73,1163 - 1172. 

Zhang, W., F. Jiang and J. Ou (2011). 

Global pesticide consumption and 

pollution: with China as a focus. 

International Academy of Ecology 

and Environmental Sciences, 1, 125 

- 144. 

© 2017 by the authors. Licensee, Editors and AIMST University, 

Malaysia. This article is an open access article distributed under the 

terms and conditions of the Creative Commons Attribution (CC BY) 

license (http://creativecommons.org/licenses/by/4.0/). 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 416



Sea Urchin - A New Potential Marine Bio-resource for 

Human Health 

M. Aminur Rahman 1, *, Fatimah Md. Yusoff 1, 2 , Kasi Marimuthu 3 and Yuji Arakaki 4 

1 Laboratory of Marine Biotechnology, Institute of Bioscience, Universiti Putra Malaysia, 

43400 UPM Serdang, Selangor, Malaysia; 2 Department of Aquaculture, Faculty of Agriculture, 

Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 3 Department 

of Biotechnology, Faculty of Applied Sciences, AIMST University, 08100 Bedong, Kedah 

Darul Aman, Malaysia; 4 Department of Tourism, Faculty of International Studies, Meio 

University, Nago, Okinawa-905-8585, Japan; 

*Correspondence: aminur1963@gmail.com; Tel: +60 3-8947-2141 

Abstract: Sea urchin gonads usually called as “Sea urchin Roe” or “Uni”, are important 

food delicacies in different parts of the world. In Asian, Mediterranean and Western Hemisphere 

countries, the roe of sea urchins is considered as a highly prized delicacy sea food 

because of its tastes and also have long been utilized as extravagance foods in Japan. Peoples 

of the Asian Pacific region have long been utilizing it for improving general body tone 

and also treatment for a number of diseases. It has been reported that, sea urchin gonads are 

found to be rich with high-quantities of bioactive compounds, such as polyunsaturated fatty 

acids (PUFAs) and β-carotenes. The PUFAs, particularly eicosapentaenoic acid (EPA, 

C20:5) (n-3)) and docosahexanoic acid (DHA C22:6 (n-3)), have profound significant effects 

on arrhythmia, cardiovascular diseases and cancer. β-carotene and some xanthophyll‟s 

have strong pro-vitamin activity and can be used to prevent tumor development and light 

sensibility. The sea urchin fisheries in recent years have extended so impressively that the 

natural populations of them have been overexploited to meet-up the increasing demand. Not 

surprisingly, the continued high demand and the decrease in supply have headed towards a 

pronounced interest for the commercial aquaculture of sea urchins. Global sea urchin harvesting, 

having peaks at 120,000 metric tons in 1995, are presently in the scale of around 

82,000 metric tons. However, these declining arrays evidently mirror the overfishing of major 

fishery grounds and focus the necessity for conservation measures, aquaculture development 

and sustainable fisheries management. Once the natural stocks decrease, the higher 

market demand for foodstuff, nutraceuticals, pharmaceuticals and cosmeceuticals, increases 

the value of the manufactured goods and therefore, culturing seems to become economically 

feasible. As per this assessment exhibits, there have been intense progresses in the aquaculture 

protocols of sea urchins during the past 15-20 years, we can come to the end that 

presently the main impediments to successful farming are actually managerial, cultural, 

conservational and economical rather than biological and ecological. Expected that demand 

is implausible to decline, the commercial value of future product will be increased. Hence, 

the fortune of sea urchin is strictly connected to those fisheries, whose prospect would 

eventually depend on the stock improvement, aquaculture production, fishery management, 

roe enhancement and market forces that will play a significant role to give a structure of 

this expanding industry. 

Keywords: Biology; culture; ecology; health; management; roe; sea urchin 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 417


Sea Urchin - A New Potential Bio-resource for Human Health 


Sea urchins (Echinodermata: 

Echinoidea) are important marine bioresources 

for conducting research in diverse 

areas of ecology, biology, biodiversity, 

aquaculture, conservation, taxonomy 

and evolution. Simultaneously, they are 

utilized as raw material to produce foodstuff, 

particularly, the product of processing 

gonads recognized as “Sea urchin 

Roe” (Kaneniwa and Takagi, 1986; 

Oshima et al., 1986; Ichihiro, 1993). It is 

also one of the highly prized seafood delicacy 

owing to the high tastes in Asian, 

Mediterranean and Western Hemisphere 

countries, for instance, Chile and Barbados 

(Kaneniwa and Takagi, 1986; Lawrence 

et al., 1997; Lawrence et al., 1997; 

Yurˈeva et al., 2003; Rahman and Yusoff, 

2010; Rahman et al., 2013a, 2014a, b; 

Parvez et al., 2016a, b). In Japan, sea urchin 

gonads (either in the state of fresh or 

processed foods) have long since been 

consumed as high-quality luxury foods 

(Shimabukoro, 1991; Rahman et al., 

2014a,b, Parvez et al., 2016a,b) and the 

roe can sell for as much as AU$450/kg 

(Richard, 2004). Due to the increasing 

demands for sea urchins, Japan imports 

big amounts from USA, South Korea, 

Thailand and other producers, thus has 

elevated concerns about overfishing, and 

hence, making it one of the valuable sea 

foods in the world (Hagen, 1996; Rahman 

et al., 2014a; Parvez et al., 2016a,b). Traditionally, 

sea urchin gonads have long 

been used by the peoples of the Asian Pacific 

Region, as a remedy for improving 

general body tones, treatment for a number 

of diseases and increasing the sexual 

potency of middle-aged men (Seifulla et 

al., 1995; Yurˈeva et al., 2003). However, 

in the recent years, the fisheries of sea 

urchins have been expanded so highly 

that the natural population in Chile, Japan, 

France, Canada and different parts of 

USA have been overexploited to meet up 

the great demand (Lawrence et al., 2001; 

Andrew et al., 2002, 2004; Rahman et al., 

2005, 2012a,b, 2013b, 2014b; Parvez et 

Rahman et al. 

al., 2016b). Not astonishingly, the continuous 

strong demand and the decline in 

supply headed to a pronounced increase 

in awareness for aquaculture of sea urchins, 

predominantly, in those parts 

wherein their natural populations have 

been dwindled (Lawrence et al., 1997; 

Lawrence, 2007). The species of sea urchins 

whose gonads have high commercial 

values could be obtained from a 

number of genera such as: Tripneustes, 

Strongylocentrotus, Paracentrotus, Loxechinus, 

Echinus, Centrostephanus, 

Hemicentrotus, Lytechinus, Diadema, 

Arbacia, Colobocentrotus, Anthocidaris, 

Psammechinus, Evechinus, Heliocidaris, 

Echinometra, Toxopneustes, Pseudocentrotus 

and Pseudoboletia (Sloan, 1985; 

Saito, 1992; Keesing and Hall, 1998; 

Lawrence, 2007; Rahman et al., 2014b). 

Nevertheless, the majority of the 

sea urchins fisheries have followed the 

same trends of quick expansion to an unmaintainable 

top, followed by a correspondingly 

speedy decline. Global sea 

urchin harvesting reached to 20,000 metric 

tons in 1995, are presently in the state 

of around 82,000 metric tons with an 

alarming decreasing rate of 32% (FAO, 

2010; Carboni et al., 2012; Rahman et al., 

2014b; Parvez et al., 2016b) (Figure 1). 

However, the newly extended sea urchin 

fishery (Loxechinus albus) from Chile 

covers half amounts of the world catch 

(Rahman et al., 2014b; Parvez et al., 

2016b). The other main sea urchin fisheries, 

landed in tonnage, are in Japan, 

Maine and California (United States), and 

British Colombia (Canada) (Andrew el al., 

2002). In case of Europe, the commercial 

sea urchin (Paracentrotus lividus) in Ireland 

and France were overfished to supply 

the French markets (Barnes et al., 

2002). There have been reported the large 

populations and abundances of edible urchins 

in Norway (Strongylocentrotus 

droebachiencis) and Scotland (Echinus 

esculentus and Psammechinus milaris), 

However, these stocks are not suitable for 

profitable fishing because their roe 

amounts are either very small or too flex- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 418



Rahman et al. 

Figure 1: Global production of sea urchin fisheries from 1950 to 2008 (FAO, 2010). 

ible (Hagen, 2000; Kelly, 2000, 2005; 

Sivertsen, 2004; Rahman et al., 2014b). 

Nonetheless, the continuous declining 

patterns noticeably reveal the overexploitation 

of most of the fishery grounds and 

focus the necessity for proper conservation 

policies, sustainable management 

strategies and appropriate aquaculture 

practices. 

2. Biology and ecology of major species 

The most parts of the world involved 

in sea urchin production are linked with 

main sites of primary productivity. In the 

subtropical and tropical regions, these are 

usually associated with seagrass beds, 

while in the temperate regions, with kelp 

forests. Urchins commonly occur at lower 

densities within the kelp communities, 

overgrazing on kelps and then lead to the 

establishment of flats dominated by encrusting 

macroalgae (called as coralline 

flats and barrens). Both of the communities 

(barrens and kelp forests) are often 

found near to each other, making either a 

mosaic of stable patches or strata. 

The biological features of sea urchins 

vary largely among species. The species 

inhabiting in temperate zones mostly have 

moderate longevity between 10 and 20 

years, even though highest longevity of 

100 years has been reported. Growth and 

production performances are flexible and 

greatly dependent on quality food and nutrition. 

In conditions with lower density, 

growth rates are usually high, while at 

high densities, growth rates are low. 

However, the density of sea urchins is not 

the single factor that starts the construction 

of barrens or flats. Inter-annual 

events that trigger warming (e.g., El 

Nino), are known to cause kelp dieback 

which then leads to overgrazing and the 

maintenance of barrens in the extended 

period. The exclusion of predators that 

would then cause moderate urchin densities 

may also endorse conditions leading 

to overgrazing. Usually, densities of sea 

urchins are the highest in barrens, but individual 

growth performance and overall 

productivity is low owing to competition 

for food and perhaps due to the deficient 

nourishment. 

The most efficient predator of sea urchins, 

particularly the sea otter and its reestablishment 

around the North is having 

main effects on sea urchin fisheries. In 

areas where populations have been re- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 419



established, predation can far exceed fishing 

pressures and urchin densities have 

been reduced to levels below that which 

is required for fishing to be profitable 

(Andrew et al., 2002). Recruitment mortality 

is also affected by different types of 

habitats. It has been reported that mortality 

of juveniles among a number of species 

is higher in kelp forests because of the 

occurrences of micro-predators in their 

habitats (Tegner, 2001). 

Given the similar habitat types, a 

number of edible sea urchin species share 

some similarities in their distribution, 

abundance and reproduction. Yet, they 

are differed in their fundamental biological 

characteristics such as growth, survival, 

production, maturity and longevity. 

2.1. Green sea urchin (Strongylocentrotus 

droebachiensis) 

Green sea urchin has a circumpolar 

distribution and occurs through the North 

Atlantic to the North Pacific. The fisheries 

of S. droebachiencis have been focused 

in Maine and the Canadian Maritimes 

but smaller fisheries are concentrated 

in Alaska, British Colombia, Washington 

and Iceland. The species is mostly 

common in the intertidal zone to a depth 

of 50 m, where it is closely related with 

kelp beds (Scheibling and Hatcher, 2001). 

Two distinct growth rates have been identified, 

but the overall growth rates are 

found to be moderate. The fast growing 

form attains the minimum legal size between 

4 and 6 years and can live for 16- 

20 years. On the other hand, the slow 

growing one inhabits for 8-12 years and 

never attains the optimum legal size (Andrew 

et al., 2002). In British Colombia, 

the minimum legal size is not more than 

55 mm TD (test diameter) and in most 

cases, the time needed to grow to this size 

is supposed to range from 4 to 8 years 

(Taylor 2004). This sea urchin attains 

sexual maturity within 1-2 years and the 

first spawning occurs between midwinters 

to early spring when it reaches to 

45-50 mm in TD. 

Rahman et al. 

2.2. Red sea urchin (Strongylocentrotus 

franciscanus) 

Strongylocentrotus franciscanus usually 

referred to as red sea urchin, is the 

biggest echinoid in the world and commonly 

occurs along the West Coast of 

North America, extending from Baja California 

to the Aleutian Archipelago and 

the coast of Siberia and northern. The 

ranges of their habitats encompass northern-wards 

up the west coast to Sitka and 

Kodiak AK at 58 o N (Tegner, 2001). This 

species is also occurs in the subtidal zone 

to a depth of 50 m seawards and is intensely 

associated with kelp forests. 

Growth performance in the earlier stage 

of urchin is comparatively fast and the 

species shows the highest longevity. TD 

at recruitment is about 90 mm, which is 

usually attains in around 6-8 years of the 

age. The maximum size is around 200 

mm and the individuals over 150 mm TD 

are older than 100 years (Tegner, 2001). 

Spawning commonly occurs over the 

spring and summer months when the urchins 

attain sexual maturity at around 50 

mm TD and the spawning usually over 

summer and spring months. 

2.3. Japanese green sea urchin (Strongylocentrotus 

intermedius) 

Strongylocentrotus intermedius commonly 

known as Japanese green sea urchin 

is the 2nd most economically important 

regular echinoid in Japan. This 

species is distributed along the Asian and 

Siberian coast of the Pacific. This species 

is common in intertidal shallow waters 

around Hokkaido and is exploited commercially 

from Aomori, Irate and Hokkaido 

(Agatsuma, 2001a). It generally 

occurs in shallower stony substratum and 

is usually associated with kelp forests 

(Agatsuma, 2001a). The matured adult of 

S. intermedius contains small reddishyellow 

gonads, which do have a good 

taste and thus listed on Tsukiji as Japanese. 

It is well-adapted to cold water and 

the growth restriction does not appear to 

be related to the temperature limits 

(Agatsuma, 2001a). Density and nutrition 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 420



are the two major factors influencing 

growth of urchins. In appropriate culture 

conditions, the urchin attains 40 mm TD 

within 2-4 years and the maximum size of 

55 mm TD at ages between 6 and 10 

years. It obtains sexual maturity at the age 

of 2 years with the size of 30-35 mm TD 

and the spawning usually occurs in autumn 

and spring. 

2.4. Strongylocentrotus nudus 

This species has been recorded on 

Tsukiji as Japanese and also considered as 

the most commonly harvested edible sea 

urchin in Japan and accounts for ~ 44% of 

the total commercial catch (Agatsuma 

2001b). It is found on inter- and sub-tidal 

rocky bottoms extending from Dalian, 

China northwards to Primorskyi Kray, 

Russia and in Japan where it is found in 

the Pacific from Sagami Bay to Cape Erimo 

on Hokkaido and in the Sea of Japan 

from Omi Island in Yamaguchi to Soya 

Cape northern Hokkaido. The urchins 

generally reach the legal size (40 mm) in 

2 to 4 years when feeding on perennial 

Laminarians whereas they take 7 to 8 

years on coralline flats. It occurs in the 

intertidal to subtidal rocky reefs and is 

strongly associated with kelp communities. 

Juveniles recruit to coralline flats and 

move to adjacent kelp forests. In kelp forests 

individuals reach 50 mm TD in 2 to 4 

years, whereas 7 to 8 years reported in 

coralline flats (Agatsuma, 2001b). Maximum 

longevity is reported as 14 to 15 

years. Sexual maturity is attained at 40 to 

45 mm TD, and spawning takes place in 

autumn. 

2.5. Purple sea urchin (Strongylocentrotus 

purpuratus) 

The sea urchin S. purpuratus, usually 

recognized as purple sea urchin, inhabits 

along the eastern edge of the Pacific coast 

of North America, extending from British 

Columbia, Canada to Ensenada, Mexico. 

It occurs abundantly in lower intertidal 

and nearshore subtidal communities but 

has been found to a maximum depth of 

150 m (Tegner, 2001). This species is 

Rahman et al. 

closely associated with kelp beds and its 

growth is extremely flexible and reliant 

on the availability of algae. The maximum 

size has been recorded to be 100 

mm TD. S. purpuratus usually attains 

sexual maturity around 2 years of age, 

and spawns during their natural breeding 

season, extending from January to March. 

2.6. Purple crowned sea urchin (Centrostephanus 

rodgersii) 

The purple crowned urchin experiences 

a subtropical distribution throughout 

the water areas of Australia and New Zealand, 

but most abundantly occurs in Eastern 

Australia. This species has also been 

reported to be extended into Bass Strait 

and the East Coast of Tasmania (Rdger, 

1999) and is possibly related with the 

warming of coastal waters around the region 

(Andrew and Byrne, 2001). It is one 

of the large urchins with long dark purple, 

black to red spines that have iridescent 

blue/green sheen. Centrostephanus rodgersii 

is usually seen in large numbers and 

plays an important ecological role in intertidal 

near shore rocky reefs by cleaning 

the areas of kelp. Growth performances 

follow the medium trends and the individuals 

attain 70-90 mm TD within the 

age between 4 and 10 years. The longest 

size (120 mm TD) achieved when the urchin 

become 20 years of age. The adult 

urchin attains sexual maturity at the size 

between 40 and 60 mm TD and spawning 

occurs during the winter months. 

2.7. Kina (Evechinus chloroticus) 

Evechinus chloroticus well-known as 

kina is a sea urchin endemic to New Zealand 

waters. The distribution of kina is 

intensely linked within kelp beds or aggregating 

in nearby barrens. It is typically 

occurs from the intertidal area to a depth 

of 14 m; even some are found in 60 m. 

Growth rate is moderate and the individuals 

reach to a size of 50 mm within the 

age of 4 years. However, E. chloroticus 

attains the maximum size of 80-100 mm 

in 8-9 years of age. Depending on the site, 

the longevity of this urchin varies be- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 421



tween 10 and 20 years (Barker, 2001). It 

is predominantly herbivorous, feeding 

mainly on brown algae, red algae and encrusting 

substrate (Barker, 2007). Kina 

usually reaches sexual maturity within 3- 

4 years of age having a size range between 

40 and 50 mm TD and spawns in 

spring, extending from November to February 

(Barker, 2007). 

2.8. Chilean red sea urchin (Loxechinus 

albus) 

The Chilean red sea urchin, Loxechinus 

albus, is one of the reasonably slowgrowing 

urchins, commonly found around 

the Pacific coasts of South America from 

Isla Labos de Afuera in Peru to the 

Southern tip of South and usually occurs 

within the depths ranged from the intertidal 

zone to a maximum of depth of 340 

m (Vasquez, 2001). This urchin attains 

sizes up to 130 mm TD and can live until 

20 years of age (Andrew et al., 2002). It is 

considered as one of the important commercial 

species along the south-west 

coast of South America due to high taste 

qualities. The distribution of L. albus is 

mostly on rocky substrates and closely 

related with the kelp beds. It is an herbivore 

and seems most likely to feed on 

whatever species of alga grow nearby. 

The urchin is comparatively slowgrowing, 

attaining a maximum size of 

130 mm TD. Spawning period differs depending 

upon its destitution patterns; 

happening in spring to summer in the 

north, summer in the south and spring in 

the extreme south. 

2.9. Variegated sea urchin (Lytechinus 

variegatus) 

The variegated sea urchin occurs in 

the shallow waters and widely distributed 

throughout the tropics and subtropics of 

the western Atlantic, from Florida, 

through the Caribbean to Brazil and Panama 

(Watts et al., 2001). Lytechinus variegatus 

is usually inhabits on seagrass 

beds and hard bottoms covered with 

macroalgae. It is a fast-growing urchin 

but the longevity is limited. Within the 

Rahman et al. 

age of 3 years, this species can reach to a 

maximum size of 92 mm TD. Average 

longevity ranged from 1 to 2 years. When 

the urchin attains a size of 40 mm TD, it 

gets sexual maturity within a year after 

metamorphosis. No reasonability of 

spawning was observed in this species. 

2.10. Rock sea urchin (Paracentrotus 

lividus) 

Paracentrotus lividus is a species of 

sea urchin belongs to the family Parechinidae 

and commonly known as rock or 

purple sea urchin. It occurs in the Mediterranean 

Sea and eastern Atlantic Ocean, 

extending from western Scotland and Ireland 

to the Azores, Canary Islands and 

Morocco, and most common in the western 

Mediterranean, the coasts of Portugal 

and the Bay of Biscay, where the water 

temperature in winter months varies within 

10 to 15 o C. This species usually inhabits 

in the shallow sub-littoral area to a 

maximum depth of 20 m. Paracentrotus 

lividus is intensely related to the seagrass 

meadows and mainly existed on encrusted 

rocky substratum where it makes permanent 

burrows to live in. It experiences 

with moderate growth rates and the individuals 

having 40 mm TD are usually 4-5 

years old, and the adults with a size of 

70+ mm TD are older than 12 years. The 

largest size of 15 mm TD is reported by 

Boudouresque and Verlaque (2001). The 

species gets sexual maturity at the size 

range between 13 and 20 mm TD and the 

spawning usually occurs during spring to 

the early summer months. 

2.11. Collector sea urchin (Tripneustes 

gratilla) 

Tripneustes gratilla is commonly recognized 

as collector, cake or Parson‟s hat 

sea urchin and has a circumtropical distribution, 

encompassing to the subtropics. 

This species is usually occurs in the Indo- 

Pacific, Hawaii, the Red Sea and Bahamas, 

and is widely distributed from Red 

sea westward to Hawaii and Clarion Island, 

eastward to Paumotu, as far south as 

Port Jackson, and at Shark‟s Bay on the 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 422



west coast of Australia. It is regarded as a 

shallow-water sea urchin and usually inhabits 

on a diversity of substrates and occurs 

in the depth range between 2 and 30 

meters (Lawrence, 2007). Tripneustes 

gratilla grazes continuously during day 

and night and its diet comprises of algae, 

periphyton and seagrass. It has higher 

growth rate with lower longevity. Within 

the age of 4 to 5 years, the urchin attains a 

maximum size of 160 mm TD. However, 

it can grow to 75 mm TD in the first year 

of age. The collector sea urchin has an 

annual reproductive cycle mediated by 

seawater temperature, length of day and 

feeding activity. Spawning mainly occurs 

during mid to later winter months, when 

water temperatures and day lengths become 

the lowest and each clutch contains 

approximately 2 million eggs. Similar to 

the other regular sea urchins, the fertilized 

eggs develop into pluteus larvae, which 

then stay in the water column for almost 

30 days. They then settle on the sea floor, 

undergo metamorphic induction and then 

become the tiny young juveniles. This 

species attains the sexual maturity at 

around 2-5 years of age to become a 

complete reproducing adult. 

2.12. Purple sea urchin (Heliocidaris 

erythrogramma) 

Heliocidaris erythrogramma or 

the purple sea urchin is usually distributed 

in the shallower coastal communities, extending 

from intertidal to a maximum 

depth of 35 m in the southern Australia. 

In the coastal waters of Tasmania, this 

species is commonly occurs in kelp communities 

and barrens, where it feeds by 

grazing and capturing drift weeds. It can 

also be occurred in high densities in association 

with sea grass beds (Keesing, 

2001). Growth rates of this sea urchin 

usually vary depending on food availability 

and nutrition, but are mostly moderate. 

Individuals of H. erythrogramma attain a 

size of 40 mm TD within one year as well 

as a harvestable size (60 mm TD) at 3 to 5 

years. It has been reported that individuals 

having maximum size of 122 mm TD 

Rahman et al. 

can live for more than 10 years (Sanderson, 

1995). It attains sexual maturity at 

TD sizes of 40–50 mm within 5–6 years 

of age (Sanderson et al., 1996). The best 

roe production was found to be 10-14% 

during August–December and spawning 

usually occurs between summer and autumn 

(Sanderson, 1994). 

2.13. Shore sea urchin (Psammechinus 

miliaris) 

Psammechinus miliaris is a species of 

sea urchin under the family Parechinidae 

and sometimes known as shore of green 

sea urchin. It shows restricted distributions 

in the southern and eastern waters of 

the North Sea, and the eastern Atlantic 

Ocean from Scandinavia south to Morocco, 

where it occurs from the low tide 

mark down to a maximum depth of 100 m. 

Its abundance is strongly associated with 

the presence of Laminaria kelp. This sea 

urchin is often found on or under Saccharina 

lastissima, a large brown seaweed 

with which it shares its range of distributions 

and also occurs in a variety of other 

habitats including under boulders and 

rocks, among seaweeds, on rough ground 

and on the rhizomes of Zostera marina in 

seagrass meadows. It is an omnivore and 

mainly feeds of macroalgae, diatoms, hydroids, 

worms, small crustaceans, mollusks 

and detritus. Longevity is relatively 

short and the growth rates are observed to 

be moderate. It has been reported that individuals 

can reach to a maximum size 

(45 mm TD) with an age between 3 and 4 

years. Psammechinus miliaris gets sexual 

maturity in the first year at 6-7 mm TD 

and usually spawns in the months of 

spring and early summer. 

2.14. White sea urchin (Salmacis 

sphaeroides) 

The short-spined white sea urchin (S. 

sphaeroides) belonging to the family 

Temnopleuridae, is considered as one of 

the rare species under the group of regular 

Echinoids. It usually occurs in the tropical 

Indo-West Pacific Ocean, extending from 

China to Solomon Islands and Australia 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 423



including Singapore and Malaysia (Tan 

and Ng, 1988; Schoppe, 2000; Miskelly, 

2002; Rahman et al., 2012b, 2013b). This 

species has almost white cloudy test of 

55-80 mm TD with plentiful small white 

spines, the size of which are between 10 

and 15 mm. Some individuals have white 

spines with marron bands, some with all 

maroon species, while the others with maroon 

and green bands. Salmacis 

sphaeroides can be found in the depth, 

ranging from 0 to 90 m seawards, and also 

be occurred in shallow intertidal zone, 

particularly among seagrass meadows, 

coral reef substrates and in muddy sublittoral 

areas or washed ashore Schoppe, 

2000). This species feeds different types 

of seaweeds, bryzoans and detritus. 

Spawning is found to be around the year. 

Following fertilization in the water column, 

the embryo develops into a blastula 

in about 9 hours. A series of larval stages 

follows, in which the larvae acquire more 

and more arms, and develops tube feet 

and spines within the larval body. Up to 

this point, the process takes about 35 days. 

Competent larvae swim near the surface 

of the substrate to determine a suitable 

site for settlement. After attachment, larval 

structures are either discarded or resorbed, 

and adult features continue to develop 

in the juvenile (Rahman et al., 

2012b). 

2.15. Rock boring sea urchin (Echinometra 

spp.) 

A number of recently diverged species 

of rock boring sea urchins belonging 

to the genus Echinometra, are widely distributed 

throughout the World‟s marine 

ecosystems. They occur commonly within 

and around coral reefs from central Japan 

in the north to southwest Australia in the 

south, from Clarion Island off Mexico in 

the east, and to the Gulf of Suez in the 

west (Rahman et al., 2000; 2005). Various 

species of Echinometra exhibits circumtropical 

distribution and usually occur 

in shallow intertidal habitats, however a 

few has been recorded at a maximum 

depth of 20. These species are usually 

Rahman et al. 

small-bodied Echinoids, having the maximum 

size of 85 mm TD and can live for 

8 to 10 years. They inhabit burrows and 

crevices and thereby defend themselves 

from strong wave action and predators. 

Echinometra spp. are active herbivorous 

in nature and without the presence of 

predators, they can occur in densities that 

exceed the primary production potential 

(McClanahan and Muthiga, 2001). Breeding 

takes place in any time throughout the 

year but they usually spawn during summer 

and autumn in warmer waters. Similar 

to the other regular Echinoids, Echinometra 

spp. release their eggs and 

sperms in the water column, where fertilization 

occurs externally and the planktonic 

echinopluteus larvae are produced 

through the embryonic and early larval 

stages. The time when the competent larvae 

gets suitable substratum, they first 

settle on the seabed then undergo metamorphosis 

to produce juvenile urchins. 

2.16. Long-spined black sea urchin (Diadema 

setosum) 

The tropical sea urchin, Diadema setosum 

commonly referred to as longspined 

black sea urchin, is a member of 

regular Echinoids under the family Diadimatidae. 

It is broadly distributed 

throughout the Indo-Pacific region, from 

Australia and Africa to Japan and Red 

Sea, extending to the Gulf of Aqaba, Gulf 

of Suez and Arabian/Persian Gulf (Lessios 

et al., 2001). This species has characteristics 

long black spines land five white 

spots on the aboral side. The distinctive 

orange ring around its anal cone completes 

the special visual features of this 

species. It is usually an omnivorous scavenger 

and detritus eater and scraps films 

of hard substrates. They are generally 

found in coral reefs and shallow rocky 

habitats at depths from 1 to 6 m. This 

species has a wide range of diets, which 

includes microalgae, seaweeds, coral 

polyps and encrusting animals (Grignard 

et al., 1996). Gametogenesis begins in 

April–May, when the seawater temperature 

rises above 25 o C in the Gulf of Suez 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 424



and spawning take place between June 

and September (Pearse, 1970). It has also 

been known to spawn both seasonally and 

thought out the year depending upon the 

locations and sites of the spawning adults. 

Temperature levels higher than 25 o C have 

been observed to be a potential spawning 

cue (Pease, 1974). Throughout the year, 

the equatorial populations are found to be 

spawn without following any particular 

times. This is eventually true for the Malaysian 

and Philippine populations of D. 

setosum (Tuason and Gomez, 1979). In 

the Persian Gulf, spawning usually occurs 

between the months of April and May 

(Alsaffar and Khalid, 2000). Some other 

cues, such as the moon phases have been 

found to influence the spawning of D. setosum. 

This urchin has also been observed 

to trigger spawning activities in concordance 

with the entrance of a full moon 

(Lessios, 1981). 

3. Culture, management and stock 

enhancement 

3.1. Aquaculture 

Mostly, the edible sea urchins are 

regular Echinoids (Lawrence, 2007), experiencing 

separate male and female sexes 

and are generally broadcast spawners. 

At the onset of the breeding season, the 

sexually matured adults release their 

gametes in the water column where fertilization 

takes place. The pluteus larvae 

form through the embryonic and early 

larval development of the fertilized eggs, 

which after a period of planktonic development, 

feed on unicellular diatom, settle 

to a suitable substratum and undergo metamorphosis 

to produce small juvenile urchins. 

At 26-28 o C, almost 1 month is required 

to complete the larval life cycle, 

comprising the feeding or 4-armed stage, 

the 6 to 8-armed stages and finally competent 

stage for settlement (Figure 2). The 

newly born metamorphosed juveniles 

grow on macroalgae until attain the marketable 

size (40–50 mm) within the age 

ranged from 1 to 3 years, depending upon 

the species (Kelly, 2005). 

Rahman et al. 

Sea urchin aquaculture has successfully 

been accomplished on a large scale in Japan 

for many decades. In order to enhance 

the natural stocks, millions of juvenile 

urchins are being produced in hatcheries, 

for releasing to the managed areas 

of seafloor on the intertidal seashore areas. 

The nationally co-ordinated reseeding 

program has been developed to the extent 

that over 66 million juveniles were released 

on the reefs within which, over 

80% were S. intermedius (Agatsuma et 

al., 2004). The contribution of released 

sea urchin juveniles to the overall catch 

has been estimated to be between 62 and 

80%. There have also been much smallscale 

reseeding programs functioning in 

South Korea and Luzon Islands in the 

Philippines (Andrew et al., 2002). The 

farm entrepreneurs and researchers 

around the southern Ireland have been 

developing techniques for commercial sea 

urchin (P. lividus) cultivation for more 

than 20 years (Leighton, 1995), and comparatively 

newly in France (Grosjean et 

al., 1998). Culture of 3 commercially important 

sea urchins (P. miliaris, E. esculentus 

and P. lividus) has been conducted 

in Scotland since 1995 and there are also 

well-established research teams in the 

east coast of North America including 

Florida, Alabama, Maine, New Henisphere, 

New Brunswick and Newfoundland 

– working on S. droebechiensis and 

L. variegatus; On the west coast of North 

America, including California and British 

Columbia (S. droebechiensis, S. franciscanus, 

S. purpuratus); in Chile (L. albus); 

in New Zealand (Evechinus chloroticus), 

Norway (S. droebachiensis) and in Israel 

(P. lividus) (Kelly, 2005). 

Sea urchin brood stocks are regularly 

collected from the wild stocks when 

they reach proper sexual maturity. Matured 

gametes are obtained by injecting 

0.5 M KCl into the coelomic cavity of 

both female and male urchins. Sperms in 

its most concentrated from are pipetted 

off the genital pores, while eggs are collected 

by inverting the female urchins 

over a glass beaker filled with filtered sea 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 425



Rahman et al. 

Figure 2: Breeding, development and complete life-cycle of ball-like white sea urchin 

(Salmacis sphaeroides) (Rahman et al., 2012b). 

water (FSW) (Rahman et al., 2000, 2001, 

2004, 2005, 2012b). At limited sperm 

concentrations, fertilization is usually 

done by mixing a few drops of diluted 

sperm (10 -4 dry sperm dilutions) with egg 

suspensions in a petri dish and the resulting 

embryos are reared. Hatching of fertilized 

eggs usually takes 10-15 hours after 

insemination, to develop into a ciliated 

blastula. When the swimming larvae 

achieve feeding stage (4-armed pluteus), 

they are reared in glass bottles on a rolling 

roller keeping a larval density of 1-2 

individual/ml of medium. The unicellular 

cultured diatoms (Chaetoceros calcitrans, 

Isochrysis galbana) are commonly used 

as supplementary larval food at the concentrations 

of 5,000, 10,000 and 15,000 

cells per ml of medium daily at 4-, 6- and 

8-armed pluteus stages, respectively until 

attaining metamorphic competence within 

around 30-35 days post-fertilization 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 426



(Rahman et al., 2016) (Figure 3). In Japan, 

partial water exchange system (Sakai et 

al., 2004) and continuous flow-through 

system (Hagen, 1996) are used for the 

large scale cultivation. The most costly 

aspect of captive larval culture is the need 

for the concurring production of the 

above microalgae as live food for larvae. 

Nevertheless, larvae of the variegated sea 

urchin (L. variegatus) have just been confirmed 

to be appropriate for culturing 

with artificial diets (Gorge at al., 2004). 

Settlement and metamorphic induction 

are considered as the most crucial 

stages for the development and culture of 

sea urchin larvae. The higher survival rate 

is always dependent on the larvae to become 

competent to metamorphose and 

then responding to the exact settlement 

cues. In small-scale culture, induction for 

metamorphosis of competent larvae has 

recently been performed on coralline red 

algal extracts + Chaetoceros calcitran 

Rahman et al. 

diatom (50:50) in petri dishes containing 

FSW (Rahman et al., 2012b). Within 1 

day after the settlement induction, majority 

of the competent larvae are found to 

metamorphose into young juvenile (Fig. 

4A). They are then reared on the encrusting 

coralline algal rocks in the aerated 

glass/plastic aquaria for at least three 

months by which they attains appropriate 

juvenile (hereafter referred to as sea urchin 

seed) sizes (Fig. 4D) for stocking in 

grow-out aquaculture system. In the countries 

like Japan, South Korea, Ireland, 

Norway, Scotland, and in British Colombia, 

Canada, sea urchin juveniles have 

been produced on a commercial or semicommercial 

scale by some welldeveloped 

hatcheries and nurseries. Almost 

all the culturists use natural biofilm 

or a specially seeded diatom substratum 

made from species locally isolated and 

then grown on a PVC wave plate. However, 

one of the most challenging areas of 

Figure 3: Developmental stages for larvae of short-spined white sea urchin, Salmacis 

sphaeroides: A) 4-arm pluteus, B) 6-arm pluteus, C) 8-arm pluteus, D) Pre-competent larva, 

E) Competent larva with complete rudiment growth, and F) Just newly metamorphosed juvenile 

with adult spines and tubefeets (Rahman et al., 2012b). 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 427



research is needed to optimize diets for 

the early juveniles and/or replacement of 

diatom biofilms. The differences in size 

and succeeding variation in growth rates 

of post-larvae remain a bottleneck in the 

supply of hatchery-reared juveniles. These 

juveniles are robust enough to survive, 

transfer to sea cages or other grow-out 

systems from a small size of 5 mm TD 

(Kelly, 2002; Sakai et al., 2004). At this 

instant, they are weaned into other diets, 

soft macroalgae or artificial diets, depending 

on the grow-out culture system. 

In the small-scale indoor aquariarearing 

system, 1-day-old juveniles are 

reared in small aquaria (25 x 20 x 20 cm) 

with aerated FSW and pieces of dead coral 

with coralline red algae are supplied as 

food (Figure 4) (Rahman et al., 2000, 

Rahman et al. 

2001, 2005). Seawater is partially 

changed twice a month and replenished 

with freshly filtered sea water. The method 

in continued for up to three months, by 

which time the juveniles reach to 9.0–10 

mm in TD. These 3-month-old juveniles 

(Figure 5A) are then transferred to 

glass/plastic aquaria (90 x 45 x 45 cm) 

provided with filtered seawater in the culture 

unit of the Laboratory of Marine Biotechnology, 

Institute of Bioscience, Universiti 

Putra Malaysia. Stocking density is 

fixed at 20 juveniles in each replicate 

aquarium and the urchins are fed with red 

alga (Amphiroa fragilissima), brown alga 

(Sargassum polysystum) and sea grass 

(Enhalus acoroides). Juveniles in all the 

treatments were fed ad libitum and seawater 

from each rearing aquarium was 

Figure 4: Juveniles of the white sea urchin, Salmacis sphaeroides: A) 1-day-old juvenile, 

B) 1-month-old juvenile, C) 2-minth-old juvenile, and D) 3-month-old juvenile for growout 

culture (Rahman et al., 2012b). 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 428



Rahman et al. 

Figure 5: Stocking juveniles and cultured adults of Salmacis sphaeroides: A) Three-monthold 

juveniles for stocking in grow-out culture, B) Sexually matured adults after the culture 

period of two years in captive aquaria-rearing system. 

Table 1: Comparison of growth and production of S. sphaeroides fed with different types 

of algae. Mean ± SE, n = 30 

Parameters 

Treatments 

T 1 (Red alga) T 2 (Brown alga) T 3 (Sea grass) 

Initial length (cm) 10.04 ± 0.70 a 10.04 ± 0.70 a 10.04 ± 0.70 a 

Final length (cm) 46.49 1.01 a 43.56 1.04 b 38.67 0.35 c 

Initial weight (g) 0.49 0.11 a 0.49 0.11 a 0.49 0.11 a 

Final weight (g) 51.17 1.17 a 31.91 1.42 b 20.80 0.65 c 

Weight gain (g) 50.67 1.93 a 31.39 1.44 b 20.31 0.43 c 

Length gain (cm) 36.46 1.01 a 33.85 0.66 b 28.63 0.35 c 

SGR (%/day) 0.73 0.01 a 0.65 0.01 b 0.58 0.01 c 

DGR (%/day) 7.92 0.30 a 4.91 0.22 b 3.17 0.10 c 

Wet gonad weight (g) 6.01 0.37 a 3.56 0.26 b 2.32 0.10 c 

Gonad index (%) 18.26 0.51 a 16.44 0.19 b 14.84 0.25 c 

Survival (%) 88.89 1.93 a 73.33 3.34 b 56.67 5.77 c 

Means sharing the same superscripts within the same row are not significantly different 

from each other (P > 0.05). 

completely changed at every 2–3 months. 

After two years of culture, the urchins 

attain sexual maturity (Figure 5B) and 

those fed red alga, performed the best 

over the brown- and sea grass-fed urchins 

with regard to body growth and edible 

gonad production (Table 1; Rahman unpublished 

data). On the contrary to the 

Japanese culture system, where hatcheryreared 

juveniles are mainly released to 

managed seafloor (Hagen, 1996; Sakai et 

al., 2004; Kelly, 2005), scientists of other 

countries have conducted research with a 

wide range of grow-out culture systems 

for the juvenile and adult urchins, ranging 

from the relocation from poor to good 

feeding grounds (Moylan, 1997) to the 

ranching of urchins caged on the seafloor 

(Cuthbert et al., 1995). Juveniles reared in 

hatcheries have been grown under suspended 

culture condition (Kelly, 2002, 

2005) in closed recirculation systems 

(Grosjean et al., 1998) and in demand 

rock pools in southern Ireland. The seacage 

culture system of stacking baskets 

suspended from a ladder-like structure 

over which a work barge or raft can operate 

has been developed by Norwegian researchers 

(Aas, 2004). The time taken for 

juveniles of most species to reach market- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 429



able size is in the range from 1 to 3 years 

(Kelly, 2005, Rahman et al., 2014b). 

3.2. Management 

The sea urchin fisheries around the world 

have consistently shown their susceptibility 

to overfishing and to the problems associated 

with readjusting effort following 

a fishing-down phase. Through the late 

1980s and 1990s, a number of fisheries 

have developed to cater for the expanding 

demand of the lucrative Japanese market. 

This has driven the development of new 

fisheries off Chile, both coasts of North 

America and Australia. These fisheries 

commenced 

A 

on virgin stocks and as a result 

have needed to deal with the issues of 

B 

rising expectations during fishing-down 

phase and adjustment of the fishery for 

long-term sustainability. 

The current position of the various 

American fisheries provides the full range 

of outcomes in dealing with this problem. 

For instance, the fisheries of Canada, 

Alaska and Washington have had active 

programs to readjust effort levels and are 

now managed on the basis of catch limits 

based on sustainable harvest strategies 

using regular population surveys. The 

Californian fishery has yet to adequately 

adjust levels of effort and there is evidence 

that the fishery is now being overfished. 

The Chilean fishery has little capacity 

to readjust effort levels and it 

would appear that once the fish-down 

phase is completed there is the potential 

for significant overfishing. Of the fisheries 

reviewed here, there are numerous examples 

of fisheries that have collapsed to 

levels of one or two magnitudes below 

their peak production. These include the 

fisheries of France (Mediterranean and 

Atlantic), Iceland, Ireland, South Korea 

and Philippines. Significant parts of the 

Chinese fishery have also collapsed. 

Those fisheries that are currently being 

managed for long term sustainability 

share a number of characteristics. They 

are: 

Rahman et al. 

• limited entry (moratoriums) followed 

by active programs to reduce latent effort, 

• resource surveys at various levels of 

complexity, 

• the use of annual Total Allowable 

Catches based on resource assessment, 

• zoning and area management, which 

may be developed to the point of rotational 

harvest, 

• the use of minimum legal sizes. 

Classical fisheries science was developed 

in consideration of offshore, open 

access and industrial fishing situations 

and the resulting management systems are 

not well adapted, or particularly robust, 

when applied to more complex spatial 

structure of small scale, inshore fishery 

resources (Orensanz and Jamieson, 1998). 

The social significance of small scale inshore 

fisheries is much greater, given the 

numbers of fishermen and other players 

involved, than the sometimes more productive 

offshore fisheries, which generally 

involve larger enterprises, higher capital 

investment and limited numbers of 

fishermen. As a result, much of the challenge 

in ensuring the sustainability of 

shellfish fisheries lies in developing and 

applying appropriate utilization, assessment 

and management models. According 

to Orensanz and Jamieson (1998), the 

management measures that explicitly 

acknowledge spatial structure of fishery 

resources, and are therefore the most suitable 

for these sorts of fisheries, include 

(but not limited to): 

i) territorial property and use rights including 

lease, traditional tenure systems 

etc.; 

ii) harvest rotation coupled with pulse 

fishing and/or thinning; 

iii) reproductive refugia and Marine Protected 

Areas; 

iv) experimental management with spatial 

control, contrasting treatments and 

replication; 

v) localized enhancement including habitat 

manipulation, seeding and predator 

control. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 430



Systems that accelerate growth to 

market size sea urchins while producing a 

uniform size class would give an economic 

advantage One probable way to obtain 

sustainable and environmentally friendly 

systems for urchin culture is to further 

examine their potential in integrated systems. 

They have already been shown to 

thrive in polyculture with Atlantic salmon 

(Kelly et al., 1998) and to have a role in 

land-based integrated systems (Shpigel et 

al., 2004). Nevertheless, many species are 

true omnivores, so the potential for their 

integration into systems where natural 

prey items, for instance, mussels and 

clams, are already produced should be 

explored. 

Rahman et al. 

3.3. Stock enhancement 

Decline in world production and overfishing 

have prompted increasing in enhancement 

as a means of maintaining 

production. It is most developed in Japan 

where the 1974 Coastal Fishing Ground 

Improvement and Development Law provide 

the basis for stock enhancement 

(Agatsuma et al., 2004). The goal of this 

program is to “develop and improve 

coastal fishing grounds systematically by 

the construction of artificial reefs and the 

release of seedlings”. Enhancement can 

comprise a number of different activities 

including direct stock enhancement 

through seeding of hatchery-raised juveniles, 

habitat improvement or restoration, 

creation of artificial reefs, predator control, 

thinning and/or roe enhancement 

through supplemental feeding to increase 

the product recoveries etc. Re-seeding has 

been especially applied in Japan since late 

1980's. The numbers have been fairly stable 

since 1997 with about 70-85 million 

juveniles reared to about 5-10 mm TD 

and released each year primarily in the 

areas with the largest historical harvest. 

Strongylocentrotus intermedius accounts 

for about 85% of the urchins released by 

the Japanese in Hokkaido (Andrew et al., 

2002). Predator removal is required as 

excess predation by sea stars etc. has been 

implicated in the few cases where the reseeding 

did not have any benefit on the 

subsequent urchin production and crabs 

and sea stars are removed from the 

grounds using baited traps prior to the 

release of the urchins to control mortality 

in the immediate period after release. 

Government has taken considerable involvement 

in the management of coastal 

fisheries, particularly in the provision of 

subsidies for enhancement and infrastructure 

development as well as management 

coordination. A couple of studies have 

been looked at the contribution of reseeding 

or habitat enhancement to the actual 

abundance of urchins in harvest areas 

in a sort of round-about way at localized 

sites around Hokkaido and estimated that 

re-seeded urchins comprised 62%, 66% 

and 80% of the total catch in 1994, 1995 

and 1996, respectively (Agatsuma, 2004; 

Rahman et al., 2014b). 

Translocation of the urchins is 

used for a number of related reasons in 

Japan. In areas where kelp forest development 

is held back by excessive urchin 

densities, urchins are sometimes removed 

and replaced with adult kelps to permit 

rapid development of complex kelp forests 

(Agatsuma, 2004). The urchins may 

then be placed into intensive sea ranching 

pens where they are fed ad libitum and 

prepared for harvest some months down 

the road. Experiments have shown that 

Green Sea Urchins at densities up to 35 

kg/m have recorded recovery increases 

from 6% to over 18% in 11 weeks on an 

artificial diet (Aas, 2004), although further 

finishing for about 6 weeks on a natural 

kelp diet is still required to get an 

acceptable taste profile at this point. The 

sea urchin Evechinus chloroticus is widely 

distributed around New Zealand but 

attempts to establish a commercial fishery 

have, like Norway, not succeeded because 

of the poor product quality and low recoveries. 

Experiments with ponding over 

2 month periods have seen recoveries to 

increase near 20% and produced other 

quality improvements that promise well 

for the future but further research is still 

needed to reach an economic breakeven. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 431



There are a number of aquaculture sites 

around New Zealand which are currently 

considered marginal for mussel farming, 

which would be suitable for urchin culture 

(Barker and Fell, 2004). Multidisciplinary 

approaches are therefore 

needed for stock enhancement and both 

scientific and user group advisors should 

be involved (Masuda and Tsukamoto, 

1998). One method of enhancement that 

apparently works very well with Green 

Sea Urchins simply requires the presence 

of a salmon net pen. The urchins can apparently 

settle out quite abundantly on 

such structures and grow quite nicely by 

feeding on the fouling organisms on the 

mesh. This has provided some opportunities 

for Canadian fishermen in British Colombia 

(BC) for some easy harvests. 

The current market system used 

for the urchin trade developed in tandem 

with the wild fishery but this will no 

doubt change dramatically once cultured 

product is available in substantial quantities. 

Cultured production is more tightly 

controlled than from the wild fishery so 

that, as with the cultured salmon, the consistent 

availability of an invariably high 

quality product throughout the year will 

have a tremendous impact on the urchin 

markets throughout the world. Traditional 

harvesters of sea urchins do not generally 

know much about the potential of aquaculture 

(Robinson, 2004) and will likely 

tend towards obstructing its development 

as opposed to recognizing the available 

advantages and applying them to their 

own benefit. This will be unfortunate because 

if the wild and cultured urchin fisheries 

could be more closely integrated, 

both would stand to benefit. For example, 

the gonad size and quality are quite easy 

to manipulate and the economic yield of 

the roe can be dramatically and fairly easily 

increased. This knowledge is probably 

directly applicable to the wild fishery and 

could contribute to an increase in quality, 

value and profitability. Already, fisheries 

and aquaculture are blurring together with 

respect to product (gonad) enhancement 

and re-seeding of juveniles is coming to 

Rahman et al. 

the fore in a number of countries (Robinson, 

2004). 

4. Bioactive compounds and human 

health benefits 

Alike many other marine invertebrates, 

sea urchins have been considered 

as a source of biologically active compounds 

with biomedical applications 

(Kelly, 2005, Rahman et al., 2014b). 

However, the full potential of echinoids 

as a source of biologically active products 

is largely unexplored (Bragadeeswaran et 

al., 2013). The marine environment is an 

exceptional reservoir of natural bioactive 

compounds, many of which exhibit structural 

and chemical features not detected in 

terrestrial derived natural products. The 

richness of diversity offers a great opportunity 

for the discovery of new bioactive 

compounds. Modern technologies have 

opened huge extents of research for the 

extraction of bioactive compounds from 

seas and oceans to treat the fatal diseases. 

The number of natural products isolated 

and purified from marine organisms increases 

rapidly and currently surpass with 

hundreds of new compounds being discovered 

every year (Proksch and Muller, 

2006). The isolated secondary metabolites 

have numerous functions; it is likely that 

some of them may be pharmacologically 

active compounds for humans and useful 

as medicines (Briskin, 2000). A number 

of such compounds have been isolated 

from echinoderms (Carballeria et al., 

1996). There has also been much valuable 

information available for new antibiotics 

and give new insights into bioactive compounds 

in sea urchins. Sea urchins shells 

are containing various polyhydroxylated 

naphtoquinone pigments, spinochromes 

(Anderson et al., 1969) as well as their 

analogous compound echinochrome A, of 

which was showed bactericidal effect as 

reported by Service et al. (1984). The 

phenolic hydroxyl groups in these molecules 

also suggested that they could participate 

in particular antioxidant activity 

as was observed in other well-known an- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 432



tioxidant polyphenols such as tea catechins. 

Similar to the structured compounds 

are also found in the shells of sea 

urchins and thus suggesting that they as 

well as echinochrome A would act as antioxidant 

substances, similar to other polyphenolic 

antioxidants in edible plants 

(Chantaro et al., 2008). While, squaric 

acid ester-based methodology was used in 

a new synthesis of chinochrome A was 

used in a new synthesis of echinochrome 

A, a polyhydroxylated napthoquinone 

pigment, commonly isolated from sea urchin 

spines (Pena-Cabrera et al., 2002). 

Gonads of sea urchins contain polyhydrxylated 

polyhydoxylated naphtoquinone 

pigments, echinochrome A, which 

highly potential in antioxidant activity 

(Lebedev et al., 2001). In our recent 

study, we found that the ovary extracts of 

long-spined black sea urchin (D. setosum) 

has profound and thereby inhibit the 

growth of pathogenic microorganisms 

(Marimuthu et al., 2015). 

Sea urchin gonads are also very 

rich in valuable bioactive compounds, 

such as polyunsaturated fatty acids 

(PUFAs) and β-carotine (Dincer and 

Cakli, 2007). PUFAs, especially eicosepentanoic 

acid (EPA, C20:5) (n-3)) and 

docosahexaenoic acid (DHA C22:6 (n-3)), 

have significant preventive effects on arrhythmia, 

cardiovascular diseases and 

cancer (Pulz, 2004). β-carotene and some 

xanthophylls have strong pro-vitamin A 

activity and can be used to prevent tumor 

development and light sensitivity (Britton 

et al., 2004). The composition of these 

valuable components, however, varies 

greatly among different urchin species 

and is influenced by their natural diets as 

well as physiological processes i.e., reproductive 

stages (Fernandez, 1998; Lawrence, 

2007). On the other hand, the high 

levels of AA and EPA recently detected 

in D. setosum and S. sphaeroides greatly 

supported the development of aquaculture 

of sea urchins (Chen et al., 2010), since 

PUFAs are important for human nutrition 

(Lawrence, 2007). 

Rahman et al. 

Based on the nutritional facts a 

100 g of sea urchin gonad, which is equal 

to 3.5 ounces, contain 172 calories and 

very little fat. In fact, the fat that a serving 

sea urchin does contain is almost all unsaturated 

fatty acids. For instance, there is 

1.75 g of polyunsaturated fat content in a 

serving sea urchin. Consumption of polyunsaturated 

fats instead of saturated fats, 

such as those found in a burger, can help 

in reducing the overall cholesterol level. 

Sea urchins also contain omega-3 fatty 

acids, which can help in lowering blood 

pressure and reducing the risk of an abnormal 

heat beat followed by heart attack 

(Rahim and Nurhasan, 2012). In addition, 

they serve as frequent model organism for 

developmental and immunological studies. 

5. Bioassays for coastal water quality 

using sea urchin embryo-larva and 

adults 

Coastal ecosystems are now matter 

to the impact of numerous human activities 

that lead to the input of a range of 

pollutants of agricultural, urban, or industrial 

origin. Sea urchins have been extensively 

used as bioindicators of marine 

pollution over the last several decades 

(Kobayashi, 1971; Phillips, 1990; Flammang 

et al., 1997). The two key life stages 

of the sea urchin most commonly studies 

and used in testing are the embryolarval 

and adult stages. Specifically, the 

early life stages of some different species 

of sea urchins have been demonstrated to 

be sensitive to metals (Kobayashi, 1973, 

1980; Kobayashi and Fujinaga, 1976; 

Plillips et al., 2003). 

Sea urchins are also useful indicator 

species for environmental contaminations 

due to the fact that their sperm, embryos 

and larvae are very sensitive to toxins 

in the water (Nacci et al., 1986; Pagano 

et al., 1986; Dinnel et al., 1989; 

Ghiradini et al., 2003; Ghiradini et al., 

2005). They are also considered as an excellent 

research species because spawning 

and gamete collection is reasonably simple, 

published literatures on echinoid em- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 433



bryological development is plentiful, the 

larvae develop quickly, animals are available 

throughout the year and are easily 

maintained under laboratory conditions 

(Hinegardner, 1969; Dinnel et al., 1989; 

Ghirardini et al., 2005). The sea urchin, S. 

sphaeroides and D. setosum are readily 

available in the Indo-Pacific including 

Malaysia and recently have documented 

embryonic and larval development stages 

(Rahman et al., 2012b; Rahman et al., 

2015). Sea urchin embryo-larva development 

test is a standard chronic toxicity 

bioassay advocated to be a cost-effective 

and useful method for use in screening 

the toxicity of specific pollutants, mixtures 

of these and natural matrices, and 

has regularly been used to assess the toxicity 

of water sediments (Beiras et al., 

2003; Cesar et al., 2009). This test consists 

of the study of teratogenic effects in 

early embryo to larval stages. The standardized 

or classical criterion for evaluating 

toxicity by means of this test involves 

distinguishing between normal larvae, i.e., 

pyramid-shaped larvae with skeletal rods 

that are half the length towards the long 

axis of the larvae, a differentiated gut and 

incipient postoral arms, and deformed 

larvae, i.e., larvae that display blocked or 

delayed embryonic development, undifferentiated 

or abnormal gut and absent or 

abnormal skeleton (USEPA, 1994; 

Warmau, et al., 1996). However, observation 

of only skeletal anomalies may be 

more rapid, sensitive and ecologically relevant 

than use of the classical criterion 

(without considering skeletal abnormalities), 

which, moreover may be affected by 

the determining role of food availability 

in the larval form, rate of growth of body 

parts and timing of development (Strathmann 

et al., 1992). 

The accumulation of pollutants in 

adult sea urchins has been used to monitor 

contaminations of many coral reef 

habitats (Phillips, 1990; Flammang et al., 

1997). Several studies have demonstrated 

metal accumulation in sea urchins adequately 

reflects abundance and availability 

in contaminated waters (Augier et al., 

Rahman et al. 

1989; Ablanedo et al., 1990; Flammang et 

al., 1997). The sea urchins, D. setosum 

and P. lividus have been used as bioindicators 

for assessing heavy metal contaminations 

in coral reef ecosystems of the 

Indo-West Pacific Ocean and the northwestern 

Mediterranean Sea, respectively 

(Warnau et al., 1995; Flammang et al., 

1997). Both the embryo-larva and adult 

D. antillarum were also found to be highly 

sensitive bioindicators for metal pollution 

in marine environments on the Caribbean 

and should be considered when 

determining ecological risks in coral reef 

environments (Bielmyer et al., 2005). 

6. Livelihood development and income 

generation 

Alike other commercially important marine 

invertebrates, Sea urchins offer important 

benefits to human beings due to 

their use in scientific research and education 

and also for food. In the economic 

point of view, sea urchin gonad either in 

the form of fresh or processed food, is 

considered as one of the most expensive 

and luxury seafood in the world (Richard, 

2004). In Japan, for example, sea urchin 

(known as “uni”) and its processed roe 

can retail for as much as AU$ 450 per kg. 

In addition, scientists and researchers can 

learn much about animal reproduction, 

fertilization, development and evolution 

by studying sea urchins, sea stars and other 

echinoderms as model species (Parvez 

et al., 2016b). 

The Bajau, Suluk, Kokos and 

Ubian tribes of Sabah (Eastern Malaysia) 

harvest the sea urchins, particularly their 

eggs, to be sold and eaten as a delicacy. 

This delicacy is usually prepared for special 

events such as Lepa-Lepa Festival, 

wedding ceremony and other cultural 

events and is being treated as valuable 

fishery resources in Malaysia (Rahim and 

Nurhasan, 2011). The sea urchins having 

long spines are known as “tayum” in Sabah, 

while the shorter spined species are 

called “tehe-tehe”. Sea urchins are usually 

sold in wet markets at different prices de- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 434



pending on their type and location. Tayum 

eggs are usually eaten raw and the 

selling price is from RM 2 to RM 5 per 

pack. Meanwhile, “tehe-tehe” is sold at 

RM 1 to RM 2 per plate i.e., containing 

three urchins with their skin intact to be 

cooked into oku-oku, a traditional Bajau 

delicacy. In comparison, price of sea urchin 

eggs is RM 36 to RM 60 for every 

80 g in America, while in Japan, an urchin 

can cost as much as RM 18 (Parvez 

et al., 2016b). Aesthetically, the diverse 

forms of the sea urchins, and their beautiful 

coloring, are often providing not only 

a source of joy and recreation but also 

increase the additional revenue to humans 

observing them. Thus, sea urchins play a 

significant role in livelihood development 

and income generation to the local coastal 

communities. 


This paper has been presented as a background 

document and review of the 

World‟s sea urchin fisheries. To summarize 

the views, reports and publications of 

other scientists/researchers, it is apparent 

that sea urchin fisheries have a poor record 

of sustainability, as evidenced by the 

declines recorded in Japan, Maine, California 

and South Korea among others, as 

well as by the ad hoc and/or ineffective 

management in many sea urchin fisheries. 

Very few stocks have been formally assessed, 

meaning it is near impossible to 

qualify declines as the fish-down of accumulated 

biomass, which does not arrest 

the productivity of the stock, or as a case 

of over-fishing in which case its productivity 

may be forced into permanent decline. 

Small-scale management is mentioned 

time and again as offering the most 

promise for ensuring long term sustainability. 

The strong and consistent spatial 

structure inherent in sea urchin stocks 

combined with excessive effort from mobile 

fleets and inappropriately large scale, 

and therefore ineffective management all 

contribute to declining production in 

many of the world‟s sea urchin fisheries. 

Rahman et al. 

This is mainly the case for the world‟s 

largest sea urchins fishery in Chile, where 

the risks of collapse cannot be discounted. 

Given that this fishery alone contributes 

upwards of 55% of the global harvest, a 

significant decline in Chile‟s fishery 

would likely lead to structural realignment 

in the market and higher prices for 

mid-range products until aquaculture production 

ramped up. There is also general 

agreement that some form of exclusive 

access as a prerequisite condition to promote 

meaningful enhancement and intelligent 

harvesting to maximize roe value 

will provide the best hedge against uncertainties 

in fisheries productivity and market 

stability. In the short term it is likely 

that global production of sea urchin roe 

from wild fisheries will decline, with the 

major production being provided by those 

fisheries that have supported active management 

strategies to readjust the effort 

and contain catches to levels that provide 

long-term sustainability. Given that demand 

is unlikely to decline; future production 

will be increasingly valuable. In 

order to make sea urchins fisheries viable 

and profitable, the following actions are 

suggested: 

Refinement of artificial diet formulations 

for juveniles and adults to maximize 

the growth rates and survivorship 

and produce gonads of the desired 

taste, texture, flavor and color. 

Optimization of grow-out facilities for 

juveniles and adults either at sea (in 

containers and „ranched‟) or landbased. 

Regulations regarding fishing methods, 

fishing areas and protection of 

company investments need to be developed. 

Better surveillance of sea urchin density 

to guarantee a steady flow of raw 

materials. 

Areas need to be thinned out to get the 

best possible product for the market, 

this is also necessary for the kelp forest 

to grow back. 

More capital needs to be directed towards 

investing in technology for 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 435



 

processing to reduce labor costs and 

preserve product quality. 

Improved cooperation between fishermen 

and processors, when marketing 

and selling the sea urchins. 


We would like to express our 

grateful thanks and appreciations to the 

Ministry of Science, Technology and Innovation 

(MOSTI), Malaysia, for providing 

financial supports through the Research 

Management Centre (RMC) of the 

Universiti Putra Malaysia (UPM) under 

the „ScienceFund‟ grant (Project No. 04- 

01-04-SF2227) for completing this work 

successfully. 

References 

Aas, K. (2004). Technology for sea-based 

farming of sea urchins. In: Lawrence 

J. M. and Guzmán O. (eds.). 

Sea Urchins: Fisheries and Ecology, 

DEStech Publications, Inc., 

Lancaster, USA. pp. 366–373. 

Agatsuma, Y. (2001a). The ecology of 

Strongylocentrotus intermedius. 

In: Lawrence, J. M. (ed.). Edible 

Sea Urchins: Biology and Ecology, 

Elsevier, Amsterdam, The Netherlands. 

pp. 333–346. 

Agatsuma, Y. (2001b). The ecology of 

Strongylocentrotus nudus. In: 

Lawrence, J. M. (ed.). Edible Sea 

Urchins: Biology and Ecology, 

Elsevier, Amsterdam, The Netherlands. 

pp. 347–361. 

Agatsuma, Y., Sakai, Y. and Andrew, 

N. L. (2004). Enhancement of Japan‟s 

sea urchin fisheries. In: 

Lawrence, J. M. and Guzman, O. 

(eds.). Sea urchins: fisheries and 

aquaculture, DEStech Publications, 

Lancaster, Pennsylvania. pp. 18– 

36. 

Alsaffar, A. H. and Khalid P. L. (2000). 

Reproductive cycles of Diadema 

setosum and Echinometra mathaei 

(Echinoidea: Echinodermata) from 

Rahman et al. 

Kuwait (Northern Arabian Gulf). 

Bulletin of Marine Science 67(2), 

845–856. 

Anderson, A. H., Mathieson, J. W. and 

Thomson, R. H. (1969). Distribution 

of spinochrome pigments in 

echinoids. Comparative Biochemistry 

and Physiology 28, 333–345. 

Andrew, N. and Byrne, M. (2001). The 

ecology of Centrostephanus rodgersii. 

In: Lawrence J.M. (ed.). Edible 

Sea Urchins: Biology and 

Ecology, Elsevier, Amsterdam, 

The Netherlands. pp. 149–160. 

Andrew N. L. et al. (2002). Status and 

management of world sea urchin 

fisheries. Oceanography and Marine 

Biology Annual Review 40, 

343–425. 

Andrew, N. L., Agatsuma, Y., Dewees, 

C. M. and Stotz, W. B. (2004). 

State of sea-urchin fisheries 2003. 

In: Lawrence, J. M. and Guzma´n, 

O. (eds.). Sea Urchin Fisheries 

and Ecology. DEStech Publications, 

Lancaster, PA, USA, pp. 

96–98. 

Barker, M. F. (2001). The ecology of 

Evechinus chloroticus. In: Lawrence, 

J. M. (ed.). Edible Sea Urchins: 

Biology and Ecology, Elsevier, 

Amsterdam, The Netherlands, 

pp. 245–260. 

Barker, M .F. (2007). The ecology of 

Evechinus chloroticus. In: Lawrence 

J. M. (ed.). Edible Sea Urchins: 


Amsterdam, The Netherlands, 

pp. 319–338. 

Barker, M. F. and Fell, J. (2004). Sea 

cage experiments on roe enhancement 

of New Zealand sea 

urchin Evachinus chloroticus. In: 

Lawrence, J. M. and Guzmán, O. 

(eds.). Sea Urchins: Fisheries and 

Ecology, DEStech Publications, 

Inc., Lancaster, pp. 375–383. 

Barnes, D. K. A., Verling, E., Crook, A., 

Davidson, I, and O’Mahoney, M. 

(2002). Local population disappearance 

follows (20 yr after) cy- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 436



Rahman et al. 

cle collapse in a pivotal ecological 

species. Marine Ecology Progress 

Series 226, 311–313. 

Beiras, R., Fernández, N., Bellas, J., 

Besada, V., González-Quijano, 

A. and Nunes, T. (2003). Integrative 

assessment of marine pollution 

in Galician estuaries using 

sediment chemistry, mussel bioaccumulation, 

and embryo–larval 

toxicity bioassays. Chemosphere 

52(7), 1209–1224. 

Boudouresque, C.-F. and Verlaque, M. 

(2001). The ecology of Paracentrotus 

lividus. In: Lawrence, J. M. 

(eds.). Edible Sea Urchins: Biology 

and Ecology, Elsevier, Amsterdam, 

The Netherlands, pp. 

177–216. 

Bragadeeswaran, S., Sri Kumaran, N., 

Prasath Sankar P. and Prabahar, 

R. (2013). Bioactive potential 

of sea urchin Temnopleurus 

toreumaticus from Devanampattinam, 

Southeast coast of India. 

Journal of Pharmacy and Alternative 

Medicine 2(3), 9–17. 

Briskin, D. (2000). Medicinal Plants and 

Phyto medicines. Linking plant 

biochemistry and physiology to 

human health. Plant Physiology 

124, 507–514. 

Britton, G., Liaaen-Jensen, S. and 

Pfander, H. (2004). Carotenoids 

handbook, Birkhauser Verlag, 

Boston, USA. 

Carboni, C., Addis, P., Cau, A. and 

Atach, T. (2012). Aquaculture 

could enhance Mediterranean sea 

urchin fishery, expand supply. 

Global Aquaculture Advocate 

15(3), 44–45. 

Carballeria, N. M., Cruz, C. and Sostre, 

A. (1996). Identification of the 

novel 7-methyl-6 octadecenoic acid 

in Holothuria mexicana. Journal 

of Natural Products 59(11), 

1076–1078. 

Cesar, A., Marín, A., Marin-Guirao, L., 

Vita, R., Lloret, J. and DelValls, 

T.A. (2009). Integrative ecotoxicological 

assessment of sediment 

in Portmán Bay (southeast Spain). 

Ecotoxicology and Environmental 

Safety 72, 1832e1841. 

Chantaro, P., Devahastin, S. and 

Chiewchan, N. (2008). Production 

of antioxidant high dietary fiber 

powder from carrot peels. 

LWT-Food Science and Technology 

41, 1987–1994. 

Chen, G. -Q., Xian, W. -Z., Lau, C. -C., 

Peng, J., Qiu, J. -W., Chen, F. 

and Jiang, Y. (2010). A comparative 

analysis of lipid and carotenoid 

composition of the gonads of 

Anthocidaris crassispina, Diadema 

setosum and Salinacis 

sphaeroides. Food Chemistry 

120(4), 973–977. 

Cuthbert, F. M., Hooper, R. G. and 

McKeever T. (1995). Sea urchin 

aquaculture phase I: sea urchin 

feeding and ranching experiments. 

Report of Project AUI-503. Canadian 

Centre for Fisheries Innovation, 

Government of Newfoundland. 

Dincer, T. and Cakli, S. (2007). Chemical 

composition and biometrical 

measurements of the Turkish Sea urchin 

(Paracentrotus lividus, Lamarck, 

1816). Critical Reviews in Food Science 

and Nutrition 47(1), 21–26. 

Dinnel, P. A., Link, J. M., Stober, Q. J., 

Letourneau, M. W. and Roberts, 

W. E. (1989). Comparative sensitivity 

of sea urchin sperm bioassays 

to metals and pesticides. Archives 


and Toxicology 18(5), 

748–755. 

Edgar, G. (1999). Tasmania. In: Andrew, 

N. L. (ed.). Under Southern Seas: 

The Ecology of Australia‟s Rocky 

Reefs, University of New South 

Wales Press, Sydney, pp. 30-39. 

FAO. (2010). The State of World Fisheries 

and Aquaculture 2010. Food 

and Agriculture Organization of 

the United Nations, Rome, Italy. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 437



Fernandez, C. (1998). Seasonal changes 

in the biochemical composition of 

the edible sea urchin Paracentrotus 

lividus (Echinodermata: Echinoidea) 

in a lagoonal environment. 

Marine Ecology 19, 1–11. 

Flammang, P., Warnau, M., Temara, 

A., Lane, D. J. W. and Jangoux, 

M. (1997). Heavy metals in Diadema 

setosum (Echinodermata 

Echinoidea) from Singapore coral 

reefs. Journal of Sea Research 38, 

35–45. 

George, S. B., Lawrence, J. M. and 

Lawrence, A. L. (2004). Complete 

larval development of the 

sea urchin Lytechinus variegatus 

fed an artificial feed. Aquaculture 

242(1-4), 217–228. 

Ghirardini, V. A, Chiaraa, L., Alessandraa, 

A. N., Alvisea, B., Hisb, E. 

and Francesco, G. (2005). Mytilus 

galloprovincialis as bioindicator 

in embryotoxicity testing to 

evaluate sediment quality in the 

lagoon of Venice (Italy). Chemistry 

and Ecology 21(6), 455–463. 

Grignard, J. C., Flammang, P., Lane, D. 

J. W. and Jangoux, M. (1996). 

Distribution and abundance of the 

echinoid Diadema setosum (Echinodermata) 

on sediment-stressed 

coral reefs in Singapore. Asian 

Marine Biology 13, 123–132. 

Grosjean, P., Spirlet, C. and Gosselin, 

P. (1998). Land-based, closedcycle 

echinoculture of Paracentrotus 

lividus (Lamarck) (Echinoidea: 

Echinodermata): a long-term 

experiment at a pilot scale. Journal 

of Shellfish Research 17(5), 

1523–1531. 

Hagen, N. T. (1996). Echinoculture: from 

fishery enhancement to closedcycle 

cultivation. World Aquaculture 

27, 6–19. 

Hagen, N. T. (2000). Echinoderm culture. 

In: Stickney, R. R. (ed.), Encyclopedia 

of aquaculture. Wiley- 

Interscience, New York, pp. 247– 

253. 

Rahman et al. 

Ichihiro, K. (1986). Breeding, processing 

and sale, Hokkai Suisan Shinbunsha, 

Sappro, Japan. 

Kaneniwa, M. and Takagi, T. (1986). 

Fatty acids in the lipid of food 

products from sea urchin. Bulletin 

of the Japanese Society of Scientific 

Fisheries 52(9), 1681–1685. 

Kessing, J. (2001). The ecology of Heliocidaris 

erythrogramma. In: Lawrence, 

J.M. (ed.). Edible Sea Urchins: 


Amsterdam, The Netherlands. 

pp. 261–270. 

Keesing, J. K. and Hall, K. C. (1998). 

Review of the status of world sea 

urchin fisheries points to opportunities 

for aquaculture. Journal of 

Shellfish Research 17(5),1597– 

1604. 

Kelly, M. S. (2000). The reproductive 

cycle of the sea urchin 

Psammechinus miliaris (Gmelin) 

(Echinodermata: Echinoidea) in a 

Scottish sea loch. Journal of the 

Marine Biological Association of 

the United Kingdom 80, 909–919. 

Kelly. M. S. (2002). Survivorship and 

growth rates of hatchery-reared 

sea urchins. Aquaculture International 

10, 309–316. 

Kelly, M. S. (2005). Echinoderms: their 

culture and bioactive compounds. 

In: Matranga, V. (ed.). Echinodermata: 

Progress in Molecular 

and Subcellular Biology, Subseries: 

Marine Molecular Biotechnology, 

Springer-Verlag, Berlin 

Heidelberg, pp. 139–165. 

Kelly, M. S., Brodie, C. C. and McKenzie, 

J. D. (1998). Somatic and 

gonadal growth of the sea urchin 

Psammechinus miliaris (Gmelin) 

maintained in polyculture with the 

Atlantic salmon. Journal of Shellfish 

Research 17(5), 1557–1562. 

Kobayashi, N. (1971). Fertilized sea urchin 

eggs as indicatory materials 

for marine pollution bioassay, preliminary 

experiment. Publications 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 438



of the Seto Marine Biological Laboratory 

18, 379–406. 

Kobayashi, N. (1973). Studies on the effects 

of some agents on fertilized 

sea urchin eggs, as a part of the 

bases for marine pollution bioassay. 

Publications of the Seto Marine 

Biological Laboratory 21(2), 

109–114. 

Kobayashi, N. (1980). Comparative sensitivity 

of various developmental 

stages of sea urchins to some 

chemicals. Marine Biology 58, 

163–171. 

Kobayashi, N. and Fujinaga, K. (1976). 

Synergism of inhibiting actions of 

heavy metals upon the fertilization 

and development of sea urchin 

eggs. Science and Engineering 

Review of Doshisha University 

17(1), 54–69. 

Lawrence, J. M. (2007). Edible Sea Urchins: 

Biology and ecology. Elsevier, 

Boston, USA. p. 380. 

Lawrence, J. M., Olave, S., Otaiza, R., 

Lawrence, A. L. and Bustos, E. 

(1997). Enhancement of gonad 

production in the Sea Urchin Loxechinus 

albus in Chile fed extruded 

feeds. Journal of the World 

Aquaculture Society 28(1), 91–96. 

Lawrence, J. M., Lawrence, A. L., 

McBride, S. C., George, S. B., 

Watts, S. A. and Plank, L. R. 

(2001). Developments in the use 

of prepared feeds in sea-urchin 

aquaculture. World Aquaculture 

32(3), 34–39. 

Lebedev, A.V., Levitskaya, E. L., 

Tikhonova, E. V. and Ivanova, 

M. V. (2001). Antioxidant properties, 

autoxidation and mutagenic 

activity of echinochrome A compared 

with its etherified derivative. 

Biochemistry 66, 885–893. 

Lessios, H. A. (1981). Reproductive periodicity 

of the echinoids Diadema 

and Echinometra on the two 

coasts of Panama. Journal of Experimental 

Marine Biology and 

Ecology 50(1), 47–61. 

Rahman et al. 

Lessios, H. A., Kessing, B. D. and 

Pearse, J. S. (2001). Population 

structure and speciation in tropical 

seas. Global phylogeography of 

the sea urchin Diadema. Evolution 

55(5), 955–975. 

Marimuthu, K., Gunaselvam, P., Rahman, 

M. A., Xavier, R., Arockiaraj, 

J., Subramanian, S., 

Yusoff, F. M. and Arshad, A. 

(2015). Antibacterial activity of 

ovary extract from sea urchin Diadema 

setosum. European Review 

for Medical and Pharmacological 

Sciences 19, 1895–1899. 

Masuda, R. and Tsukamoto, K. (1998). 

Stock enhancement in Japan: Review 

and perspective. Bulletin of 

Marine Science 62(2), 337–358. 

McClanahan, T. R. and Muthiga, N. A. 

(2001). The ecology of Echinometra. 

In: Lawrence, J. M. (ed.). Edible Sea 

Urchins: Biology and Ecology. Elsevier, 

Amsterdam, the Netherlands, pp. 

225–243. 

Miskelly, A. (2002). Sea Urchins of and 

Indo-Pacific, Capricornica Publications, 

Sydney, Australia. 

Moylan, E. (1997). Gonad conditioning 

and wild stock enhancement of the 

purple sea urchin Paracentrotus 

lividus on the west coasts of Ireland. 

Bulletin of the Aquaculture 

Association of Canada 97(1), 38– 

45. 

Nacci, D., Jackim, E. and Walsh, R. 

(1986). Comparative evaluation of 

three rapid marine toxicity tests: 

sea urchin early embryo growth 

test, sea urchin sperm cell toxicity 

test and microtox. Environmental 

Toxicology and Chemistry 5, 521– 

525. 

Orensanz, J. M. and Jamieson, G. S. 

(1998). The assessment and management 

of spatially structured 

stocks: an overview of the North 

Pacific Symposium on Invertebrate 

Stock Assessment and Management. 

In: Jamieson, G. S. and 

Campbell, A. (eds.). Proceedings 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 439



Rahman et al. 

of the North Pacific Symposium 

on Invertebrate Stock Assessment 

and management. Canadian Special 

Publication of Fisheries and 

Aquatic Sciences 125 pp. 441–445. 

Oshima, T., Wada, S. and Koizumi, C. 

(1986). Lipid deterioration of salted 

gonads of sea urchin during 

storage at 5 o C. Bulletin of the 

Japanese Society of Scientific 

Fisheries 52(3), 511–517. 

Pagano, G., Cipollaro, M., Esposito, A., 

Ragucci, E., Giordano, G. G. and 

Trieff, N. M. (1986). The sea urchin: 

Bioassay for the assessment 

of damage from environmental 

contaminants. In: Cairns, J. Jr. 

(ed.). Community Toxicity Testing, 

Association for Standard Testing 

and Materials, Philadelphia, pp. 

67–92. 

Parvez, M. S., Rahman, M. A. and 

Yusoff, F. M. (2016a). Sea urchin 

fisheries in Malaysia: status, potentials 

and benefits. In: Rahman, 

M. A. and Monticolo, D. (eds.). 

Proceedings of the 5th International 

Conference on Chemical 

Engineering and Biological Sciences 

(ICCBS-16), International 

Scientific Academy of Engineering 

and Technology, Kuala Lumpur, 

Malaysia, pp. 14–16. 

Parvez, M. S., Rahman, M. A., Yusoff, 

F. M. and Arshad, A. (2016b). 

Status, prospects and potentials of 

the commercially important species 

of sea urchin, Tripneustes 

gratilla (Linnaeus 1758) in Malaysia. 

International Journal of 

Biological, Ecological and Environmental 

Sciences 5(1), 50–54. 

Pearse, J. S. (1970). Reproductive periodicities 

of Indo-Pacific invertebrates 

in the Gulf of Suez III. The 

echinoid Diadema setosum (Leske). 

Bulletin of Marine Science 20, 

697–720. 

Pearse, J. S. (1974). Reproductive patterns 

of tropical reef animals: 

three species of sea urchins. Proceedings 

of the 2nd International 

Coral Reef Symposium. Brisbane, 

ICRS, Australia, 1, 235–240. 

Pena-Cabrera, E. and Liebeskind, L. S. 

(2002). Squaric acid ester-based 

total synthesis of Echinochrome 

A. Journal of Organic Chemistry 

67, 1689–1691. 

Phillips, D. J. H. (1990). Use of 

macroalgae and invertebrates as 

monitors of metal levels in estuaries 

and coastal waters. In: Furness, 

R. W. and Rainbow, P. S. (eds.). 

Heavy Metals in the Marine Environment, 

CRC Press, Boca Raton, 

FL, pp. 81–99. 

Phillips, B. M., Nicely, P. A., Hunt, J. 

W., Anderson, B. S., Tjeerdema, 

R. S., Palmer, S. E., Palmer, F. 

H. and Puckett, H. M. (2003). 

Toxicity of cadmium–copper– 

nickel–zinc mixtures to larval 

purple sea urchins. Bulletin of Environmental 

Contamination and 


Proksch, P. and Muller, W. E. G. 

(2006). Frontiers in Marine Biotechnology. 

Horizon Bioscience, 

Norfolk, UK. 

Pulz, O. and Gross, W. (2004). Valuable 

products from biotechnology of 

microalgae. Applied Microbiology 

and Biotechnology 65(6), 635–648. 

Rahim. S. A. K. A. and Nurhasan, R. 

(2012). Edible sea urchin species 

in Sabah waters. Research Bulletin, 

Faculty of Resource Science 

and Technology (FRST), Universiti 

Malaysia Sarawak, 1, 2–3. 

Rahman, M. A. and Yusoff, F. M. 

(2010). Sea urchins in Malaysian 

coastal waters. The Oceanographer, 

4(1), 20–21. 

Rahman, M. A., Uehara, T. and Aslan, 

L. M. (2000). Comparative viability 

and growth of hybrids between 

two sympatric species of sea urchins 

(genus Echinometra) in 

Okinawa. Aquaculture 183, 45–56. 

Rahman, M. A., Uehara, T. and Pearse, 

J. S. (2001). Hybrids of two close- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 440



ly related tropical sea urchins (genus 

Echinometra): evidence 

against postzygotic isolating 

mechanisms. Biological Bulletin 

200, 97–106. 

Rahman, M. A., Uehara, T. and Pearse, 

J. S. (2004). Experimental hybridization 

between two recently diverged 

species of tropical sea urchins, 

Echinometra mathaei and 

Echinometra oblonga. Invertebrate 

Reproduction and Development 

45, 1– 14. 

Rahman, M. A., Uehara, T. and Lawrence, 

J. M. (2005). Growth and 

heterosis of hybrids of two closely 

related species of Pacific sea urchins 

(genus Echinometra) in 

Okinawa. Aquaculture 245, 121– 

133. 

Rahman, M. A., Amin, S. M. N., Yusoff, 

F. M., Arshad, A., Kuppan, P. 

and Shamsudin, M. N. (2012a). 

Length weight relationships and 

fecundity estimates of long-spined 

sea urchin, Diadema setosum, 

from the Pulau Pangkor, Peninsular 

Malaysia. Aquatic Ecosystem 

Health and Management 15, 311– 

315. 

Rahman, M. A., Yusoff, F. M., Arshad, 

A. Shamsudin, M .N. and Amin, 

S. M. N. (2012b). Embryonic, larval, 

and early juvenile development 

of the tropical sea urchin, 

Salmacis sphaeroides (Echinodermata: 

Echinoidea). The Scientific 

World Journal 2012, 1–9. 

Rahman, M. A., Arshad, A., Yusoff, F. 

M. and Amin, S. M. N. (2013a). 

Hybridization and growth of tropical 

sea urchins. Asian Journal of 

Animal and Veterinary Advances 

8(2), 177–193. 

Rahman, M. A., Yusoff, F. M., Arshad, 

A., Amin, S. M. N. and 

Shamsudin, M. N. (2013b). Population 

characteristics and fecundity 

estimates of short-spined 

white sea urchin, Salmacis 

sphaeroides (Linnaeus, 1758) 

Rahman et al. 

from the coastal waters of Johor, 

Malaysia. Asian Journal of Animal 

and Veterinary Advances 8(2), 

301–308. 

Rahman, M. A., Yusoff, F. M. and Arshad, 

A. (2014a). Potential and 

prospect for sea urchin resource 

development in Malaysia. Fishmail 

21, 16–18. 

Rahman, M. A., Arshad, A. and Yusoff, 

F. M. (2014b). Sea urchins (Echinodermata: 

Echinoidea): Their biology, 

culture and bioactive compounds. 

In: Kao, J. C. M. and 

Rahman, M. A. (eds.). Proceedings 

of the International Conference 

on Advances in Environment, 

Agriculture and Medical Sciences 

(ICAEAM‟14), International 

Academy of Arts, Science & 

Technology, Kuala Lumpur, Malaysia, 

pp. 23–27. 

Rahman M. A., Yusoff, F. M. and Arshad, 

A. (2015). Embryonic, larval 

and juvenile development of 

tropical sea urchin, Diadema setosum. 

Iranian Journal of Fisheries 

Sciences 14(2), 409–424. 

Rahman, M. A. Yusoff, F. M., Arshad, 

A. and Ara, R. (2016). Growth 

and survival of the tropical sea urchin, 

Salmacis sphaeroides fed 

with different macroalgae in captive 

rearing condition. Journal of 

Environmental Biology 37, 855– 

862. 

Richard, M. (2004). The little urchins 

that can command a princely price. 

The Sydney Morning Herald. 

Robinson, S. M. (2004). The evolving 

role of aquaculture in the global 

production of sea urchins. In: Sea 

Urchins: Fisheries and Ecology 

proceedings of the international 

Conference on Sea-Urchin Fisheries 

and Aquaculture. Puerto Varas, 

Chile, March 25-27, 2003. DEStech 

Publications. Lancaster, PA. 

Saito, K. (1992). Sea urchin fishery of 

Japan. In: Anonymous, The management 

and enhancement of sea 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 441



urchins and other kelp bed resources: 

a Pacific rim perspective. 

California Sea Grant College, La 

Jolla, Californoa, Report No. T- 

CSGCP-028. 

Sakai, Y., Tajima, K. I. and Agatsuma, 

Y. (2004). Mass production of 

seed of the Japanese edible sea urchins 

Strongylocentrotus droebachiensis 

and Strongylocentrotus 

nudus. In: Lawrence, J. M. and 

Guzman, O. (eds.). Sea urchins: 

fisheries and aquaculture, DEStech 

Publications, Lancaster, 

Pennsylvania, pp 287–298. 

Sanderson, C. J. (1994). Interim Sea Urchin 

Report June 1994. Department 

of Primary Industry and 

Fisheries Marine Research Laboratory 

International Report 8 

Sanderson, C. J. (1995). Interim Sea Urchin 

Report February 1995. Department 

of Primary Industry and 

Fisheries Marine Research Laboratory 

International Report 21. 

Sanderson, C. J. Le Rossignol, M. and 

James, W. (1996). A pilot program 

to maximize Tasmania‟s sea 

urchin 

(Heliocidaris 

erythrogramma) resource. Fisheries 

Research and Development 

Corporation Final Report 93/221. 

Scheibling, R. E. and Hatcher, B. G. 

(2001). The ecology of Strongylocentrotus 

droebachiensis. In: 

Lawrence, J. M. (ed.). Edible Sea 

Urchins: Biology and Ecology. 

Elsevier, Amsterdam, The Netherlands, 

pp. 271–297. 

Schoppe, S. (2000). A guide to common 

shallow water sea stars, sea urchins, 

sea cucumbers and feather 

stars (Echinoderms) of the Philippines. 

Times Editions, Singapore, 

p. 144. 

Seifullah, R. D., Ankudinova, I. A. and 

Kim, E. K. (1995). Seksual‟noe 

provedenie muzhchin (sexual behavior 

of men). Izd. Yaguar, Moscow. 

Rahman et al. 

Service, M. and Wardlaw, A. C. (1984). 

Echinochrome-A as a bactericidal 

substance in the coelomic fluid of 

Echinus esculentus. Comparative 

Biochemistry and Physiology Part 

B 79, 161–165. 

Shpigel, M., McBride, S. C., Marciano, 

S. and Lupatsch, I. (2004). Propagation 

of the European sea urchin 

Paracentrotus lividus in Israel. In: 

Lawrence, J. M. and Guzman O. 

(eds.). Sea urchins: fisheries and 

aquaculture, DEStech, Publications, 

Lancaster, Pennsylvania, p. 

85. 

Shimabukuro, S. (1991). Tripneustes 

gratilla (sea urchin). In: Shokita, 

S., Kakazu, K., Tomomi, A., Toma, 

T., Yamaguchi, M. (eds.). 

Aquaculture in Tropical Areas. 

Midori Shobo Co. Ltd. Tokyo, pp. 

313–328. 

Sivertsen, K. (2004). Harvestable sea 

urchin Strongylocentrotus droebachiensis 

resources along the 

Norwegian coast. In: Lawrence, 

J.M. and Guzman, O. (eds.). Sea 

Urchins: Fisheries and Aquaculture, 

DEStech Publications, Lancaster, 

Pennsylvania, p. 85. 

Sloan, N. A. (1985). Echinoderm fisheries 

of the world: a review. In: 

Keegan, B. F. and O' Connor, B. 

D. S. (eds.). Echinoderms, A.A. 

Bolkema, Rotterdam, pp. 109– 

124. 

Strathmann, R. R., Fenaux, L. and 

Strathmann, M. F. (1992). Heterochronic 

developmental plasticity 

in larval sea urchins and its implications 

for evolution of nonfeeding 

larvae. Evolution 46, 972– 

986. 

Tan, L. W. H. and Ng, P. K. L. (1988). 

A Guide to Seashore Life. Singapore 

Science Centre, Singapore, p. 

160. 

Taylor, P.H. (2004). Green Gold: Scientific 

findings for management of 

Maine‟s Green Sea Urchin fishery. 

Maine Department of Marine 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 442



Resources, Boothbay Harbour, 

Maine, USA. 

Tegner, M. (2001). The ecology of 

Strongylocentrotus fransiscanus 

and Strongylocentrotus purpuratus. 

In: Lawrence, J. M. (ed.). 

Edible Sea Urchins: Biology and 

Ecology. Elsevier, Amsterdam, 

The Netherlands, pp. 307–331. 

Tuason, A. Y. and Gomez, E. D. (1979). 

The reproductive biology of Tripneustes 

gratilla Linnaeus (Echinodermata: 

Echinoidea), with 

some notes on Diadema setosum 

Leske. Proceedings of the International 

Symposium of Marine Biogeography 

and Evolution in the 

Southern Hemisphere. Auckland, 

New Zealand. 2, 707–716. 

USEPA. (1994). Methods for Derivation 

of Inhalation Reference Concentrations 

and Application of Inhalation 

Dosimetry EPA/600/8- 

90/066F. United States Environmental 

Protection Agency 

(USEPA), Washington DC, USA. 

Vasquez, J. (2001). The ecology of Loxechinus 

albus. In: Lawrence, J. M. 

Rahman et al. 

(ed.). Edible Sea Urchins: Biology 

and Ecology, Elsevier, Amsterdam, 

The Netherlands, pp. 161– 

175. 

Watts, S. A., McClintock, J. B. and 

Lawrence, J. M. (2001). The 

ecology of Lytechinus variegatus. 

In: Lawrence, J. M. (ed.). Edible 

Sea Urchins: Biology and Ecology. 

Elsevier, Amsterdam, The 

Netherlands, pp. 375–394. 

Warnau, M., Temara, A., Jangoux, M., 

Dubois, P., Iaccarino, M., De Biase, 

A. and Pagano, G. (1996). 

Spermiotoxicity and embryotoxicity 

of heavy metals in the echinoid 

Paracentrotus lividus. Environmental 

Toxicology and Chemistry 

15, 1931e1936. 

Yur’eva, M. I., Lisakovskaya, O. V., 

Akulin, V. N. and Kropotov, A. 

V. (2003). Gonads of sea urchins 

as the source of medication stimulating 

sexual behavior. Russian 

Journal of Marine Biology 29, 

189–193. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 443




Thavasimuthu Citarasu* and Mariavincent Michael Babu 

Centre for Marine Science and technology, Manonmaniam Sundaranar University, 

Rajakkamangalam- 629 502, Tamilnadu, India; michaelmsu@live.com (MMB); 

*Correspondence: citarasu@gmail.com 

Abstract: Marine environments are seriously affected by different pollutants created by 

human activities. The main entry of pollutants to marine environment is through atmosphere, 

water bodies, ships and other human activities. The pollutants seriously affect the 

marine flora, fauna and disturb the food supply chains. Several synthetic chemical residues 

including carbon tetrachloride, polychlorinated biphenyls, trichloroethylene and vinyl chloride 

are found in the marine sediments and flora and fauna. This type of marine pollution 

does have direct or indirect effect on human health. The biotechnological approaches such 

as bioremediation, probiotics, waste treatments by micro algae and seaweeds are useful to 

restrict and reduce the pollutants in the effluents before they reach the marine water bodies. 

This article highlights various aspects of marine pollution and its impacts on living organisms.Several 

pollution preventive measures and awareness programs are also discussed in 

this chapter. 

Keywords: Marine biotechnology; marine ecosystem; marine pollution; microplastics; pollution 

awareness 

1. Marine ecosystem and its resources 

The ocean occupy 71 percentages 

of earth‟s surface; they are interconnected 

and traditionally divided into four large 

basins including North and South Atlantic, 

North and South Pacific, Arctic and 

Indian oceans. The average depths are 

13,216, 10,932, 12,786 and 3,665 feet for 

Pacific, Atlantic, Indian and Arctic 

oceans, respectively. Marine environment 

is most important for life on earth, the 

living organisms originated in marine and 

they emigrated to terrestrial and freshwater 

bodies. Oceans are the main regulators 

of climate and temperature to the terrestrial 

ecosystem. Phytoplanktons are important 

for oxygen production; they yield 

around eighty percentages of oxygen 

which is used by the animals and plants 

for breathing in terrestrial and aquatic 

ecosystems (Bigg et al., 2003). Marine 

ecosystem is the largest ecosystem with 

intertidal zones, coral reefs, estuaries, lagoons, 

salty marshes, mangroves, deep 

sea and sea floor ecosystems which are 

important for marine (and terrestrial) living 

organism (Barange et al., 2010). They 

provide goods and various services to the 

human society such as good climate, vital 

foods, medicines, bio processing and employments 

including fishing, process industries, 

aquaculture and coastal tourism 

etc. 

2. Marine pollution 

The oceans are susceptible for 

pollution ever by human activities by polluting 

with different agents and physical 

destruction ocean environments. The definition 

of Marine Pollution as “Introduction 

by man, directly, or indirectly, of 

substances or energy to the marine environment 

resulting in deleterious effects 

such as: hazards to human health, hindrance 

to marine activities, impairment of 

the quality of seawater for various uses 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 444



and reduction of amenities”. Marine pollution 

is mainly classified to coastal 

sources by riverine inputs, deposition 

from atmosphere and offshore inputs etc. 

Coastal diffuse sources are contaminants 

via coastal industry, sewages and development 

sites (Clark, 2001). Alternative 

inputs are site-specific discharges, agriculture 

lands and forests, mainly for nutrients 

leakage to groundwater bodies, 

further they transported into the marine 

ecosystem by backwater bodied and finally 

huge quantities of contaminants deposited 

marine environments (GESAMP, 

1993). Municipal wastes and sewage water 

effluents are not properly treated and 

discharged in to sea, these problems 

mainly happened in developing countries. 

Emissions through air craft‟s are main 

sources of atmospheric pollution to sea 

and the pollutants are dispersed a vast areas 

by the wind flow and weather changes. 

Marine pollution in offshore caused by 

discharges of pollutants from vessel based 

at least 10 % of the total marine pollution 

and they contribute by different ways 

(Anon, 2005). The other sources are including 

crude oil extraction and of mineral 

extraction etc. 

3. Pollutant Sources 

3.1. Oil pollutants 

Hydrocarbons are classified into 

alkanes, naphthenes and aromatics. The 

crude oil also contains nitrogen, oxygen 

and vanadium compounds. The spillage 

of oils from the ships/ cargo, platforms of 

offshore oil and on-shore refineries, it 

may be produced serious effects to the 

marine environments in multiple ways. 

The causes of oil pollution producealterations 

in physic chemical levels, also involved 

toxication of marine habitats and 

the flora and fauna seriously affected aftermath 

of large spills. The turbidity of oil 

prevents the light penetrations and lead to 

photosynthesis processes for phytoplanktons. 

Lose of waterproofing qualities 

faced by the larger animals, aquatic birds 

getting lost of waterproofing by losing 

Citarasu and Babu 

their feathers (Kachel, 2008). The crude 

oil is highly toxic to the marine organisms 

because of it contains toluene, xylene, 

benzene and polycyclic aromatic hydrocarbons 

and these chemicals bioaccumulated 

to planktons, fishes, shellfishes, sediments 

constitute a long lasting threatening 

to the benthic animals. Polycyclic Aromatic 

Hydrocarbons (PAHs) is having 

many pathetic effects to the living organisms 

including mutagenic, carcinogenic 

and act as a teratogens (Kachel, 2008). 

3.2. Persistent toxic substances (PTS) 

Persistent Toxic Substances” 

(PTS) also called Persistent Organic Pollutants 

(POPs) are noxious, long-lived 

and less persistent. The prolonged usage 

and dispersion of PTS may cause serious 

problems and responsible for chemical 

and biological degradation. Their physical 

characteristics are chlorinated or halogenated 

affected to water solubility levels and 

high lipid solubility, no degradability 

leading to fatty tissues bioaccumulationsn 

(El-Shahawi et al., 2010). POPs constitute 

remarkable societal advantages, or not 

planned by-products of burning processes, 

such as dioxin. The halogenated hydrocarbons 

derivatives of tributyl tin 

(TBT), dibutyl tin and monobutyltin that 

are the disruptors of endocrine organs 

(Kachel, 2008). 

3.3. Heavy metal pollution 

Through riverine input, heavy 

metals are entering the sea and accumulate 

in marine sediments as well as flora 

and fauna. The important heavy metals 

are mercury (Hg), cadmium (Cd) and lead 

(Pb), accumulations are important for toxicity 

(Burger and Gochfeld, 2002). They 

generally share the features of PTS, because 

they are bioaccumulate, nondegradable 

and generate stringent or long 

standing toxic effects. The toxicity effect 

are vary based on the heavy metal types, 

for example if absorption of mercury as in 

very little doses, cause severe harmto 

brain and the central nervous system. The 

main sources of oceanic and atmospheric 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 445



contamination heavy metal contaminations 

provided by electric utilities, fuel 

combustion, iron manufacturing, incineration 

of urban refuse and fuel additives. 

Marine anti-fouling paints are very dangerous 

to marine organisms because it 

contains. Lead is another big poison creates 

the problems like neurotoxicity, mental 

health problems, lead is mainly released 

from batteries, sewage and fuel 

combustions. Electroplating factories, 

batteries and sewages are also responsible 

for cadmium toxicity and it affects bones 

by deformities and kidney dysfunction. 

Nickel poisoning is also a potent carcinogen 

that is toxic at relatively low concentration. 

Crude oil contains selenite with 

sulfur, reported as a toxic metal found out 

in marine environment at a huge level. 

The other dangerous heavy metals contaminations 

released through various 

routes including rain of pollutants from 

the atmospheric air, fallout from ship destruction 

and contaminated land runoff 

creating dangerous environmental problems 

(Kennish, 1998). 


3.4. Thermal pollution 

Nuclear reactors release huge 

amount of heat to the marine environments 

leading to decreased level of oxygen 

can disastrous effects on ecosystems 

and its communities.The decreased level 

of oxygen in marine water, degrade the 

quality of wildlife animals that lives underwater. 

The increased temperature 

holds less oxygen and creates suffocation 

to marine fauna including copepods and 

amphibians (Gautam et al., 2016). Industries 

are responsible for decrease the quality 

marine life and destroy habitats by altering 

the natural environments. Natural 

destruction including geothermal activity 

and volcanoes under the marine can induce 

warm lava to increase the temperature 

of marine waters. Thunders and 

lightening is also responsible for tremendouslevel 

of heat into the marine that 

leads to raise the temperature of marine 

water. The high temperature discharge 

from industries is responsible for inducing 

the toxin secretion from the microbes 

and harmful algal blooms in the marine 

water create other environmental problems. 

Environmental changes causesparticular 

animals can change their living 

place to alternate place due to warmer 

waters and reproduction also affected by 

increasing temperature. Marine organisms 

lay of undeveloped eggs or the temperature 

prevents the normal development of 

particular eggs due to rise of temperature 

level in marine waters. Enzymatic activity 

and metabolic rate also raised and increased 

food consumption due to increase 

temperature. This is seriously affecting 

the stability of food chain and modifies 

the balance of species composition (Brett, 

1970). 

3.5. Nuclear radiation 

In marine environments, radiations 

may classified in to natural and human 

activities, natural radiations are 

emission of cosmic rays, potassium-40 

by earth's crust and decay products of 

uranium etc. Human activities including 

nuclear power plants and reprocessing, 

nuclear weapons testing and accidents, 

fallouts of nuclear wastes, radiation using 

food conservation, medical diagnosis 

combustion, land-based mining, phosphate 

production, and oil exploration etc 

(Shinde and Gawande, 2015). High level 

radioactive waste dumping is not permitted 

in the ocean, even low level wastes is 

still permitted to the marine trenches, because 

low level wastes containing low 

radioactivity than high level waste. High 

level nuclear waste had half-life for 

24,100 years whereas the short-lived radioactive 

elements had the half life of 30 

years. The radioactivity may absorbed by 

micro algae, zooplankton, and other small 

marine organisms and then this will be 

transmitted or biomagnified to the food 

chain, to fish, marine mammals, and humans. 

In human concerns, the radiations 

cause cancer, changes in „DNA‟ that ensures 

cell repair (Kachel, 2008). 

3.6. Excess nutrients 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 446



Emissions and discharges of agriculture 

sources are a significant pollutions 

to 

the coastal 

zone particularly affects biogeochemical 

cycles and primary producers. Eutrophication 

is created by the pollutants including 

discharge of ammonia and methane, 

use of insecticides and other pesticides 

affecting marine flora and fauna. Excess 

nutrients discharges including phosphorus 

and compounds are responsible to eutrophication. 

High concentrations of nutrients, 

based on the physico-chemical 

properties of the affected marine environments, 

may lead to increased growth 

of phytoplanktons (Sarkar and Malchow, 

2005). Hydrogen sulphides also induce 

the negative impacts, if the oxygen level 

decreases, hydrogen sulphides levels also 

increased. Hydrogen sulphides induced 

low resistance to the aquatic living organisms 

leading to die off. The dead micro 

algae due to hydrogen sulphide toxicity 

floats on the sea surface leading to interfere 

the penetration of sunlight. Fertilizer 

runoffduring heavy rainthe organic fertilizers 

run off from the agricultural field 

and it affects the marine environment and 

back water bodies. 

3.7. Microbial contamination 

Generally microorganisms are released 

from waste water, waste products 

and sometimes human and animal wastes 

into the environment seriously spoiled the 

marine ecosystem. The viruses do not 

replicate without the help of host animals. 

First it may infect some other host cells 

and multiply. Seafood contaminations are 

the important pollution. The effluents 

from fish processing units, shrimp farms, 

hatcheries are creating big problems. The 

effluents contain serious bacterial pathogens 

such as salmonella, pathogenic Vibrio, 

WSSV and the fungal pathogen 

Fusarium. Due to the broader host range 

of these pathogens it may infect very easily 

in the marine animals and affect the 

food chain. This also affects the consumer 

levels very easily and there is an easy to 


get the diseases such cholera, typhoid etc 

(Yazhini et al., 2015). 

3.8. Noise pollution 

Life in marine can be susceptible 

for noises from different noise pollutants 

including oil exploration, seismic surveys, 

passing ships, and naval low-frequency 

sonar waves. Sound is very fast in water 

than in the atmosphere and the noise pollution 

in marine are increased at ten folds 

during the period of 1950 to 1975. Marine 

mammals including dolphins, porpoises 

and whales are using sound as communication 

and sensation because sound is not 

or less effective for their communications. 

Also the particulates scattering the light 

and it interferes the communications in 

marine mammals. Also it is impossible to 

using smell as communications, because 

the chemical molecules in water diffuse 

more slowly than in air are not possible 

(Payne, 1983). Sound speed is four times 

higher in water than atmosphere so it is 

opt to communicate the marine mammals. 

Sonar waves is very dangerous to marine 

mammals it interfere the sound wave 

communications and causes severe injuries 

including lose of sense organs leading 

to death. Also the sounds emanated from 

the passing ships other instruments for 

marine mining activities also disturbed 

the dolphin and whale populations. 

3.9. Ship based threatening 

Ship based threatening mainly 

caused by operationally or accidentally 

and releases the pollutants to the marine 

ecosystem and these pollutants mainly 

damage the flora and fauna. The pollutants 

also somewhat released more in ship 

are on voyage than accidental ships. The 

pollutants released including tank residues, 

bunker oils, chronic discharge of 

sewage, garbage, exchange of ballast water, 

other emissions from vessel and antifouling 

paints etc. The sewage discharges 

causes severe problems including microbial 

pollution especially bacterial infections 

it will be seriously damage the biodiversity, 

fisheries and food chains etc. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 447



The excess nutrient discharges also affect 

the primary production etc (Katchel, 

2008). Pollutants are released by accidental 

pollution including contacts with 

external objects, due to collisions, cargotransfer 

failures, sinking or loss of cargo, 

explosions and groundings etc. It is also 

very hazardous, the toxic materials spilling 

from cargoes which carry large quantities. 

The oil tankers happened in accident 

may spill larger quantities of crude 

oil to the marine environments. The oil 

may drift to the sea shore and seriously 

affected the ecosystem particularly the 

rocky animals including oyster and mussel 

beds, backwater etc. The places which 

affected by oil spills may suffer a long 

times, until the oil pollution completely 

degraded or solved (Katchel, 2008). The 

hydrocarbons form the oil spills penetrates 

the marine and related ecosystems 

sediments alter the community structure 

especially the phytoplanktons and worms. 

Ship can also harm to the marine habitats 

and wildlife by damaging the marine animals 

by physical destruction by anchoring. 

Anchoring may seriously affected by 

the coral and sponge beds. Another physical 

hindrance including collisions by 

ships, ship‟s propellers and ship strikes 

are cause the damages and death to the 

marine mammals including whales etc. 

3.10. Harmful algal blooms (HABs) 

Harmful algal bloom (HABs) 

causes pathetic effects to other marine 

animals by mechanical damage through 

the secretion of toxins.HABs are involved 

in large-scale marine mortality of marine 

animals and they secreted with different 

toxins responsible for shellfish poisonings 

(Nancy, 2012). Red tides caused by 

HABs that can affect estuarine and salt 

lakhs areas by depletion of oxygen, increasing 

the carbon dioxide and secrete 

toxins. Thousands of harmful algal species 

were identified among that one hundred 

species ware produce toxins that will 

seriously affect the shellfish species by 

ingested the HABs by filter feeding. The 

excess amount algal bloom may dies off 


and deposited the sediments may add 

dead organic matter load. Further dead 

organic matter may cause bad effects to 

the normal microbial flora and depleted 

the oxygen and this will affect the decomposition 

process. Shrimp farm effluents 

including algal bloom die off, large 

amount of dead organic load accumulate, 

affect filter feeding animals, shrimps and 

lobsters. 

3.11. Ocean mining 

Deep sea mining is the mineral retrieval 

process from the ocean floor by 

disturbing the marine organism from benthic 

regions. Ocean mining takes place 

about from 1,400 to 3,700 meters below 

the oceans‟ surface for extinct or active 

hydrothermal vents (Ahnert and Borowski, 

2000). The mine deposits are drill 

out from the ocean deeps by hydraulic 

pumps or buckets that take ore to the surface. 

Removal of marine floors disturbs 

the benthic habitats. Removing parts of 

the seafloor disturbs the habitat of benthic 

organisms depending upon the places and 

type of mining; sometimes it may disturbances 

to the marine benthos permanently. 

Leakage, corrosion and spilling might be 

altering the area of mining bycontaminationof 

chemicals. Surface plumes may 

cause more serious effects and based on 

the particles size and water currents by 

the plumes could spread over vast areas 

of the floor.Sand mining is a practice 

mined from beaches, inland dunes 

and dredged from ocean beds and river 

beds. Sand mining also emits radiation 

problems and pathetic effects to the living 

organisms in sea shore peoples (Pitchaiah, 

2017). 

3.12. Plastics and polythenes 

Plastic pollution is one of the serious 

concerns in recent years because the 

plastic pollutions in ocean become significant 

environmental issues related to governmental 

and nongovernmental organizations, 

scientists and members of the 

public worldwide. The main risk to marine 

biota posed by different activities in- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 448




cluding marine vessels, commercial fishing, 

marine-industries and recreational 

coastal tourism. These are the major 

sources for generating the plastic and polythene 

wastes hat can directly enter the 

marine environment. During World War 

II end, approximately eighty percent of 

the marine debris is considered as plastic 

wastes. 

Marine vessels have been reported as a 

major contributor of marine litter and it is 

estimated that the commercial fishing 

fleet dumped more than 23,000 tons of 

plastic packaging materials during 1970s 

(Pruter, 1987). The waste materials including 

nylon netting, plastic monofilament 

line and discarded or waste fishing 

gears are neutrally buoyant can therefore 

drift at variable depths within the oceans. 

The waste plastic materials are problematic 

to marine animals because they cause 

entanglement of marine biota, known as 

„„ghost fishing‟‟ (Lozano and Mouat, 

2009). 

Waste six pack rings, plastic bags 

and other forms of plastic waste cause 

dangerous effects to wildlife and fisheries. 

Plastic fishing nets can be lost or left 

by fishermen to the ocean are dangerous 

and create so many problems including 

entangle to fishes, sea turtles, sharks, dolphins, 

crocodiles, crabs, seabirds, and 

other creatures, restricting movement, 

causing starvation and infection etc. Plastic 

waste debris during bulky or tangled, it 

is very difficult to move, and may become 

permanently trapped in the digestive 

tracts of marine animals finally leading to 

blocking the food passage and causing 

death through infection or starvation. 

Moser and Lee (1992) conducted a study 

for plastic pollution in sea birds, they collected 

1033 birds and among the birds, 

fifty five percentages of the birds guts 

containing plastic particles. Carpenter et 

al. (1972) observed different species of 

fishes had plastic wastes in their guts especially 

white plastic spherules had been 

ingested, indicating that they feed selectively. 

The same pattern of white plastic 

debris ingestion also observed in loggerhead 

sea turtles, Caretta caretta from 

Central Mediterranean sea (Gramentz, 

1988). Polythenes is the another dangerous 

pollutants Generated from household, 

industrial wastes and Recreational beaches. 

Sea turtles mistakenly easting the polythene 

bags. During degradation of polythenes, 

it releases phenol, bisphenol, 

phathalate and gases it causing cancer, 

heart failures in humans. 

3.13. Microplastics 

Microplastic contamination in 

ocean has been a serious issue nowadays 

with harmful effects to marine biota. 

They are very tiny plastic granules derived 

from the breakdown of macroplastics 

and the plastic particles used in cosmetics 

and detergents. Primary microplastics 

in personal care and cosmetic products 

are a minor source of releases of microplastics 

to the environment. Microplastics 

which used in detergents, cosmetics 

and also in air-blasting media are entering 

the freshwater bodies and reached 

to domestic or industrial drainage systems 

(Derraik, 2002). Most of the microplastic 

granules are trapped the waste-water 

treatment plants unfortunately the small 

microplastic granules from cosmetics and 

detergents are passing through such filtration 

systems and finally reached the marine 

environments (Browne et al., 2007). 

The small size nature, microplastics are 

considered bio-available to marine organisms 

throughout the food-web. Microplastics 

in intertidal sediments have been 

shown to reduce the thermal conductive 

properties and alter drainage. Due to the 

tinny size and availability of microplastics 

in pelagic and benthic ecosystems, 

they have easily ingested by the marine 

biota including microalgae, crustaceans, 

fishes and sea birds etc (Blight and Burger, 

1997). Devriese et al. (2015) studied 

the microplastic contamination in the 

brown shrimp, Crangon crangon sampled 

Europe and the study revealed that, the 

shrimps are heavily contaminated with 

microplastics. Ingestion of microplastics 

may release toxins to the food chain and 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 449



lead to the bioaccumulation and biomagnifications. 

Additives used in microplastics 

such aspolybrominateddiphenyl 

ethers, phenol and bisphenol-A are inhibit 

the synthesis of endogenous hormones 

leading to affect the reproduction. 

Phthalates are associated with broad 

range of genotoxic damage including micronuclei 

and apoptosis in mussel haemocytes, 

suppressed the locomotion in invertebrates 

and intersex conditions in fishes 

(Oehlmann et al., 2009). Consumption of 

microplastics and nano-plastics by humans 

through marine foods including 

shrimps, shellfish and small fishes may 

cause health problems. Ingestion of microplastics 

by young fishes causing 

deaths, stunted growth, altering behavior, 

sometimes killed and prevented from 

reaching maturity. 

3.14. Ocean acidification 

The oceans are act as a carbon 

sink, because they absorbing more carbon 

dioxide from atmosphere. If the carbon 

dioxide levels increased in the oceans the 

Ocean become more acidic. The acidic 

nature is seriously affected by the formation 

of calcium carbonate in corals, 

shellfishes including shrimps, oysters, 

mussels, clams and other molluscs etc 

shells (Caldeira and Wickett, 2003). The 

higher acidity in the seawater also creates 

more pathetic effects to the marine organisms 

in combinations with environmental 

stressors including higher ocean temperature 

and other pollutants etc. Marine organisms 

like algae and zooplanktons using 

carbonate ions to produce calcium 

carbonate shells and skeletons. Unfortunately, 

the acidification leads the less 

availability of carbonate ions in marine 

waters and it interfere the formation of 

shells. Also the ocean acidification interfere 

the iron, phosphorous, nitrogen and 

other elements absorption from marine 

waters by the marine organisms for their 

vital growth. More acidity interfere the 

attachment of iron to other organic compounds 

may hindrance to the normal marine 

life. 


3.15. Coastal tourism 

Coastal tourism creates so many 

harmful effects to the marine environments 

adding pollution by waste disposal, 

input of local waste structures and habitats 

under enormous pressure. For new 

tourists, over developments including 

construction of resorts, marinas, golf 

courses and airports etc are not advisable 

to clean environments. For the new developments 

peoples removed the mangrove 

forests and sea grass meadows. 

Piers and other related structures are built 

directly from the top of coral reefs related 

to the tourist developments is not advisable 

and spoil the marine environments. 

The overcrowding of tourists in the 

beaches may affect the nesting sites of the 

marine endangered turtles by destruction. 

Resorts from beaches may discharges 

their sewage effluents directly to the marine 

waters which is seriously affect the 

coral reefs and other sensitive marine 

habitats. Coral reefs also damaged by different 

tourist‟s activities including fishing, 

snorkeling, diving and careless boating 

etc. Boating and other human activities 

also disturbed the marine animals including 

seals, dugongs, whales, whale 

sharks, dolphins and marine birds. Overfishing 

in a particular area for seafood 

consumption affected local fish populations(Sunulu, 

2003).Tourists also discharges 

the polythene bags, bottles and 

other plastic materials to the beach by 

improper disposal and it reaches to the 

sea create so many environmental problems. 

Tourism can cause pollutions including 

solid waste and littering, releases 

of sewage, oil and chemicals, air emissions, 

noise and even architectural activities. 

Tourism development of marinas and 

breakwaters also can changes in currents 

and coastlines. 

3.16. Ballast water 

Ships need ballast to maintain the 

balance successfully and safely. Discharges 

of ballast waters from ships to 

marine environment have negative im- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 450



pacts to various ways. Huge quantities of 

waters are loaded by cruise ships, cargos 

and large tankers from one place for balancing 

and discharge to some other places. 

Ballast water have various biological 

materials such as seaweeds, sea grass, 

phytoplanktons, zooplanktons, small fishes, 

invertebrates, microbes including bacteria, 

fungi and virus etc. The biological 

materials are nuisance, non-native and 

exotic species can create extensive ecological 

and economic problems to the 

aquatic ecosystem with human health issues 

etc. The important preventive 

measures for treating the ballast water 

bytreating with ultraviolet irradiation, filtration, 

heat, gas super saturation and 

electrical fields etc (Silva et al., 2004), 

chemical treatments byiodides, borates, 

chlorates, ozone, ionization, copper ions, 

hydrogen peroxide, adjustment of 

pH,changes in salinity for eradication of 

organisms, ozonization and deoxigenization 

etc (Wright et al., 2003). 

3.17. Natural calamities 

Hurricanes and floods can induce 

waste transportation from land to the marine 

environment. Earthquakes and tsunami 

can cause ground, air, and water 

pollution (Shaw, 2006). The pollutants 

including detergents, chemicals from processing 

plants, farm wastes and fertilizers 

from crops are swept downstream and 

deposited to marine through large river 

floods. Volcanic eruptions have also been 

creating fluorine-containing compounds 

deposition to sea and also harm to the marine 

flora and fauna. High amount of minerals 

are accumulated to the marine environments 

from demolishing of the buildings, 

bridges during natural disasters and 

small amount of hazardous materials 

reached to marine environments that 

threatening to marine living organisms. 

Heavy floods in rivers also washed out 

the sewages to the accumulated in the marine 

environments that contains pathogenic 

bacteria fungi and virus which causes 

serious pathological effects to the marine 


living organisms and passes through the 

food chain. 

3.18. Marine littering 

Littering to marine environment is 

a global concern, they affect ecosystem 

very seriously. Several million tons of 

litters are discharged to the worldwide 

every year creating economic, environmental, 

health and aesthetic issues 

(Strieker, 1998).The important land based 

disposals including industrial outfalls, 

rivers and floodwaters, discharge from 

storm water drains, untreated municipal 

sewerage, land-fills and littering from 

coastal tourism. The marine based litters 

including shipping transport, offshore 

mining and extraction, fishing industry, 

illegal dumping at sea and discarded fishing 

gear etc. The vast majority of marine 

litter is plastic, which never truly breaks 

down. Another serious problem is nuclear 

waste disposal to the marine trenches, 

Mariana trench is the main site 

for nuclear waste disposal because of its 

huge depth 36, 201 feet which is situated 

in the western Pacific Ocean, to the east 

of the Mariana Islands (Sheavly and Register, 

2007). Ocean is used as a dumping 

ground for disposing the nuclear materials 

for many decades after Second World 

War II, and then it was banned internationally. 

Thirteen nuclear capable countries 

are dumped their nuclear/radioactive 

waste including started from 1946 to 

1993. The important nuclear waste materials 

are medical products, industrial 

products, weapons, house hold containers, 

reactor vessels, with and without spent or 

damaged nuclear fuel etc. The discarded 

wastes emit nuclear radiation leading to 

health issues to the marine living organisms. 

4. Impact of pollutants to marine life 

The pollutants which are affect a 

broad range to the environments as well 

as the living organism in the entire world 

directly or indirectly. Climate changes 

are spoiled by extreme weather conditions 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 451



as well as rising of sea level, sea surface 

warming by temperatures and ocean acidification. 

Corals reefs are important for 

preventing acidification etc, loss by anthropogenic 

stress activities, collection 

and recreational activities and coral beds 

may be affected. Mangrove forest is important 

for preventing barrier for the natural 

calamities like tsunami. Degradation 

of mangroves by over exploitation may 

affect the marine environments. Illegal 

and over exploitation of capture fisheries 

by trawlers may spoil the fishery resources 

leading to decline the particular 

fish species. The pollution in beaches by 

coastal tourism causes several environmental 

impacts and the oil pollution causes 

reduction in benthic organisms. Biodiversity 

is important for produce organic 

material, decompose organic material, 

capture and store energy, cycling water 

and nutrients and helps to regulate climate 

and atmospheric gases. The pollutants 

seriously interrupt the food chains leading 

to several problems. In concern with the 

human health point of view, the chemical 

poisoning in food chain creates the bioaccumulation 

and biomagnifications problems 

(Table 1). The harmful chemical 

contamination in seafood crate several 

problems to humans. The pathogenic bacteria 

including pathogenic vibrios, salmonella 

contaminations from waste water 

disposal from fish processing industry 

may cause the disease problems in the 

fishes/ shrimps from the sea. The consumption 

of infected fish/ shrimp may 

cause diseases to humans. The residue of 


pesticides may change hormonal system, 

reduce fertility, weaken immune system 

and create cancer. The degraded derivatives 

like phenol, bisphenols and phathalates 

also creating heart diseases and 

cancers to humans by consuming sea 

foods. 

5. Remedies to solve the pollutants by 

biotechnological approach 

Bioremediation is defined as“The 

act of adding materials to contaminated 

environments such as oil spill sites, to 

cause an acceleration of the natural biodegradation 

process”. The microbes utilize 

the nutrients from the polluted water 

bodies and reduce or neutralize the pollutants. 

The extremophilic microbes including 

halophilic, thermophilic, acidophilic 

and alkaliphilic are useful to bioremediation 

purposes for to neutralize the pollutants, 

because they withstand high temperature, 

low and high pH and higher salinity. 

The polluted effluents/ water bodies, 

fish farm effluents, effluents from any 

waste sources may remediate with microbes 

before reaching the water bodies 

like river, back water may help to reduce 

or remove the pollutants and solve the 

pollution problems. Genetically modified 

microbes like super bug also helps degrade 

the oil from oil contaminated water 

bodies. 

The water probiotic microbes like 

Lactobacillus sp and Bifidobacterium sp 

also clean the aquaculture ponds by reducing 

or removal pathogenic microbes 

Table 1: Impact on human heath by some synthetic chemicals detected in ocean 

No Chemical residues Human health impacts 

1. Benzene Anemia, blood disorders and chromosomal damage 

2. Carbon tetrachloride Cancer; central nervous system, liver, kidney and 

lung damages 

3. Dioxin Cancer and skin disorders 

4. Ethylene dibromide Sterility and Cancer 

5. Polychlorinated biphenyls Kidney, lung and liver damages 

6. Trichloroethylene CNS, cancers, liver and kidney damage and skin 

problems 

7. Vinyl chloride Cardiovascular problems, liver, kidney, and lung 

damages and gastrointestinal problems 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 452



and unwanted physicochemical parameters 

which solve the effluent contamination 

to the marine environments. The micro 

algae like Chlorella sp., Ankistrodesmus 

sp. are also useful to removal 

of excess nutrients and Co 2 in waste 

water systems, Solve BOD problems by 

releasing oxygen which will help to effluent 

treatment. The seaweeds like Sargassum 

sp also helpful to chelate the heavy 

metal contaminates in the water bodies 

especially the effluents. These types of 

treatments which restrict the polluted effluent 

water to the marine environments. 

6. Suggestions to protecting marine environment 


Several measures for protecting 

the marine environments from the pollutants 

including legislation for plastics and 

polythenes, set standards protocols for 

sewage and other effluent discharges, low 

level use of pesticides, coastal cleaning 

programmes, public awareness, restrictions 

of polluter pays principal and 

implementation of laws pertaining to prevention 

by strictly and coastal zone management 

activities before constructing 

new industries on the coast. Also strictly 

advice to the peoples for following the 

marine act s including National Marine 

sanctuaries Act of 1972, Fisheries Management 

and Conservation Act – 1976, 

Clean Water Act of 1977, Endangered 

Species Act, Oceans Act of 2000, Estuaries 

and Clean Waters Act of 2000 and Estuaries 

and Clean Waters Act of 2000. It 

is also important to conserve the marine 

ecosystem and marine animals we must 

follow the international marine legislation 

such as Convention on the Prevention of 

Marine Pollution by Dumping wastes and 

Other Matter. One of the important legislation, 

Protocol to the International Convention 

for the Prevention of Pollution 

from Ships (MARPOL) address the marine 

pollution problem caused by marine 

vessels to the marine environment in 1978 

(Lentz, 1987). Education is also a very 

important and powerful criterion to address 

the pollution problems, especially 

for school students and create awareness. 

Because school students and youngsters 

take their responsibility and awareness to 

the public peoples, families and related 

communities related to the marine pollutions 

and the future conservations etc. 

Coastal cleanup is the major awareness 

programme, the International Coastal 

Cleanup is one of the largest volunteer 

program in worldfor cleaning the coastal 

areas. Volunteers are clean or remove 

trash from the coastal areas especially 

beaches the entire world. Waste management 

is also an important strategy for restrict 

the entry of pollutants through effluents 

to the clean water bodies. Industries 

are advised to set up waste proper 

treatment plants, minimize waste for 

adopting suitable measures, waste recycling 

and reuse and recovery of waste water 

effluents. 


The entry of different pollutants 

from atmosphere, through water bodies, 

by ships, from beaches and other human 

activities are very harmful to the marine 

environments and affect all marine flora 

and fauna. The pollutants also directly or 

indirectly affect the human community 

through food chain. Among the pollutants 

plastics and micro plastics are the serious 

concerns to the marine environments and 

affect lower to higher organisms and indirectly 

affect human‟s health. Restriction 

or reducing the pollution is in our hand, 

we can take care of the environments by 

avoiding pollution to save our environments 

for future. Several pollution preventive 

measures can be adopted including 

picking up and disposing various 

types of litters at proper place; by reducing, 

reusing and recycling the waste materials; 

bring reusable, biodegradable bags 

to the grocery stores; disposing properly 

the trash; promoting awareness among 

your friends and family; restrict the usage 

of plastic bags; awareness programs to the 

public especially the school children, un- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 453



educated publics to restrict / stop the pollution 

will definitely help in minimizing 

the pollution. 

References 

Ahnert, A. and Borowski, C. (2000). 

Environmental risk assessment of anthropogenic 

activity in the deepsea". 

Journal of Aquatic Ecosystem 

Stress and Recovery 7 (4), 299–315. 

Anonn (2005). Land-based activities account 

for roughly 80 per cent of released 

pollutants; cf. UN Doc. 

A/60/63, Oceans and the Law of the 

Sea – Report to the 60th session of 

the General Assembly, 4 March 

2005, p. 104. 

Barange, M., Cheung, W. W. L., Merino, 

G. and Perry, R. I. (2010). 

Modelling the potential impacts of 

climate change and human activities 

on the sustainability of marine resources. 

Current Opinion in Environmental 

Sustainability 2(5-6), 

326–333. 

Bigg, G.R., Jickells, T.D., Liss , P.S. 

and Osborn, T.J. (2003). The role 

of the oceans in climate. Int. J. Climatol. 

23, 1127–1159 

Blight, L.K. and Burger, A.E. (1997). 

Occurrence of plastic particles in 

seabirds from the eastern North Pacific. 

Marine Pollution Bulletin 34, 

323–325 

Brett, J.R. (1970). Temperature- 

Animals-Fishes. Marine Ecology, 

Vol.1. Environmental Factors, Part 1. 

pp. 515-560. 

Browne, M.A., Galloway, T. and 

Thompson, R. (2007). Microplastic 

– an emerging contaminant of potential 

concern?.Integrated Environmental 

Assessment and Management 3, 

559–561 

Burger, J. and Gochfeld, M. (2001). 

Heavy metals in commercial fish in 

New Jersey. Environ. Res. 99, 403- 

412 


Caldeira, K. and Wickett, M. E. (2003). 

Anthropogenic carbon and ocean 

pH". Nature 425 (6956), 365–365. 

Carpenter, E.J., Anderson, S.J., Harvey, 

G.R., Miklas, H.P. and Peck, 

B.B. (1972). Polystyrene spherules in 

coastal waters. Science 178, 749–750 

Clark, R.B. (2001). Marine Pollution. 

GESAMP, Protecting the Oceans 

from Land-Based Activities, 

GESAMP Report and Studies No. 71 

.p. 17. 

Derraik, J.G.B. (2002). The pollution of 

the marine environment by plastic 

debris: a review. Marine Pollution 

Bulletin 44, 842–852 

Devriese, L.I. , van der Meulen M.D, , 

Maes , T. , Bekaert, K.B , Pont, 

I.P. , Frère, L., Robbens, J. and 

Vethaak, A.D. (2015). Microplastic 

contamination in brown shrimp 

(Crangon crangon, Linnaeus 1758) 

from coastal waters of the Southern 

North Sea and Channel area. Marine 

Pollution Bulletin 1(2), 179-87 

El-Shahawi, M.S., Hamza, A., Bashammakhb, 

A.S. and Al-Saggaf, 

W.T. (2010). An overview on the 

accumulation, distribution, transformations, 

toxicity and analytical 

methods for the monitoring of persistent 

organic pollutants. Talanta 80, 

1587–1597. 

Gautam, N., Rai, P. and Chandra, H. 

(2016). A case study on environmental 

air pollution and control methods, 

3 rd International conference on science, 

technology and management, 

India international centre , New Delhi. 

GESAMP (1993). Supra note 106, p.50. 

Gramentz, D. (1988). Involvement of 

loggerhead turtle with the plastic, 

metal, and hydrocarbon pollution in 

the Central Mediterranean. Marine 

Pollution Bulletin 19, 11–13. 

Kachel, M.J. (2008). Particularly sensitive 

Sea Areas, the IMO‟s Role in 

protecting vulnerable marine areas, 

International Max Plank research 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 454



school of marine affairs at the university 

of Hamburg. 

Kennish M.J. (1998). Pollution impacts 

on marine biotic environments; CRC 

Press. pp. 310. 

Lentz, S.A. (1987). Plastics in the marine 

environment: legal approaches for international 

action. Marine Pollution 

Bulletin 18, 361–365 

Lozano, R.L. and Mouat, J. (2009). Marine 

litter in the North-East Atlantic 

Region: Assessment and priorities 

for response. KIMO International. 

Moser, M.L. and Lee, D.S. (1992). A 

fourteen-year survey of plastic ingestion 

by western North Atlantic seabirds. 

Colonial Water birds15, 83–94 

Nancy, D. (2012). Phytoplankton 

Blooms: The Basics. NOAA 

FKNMS. 

Oehlmann, J., Schulte-Oehlmann, U., 

Kloas, W., Jagnytsch, O., Lutz,I., 

Kusk, K.O, Wollenberger, L, Santos, 

E.M., Paull, G.C., Van Look, 

K.J.W. and Tyler, C.R. (2009). A 

critical analysis of the biological impacts 

of plasticizers on wildlife.Phil. 

Trans. R. Soc. B. 364, 2047–2062 

Payne, R. (1983).Communication and 

behaviour of whales. Westview 

Press. 

Pitchaiah, P.S. (2017). Impacts of Sand 

Mining on Environment – A Review. 

SSRG International Journal of Geo 

informatics and Geological Science. 

4(1): 1. 

Pruter, A.T. (1987). Sources, quantities 

and distribution of persistent plastics 

in the marine environment.Marine 

Pollution Bulletin 18, 305–310 

Sarkar, R.R and Malchow, H. (2005). 

Nutrients and toxin producing phyto- 


plankton control algal blooms – a 

spatio-temporal study in a noisy environment. 

J. Biosci. 30(5), 749–760 

Shaw, R. (2006). Indian Ocean tsunami 

and after-math: need for environment-disaster 

synergy in the reconstruction 

process. Disaster Prevention 

and Management 7(1), 5-20. 

Sheavly, S. B. and Register, K. M. 

(2007). Marine Debris & Plastics: 

Environmental Concerns, Sources, 

Impacts and Solutions. Journal of 

Polymers and the Environment. 

15 (4), 301–305. 

Shinde, R. and Gawande, S. 

(2015).Ocean pollution and its perspective. 

International journal of engineering 

and general science, 3,4. 

Silva J.S.V., Fernandes, F.C., Souza, 

R.C.C.L., Larsen, K.T.S., Danelon, 

O.M. (2004). Ballast Water and 

Bioinvasion. Rio de Janeiro: 

Interciencias, pp.1-10. 

Strieker , G. (1998). Pollution invades 

small Pacific island. CNN. 

Sunlu U. (2003). Environmental impacts 

of tourism.CIHEAM, pp. 263-270 

Wright, D.A., Dawson, R., Mackey, 

T.P., Cutler, H.G., Cutler, S.J. 

(2003).Some shipboard trials of ballast 

water treatment systems in the 

United States. International Ballast 

Water Treatment Symposium, 

Globallast Monograph Series, London, 

pp. 15. 

Yazhini , R., Pavithra , M., Suganthy , 

C., Elumalai, E.K. and Ganeshkumar, 

G. (2015). Microbial contamination 

of sea water at Puducherry 

sea shore. Malaya Journal of Biosciences, 

2(2), 115-118. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 455



Ecology, Distribution and Diversity of Bioluminescent 

Bacteria in Palk Strait, Southeast Coast of India 

Srinivasan Rajendran 1 , Ganapathy selvam Govindarasu 2 and Govindasamy 

Chinnavenkataraman 1, * 

1 Department of Oceanography and Coastal Area Studies, School of Marine Sciences, Alagappa 

University, Thondi Campus, Thondi – 623 409, Ramanathapuram District, Tamil 

Nadu, India; 2 Division of Algal Biotechnology, Department of Botany, Annamalai University, 

Annamalainagar, Chidambaram, Tamil Nadu, India; 

*Correspondence: sandalsrini@gmail.com; Tel.: +91 9788780266 

Abstract: The present study was carried out to determine the influence of ecological characteristics 

and bioluminescent bacterial distribution in the seawater and sediment of Palk 

Strait, Southeast coast of India during July 2010 - June 2012. The physico-chemical parameters 

such as., atmospheric temperature range was varied from 24.3 - 35.3°C, surface water 

temperature 23.2 - 33.5°C, pH (6.2 - 8.91), dissolved oxygen concentration ranged from 

3.84 - 7.73ml l -1 , salinity fluctuated between 23.12 and 39 ppt, POC concentration ranged 

from 0.78 - 2.56mg dry wt.l -1 . The seawater was analysed and results suggest that it contains, 

inorganic phosphate (4.12 - 21.6μM), reactive silicate (4.3 - 19.26μM), inorganic nitrate 

(1.95 - 12.25μM), inorganic nitrite (1.1 - 5.5μM), total nitrogen (1.5 - 10.39μM), calcium 

(120 - 990 mg l -1 ) and magnesium (730 - 2460mg l -1 ). Bottom water temperature 

ranged from 20.4 to 27.1ºC, sediment temperature (17 to 25ºC), sediment pH (6.2 to 9.1), 

concentration of sediment nutrients and heavy metals phosphorus concentration ranged 

from 0.065 to 0.315%, potassium (3.18 to 8.57%), calcium (4.08 to 25.44%), magnesium 

(0.29 to 1.7%), silicon (33.2 to 56.53%), sodium (1.56 to 3.35%), sulphur (0.32 to 2.06%), 

chloride (1.3 to 5.71%), aluminium (4.63 to 11.87%), titanium (1.55 to 9.15%), manganese 

(0.08 to 0.26% ), iron (3.2 to 11.13%), cobalt (3 to 11ppm), copper (4 to 29ppm), chromium 

(26 - 85ppm), nickel (5 to 18ppm) and lead (11 to 27ppm). Seawater colony forming unit 

(CFU) for the bioluminescent bacterial population density was varied from 1.06 x 10 4 to 

9.44 x 10 4 CFU/ml and sediment (2.6 x10 4 to 23.2 x10 4 CFU/g). Statistical analysis seawater 

colony forming unit (CFU) of bioluminescent bacteria showed a positive correlation 

with salinity and water pH. Sediment colony forming unit of bioluminescent bacteria 

showed a positive correlation sediment temperature, sediment pH, sediment silicon, sodium 

and chloride. It can be concluded that the ecological parameters were observed in water and 

sediment which highly influence the bioluminescent bacterial populations and their diversity. 

Keyword: Bioluminescent bacteria; colony formation Palk Strait; diversity; physicochemical 

parameters 


Bioluminescence is a ubiquitous 

feature of the world oceans and originates 

from organisms representing all tropic 

levels. They are ecologically versatile, 

present in water, sediment and also harbored 

in the light organs of some fish and 

gut of many marine organisms (Hastings, 

1986; Govindasamy and Srinivasan, 

2012). Bioluminescence is also subject to 

control by a number of environmental 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 456


Ecology, Distribution and Diversity of Bioluminescent Bacteria 

Srinivasan et al. 

factors. Low oxygen tension may increase 

luminescence and luciferase levels (Ruby 

and Nealson, 1978; Nealson and Hasting, 

1979). Although on large scales bioluminescence 

may be correlated with hydrographic 

features (Swift et al., 1995). The 

marine environment contains an immense 

diversity of microbes that have evolved to 

perform equally diverse functions in their 

respective environments and ecological 

niches. Particularly when their populations 

are dense, disturbance of the water 

during the night causes bright blue bioluminescent 

display that have been reported 

(Harvey, 1952) and are now known to 

occur globally (Lynch, 1981). Its widespread 

distribution, bioluminescence is 

clearly a predominant form of communication 

in the sea, with important effects 

on the immense daily vertical migration, 

predator-prey interactions and the flow of 

material through the food web. Bioluminescent 

bacteria emit light continuously, 

whereas higher organisms usually emit 

light in pulses, accomplished by localizing 

the systems into organelles that are 

regulated by pH change, calcium flux and 

oxygen (Nelson and Hastings, 1979). 

These are among the most extensively 

studied group of marine bacteria with regard 

to ecology, taxonomy and phylogeny. 

Many studies have suggested that the 

distribution and species composition of 

luminous bacteria are influenced by season, 

temperature, nutrient concentration, 

depth and geographical location (Nair et 

al. 1979; Ramaiah and Chandramohan, 

1988). Furthermore, the chemical reaction, 

which produces light in bioluminescent 

bacteria, is essentially the same for 

all species (Nealson and Hastings, 1979). 

In spite of the fact that a wide 

range of habitats are occupied by luminous 

bacteria, very little information concerning 

their distribution of luminous bacteria 

in Palk Strait, coastal region especially 

in seagrass meadows sediments. 

Hence, the present study was undertaken 

to understand the ecology, distribution 

and diversity of bioluminescent bacteria 

in this seagrass ecosystem along with environmental 

parameters in Devipattinam, 

Thondi and Manamelkudi region of Palk 

Strait, Southeast coast of India. 

2. Materials and methods 

A total of 144 water and sediment 

samples were collected from three different 

places of Palk Strait viz., Devipattinam 

(Station I; Lat. 9˚28N; Long. 

78˚54’E), Thondi (Station II; Lat. 9˚45’N; 

Long. 79˚3’E) and Manamelkudi (Station 

III; Lat. 10˚3’N; Long. 79˚13’60’E) during 

July 2010 - June 2012. The surface 

water sample was collected with sterile 

polypropylene bottle and sediment sample 

were also collected using alcohol-rinsed, 

air dried, Peterson grab sampler. The central 

portion of the top 2cm sediment samples 

was taken out with the help of a sterile 

spatula and the samples were then 

transferred into a sterile polythene bag. 

All samples were transported to the laboratory 

within 1-3 hours of collection under 

ice cold condition. 

Atmospheric, surface water, bottom water 

and sediment temperatures were measured 

using standard mercury filled centigrade 

thermometer. Salinity was estimated 

with the help of hand refractometer 

(Model 2491 Master-S/Milla). Seawater 

pH was measured using high configuration 

digital pH pen. Soil pH was measured 

at a soil/water ratio of 1:2.5 (w/w). 

Air-dried soil (10g, 2.8 mm) and 25 ml 

distilled water were shaken together for 2 

min and left to settle for 30 min, this procedure 

was repeated once and then the pH 

was determined using high configuration 

digital pH pen (Model No: Reed 8690; 

±0.001) (Rousk et al., 2009). Dissolved 

Oxygen, Particulate Organic Carbon 

(POC), inorganic Phosphate (PO 4 ), reactive 

Silicate (SiO 3 ), inorganic Nitrate 

(NO 3 ), inorganic Nitrite (NO 2 ), Total Nitrogen 

(TN), and Calcium (Ca) and Magnesium 

(Mg) were analyzed as described 

by Parsons et al. (1984). All sediment 

samples were air-dried at 25°C in the laboratory 

of Department of Oceanography 

and Coastal Area Studies, Alagappa Uni- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 457



versity, India. The soil particles were disaggregated, 

crushed and sieved through 

10mm nylon sieve and then stored in polythene 

bags and labelled at 4°C until the 

analysis. All the elemental present in the 

sediments were analyzed by using 

Bruker S4-Pioneer model wavelength 

dispersive X-ray fluorescence spectrophotometer 

(WD-XRF). The samples were 

ground to particle size well below 100µm 

using ball mill in order to minimize the 

grain size interference on XRFmeasurement. 

The major elements viz., 

Phosphorus (P), Potassium (K), Calcium 

(Ca), Magnesium (Mg), Silicon (Si), Sodium 

(Na), Sulphur (S), Chloride (Cl), 

Aluminium (Al), Titanium (Ti), Manganese 

(Mn), Iron (Fe), Cobalt (Co), Copper 

(Cu), Chromium (Cr), Nickel (Ni) and 

Lead (Pb) were analyzed. 

Then, 1ml seawater/ 1g sediments 

of samples were mixed with 99/100ml of 

50% aged sterile seawater mixed vigorously 

and used for serial dilution in test 

tubes upto 10 -2 dilutions up to 10 -7 . About 

0.1ml from the each dilution was spread 

in luminescent agar (LA) medium (Dehydrated 

Nutrient broth 8g, Sodium Chloride 

30g, Glycerol 3g, Calcium Carbonate 


5g, Agar 15g, Seawater 1000ml, pH 

7.2±01). The plates were incubated at 

32°C for 24 hrs. After incubation, the 

colonies were counted in the dark room 

and expressed in CFU ml/g and identified 

by the standard procedure (Nealson, 

1978). The isolated bioluminescent colonies 

were purified and stored at 4°C with 

40% glycerol until analysis (Kita- 

Tsukamoto et al., 2006). All the data were 

computed using SPSS v 16.0 statistical 

software to obtain Pearson’s correlation 

co-efficient (r) for the statistical interpretation. 

The correlation coefficient and 

standard deviation (SD ±) were calculated 

between the physico-chemical variables 

and CFU of bioluminescent bacterial 

population in all samples collected from 

all the stations from June 2010 to May 

2012. 

3. Results 

Atmospheric temperature was varied 

from 24.3 to 35.3°C (Figure 1), the 

surface water temperature was varied 

from 23.2 to 33.5°C (Figure 2). The pH 

range was from 6.2 to 8.91 (Figure 3), the 

Figure 1: Monthly variations of atmospheric temperature (°C) recorded at stations I, II and 

III from July 2010 to June 2012. 

Figure 2: Monthly variations of surface water temperature (°C) recorded at stations I, II 

and III from July 2010 to June 2012. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 458




Figure 3: Monthly variations of hydrogen ion concentration (pH) in seawater recorded at 

stations I, II and III from July 2010 to June 2012. 

Figure 4: Monthly variations of dissolved oxygen (ml l -1 ) recorded at stations I, II and III 

from July2010 to June 2012. 

Figure 5: Monthly variations of salinity (S % o) in seawater recorded at stations I, II and III 

from July 2010 to June 2012. 

Figure 6: Monthly variations of particulate organic carbon (mg dry wt.l -1 ) recorded at stations 

I, II and III from July 2010 to June 2012. 

dissolved oxygen concentration ranged 

from 3.84 to 7.73ml l -1 (Figure 4) and the 

salinity was fluctuated between 23.12 and 

39 ppt (Figure 5). Particulate organic carbon 

concentration ranged from 0.78 to 

2.56mg dry wt.l -1 during the study period 

at all the stations (Figure 6). The results 

showed that seawater contains nutrients 

viz., inorganic phosphate (4.12 to 

21.6µM; Figure 7), reactive silicate (4.3 

to 19.26µM; Figure 8), inorganic nitrate 

(1.95 to 12.25µM; Figure 9), inorganic 

nitrite (1.1 to 5.5µM; Figure 10), total nitrogen 

(1.5 to 10.39µM; Figure 11), 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 459




Figure 7: Monthly variations of inorganic phosphate (µM) concentration in seawater recorded 

at stations I, II and III from July 2010 to June 2012. 

Figure 8: Monthly variations of reactive silicate (µM) concentration in seawater recorded 


Figure 9: Monthly variations of inorganic nitrate (µM) concentration in seawater recorded 


Figure 10: Monthly variations of inorganic nitrite (µM) concentration in seawater recorded 


Figure 11: Monthly variations of total nitrogen (µM) concentration in seawater recorded at 


ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 460



calcium (120 to 990 mg l -1 ; Figure 12) 

and magnesium (730 to 2460mg l -1 ; Figure13). 

The bacterial diversity revealed 

that, the bioluminescent bacterial population 

density was varied from 1.06 x 10 4 to 

9.44 x 10 4 CFU/ml in seawater (Figure 

14). 

Bottom water temperature was in 

the range of 20.4 to 27.1ºC during the 

study period at all three stations (Figure 

15). Sediment temperature varied from 17 

to 25ºC (Figure 16). Sediment pH varied 

from 6.2 and 9.1 at all stations (Figure 

17). The results for sediment nutrients 

showed that phosphorus (0.065 to 

0.315%; Figure 18), potassium (3.18 to 

8.57%; Figure 19), calcium (4.08 to 


25.44%; Figure 20), magnesium (0.29 to 

1.7%; Figure21), silicon (33.2 to 56.53%; 

Figure 22), sodium (1.56 to 3.35%; Figure 

23), sulphur (0.32 to 2.06%; Figure 

24), chloride (1.3 to 5.71%; Figure 25), 

aluminium (4.63% to 11.87%; Figure 26), 

titanium (1.55 to 9.15%; Figure 27), manganese 

(0.08 to 0.26%; Figure28), iron 

(3.2 to 11.13%; Figure 29), cobalt (3 to 

11ppm; Figure 30), copper (4 to 29ppm 

Figure 31), chromium (26 to 85ppm; Figure 

32), nickel (5 to 18ppm; Figure 33) 

and lead (11 to 27ppm; Figure 34). In sediment, 

the bioluminescent bacterial population 

density varied from 2.6 x10 4 to 23.2 

x10 4 CFU/g (Figure 35). 

Figure 12: Monthly variations of calcium (mg l -1 ) concentration in seawater recorded at 


Figure 13: Monthly variations of magnesium (mg l -1 ) concentration in seawater recorded at 


Figure 14: Monthly variations of bioluminescent bacteria (CFU/ml x 10 4 ) populations in 

seawater recorded at stations I, II and III from July 2010 to June 2012. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 461




Figure 15: Monthly variations in bottom water temperature (ºC) recorded at stations I, II 

and III from July 2010 to June 2012. 

Figure 16: Monthly variations in sediment temperature (ºC) recorded at stations I, II and III 

from July 2010 to June 2012. 

Figure 17: Monthly variations of hydrogen ion concentration (pH) in sediment recorded at 


Figure 18: Monthly percentage variations of phosphorus (%) concentration in sediment 

recorded at stations I, II and III from July 2010 to June 2012. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 462




Figure 19: Monthly percentage variations of potassium (%) concentration in sediment recorded 


Figure 20: Monthly percentage variations of calcium (%) concentration in sediment recorded 


Figure 21: Monthly percentage variations of magnesium (%) concentration in sediment 


Figure 22: Monthly percentage variations of silicon (%) concentration in sediment recorded 


ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 463




Figure 23: Monthly percentage variations of sodium (%) concentration in sediment recorded 


Figure 24: Monthly percentage variations of sulphur (%) concentration in sediment recorded 


Figure 25: Monthly percentage variations of chloride (%) concentration in sediment recorded 


Figure 26: Monthly percentage variations of aluminum (%) concentration in sediment recorded 


Figure 27: Monthly percentage variations of titanium (%) concentration in sediment recorded 


ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 464




Figure 28: Monthly percentage variations of manganese (%) concentration in sediment 


Figure 29: Monthly percentage variations of iron (%) concentration in sediment recorded at 


Figure 30: Monthly variations of cobalt (ppm) concentration in sediment recorded at stations 


Figure 31: Monthly variations of copper (ppm) concentration in sediment recorded at stations 


ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 465




Figure 32: Monthly variations of chromium (ppm) concentration in sediment recorded at 


Figure 33: Monthly variations of nickel (ppm) concentration in sediment recorded at stations 


Figure 34: Monthly variations of lead (ppm) concentration in sediment recorded at stations 


Figure 35: Monthly variations of bioluminescent bacteria (CFU/g x 10 4 ) populations in 

sediment recorded at stations I, II and III from July 2010 to June 2012. 

4. Discussion 

Based on the results, the physicochemical 

parameters, heavy metals in water 

and sediments would form a useful 

tool for the assessment and monitoring of 

coastal ecosystems. The similar results 

and trend was observed in Muthupattai 

mangroves, Southeast coast of India 

(Ashokkumar et al., 2009; Senthilnathan 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 466




and Balasubramanian, 1999). The higher 

concentration of metals were recorded 

during monsoon season, which could be 

mainly due to land runoff and influx of 

metal rich freshwater that in turn reflects 

in the metal concentration in sediment 

(Athalye and Gokhale, 1989). The assessment 

of trace metal concentrations 

and distribution in marine water and sediment 

leads to an understanding of their 

behaviour and detects the pollution source 

in marine environment (Forstner and 

Wittman, 1979). Besides substantiating 

higher biological productivity, higher 

densities of luminous bacteria in this bay 

also signify pollution free environment in 

this region. 

Bioluminescent bacteria are being 

found in marine environment. Microbial 

activities play an important role in marine 

food webs, nutrient mineralization and 

recycling. The ecological importance of 

bioluminescence in the ocean is evident in 

the dominance of light emitters in open 

waters. Ecology of bioluminescent bacteria 

has focused primarily on distribution 

of these organisms in marine environment 

(Nealson and Hastings, 1979); Atalntic 

Ocean (Ramaiah and Chandramohan, 

1988), Indian Ocean (Lynch, 1981) and 

near shore water Porto Nova (Ramesh et 

al., 1989). 

The present study was carried out 

to understand the role of ecological parameters 

of the bioluminescent bacteria 

(during July 2010 - June 2012) at different 

stations of Palk Strait region, India. 

The maximum counts of bioluminescent 

bacteria in seawater/ sediments samples 

was recorded during summer season (May 

2011) at station I; whereas, minimum 

counts of bioluminescent bacteria seawater/ 

sediment samples was recorded at 

monsoon season (2010) at station III. The 

CFU values suggested that the higher 

population counts were recorded during 

the summer months during the study periods 

at all the stations. This variation 

might be due to the high-saline relativity. 

The statistical analysis revealed that seawater 

colony forming unit (CFU) of bioluminescent 

bacteria showed a positive 

correlation with salinity at station-I (r= 

0.773; P< 0.001), station-II (r= 0.903; P< 

0.001) and station-III (r=0.837; P< 0.001) 

and water pH at station-I (r=0.726; P< 

0.01), station-II (r= 0.631; P< 0.01) and 

station-III (r=0.874; P< 0.001) The counts 

were at low levels during the active monsoon 

period because of high rainfall. The 

monsoonal flood water may have altered 

the luminous bacterial population from 

sediment as the Vellar estuary is shallow 

(Ramesh et al., 1989). Further, the maximum 

atmospheric, surface water and sediment 

temperature were recorded during 

summer at station I and minimum was 

recorded during monsoon at station III. 

Maximum bottom water temperature was 

recorded during pre-monsoon at station I 

and minimum was recorded during monsoon 

at station III. Surface water temperature 

was slightly higher than the bottom 

water at all the stations. Surface and bottom 

water temperature of all stations are 

slightly varied monthly. Temperature is 

an important environmental factor, can 

influence the diversity of luminous bacteria 

(Ruby and Nealson, 1978; Yetinson 

and Shilo, 1979; Ruby et al., 1980). Dunlap 

(2009) reported that the temperature 

relationships of luminous bacteria appear 

to be a specific to Vibrio species. According 

to Govindasamy et al. (2000), the surface 

water temperature could be changed 

by the intensity of solar radiation; evaporation, 

freshwater influx, cooling and it 

might mix up with ebb and flow form adjoining 

neritic water. It is further evident 

that the atmospheric temperature showed 

positive correlation at station-II (r= 0.663; 

P< 0.01) and at station - III (r= 0.685; P< 

0.01) to seawater with CFU microbial 

counting at all the stations. Surface water 

temperature was found low during monsoon 

because of rainfall but the maximum 

temperature was observed during summer 

(Kannapiran et al., 2008). This could be 

attributed due to high solar radiation as 

reported by Ashok Prabu et al. (2008). 

Lower temperature was observed due to 

cloudy sky and rainfall that brought down 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 467




temperature to minimum (Kannan and 

Kannan, 1996). In addition, the surface 

water temperature showed positive correlation 

with colony forming unit (CFU) at 

station - II (r= 0.635; P< 0.02) and at station 

- III (r= 0.627; P< 0.02). Further, the 

statistical analysis revealed that the sediment 

colony forming unit of bioluminescent 

bacteria showed a positive correlation 

with sediments temperature at station 

- I (r= 0.806; P< 0.001), station-II (r= 

0.842; P< 0.001) and station-III (r= 0.784; 

P< 0.001). 

Surface water salinity was reduced 

greatly during the monsoon and it was 

gradually increased from postmonsoon to 

summer at all stations. The maximum salinity 

could be due to low amount of rainfall 

and higher rate of evaporation in the 

shallow coastal area owing to high atmospheric 

temperature (Govindasamy 

and Kannan, 1991). Significantly positive 

correlation was observed between seawater 

salinity and CFU of bioluminescent 

bacteria at station-I (r= 0.773; P< 0.001), 

station-II (r= 0.903; P< 0.001) and station-III 

(r= 0.837; P< 0.001). The present 

study results are in line with the research 

findings of Abraham et al. (2003). 

The high pH values recorded during 

summer and this might be due to the 

influence of seawater penetration and 

high biological activity. These findings 

are in accordance with the previous report 

(Smith and Key, 1975). The statistical 

analysis shows the positive correlation 

between pH and CFU station - I (r=0.726; 

P< 0.01) and station - III (r= 0.874; P< 

0.001) to seawater colony forming unit. 

The statistical analysis shows the positive 

correlation with sediments pH station-I 

(r= 0.692; P< 0.001), station-II (r= 0.641; 

P< 0.001) and station-III (r= 0.813; P< 

0.001) to sediment colony forming unit. 

Dissolved oxygen is one of the most important 

abiotic parameters influencing the 

life in the coastal environment. In the present 

study, the maximum dissolved oxygen 

was recorded during monsoon and 

this might be due to the cumulative effect 

of higher wind velocity coupled with rainfall 

and the resultant freshwater mixing. 

The minimum dissolved oxygen was 

found during summer months, which 

could be mainly due to reduced agitation 

in the coastal and estuarine water (Ruby 

and Nealson, 1978; Nealson and Hasting, 

1979). Further, this is evidenced by the 

negative correlation between the dissolved 

oxygen and seawater CFU at station-III 

(r= -0.823; P< 0.001). 

Maximum particular organic carbon 

(POC) was recorded during the 

month of November (2010) at station-II 

and minimum POC was observed during 

the month of May (2012) at station-I. The 

maximum POC content in water was 

mainly due to the organic matter brought 

in from the land through run-off. Further, 

it could be also due to the presence of 

plant (seagrass and seaweeds) and animal 

organic matter within the seagrass ecosystem 

and exported from the adjacent ecosystem 

by wind and wave action. Further, 

the seasonal variation in POC content in 

the water could be related to the plankton 

productivity (Kannapiran et al., 2008). 

This is further evidenced by the negative 

correlation between POC and seawater 

CFU at station-I (r= -0.786; P< 0.001), 

station-II (r= -0.841; P< 0.001) and station-III 

(r= -0.832; P< 0.001). 

Maximum inorganic phosphate 

was observed during the monsoon season 

(December 2010) at station-III and minimum 

was recorded during the summer 

season (May 2011) at station-I. Possibly, 

the maximum concentration of phosphate 

was due to invasion of upwelling of water, 

which increased the level of phosphate. 

Low values of phosphate observed 

to utilization by phytoplankton, seagrasses 

and other primary producers (Rajasegar, 

2003). The statistical analysis shows 

the negative correlation to seawater colony 

forming unit with inorganic phosphate 

at station - I (r= - 0.734; P< 0.01), station- 

II (r= -0.832; P< 0.001) and station-III (r= 

-0.828; P< 0.001). 

Minimum concentration of silicate 

was observed during the summer season 

(May 2012) at station II and maximum 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 468



during the monsoon season (November 

2010) at station III. It might be due to the 

heavy rain, land runoff water mixing was 

high level. It has been reported that the 

silicate from the bottom sediment might 

have been exchanged with overlaying water 

in this mangrove environment (Govindasamy 

and Kannan, 1996). The low 

value observed in summer and postmonsoon 

season could be attributed to 

uptake of silicates (Saravanakumar et al., 

2008). The statistical analysis of silicate 

showed negative correlation with seawater 

colony forming unit at station-II (r= 

-0.786; P< 0.001) and station-III (r= - 

0.841; P< 0.001). 

Nitrate concentration was found 

lower than that of nitrate and same trend 

of fluctuation was reported in Muthpettai 

mangrove (Ashokkumar et al., 2011). 

The statistical analysis showed nitrate a 

negative correlation with seawater colony 

forming unit at station-I (r= -0.868; P< 

0.001), station-II (r= -0.888; P< 0.001) 

and station-III (r= -0.926; P< 0.001). 

Maximum level of nitrite was recorded 

during December 2011 at station-II 

and minimum was observed during the 

May 2011 at station-I. It might be due to 

the minimum nitrite were recorded during 

the summer may be due to high salinity. 

The higher nitrate value in monsoon season 

could be due to the increased phytoplankton 

excretion, oxidation of ammonia, 

reduction of nitrate, the recycling of 

nitrogen and bacterial decomposition of 

planktonic detritus (Govindasamy et al., 

2000; Asha and Diwakar, 2007) and also 

due to denitrification and air-sea interaction 

exchange of chemicals were also responsiple 

for this increased values (Rajasegar, 

2003; Ashok Prabu et al., 2008). 

The statistical analysis which showed nitrite 

a negative correlation with seawater 

colony forming unit at station-I (r= - 

0.767; P< 0.001), station-II (r= -0.834; P< 

0.001) and station-III (r= -0.882; P< 

0.001). 

Maximum level of total nitrogen 

was recorded during the monsoon (December 

2010) at station III and minimum 


was recorded during the summer (May 

2011) at station I. It might be due to 

freshwater inflow was high in the monsoon 

season so high level of nitrogen was 

recorded. The statistical analysis showed 

total nitrogen a negative correlation with 

seawater CFU of bioluminescent bacteria 

at station-I (r= -0.829; P< 0.001), station- 

II (r= -0.929; P< 0.001) and station-III (r= 

-0 .896; P< 0.001). 

Maximum and minimum level of 

seawater calcium was observed during the 

monsoon (December 2010) and summer 

seasons (May 2011) at station-III. Maximum 

level of sediment calcium was observed 

during the monsoon season (December 

2010) at station-II and minimum 

level of calcium was observed during the 

summer (May 2011) at station-II. Maximum 

level of seawater magnesium was 

recorded during the monsoon season December 

(2010) at station I and minimum 

was recorded during the summer season 

May (2011). Maximum level of sediment 

magnesium was observed during the post 

monsoon season (January 2011) at station-I 

and minimum level of magnesium 

was recorded during the summer season 

(May 2011) at station-II. The statistical 

analysis showed seawater calcium a negative 

correlation to seawater colony forming 

unit at station-II (r= -0.816; P< 0.001) 

and station-III (r= -0.762; P< 0.001). In 

addition, to that the seawater magnesium 

a negative correlation with seawater colony 

forming unit at station-II (r= -0.829; 

P< 0.001) and station-III (r= -0.840; P< 

0.001). Sediment calcium a negative correlation 

with sediment colony forming 

unit at station-I (r= -0.843; P< 0.001), station-II 

(r= -0.832; P< 0.001) and station- 

III (r= -0 .909; P< 0.001); Sediment magnesium 

showed a negative correlation 

with sediment colony forming unit at station-I 

(r= -0.710; P< 0.001), station-II (r= 


0.721; P< 0.001). It might be due to the 

calcium and magnesium higher level was 

recorded in monsoon and post monsoon 

seasons. Calcium and magnesium concentrations 

could be increased due to the in- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 469




put of freshwater (Sundararajan and Natesan, 

2010). 

Maximum sediment total phosphorus 

was observed during the month of 

December-2010 at station-I and minimum 

was recorded during the month of May- 

2011 at station-III. and this might be due 

to the domestic, municipal and agricultural 

waste (non-pointed sources). The regeneration 

and releases of total phosphorus 

from bottom mud into the water column 

by turbulence and mixing was also 

attributed to recorded higher values 

(Chandran and Ramamoorthy, 1984). The 

statistical analysis showed sediment total 

phosphorus a negative correlation with 

sediments colony forming unit at station-I 

(r= -0.848; P< 0.001), station-II (r= - 

0.843; P< 0.001) and station-III (r= - 

0.861; P< 0.001). Maximum concentration 

of sediment potassium was observed 

during the monsoon season (December 

2010) at station-I and minimum concentration 

of potassium was recorded during 

the summer (June 2010) at station-II. It 

might be due to high concentration of potassium 

are inflected by heavy rainfall 

inflow on in peak values was recorded 

during the monsoon seasons. Further, evidenced 

by the statistical analysis showed 

potassium a significant negative correlation 

with sediments colony forming unit 

at station-II (r= -0.656; P< 0.001) and station-III 

(r= -0.751; P< 0.001). 

Maximum and minimum sediment 

silicon was observed during the May - 

2012 at station II and November -2011 at 

station-III. This might be due to its biological 

significance; the flux of particulate 

silica from surface waters plays an important 

role in the cycling of other elements 

in the marine environment such as 

radium, barium and germanium (Li et al., 

1973; Froelich and Andreae, 1980). The 

statistical analysis showed silicon a significant 

positive correlation with sediment 

colony forming unit at station-I (r= 0.745; 

P< 0.001), station-II (r= 0.824; P< 0.001) 

and station-III (r= 0.836; P< 0.001). The 

statistical analysis showes sulphur 

showed a negative correlation with sediment 


0.661; P< 0.001), station-II (r= -0.856; P< 


0.001). 

Maximum concentration of sediment 

sodium was observed during the 

month of 2011 at station-III and minimum 

was recorded at station-II and this might 

be due to the high counts of microbial 

populations depending on the sodium 

concentration and it was varied spatially. 

The statistical analysis which showed sediment 

sodium a positive correlation with 

sediment colony forming unit at station-I 

(r= 0.834; P< 0.001), station-II (r= 0.824; 

P< 0.001) and station-III (r= 0.845; P< 

0.001). Maximum and minimum level of 

chloride was recorded during the summer 

and post monsoon seasons at station III 

and station I respectively. The statistical 

analysis of sediments chloride shows a 

positive correlation with sediment colony 

forming unit station-I (r= 0.665; P< 

0.001), station-II (r= 0.840; P< 0.001) and 

station-III (r= 0.811; P< 0.001). 

The maximum sediment aluminium 

was observed recorded during the post 

monsoon month of January 2012 at station-I 

and minimum was observed during 

the month of April 2011 at station-III. It 

might be due to contribution from detrital 

mineral grains supplied from through the 

rivers in addition to the precipitation of 

their dissolved species under prevailing 

estuarine condition. The statistical analysis 

which showed a negative correlation 

to sediment colony forming unit with aluminium 

at station-II (r= -0.706; P< 

0.001). Removal of dissolved river-borne 

aluminum by coagulation in the coastal 

areas is common (Holliday and Liss, 

1976). 

In sediment, the maximum and 

minimum titanium level was recorded 

during the monsoon 2010 and summer 

April 2011 at stations III and I respectively. 

Moreover, the statistical analysis revealed 

that the sediment titanium shows a 

negative correlation to sediment colony 


0.001), station-II (r= -0.792; P< 0.001) 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 470



and station-III (r= -0.888; P< 0.01). Maximum 

and minimum level of sediment 

manganese was recorded during the monsoon 

(2010) at station III and summer 

(2012) seasons at station I. The statistical 

analysis showed sediment manganese a 

negative correlation with sediment colony 


0.001), station-II (r= -0.819; P< 0.001) 

and station-III (r= -0.800; P< 0.001). 

Maximum and minimum concentration 

of sediment iron was recorded during 

the post monsoon (2011) and summer 

(2012) seasons at station I and III. In general, 

marine environment and discharge of 

aquatic ponds, domestic wastes, land and 

agricultural drainage, boating activities 

such as loading and unloading of materials, 

antifouling paints from boating activities 

contribute to enhance the metal level 

in marine environment (Govindasamy and 

Azariah, 1999; Ashokkumar et al., 2009). 

The statistical analysis showed sediment 

iron a negative correlation with sediment 


0.643; P< 0.01), station-II (r= -0.695; P< 


0.001). 

Maximum and minimum level of 

copper was observed during the month of 

January 2012 at station III and summer 

month of May (2012) at station I. It might 

be due to the discharge of maximum level 

of freshwater in the central west coast of 

India (Sankaranarayanan and Reddy, 

1973). The statistical analysis showed 

sediment copper a negative correlation 

with sediment colony forming unit station-I 

(r= -0.675; P< 0.001), station-II (r= 


0.770; P



lation in the Palk Strait region. Most of 

the parameters such as the atmospheric 

temperature, salinity, pH were significantly 

correlated with the bioluminescent bacterial 

population in all the three stations 

studied. 

References 

Abraham, T.J., S.A. Shanmugam, R. 

Palaniappan and K. Dhevendaran. 

(2003). Distribution and 

abundance of luminous bacteria 

with special reference to shrimp 

farming activities. Indian Journal 

of Marine Sciences 32(3), 208 - 

213. 

Asha, P.S and K. Diwakaran. (2007). 

Hydrobiology of the inshore waters 

off Tuticorin Bay, Gulf of Mannar. 

Journal of Marine Biologycal Association 

of India 49(1), 07-11. 

Ashok Prabu, V., M. Rajkumar and 

P. Perumal. (2008). Seasonal variation 

in physicochemical characteristics 

of Picavaram mangroves, 

southeast coast of India. Journal of 

Environmental Biology 29, 945- 

950. 

Ashokkumar, A., V.S. Reddy Belum, 

B. Ramaiah, P. Sanjana Reddy, 

K.L. Sahrawat and H.D. Upadhyaya. 

(2009). Genetic variability 

and plant character association of 

grain Fe and Zn in selected core 

collections of Sorghum germplasm 

and breeding lines. Journal of SAT 

Agricultural Research 7, 1-4. 

Ashokkumar, S., G. Rajaram, P. 

Manivasagam, S. Ramesh, P. 

Sampathkumar and P. Mayavu. 

(2011). Studies on hydrographical 

parameters, nutrients and microbial 

population of Mullipallam Creek in 

Muthupettai Mangrove (Southeast 

coast of India). Research Journal 

of Microbiology 6(1), 71-86. 

Athalye, R.P and K.S. Gokhale. 

(1989). Study of selected trace 

metals in the sedimends of Thane 

creek near Thane city- Antagonistic 


behavior of zinc and copper. Mahasagar 

Bulltion National Institute 

of Ocanography 22, 185-191. 

Chandran, R and K. Ramamoorthi. 

(1984). Hydrobiological studies in 

the gradient zone of the Vellar estuary. 

I. Physicochemical parameters. 

Mahasagar Bulletin. National 

Institute of Oceanography 17, 69- 

78. 

Chatterjee, M., E.V. Silva Filho, S.K. 

Sarkar, S.M. Sella, A. 

Bhattacharya, K.K. Satapathy, 

M.V.R. Prasad, S. Chakraborthy 

and B.D. Bhattacharya. (2006). 

Distribution and possible source of 

trace elements in the sediment 

cores of tropical macrotital estuary 

and ecotoxicological significance 

33(3), 346-356. 

Dunlap, P.V. (2009). Bioluminescence. 

Microbial Physiology. 48-61. 

Forstner, V and G.T.W. Wittmann. 

(1979). Metal pollution in the 

aquatic environment. Springer- 

Verlag, Berlin, 486. 

Froelich, P.N and M.O. Andreae. 

(1980). Germanium in the oceans: 

eka-silicon. Trans Am Geophys. 

Union 61, 987. 

Govindasamy, C and R. Srinivasan. 

(2012). Association of bioluminescent 

bacteria from blue swimmer 

crab Portunus pelagicus (Linneaus, 

1758). Asian Pacific Journal of 

Tropical Disease S699-S702. 

Govindasamy, C and J. Azariah. 

(1999). Seasonal Variation of Dissolved 

Metals in Coastal Water of 

the Coromandel Coast, India. Indian 

Journal of Marine Sciences 28, 

249- 256. 

Govindasamy, C and Kannan, L. 

(1991). Rotifers of the Pitchavaram 

Mangroves (Southeast Coast of India): 

A Hydrographical Approach. 

Mahasagar- Bulletin National Institute 

of Oceanography 24(1), 39- 

45. 

Govindasamy, C and L. Kannan. 

(1996). Ecology of Rotifers of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 472



Pichavaram mangroves, southeast 

coast of India. Indian Hydrobiology 

1, 69-76. 

Govindasamy, C., L. Kannan and Jayapaul 

Azariah. (2001). Seasonal 

variation in physico-chemical 

properties and primary production 

in the coastal water biotopes of 

Coromandal coast. Indian Journal 

of Environmental Biology. 21, 1-7. 

Harvey, E. N. (1952). Bioluminescence. 

Academic Press Inc., New 

York, p. 649 

Hastings, J. W. (1986). Bioluminescence. 

Annual Review of Biochemistry 

37, 597-630. 

Holliday, L.M and P.S. Liss. (1976). 

The behaviour of dissolved iron, 

manganese and zinc in the Beaulieu 

estuary. Estuarine Coastal 

Marine Sciences 4, 349-353. 

Kannan, R and L. Kannan (1996). 

Physico-chemical characteristics of 

seaweed beds of the Palk Bay, 

Southeast coast of India. Indian 


358-362. 

Kannapiran, E., L. Kannan, A. 

Purushothaman and T. Thangarajdou. 

(2008). Physico-chemical 

and microbial characteristics of the 

coral reef environment of the Gulf 

of Mannar marine biosphere reserve, 

India. Journal of Environmental 

Biology 29(2), 215- 222. 

Karuppasamy, P.K and P. Perumal. 

(2000). Biodiversity of zooplankton 

at Pichavaram mangroves, 

South India. Advanced in Biological 


Kita-Tsukamoto, K., K. Yao, A. Kamiya, 

S. Yoshizawa, N. Uchiyama, 

K. Kogure and M. Wada. 

(2006). Rapid identification of marine 

bioluminescent bacteria by 

amplified 16S ribosomal RNA 

gene restriction analysis. FEMS 

Microbiology Letters 258(2), 320. 

Li, Y., T.L. Ku, G.G. Mathieu and K. 

Wolgemuth. 1973. Barium in the 

Antartic Ocean and implications 


regarding the marine geochemistry 

of Ba and Ra. Earth Planet Sci Let. 

19, 352-361. 

Lynch, R.V. (1981). Distribution of 

luminous marine organisms: a literature 

review, In Bioluminescence: 

current perspectives, (Eds) 

K.H. Nealson, Burgess Publising 

Co, Minnesota, 153-159. 

Mannan, M., B. Zourarah, C. Carruesco, 

A. Aajjane and J. Nadu. 

(2004). Distribution of heavy metals 

in Sidi Moussa lagoon sediment 

(Atlantic Moroccan Coast). Journal 

of American Earth Sciences 39, 

473-484. 

Nair, G.B., M. Abraham and R. Natarajan. 

(1979). Isolation and identification 

of luminous bacteria from 

Porto Novo estuarine environs. Indian 


46-48. 

Nealson, K.H and J.W. Hastings. 

(1979). Bacterial bioluminescence: 

its control and ecological significance. 

Microbiological Reviews 43, 

496-518. 

Nealson, K.H. (1978). Isolation, identification 

and manipulation of luminous 

bacteria. Methodes in Enzymology 

57,153-166. 

Parsons, T.R., Y. Maita and C.M. 

Lalli, (1984). A Manual of Chemical 

and Biological Methods Seawater 

Analysis. Pergamon Press, 

Oxford, New York, Toronto, Sydney, 

Frankfurt, 1-173. 

Rajasegar, M. (2003). Physicochemical 

characteristics of the Vellar 

estuary in relation to shrimp 

forming. Journal of Environmental 

Biology 24, 95-101. 

Ramachandran, S., S. Anitha, V. Balamurugan, 

K. Dharaanirajan, 

K.E. Venthan and M.I.P. Divien. 

(2005). Ecological impact of tsunami 

on Nicobar Islands (Camrta, 

Katchal, Nancowry and Trinkat). 

Current Sciences 89(1), 195-200. 

Ramaiah, N and D. Chandramohan. 

(1988). Distribution of luminous 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 473



bacteria and bacterial luminescence 

in the equation region of the Indian 

Ocean. Microbiologica 11, 243- 

254. 

Ramesh, A., B.G. Loganathan and K. 

Venugopalan. (1989). Seasonal 

distribution of luminous bacteria in 

the sediments of a tropical estuary. 

Journal of Genetics and Applied 


Rousk, J., P.C. Brookes and E. Baath. 

(2009). Contrasting Soil pH Effects 

on Fungal and Bacterial Growth 

Suggest Functional Redundancy in 

Carbon Mineralization. Applied 

and Environmental Microbiology 

75(6), 1589-1596. 

Ruby, E.G and K.H. Nealson (1978). 

Seasonal changes in the species 

composition of luminous bacteria 

in near shore seawater. Luminology 

and Oceanography 23, 530-533. 

Ruby, E.G., E.P. Greenberg, and 

J.W. Hastings. (1980). Planktonic 

marine luminous bacteria: species 

distribution in the water column. 

Applied Environmental Microbiology 

39,302-306. 

Sankaranarayanan, V.N. and C.V.G. 

Reddy. (1973). Copper content in 

the inshore and estuarine waters 

along the central west coast of India. 

Current Sciences 4, 223-224. 

Saravanakumar, A., M. Rajkumar, J. 

Sesh Serebiah and G.A. 


Thivakaran. (2008). Seasonal variations 

in physicochemical characteristics 

of water, sediment and soil 

texture in arid zone mangrove of 

Kachchh Gujarat. Journal of Environmental 

Biology 29, 725-732. 

Senthilnathan. S and T. Balasubramanian. 

(1999). Heavy metal distribution 

in Pondicherry harbour, 

southeast coast of India. Indian 

Joumal of Marine Sciences 28, 

380- 382. 

Sundararajan, M and U. Natesan. 

(2010). Environmental significance 

in recent sediments along Bay of 

Bengal and Palk Strait, East Coast 

of India: A geochemical approach. 

International Journal of Environmental 

Research 4(1), 99-120. 

Swift, E., J.M. Sullivan, H.P. 

Batchelder, J. Van Keuren, R.D. 

Vaillancourt and R.R. Bidigare. 

(1995). Bioluminescent organisms 


bioluminescence 

measurements in the North Atlantic 

Ocean near latitude 59.5ºN, 

longitude 21º. World Journal of 

Geophysis and Reserach 100, 

6527-6647. 

Yetinson, T and M. Shilo. (1979). Seasonal 

and geographic distribution 

of luminous bacteria in the eastern 

Mediterranean and the Gulf of Elat. 

Applied Environmental Microbiology 

37, 1230-1238. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 474



Synthesis of Biocompatible Silver Nanoparticles Using 

Green Alga (Ulva reticulata) Extract 

Ganapathy Selvam Govindarasu 1 , Srinivasan Rajendran 2 and Sivakumar 

Kathiresan 1, * 

1 Division of Algal Biotechnology, Department of Botany, Annamalai University, 

Annamalainagar-608 002, Tamil Nadu, India; 2 Department of Oceanography and Coastal 

Area Studies, School of Marine Sciences Alagappa University, Thondi Campus, Thondi – 

623 409, Ramanathapuram District, Tamil Nadu, India; *Correspondence: 

vgs.biot@gmail.com / kshivam69@gmail.com; Tel: +91 9786330511 

Abstract: The synthesis of nanoparticles has become a matter of great interest in recent 

times due to their various advantageous properties and applications in a variety of fields. 

We have reported biological synthesis of nano-sized silver particles. The nanoparticles of 

silver were formed by the reduction of silver nitrate to aqueous silver metal ions while effect 

of marine seaweed U. reticulata extract was investigated; silver nanoparticles were 

characterized using UV-visible absorption and room temperature photoluminescence. The 

X-ray diffraction results revealed that the synthesized silver nanoparticles were in the cubic 

phase. The existence of functional groups was identified using Fourier transform infrared 

spectroscopy. The morphology and size of the synthesized particles were studied with 

atomic force microscope. Further, the photocatalytic degradation of methyl orange was 

measured spectrophotometrically by using silver as nanocatalyst under visible light illumination. 

Silver nanoparticles synthesized from U. reticulata by facile method from can able 

to degrade dyes in the presence of visible light and paves way for ecological health and environmental 

bioremediation. Despite numerous studies conducted over the last decade, 

there are still considerable gaps in our knowledge about the biotechnological potential of 

green-synthesized nanoparticles. Furthermore, the precise basis of their antibiotic activity 

has yet to be defined. The biological methods are generally cost effective, nontoxic, and 

ecofriendly. This chapter focuses on the methods involved in algal-synthesized nanoparticles 

and its applications. 

Keywords: AFM; Ag nanoparticles; antibacterial activity; cubic phase; methyl orange; photocatalytic 

degradation; Ulva reticulata 


Nanotechnology deals with the 

synthesis of nanoparticles that exhibit 

completely new or improved properties 

based on specific characteristics such as 

size, distribution and morphology. New 

applications of nanoparticles and nanomaterials 

are emerging rapidly (Jahn 

1999; Naiwa 2000; Murphy 

2008).Currently, there is a growing need 

to develop eco- friendly and sustainable 

methods for the synthesis of nanomaterials 

that do not use toxic chemicals in the 

synthesis protocols so as to avoid adverse 

effects in medical applications. Among 

the nanoparticles, silver nanoparticles 

(AgNPs) have received considerable attention 

due to their attractive physicochemical 

properties (Elechiguerra et al., 

2005). The metallic nanoparticles are 

most promising and considered as remarkable 

biomedical agents. Due to their 

large surface volume ratio, they govern 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 475


Synthesis of Biocompatible Silver Nanoparticles… 

interest of researchers on microbial resistance. 

Among the developed nanoparticles, 

silver (Ag) nanoparticles are pertaining 

to have a wide range of application 

in the fields of physical, chemical 

and biological sciences. In the past decade, 

several kinds of the biological organisms 

(microbes, plants and seaweeds) 

have been employed and well-studied for 

the ability of silver nanoparticles (Ag- 

NPs) synthesis (Ramanathan et al., 2011; 

Ahmad et al., 2003; Shankar et al., 2003; 

Mohanpuria et al., 2008; Kumar et al., 

2012a). Application of green chemistry in 

synthesizing nanomaterials has vital role 

in medicinal and all technological aspects 

(Mondal et al., 2011, Begum et al., 2009). 

Biologically synthesized Ag-NPs have 

wide range of applications because of 

their remarkable physical and chemical 

properties. The literature on the extra cellular 

biosynthesis of Ag-NPs using plants 

and pure compounds from plants are insignificant 

(Kattumuri et al., 2007; Song 

and Kim 2008; Gilaki 2010). 

In this article, we describe a simple 

one step method for the synthesis of 

Ag-NPs by the reduction of aqueous Agions 

using extracts of green seaweed, at 

direct sunlight conditions. 


2.1. Screening and selection of sample 

Fresh sample of Ulva reticulata 

green seaweed was collected in the month 

of January 2013 from Pudumadam coastal 

region (78.99°’E, 9.27°’N), in Gulf of 

Mannar, Tamil Nadu, India. Sample was 

immediately brought to the laboratory in 

polythene bags and cleaned thoroughly 

with fresh water to remove adhering debris 

and associated biota. The alga sample 

was cleaned with distilled water using 

brush for the removal of the epiphytes. 

2.2. Preparation of aqueous extract 

The whole U. reticulata samples 

were initially rinsed thrice in distilled water 

and dried on paper toweling. Twenty 

five (25) gram sample was cut into fine 

Ganapathy Selvam et al. 

pieces and boiled in 100 ml of sterile distilled 

water for 5 min. The crude extract 

was passed through Whatman No.1 filter 

paper and the filtrate was stored at 4°C 

for further use the methods suggested by 

Jha et al. (2009). 

2.3. Synthesis of silver nanoparticles 

Analytical grade (AR) Silver nitrate 

(AgNO 3 ) was purchased from E. 

Merck (India). In the typical synthesis of 

silver nanoparticles, 10 ml of the aqueous 

extract of Ulva reticulata was added to 90 

ml of 1 mM aqueous AgNO 3 solution in 

250 ml conical flask and incubated at 

room temperature for 72 h by agitating at 

120 rpm. Suitable controls were maintained 

throughout the experiments (Parashar 

et al., 2009). The bio-reduction of 

AgNO 3 into AgNPs can be confirmed 

visually by the change in colour from 

light yellow to brown indicating the formation 

of silver nanoparticles (Figure 1). 

2.4. Characterization techniques 

The colour change in reaction 

mixture (metal ion solution + seaweed 

extract) was recorded through visual observation. 

The bio reduction of silver ions 

in aqueous solution was monitored by periodic 

sampling of aliquots (0.5 ml) and 

subsequently measuring UV-Vis spectra 

of the solution. UV-vis spectra of these 

aliquots were monitored as a function of 

time of reaction on UV-Vis spectrophotometer 

UV-2450 (Shimadzu). 

The Ag-NPs solution thus obtained 

was purified by repeated centrifugation 

at 5000 rpm for 20 min followed 

by resuspention of the pellet of Ag-NPs in 

10 ml of deionized water. After freeze 

drying of the purified Ag-NPs, its structure 

and composition was analyzed by 

XRD. The dried mixture of Ag-NPs was 

collected for the determination of the 

formation of Ag-NPs by an X’Pert Pro x- 

ray diffractometer (PAN analytical BV, 

The Netherlands) operated at a voltage of 

40 kV and a current of 30 mA with Cu Kα 

radiation in θ- 2 θ configurations. The 

crystallite domain size was calculated 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 476




Figure 1: Silver nitrate (AgNO 3 ) solution and others colour changes during the reduction of 

AgNO 3 into AgNPs by the extract of U. reticulata after 20 min of incubation. 

from the width of the XRD peaks, assuming 

that they are free from non-uniform 

strains, using the Scherrer’s formula. 

D= 0.94 λ / β Cos θ 

where D is the average crystallite domain 

size perpendicular to the reflecting planes, 

λ is the X-ray wavelength, β is the full 

width at half maximum (FWHM), and θ 

is the diffraction angle. To eliminate additional 

instrumental broadening the 

FWHM was corrected, using the FWHM 

from a large grained Si sample. 

β corrected = (FWHM 2 sample- 

FWHM 2 si) 1/2 

This modified formula is valid only when 

the crystallite size is smaller than 100 nm. 

The silver nanoparticles were observed 

using SEM. Sample was prepared 

by placing a drop of AgNPs on carbon 

coated copper stuff and subsequently drying 

air, before transferring it to the microscope 

operated at an accelerated and voltage 

of 120KV (JOEL Model JSM-5010 

LV with INSA EDS) and followed for 

Energy Dispersive Spectrophotometer 

analysis. 

The morphology of the product 

was observed by Nano Surf Easy Scan 2 

Atomic Force Microscope (AFM) measurement 

to study the morphology and size 

of the Ag-NPs (Al-Warthan et al., 2010). 

2.5. Photocatalytic degradation 

The photocatalytic degradation of 

methyl orange was evaluated by biosynthesized 

Ag-NPs (Rashed and El-Amin 

2007). All the experiments were performed 

outdoor with sun as the main 

source of light (Wang et al., 2000). Prior 

to the experiment, a suspension was prepared 

by adding 20 mg of Ag-NPs to 50 

ml of methyl orange solution (Fisher Scientific). 

Later, the mixture was allowed to 

stir constantly for about 30 min in darkness 

to ensure constant equilibrium of Ag- 

NPs in the organic solution. During the 

reaction, the mixture was kept under sunlight 

within a Pyrex glass beaker and 

stirred constantly. The mean temperature 

was found to be 29˚C with 10 h mean 

shine duration. The absorption spectrum 

of the suspension mixture was measured 

periodically using a UV–visible spectrophotometer 

(Shimadzu, UV-2450, Japan) 

after centrifugation to ensure the degradation 

of methyl orange solution. 

2.6. Antibacterial activities 

Experimental pathogens namely, 

Gram positive bacterium Staphylococcus 

aureus, Gram negative bacteria Pseudomonas 

aeruginosa, Escherchia coli, Proteus 

mirabilis and Proteus vulgaris were 

obtained from Raja Sir Muthaiya Medical 

College, Annamalai University. Pathogens 

were loaded in inoculated in medium 

(Muller Hinton Agar medium) [100 

µg/ml, 50 µg/ml, 25 µg/ml (Ag-NPs)] and 

AgNO 3 was used as negative control. 

Each culture was spread on to Muller 

Hinton Agar plates. Sterile paper discs of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 477



6mm diameter along with streptomycin 

(Positive control) antibiotic containing 

discs were placed in each plate. Bacterial 

growth inhibition was determined as the 

diameter of the inhibition zones around 

the discs. All tests were performed in triplicate. 

Then, Petri dishes were incubated 

at 37°C for 18–24 h aerobically, inhibition 

zone were measured and data was 

recorded (Bauer et al., 1966). 

3. Results and discussion 

3.1. UV–Visible spectroscopy 

UV–visible absorption is one 

among the most important techniques to 

identify the formation of metal nanoparticles, 

provided surface plasmon resonance 

exists for metal (Binupriya et al., 2010). It 

is well known that silver nanoparticles 

exhibit yellowish brown color in aqueous 

solution due to excitation of surface 

plasmon vibrations in silver nanoparticles 

(Shankar et al., 2004). As the extract was 

mixed in the aqueous solution of the silver 

ion complex, it started to change the 

color from watery to yellowish brown due 

to reduction of silver ion which indicated 

formation of silver nanoparticles. It is 

generally recognized that UV–Vis spectroscopy 

could be used to examine size 

and shape controlled nanoparticles in 

aqueous suspensions (Wiley et al., 2006). 

The UV-Vis spectra recorded from the 

reaction medium after 4 hours is shown in 

Figure 2. A strong silver plasmon absorption 

maximum was recorded at 410-420 

nm in UV-Vis spectroscopy. The observed 

band in this range has been associated 

with Ag-NPs confirming the synthesis 

of spherical Ag-NPs with narrow size 

distribution has been revealed (Henglein 

1993 and similar observation were also 

made by Kumar et al., 2012b). Elevation 

in temperature results in formation of 

spherical and octahedral shaped nanoparticles 

of size 5-100 nm (Lengke et al., 

2007). Similarly, shape-controlled Ag- 

NPs can be also synthesized using biological 

system under varying temperature 

(Bansal et al., 2012). 


3.2. FTIR spectrum 

FTIR analysis was used for the 

characterization of the extract and the resulting 

nanoparticles. FT-IR measurements 

were carried out to identify the 

possible biomolecules responsible for the 

reduction of Ag + ions and the capping of 

the bioreduced AgNPs synthesized. FT- 

IR spectrum (Figure 3) showed different 

major peaks positioned at 3405.37, 

2955.75, 2922.87, 2851.82, 1654.15, 

1637.15, 1512.61, 1457.45, 1418.38, 

132.90, 1250.54, 1175.74, 1109.93, 

812.05 and 699.73 cm -1 . The presence of 

peak at 3405.37 cm -1 could be due to O-H 

group in alcohols and phenols. A small 

peak observed at 2955.75, 2922.87, and 

2851.82 cm -1 is due to C-H stretching of 

alkanes. Sharp and intense bands observed 

at 1654.15, 1637.15, 1560.38, 

1512.61, and 1490.30 cm -1 are due to – 

C=C- stretch, N-H bend, NO 2 asymmetrical 

stretch and nitro compounds, respectively. 

Another bands were positioned at 

1457.45 (C-H bend alkanes), 1418.32 (C- 

C stretch (in-ring) aromatics), 1362.90 

(C-H rock alkanes) and 1250.74 (C-N 

stretch aromatic compounds). The observed 

bands ranging between 1109.93 

and1032.31 cm -1 are due to C-N stretch 

band of aliphatic amines. Bands observed 

at 812.05 and 743. 34 cm -1 are due to N-H 

wag band of primary and secondary 

amines. A band positioned at 699.73 cm -1 

is due to –C (triple bond) C-H; C-H bond 

alkynes. After bio-reduction, there is a 

shift in the absorption and band at 

2955.75, 1457.45, 1362.90, 812.05 and 

743.34 cm -1 may be due to the binding of 

(NH) C=H and N-H wag group with the 

nanoparticles. The (NH) C=H groups 

within the cage of cyclic peptides are involved 

in stabilizing the nanoparticles. 

Thus, the peptides may play an important 

role in the reduction of Ag-NPs. This 

could be due to the ability of reducing and 

capping agents present in U. reticulata 

which were revealed by FT-IR studies. 

Fourier Transform Infra-Red (FT-IR) 

spectroscopy analysis showed that the 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 478




Figure 2: UV–visible absorption spectra of silver nanoparticles after 30 min of incubation. 

Figure 3: FT-IR spectra of AgNPs synthesized by U. reticulata. 

synthesized nano-Ag was capped with 

bimolecular compounds which are responsible 

for reduction of silver ions 

(Jegadeeswaran et al., 2012). The abovementioned 

shift was also observed in 

Codium capitatum (Kannan et al., 2013). 

3.3. Crystal structures analysis and determination 

of crystallite size 

The XRD pattern showed three 

intense peaks in the whole spectrum of 2θ 

value ranging from 10 to 80. Average size 

of the synthesized particles was 10 nm 

with size range 10 - 50nm with cubic and 

hexagonal shape. The typical XRD pattern 

revealed that the sample contains a 

mixed phase (cubic and hexagonal) structures 

of silver nanoparticles. The average 

estimated particle size of this sample was 

10 nm derived from the FWHM of peak 

corresponding to 90 plane (Figure 4). X- 

ray diffraction showed the average particle 

size of 15 nm as well as revealed their 

cubic structure (Geethalakshmi and Sarada 

2010). 

3.4. Particles morphology (SEM and 

AFM measurements) 

The SEM image (Figure 5 a and b) 

depicts the high density Ag-NPs synt - 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 479




Figure 4: XRD patterns of silver nanoparticles synthesized after 120 h of incubation. 

Figure 5: (a) SEM micrograph of Silver nanoparticles synthesized from the extracts of U. 

reticulata; (b) energy dispersive spectrometer analysis 

-hesized by the U. reticulata and confirms 

the development of silver nanostructures 

with energy dispersive spectrometer. The 

SEM micrographs of nanoparticle obtained 

in the filtrate showed that Ag-NPs 

are spherical shaped and well distributed 

without aggregation in solution. Ag-NPs 

predominantly spherical well distributed 

with an average size 15 nm (Saraniya Devi 

et al., 2013). It is known that the shape 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 480



of metal nanoparticles considerably 

changes their optical electronics properties 

(Xu and Kall, 2002). Similar phenomenon 

was reported by Chandran et al. 

(2006). 

The surface morphology and size 

of the Ag-NPs harvested after 120 h of 

incubation were studied by AFM. The 

two- and three-dimensional images of the 

nanoparticles are shown in Figure 6a and 

6b. From the 2D view, well-separated 

spherical particles are seen. The sizes of 

the particles are in the range of 1–40 nm. 

However, most of the particles are in the 

range of 10 nm. The 3D view revealed 

that the growth direction of all the particles 

was almost same confirming the single 

crystalline nature of the cubic phase 

of Ag-NPs. Williams, (2008) reported that 

nanoparticles are clusters of atoms in the 

size range of 1–100 nm. Morphology and 

size of the synthesized particles were 

studied with atomic force microscope 

(shanmugam et al., 2014). 

3.5. Photocatalytic degradation 

Photocatalytic degradation of methyl 

orange dye was investigated using 

biometrically synthesized silver nanocatalysts 

by solar irradiation technique at different 

time intervals as shown in Figure 7. 

The characteristic absorption peak of methyl 

orange solution was found to be 420 

nm. Degradation of methyl orange was 

visualized by decrease in peak intensity 


within 10 h of incubation time. There is 

no considerable shift in peak position for 

methyl orange solution without exposure 

to Ag-NPs. Kansal et al. (2006) have reported 

that compared to other irradiation 

techniques, solar light was found to be 

faster in decolorizing methyl orange in 

the presence of metal catalyst. The adsorption 

of Ag-NPs on to the methyl orange 

solution was initially low and further 

increased with constant increase in time. 

Altogether, the photocatalytic properties 

of Ag-NPs in visible light may be well 

due to excitation of SPR, which is nothing 

but oscillation of charge density that can 

propagate at the interface between metal 

and dielectric medium (Garcia, 2012). 

Ag-NPs are potential, highly efficient and 

stable photocatalysts under ambient temperature 

with visible light illumination for 

degrading organic compounds and dyes 

(Wang et al., 2008). 

3.6. Antibacterial activities 

Highest inhibition zone (10mm) in 

Proteus mirabilis was observed in U. reticulata 

at 100 µg/ml, lowest inhibition 

zone (7 mm) was observed in U. reticulata 

25 µg/ml. U. reticulata at 100 µg/ml 

exhibits high inhibition zone of 8 mm in 

Escherichia coli and lowest inhibition 

zone (7 mm) was present in U. reticulata 

at 25 µg/ml. Ag-NPs from U. reticulata 

was compared effectively with silver nitrate 

solution and standard antibiotic 

Figure 6: (a). AFM images of synthesized silver nanoparticles using extract of U. reticulata; 

(b) corresponding 3D view. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 481




Figure 7: Photocatalytic degradation of methyl orange using silver nanoparticles synthesized 

from Ulva reticulata. 

Figure 8: Antibacterial 

activity of silver 

nanoparticles 

synthesized from U. 

reticulata against 

human pathogens. 

Con, Control; A, 100µg/ml; B, 50µg/ml; 

C, 25 µg/ml; S, Ampicillin; (1) Staphylococcus 

aureus; (2) Pseudomonas aeruginosa; 

(3) Escherichia coli; (4) Proteus mirabilis, 

and (5) Proteus vulgaris. 

streptomycin, Ag-NPs exhibited more 

activity than silver nitrate solution. Maximum 

inhibitory activity was observed in 

Ag-NPs from U. reticulata, when compared 

to control. Raimondi et al. (2005) 

and Morones et al. (2005) corroborated 

that the bactericidal effect of silver nanoparticles 

is size dependent, the antimicrobial 

efficacy of the nanoparticle depend 

on the shapes of the nanoparticles also, 

this can be confirmed by studying the inhibition 

of bacterial growth by differentially 

shaped nanoparticles. 

4. Conclusion 

In this present investigation, the 

environmental friendly synthesis of Ag- 

NPs using fresh extract of the green seaweed 

U. reticulata is described. Despite 

numerous studies conducted over the last 

decade, there are still considerable gaps in 

our knowledge about the biotechnological 

potential of green-synthesized nanoparticles. 

Furthermore, the precise basis of 

their antibiotic activity has yet to be defined. 

In addition, improvements in the 

way that green-synthesized nanoparticles 

are incorporated into medical devices 

could increase their efficacy and diminish 

any side effects; but, further research is 

required to perfect this technology. The 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 482



morphology of silver nanoparticles was 

characterized by SEM and AM. The nanoparticles 

were found to be active in degrading 

methyl orange solution with visible 

light illumination. The antimicrobial 

activity of synthesized Ag-NPs is promising. 

In a nutshell, synthesis and characterization 

of Ag-NPs with regard to novel 

morphology are of great interest in the 

fabrication of antibacterial materials. 

Acknowledgments 

The authors wish to thank Professor 

and Head, Department of Botany, Annamalai 

University. We also thank Dr. S. 

Barathan, Professor and Head, Department 

of Physics. Authors cordially thank 

Dr. B.Shanthi and Dr. G. Sivakumar, 

CISL Lab, Department of Physics, Annamalai 

University, for their help in 

providing access to SEM and AFM and 

for his suggestions while performing the 

research work. 

References 

Ahmad A, Mukherjee P, Senapati S, 

Mandal D, Khan MI, Kumar R, 

Sastry M. (2003). Extracellular biosynthesis 

of silver nanoparticles using 

the fungus Fusarium oxysporium. 

Colloids Surf B Interface. 28, 

313–318. 

Al-Warthan A, Kholoud MM, El-Nour 

A, Eftaiha A, Ammar RAA. 

(2010). Synthesis and applications 

of silver nanoparticles. Arabian 

Journal of Chemistry. 3, 135–140. 

Bansal V, Bharde A, Ramanathan R, 

Bhargava SK. (2012). Inorganic 

materials using ‘unusual’ microorganisms. 

Advances in Colloid and 

Interface Science. 179–182 (1), 

150–168. 

Bauer AW, Kirby WMM, Sherris JC 

and Turck M. (1966). Antibiotic 

susceptibility testing by a standardized 

single disk method. Journal of 

Clinical Pathology. 45(4), 493-496. 


Begum, N.A., Mondal, S., Basu,S., Laskar, 

R.A. and Mandal, D. (2009). 

Biogenic synthesis of Au and Ag 

nanoparticles using aqueous solutions 

of Black Tea leaf extracts. 

Colloids Surf B Biointerfaces. 71, 

113–118. 

Binupriya AR, Sathishkumar M, Vijayaraghavan 

K, Yun SI. (2010). 

Bioreduction of trivalent aurum to 

nano-crystalline gold particles by 

active and inactive cells and cellfree 

extract of Aspergillus oryzae 

var. viridis. Journal of Hazardous 

Materials. 177, 539-545. 

Chandran SP, Chaudhary M, Rasricha 

R, Ahmad M, and Sastry. (2006). 

Synthesis of gold nanotriangales 

and silver nanoparticles using Aloevera 

plant extract. Biotechnology 

Progress. 22, 577. 

Elechiguerra JL, Burt JL, Morones JR, 

Bragado AC, Gao X, Lara HH, 

Yocaman M. (2005). Interaction of 

silver nanoparticles with HIV- 1. 

Journal of Nanobiotechnology. 3, 6. 

Garcia M.A. (2012). Surface plasmons in 

metallic nanoparticles: fundamentals 

and applications. Journal of 

Physics D: Applied Physics. 44(28), 

389501. 

Geethalakshmi R, Sarada DVL. (2010). 

Synthesis of plant-mediated silver 

nanoparticles using Trianthema 

decandra extract and evaluation of 

their antimicrobial activities. International 

Journal of Engineering 

Science and Technology. 2(5), 970- 

975. 

Gilaki, M. (2010). Biosynthesis of silver 

nanoparticles using plant extracts. 

Journal of Biological Sciences. 

10(5), 465-467. 

Henglein A. (1993). Physicochemical 

properties of small metal particles in 

solution: ‘‘microelectrode’’ reactions, 

chemisorptions, composite 

metal particles, and the atom-tometal 

transition. The Journal of 

Physical Chemistry. 97, 5457–5471. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 483




Jahn, W. (1999). Chemical aspects of the 

use of gold clusters in structural biology. 

Journal of Structural 

Biology. 127, 106-112. 

Jegadeeswaran P, Rajeshwari Shivaraj, 

Venckatesha R. (2012). Green synthesis 

of silver nanoparticles from 

extract of Padina tetrastromatica 

leaf. Digest Journal of Nanomaterials 

and Biostructures. 7(3), 991 – 

998. 

Jha B, Reddy CR, Thakur MC, Rao 

MU. (2009). Dordrecht, Netherlands: 

Springer; Seaweeds of India: 

The diversity and distribution of 

seaweeds of Gujarat coast; 89. 

Kannan RRR, Stirk WA, Van Staden 

J. (2013). Synthesis of silver nanoparticles 

using the seaweed Codium 

capitatum P.C. Silva (Chlorophyceae). 

South African Journal of 

Botany. 86, 1–4. 

Kansal SK, Singh M, Sudo D. (2006). 

Studies on TiO 2 /ZnO photocatalysed 

degradation of lignin. Journal 

of Hazardous Materials. 153, 412- 

417. 

Kattumuri V, Katti K, Bhaskaran S, 

Boote EJ, Casteel SW and Fent 

GM. (2007). Gum Arabic as a phytochemical 

construct for the stabilization 

of gold nanoparticles. In Vivo 

Pharmacokinetics and X- 

RayContrast-Imaging Studies. 3(2), 

333-341. 

Kumar P, Senthamilselvi S, Lakshmipraba 

A, Premkumar K, Muthukumaran 

R, Visvanathan P, 

Ganeshkumar RS, Govindaraju 

M. (2012a). Efficacy of biosynthesized 

silver nanoparticles using Acanthophora 

spicifera to encumber 

biofilm formation. Digital Journal 

of Nanomaterial Bioscience. 7, 511– 

522. 

Kumar P, Senthamilselvi S, Lakshmiprabha 

A, Premkumar K, 

Ganeshkumar RS, Govindaraju 

M. (2012b). Synthesis of silver nanoparticles 

from Sargassum tenerrimum 

and screening phytochemicals 

for its anti-bacterial activity. 

Nano-Biomedical Engineering. 4(4), 

12-16. 

Lengke MF, Fleet ME, Southam G. 

(2007). Biosynthesis of silver nanoparticles 

by filamentous cynaobacteria 

from a silver (I) nitrate complex. 

Langmuir. 23, 2694-2699. 

Mohanpuria P, Rana NK, Yadav SK. 

(2008). Biosynthesis of nanoparticles: 

technological concepts and future 

applications. Journal of Nanoparticle 

Research. 10, 507–517. 

Mondal S, Roy N, Laskar RA, Sk I, 

Basu S, Mandal D. (2011). Biogenic 

synthesis of Ag, Au and bimetallic 

Au/Ag alloy nanoparticles using 

aqueous extract of mahogany 

(Swietenia mahogani JACQ.) 

leaves. Colloids interfaces B. Biointerfaces. 

82, 497-504. 

Morones JR, Elechiguerra JL, 

Camacho A and Ramirez JT. 

(2005). The bactericidal effect of 

silver nanoparticles. Nanotechnology. 

16, 2346-2353. 

Murphy C.J. (2008). Sustainability as a 

design criterion in nanoparticle synthesis 

and applications. Journal of 

Materials Chemistry. 18, 2173– 

2176. 

Naiwa H.S. (2000). Ed., Hand Book of 

Nanostructural Materials and Nanotechnology 

Academic Press New 

York. 1-5. 

Parashar, U.K., Saxenaa, P.S. and Srivastava, 

A. (2009). Bioinspired 

synthesis of silver nanoparticles. 

Digest Journal of Nanomaterial and 

Biostructure, 4, 159-166. 

Raimondi F, Scherer G.G, Kotz R and 

Wokaun A. (2005). Nanoparticles 

in energy technology: examples 

from electrochemistry and catalysis. 

Angewandte Chemie International 

Edition in English. 44, 2190-2209. 

Ramanathan R, O’Mullane AP, Parikh 

RY, Smooker PM, Bhargava SK, 

Bansal V. (2011). Bacterial kinetics-controlled 

shape-directed biosynthesis 

of silver nanoplates using 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 484



Morganella psychrotolerans. Langmuir. 

27(2), 714–719. 

Rashed MN, El-Amin AA. (2007). Photocatalytic 

degradation of methyl 

orange in aqueous TiO 2 under different 

solar irradiation sources. International 

Journal of Physical Sciences. 

2, 73-81. 

Saraniya Devi J, Valentin Bhimba B, 

Magesh peter. (2013). Production 

of biogenic Silver nanoparticles using 

Sargassum longifolium and its 

application. Indian Journal of Geomarine 

Science. 42(1), 125-130. 

Shankar SS, Ahmad A, Sastry M. 

(2003). Geranium leaf assisted biosynthesis 

of silver nanoparticles. Biotechnological 

Progress. 19, 1627– 

1631. 

Shankar SS, Rai A, Ankamwar B, 

Singh A, Ahmad A, Sastry M. 

(2004). Biological synthesis of triangular 

gold nanoprisms. Nature 

materials. 3(7), 482-488. 

Shanmugam N, Rajkamal P, Cholan 

S, Kannadasan N, Sathishkumar 

K, Viruthagiri G, Sundaramanickam 

A. (2014). Biosynthesis of 

silver nanoparticles from the marine 

seaweed Sargassum wightii and 

their antibacterial activity against 

some human pathogens. Applied 

Nanoscience. 4(7), 881–888. 


Song JY, Kim BS. (2008). Biological 

synthesis of bimetallic Au/Ag nanoparticles 

using Persimmon (Diopyros 

kaki) leaf extract. Korean Journal 

of Chemical Engineering. 25(4), 

808-811. 

Wang G, Liao C, Wu F. (2000). Photodegradation 

of humic acids in the 

presence of hydrogen peroxide. 

Chemosphere. 42, 379-387. 

Wang P, Huang B, Qin X, Zhang X, 

Dai Y, Wei J, Whangbo MH. 

(2008). Efficient and stable photocatalyst 

under visible light. Chemical 

Engineering Journal. 14, 10543- 

10546. 

Wiley BJ, Im SH, McLellan J, Siekkinen 

A. Xia Y. (2006). Maneuvering 

the surface plasmon resonance 

of silver nanostructures through 

shape-controlled synthesis. The 

Journal of Physical Chemistry. 

110(32), 15666- 15675. 

Williams D. (2008). The relationship between 

biomaterials and nanotechnology. 

Biomaterials. 29(12), 1737- 

1738. 

Xu H, and Kall M. (2002). Morphology 

effects on the optical properties of 

nanoparticles. Journal of Nanoscience 

and Nanotechnology. 4, 254- 

259. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 485



Diversity and Ethno-Botanical Potential of Tree Plants of 

Katarniaghat Wildlife Sanctuary, Bahraich (UP) India: 

An Overview 

Tej Pratap Mall* 

Postgraduate Department of Botany, Kisan PG College, Bahraich-271 801, Uttar Pradesh, 

India; *Correspondence: drtejpratapmall@gmail.com; Te: +91 945 042 5622 

Abstract: Plants are of variable purpose having efficiency as ethno-botanical, ethnomedicinal, 

ethno-veterinary and even in agro-forestry wherein they act as shelter, source of 

habitat for several organisms, etc. They even have a role in improving the soil conditions. 

Many useful products such as fruits, timber, fire wood and variety of metabolic chemicals 

are also obtained from plants. In Katarniaghat Wildlife Sanctuary (KWS), there are fifty 

five tree plant species representing forty five genera belonging to thirty one families. Moraceae 

was found to be the largest family with seven plant species; whereas Euphorbiaceae 

and Mimosaceae with five; Anacardiaceae, Myrtacea and Rubiaceae with three; Caesalpiniaceae, 

Ehretiaceae, Papilionaceae and Louraceae with two and rest twenty one families, 

viz., Rutaceae, Apocyanaceae, Baringtoniaceae, Bombocaceae, Dilleniaceae, Ebenaceae, 

Tiliaceae, Ulmaceae, Malvaceae, Lythraceae, Sapotaceae, Annonaceae, Rutaceae, 

Sapindaceae, Dipterocarpaceae, Sterculeaceae, Bignoniaceae, Verbinaceae, Combretaceae, 

Meliaceae and Rhamnaceae with single plant species only. This chapter is an attempt 

to summarise the information available on plant species found in KWS which are yet not 

popular due to limited research. 

Keywords: Ethnobotanical; ethnomedicinal; ethnoveterinary; Katarniaghat wildlife sanctuary; 

nutrimental tree 


We cannot survive without plants. 

We depend on plants for food: directly in 

the form of grains, roots and tubers, fruits, 

vegetables, spices, oil and beverages. 

Much of our food also comes indirectly 

form plants. We get our meat and milk 

from animals that are dependent on plants 

for food. Plants provide fuel, either as 

firewood or in the form of fossil fuel, to 

cook our food, keep us warm, run our 

machinery and light up our homes and 

cities. We also depend on trees for construction 

materials to build our houses 

and to craft our furniture. From cotton 

and flax we get fibres for our clothes. 

Plant dyes colour our clothes, at least before 

synthetic dyes were developed. In 

cities and towns, trees provide shade and 

shelter, and their flowers brighten the 

surroundings, Plants in parks and gardens 

contribute to the serene and 

peaceful environment, making such places 

favourite retreats (Chin, 2005). 

The knowledge of utilizing wild 

plants was painstakingly passed on 

from generation to generation database 

of valuable information of the plants 

around him. It is natural to assume that 

certain members of the tribe were gradually 

entrusted with such knowledge. These 

individuals were known as shamans, 

bomohs, healers or witchdoctors. As 

communications between settlements was 

then poor, it is likely that such knowledge 

was developed independently in different 

locations (Chin, 2005). The primitive 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 486


Diversity and Ethno-Botanical Potential of Tree Plants… 

man, through his trial and error, has 

selected many wild fruits which are 

edible and subsequently domesticated 

them which played a very vital part 

in supplementary diet knowingly or 

unknowingly. Although due to the ignorance 

of modern generation the importance 

of wild plants were recently 

have been decreasing yet many people 

especially in rural areas still use them 

extensively as a supplementary to their 

basic food requirement. A scientific study 

of wild fruits is important for the potential 

sources which are protective 

foods. The nutrients/pigments present 

in the fruits prevent different degradative/ageing 

process in our body and thus 

via restoring health offer longevity 

(Singh, 2011). These wild fruits would be 

utilized at the time of scarcity or cultivated 

as a source of food material for 

ever increasing population (Rashid et 

al., 2008). 

India has been considered as one of 

the 17 mega-diversity centers of the word 

with a wide range of phyto-geographical variations. 

It consists of about 64 million hectares 

forest covers out of which 86% is tropical 

forest comprising 54% dry deciduous, 

37% moist deciduous and 9% wet evergreen 

& semi-evergreen (Kaul and Sharma, 1971). 

As a characteristic feature, the tropical forest 

shows a huge variation in tree species diversity 

place to place (Pitman et al., 2002). 

Among the different phyto diverse regions 

found in the country, the Terai region is one 

of them existing from Uttarakhand to West 

Bengal. It is the transition zone between two 

eco-climatic zones, the Gangetic plain towards 

south and Bhabhar towards north, 

along with the sub- Himalayan tracts (Tripathi 

and Singh, 2009). The region has lost majority 

of its natural forest due to deforestation 

chiefly for agriculture and lack of sustainable 

forest management in last many centuries 

(Bajpai et al., 2012a, b). Now the natural forests 

of the region have been restricted to the 

wildlife protected areas only. Katerniaghat 

Wildlife Sanctuary (KWS) is also one of 

them. 

Traditional medicines are used by 

about 60 percent of the world’s population. 

These are not only used for primary health 

care just in rural areas, in developing coun- 

Mall 

tries, but also in developed countries, where 

modern medicines are predominantly used. 

While the traditional medicines are derived 

from medicinal plants, minerals, and organic 

matter, the herbal drugs are prepared from 

medicinal plants only. Use of plants as a 

source of medicines has been inherited and is 

an important component of the health care 

system in India. There are about 45,000 plant 

species in India, with high concentration in 

the region of Eastern Himalayas, Western 

Ghats and Andman and Nicobar Island. The 

officially documented plants with medicinal 

potential are 3,000 but traditional practioners 

use more than 6,000. India is the largest producer 

of medicinal herbs and is appropriately 

called the botanical garden of the world. In 

rural India, 70 percent of the population is 

dependent on the traditional system of medicine, 

the Ayurveda, which is the ancient Indian 

therapeutic measure renowned as one of 

the major systems of the alternative and complementary 

medicine (Bhatia, et al., 2013). 

In Katarniaghat Wildlife Sanctuary 

there are fifty five tree plant species representing 

forty five genera belonging to thirty 

one families. Moraceae was found to be the 

largest family with seven plant species 

whereas Euphorbiaceae and Mimosaceae 

with five; Anacardiaceae, Myrtacea and Rubiaceae 

with three; Caesalpiniaceae, Ehretiaceae, 

Papilionaceae and Louraceae with 

two and rest twenty one families, viz., Rutaceae, 

Apocyanaceae, Baringtoniaceae, 

Bombocaceae, Dilleniaceae, Ebenaceae, Tiliaceae, 

Ulmaceae, Malvaceae, Lythraceae, 

Sapotaceae, Annonaceae, Rutaceae, Sapindaceae, 

Dipterocarpaceae, Sterculeaceae, 

Bignoniaceae, Verbinaceae, Combretaceae, 

Meliaceae and Rhamnaceae with single plant 

species only. The available literature reveals 

that most of the tree plants found in Katarniaghat 

Wildlife Sanctuary (KWS) are multipurpose, 

ethno-botanical, nutrimental, ethnomedicinal, 

ethno-veterinary and of environmental 

use in agro-forestry which provide 

shade, habitat for organisms, soil improvement, 

etc., many useful products are also obtained 

such as fruits, timber, fire wood and 

variety of metabolic chemicals which may be 

used in the form of home remedies and for 

traditional medicine. Considering the multipurpose 

importance of these trees of KWS, 

the present overview is an attempt to summarize 

the information’s available on these 

plants which are yet not popular due to one 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 487



Mall 

reason or the other despite providing an array 

of benefits. 

2. Study area 

The study area Katerniaghat Wildlife 

Sanctuary (KWS) is situated in Bahraich 

district of Uttar Pradesh in India. It 

lies along Indo-Nepal international boarder 

and is situated between 27° 41’ – 27° 

56’ N and 81° 48’ – 81° 56’ E covering 

an area of 440 km 2 with 116 to 165 m elevation. 

The sanctuary comes under the 

tropical moist deciduous forest of the 

Himalayan Terai-Bhabar region (Champion 

and Seth, 1968; Rodgers and 

Panwar, 1988). The forest of the sanctuary 

area has been classified into two major 

forest types (i) The Sal forest and (ii) 

The miscellaneous forest (Champion and 

Seth, 1968). Pedagogically the study area 

is made up of the alluvial soil of the 

Kaudiyala and Saryu rivers and its tributaries 

flowing adjoining to it. Geologically 

the sanctuary area has been divided 

into high and low land areas. 

3. Climate 

A typical tropical monsoonal climate 

with three distinct seasons, i.e., 

summer (April to June), winter (November 

to February) and warm-rainy (July to 

September) prevails in the study area. 

March and October are considered as 

transition months between the seasons. 

The mean maximum temperature ranges 

from 22 °C in January to 40 °C in May and 

the mean minimum temperature ranges 

from 8 °C in January to 27 °C in June. The 

annual rainfall ranges from 36 to 142 cm 

in winter, 34 to 662 cm in summer and 

1294 to 1689 cm in warm-rainy seasons 

(Bajpai et al., 2012). 

4. Observations 

At present the KWS has been divided 

in to three types of forests- miscellaneous 

forest, sal forest and teak plantation 

forest. The IVI of the plants in all the 

three forests are presented respectively 

while description of the plant. Since the 

list of the trees is big, the description of 

all is beyond the scope of this manuscript 

so we have taken thirteen plants, viz., 

Acacia catechu, Acacia concinna, Aegle 

marmelos, Albizia lebbeck, Albizia 

procera, Alstonia scholaris, Bombax ceiba, 

Diospyros cordifolia, Ficus racemosa, 

Madhuca latifolia, Shleichera oleosa, 

Syzygium cumuni and Ziziphus mauritiana 

for detail description. 

4.1. Acacia catechu Willd.Khair, Catechu 

(Mimosaceae) 

It is a moderate size tree. Leaves 

are pinnate. Flowers yellow in globose, 

peduncled axillary heads. Pods strap 

shaped, dark brown. Phenology - August 

to February. In Katarniaghat Wildlife 

Sanctuary it is found only in miscellaneous 

forest with IVI value 18.3. 

Ethnobotanical potential 

Catechu, a multipurpose tree species 

is widely used by the inhabitants for 

fodder, fuel, building material and in 

health care. 

The heartwood of the tree is mainly 

used for extracting Katha and Cutch 

(decoction obtained after filtration) 

which are sold in the market. 

Katha is commonly used in ayurvedic 

preparations. 

Katha serves as one of the major 

components in masticatory, i.e., 

chewing of betel leaf (pan) in India. 

catechu is a valuable bio-resources 

and has been exploited commercially 

in tannin and Katha industry for decades. 

Besides its commercial importance, 

it is equally significant for the people 

particularly rural communities living 

in the vicinity of catechu forests as it 

is a subsidiary source of income to 

them. To a certain extent, these people 

are dependent on this plant to fulfill 

their day to day need of fuel, fodder, 

building material, etc. 

Ethno-medicinal potential 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 488



 

 

 

 

 

 

 

Fuel 

 

The decoction of bark mixed with 

milk is taken to cure cold and cough. 

The bark decoction is either alone or 

used in combination with opium to 

cure severe diarrhea. 

Katha after drying is applied on lemon 

slice and taken regularly with 

empty stomach to cure piles. 

Heartwood of khair is boiled with 

other ingredients to prepare the decoction. 

It is taken as tea by the 

pregnant ladies to keep warm their 

body. It is also given to cure fever 

due to cold during the pregnancy. 

A decoction is served to women after 

2-3 days of child delivery, prepared 

by boiling Katha along with cardamom. 

It is believed that it provides 

strength to the body and also helps in 

secretion of milk. 

The water boiled with the heartwood 

chips of Khair, is used to take bath 

by women after delivery. It is considered 

beneficial to cure the body 

pains. 

Katha or decoction of heartwood is 

applied in mouth and on tongue to 

cure mouth ulcer. It is also applied 

externally on ulcers, boils, skin eruptions 

and on gums as disinfectant. 

The dried logs, twigs and branches 

are largely used as fuel. 

Fodder 

The trees are lopped heavily for 

their leaves used as fodder particularly 

for sheep and goats. 

Building and furniture material 

The wood is considered durable and 

widely used by the inhabitants for 

house building material as pole and 

to prepare furniture like bed poles, 

tables etc. 

House hold articles 

Wood of khair is preferred to prepare 

various parts of local plough, 

Mall 

handles of axe, saw, sickle, hammer, 

spade and combs. 

Socio-religious beliefs 

Khair is considered one of the sacred 

trees by the natives and wood 

is used in the religious ceremonies 

at the time of havans (yagya). 

Wood is considered sacred and used 

as one of the religious plants along 

with bhoj patra (Betula utilis) at the 

funeral ceremony. It is believed to 

provide mukti or moksha (peace to 

the heavenly soul). 

Fencing 

Cut branches are extensively used 

for fencing purpose by the farmers 

to protect agricultural fields and local 

grasslands from domestic livestock 

and wild animals. 

Tanning 

The cutch is used locally for tanning 

leather and as dye to a great extent. 

Economic Importance 

Besides traditional utility, A. catechu 

is widely utilized commercially 

for extracting Katha from the heart 

wood which costs around US $ 4-6 

per kilogram in Indian markets. 

Cutch is used as adhesive in plywood 

industry and it is also used in 

preparing polishes and paints (Singh 

and Lal, 2006). 

From the present study, it is envisaged 

that A. catechu has a great socioeconomic 

importance as it is widely used 

for different purposes by the natives. Besides, 

traditional and commercial importance, 

it has tremendous ecological 

significance. Because of its leguminous 

nature and soil binding abilities, it is a 

suitable species for wasteland development. 

4.2. Acacia concinna (Willd.) DC 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 489



Mall 

Synonyms: Acacia sinuata (Lour.) 

Merrill, Acacia rugata Lamk., Mimosa 

sinuate Lour. 

Acacia concinna is a prickly bush or 

climbing shrub and grows in tropical forests 

of India, common in the warm plains 

leaflets membranous. The leaves are bipinnate 

and the thorns are soft and small. 

Flowers are white and pink in copiously 

panicled globose heads in the axils of 

leaf. Pods strap shaped, straight, thick, 

succulent when dry shriveled and rough 

with straight waved sutures. There are 

about 6-10 seeds in a pod. Phenology is 

January-March & November to February. 

The tree is food for the larvae of the butterfly 

Pantoporia hordonia. Alkaloids are 

found in the tree's fruit. In Katarniaghat 

Wildlife Sanctuary it is found only in 

miscellaneous forest with IVI value of 1.0 

Ethno-botanical potential 

The leaves, pods has astringent action 

and useful in treating cuts, 

wounds and oral problems. The decoction 

of pods is prepared and used 

for washing and cleaning of wound 

for quick healing. Acacia concinna 

is a good herbal remedy for hair. It 

is an advantageous herb for baldness. 

Its usage helps in preserving 

the natural oil of our hair and nurtures 

the scalp. It encourages the 

growth of hair and strengthens 

them. It is an effective hair cleanser. 

It prevents dandruff and lice. 

It is used in the manufacture of 

shampoos, soaps and hair packs. 

It is a good herbal treatment for 

black fever (Visceral Leishmaniasis) 

and fever due to Malaria. 

Acacia concinna is a herbal treatment 

for skin ailments. It is advantageous 

in curing psoriasis (genetic 

disease) and the spreadable diseases 

like eczema. It provides a relief in 

scabies, rashes, cuts, bruises and 

cures them. 

It is effectual in curing oral ailments. 

It helps in suppressing bad 

breath. It is helpful in treating 

mouth ulcers, gumboils and pain in 

the throat. 

It aids in the prevention of tooth 

degradation and formation of 

plaque. 

It is also helpful in lowering the 

chances of encountering diabetes. 

Acacia concinna is a good herbal 

treatment for lowering the body 

cholesterol. 

It is a fruitful remedy in curing digestive 

disorders and relieving constipation. 

It facilitates proper bowl 

movement and improves the flow of 

urine. 

It also possesses the attribute of being 

a contraceptive which helps in 

birth control. 

It is utilized in the preparation of 

savoury jams (chutneys). It adds to 

the flavor. 

Acacia concinna has been used traditionally 

for hair care in the Indian 

Subcontinent since ancient times. 

It is one of the Ayurvedic medicinal 

plants. 

Fruit for hair are being used as a 

traditional shampoo. In order to 

prepare it the fruit pods, leaves and 

bark of the plant are dried, ground 

into a powder then made into a 

paste. While this traditional shampoo 

does not produce the normal 

amount of lather which are found in 

sulfate-containing shampoo, it is 

considered a good cleanser. It is 

mild, having a naturally low pH, 

and doesn't strip hair of natural oils. 

Usually no conditioner is needed, 

for shikakai which also acts as a detangler. 

An infusion of the leaves has been 

used in anti-dandruff preparations. 

Since A. concinna extracts are used 

in natural shampoos or hair powders 

so the tree is now grown commercially 

in India. 

The plant parts used for the dry 

powder or the extract are the bark, 

leaves or pods. The bark contains 

high levels of saponins, which are- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 490



 

 

foaming agents found in several 

other plant species used as shampoos 

or soaps. Saponin-containing 

plants have a long history of use as 

mild cleaning agents. 

Saponins from the plant's pods have 

been traditionally used as a detergent, 

and in Bengal for poisoning 

fish; they are documented to be potent 

marine toxins. 

The leaves have an acidic taste and 

are used in chutneys. 

Chemical constituents 

In commercial extracts, when the 

plant is hydrolyzed it yields lupeol, 

spinasterol, acacic acid, lactone, and 

the natural sugars glucose, arabinose 

and rhamnose. It also contains hexacosanol, 

spinasterone, oxalic acid, 

tartaric acid, citric acid, succinic acid, 

ascorbic acid, and the alkaloid 

scalyctomine and nicotine. 

Acacia concinna is a thorny medicinal 

plant, native to south Asia, 

widely known for the organic shampoo 

derived from its fruit, shikakai. 

The pods (shikakai), have medicinal 

properties and are used for hair 

cleansing and enhancement. Shikakai 

is traditionally preferred over 

commercially available shampoo, 

across the Indian Subcontinent. 

Shikakai is also used for manufacturing 

body and facial care creams, 

across the personal hygiene industry. 

A particular extract from Acacia 

concinna leaves has shown to be effective 

in treatment of malarial fever. 

Ethno-medicinal potentialty 

Shikakai is also used in traditional 

medicine to treat jaundice, constipation 

and skin problems. 

Acacia concinna for pain 

Apply some castor oil on the affective 

area. Heat with a hot water 

bag. Now massage with powdered 

Mall 

Acacia concinna for 15 minutes. 

Wash the area with hot water and 

wipe it. 

Acacia concinna for constipation: 

Discard the Acacia concinna 

seeds after crushing the fruit. 

Soak it in 1 glass of water for 

one hour. Take the infusion. 

Acacia concinna (sikakai) for jaundice 

Take out the Acacia seeds after 

crushing the fruit. Soak it in a 

glass of water for an hour. Take 

one fourth of the glass infusion. 

Acacia concinna (shikakai) for dandruff 

Acacia concinna (shikakai) is a 

boon for getting rid of dandruff. 

Boil a handful of coarsely crushed 

Acacia concinna (Shikakai) in a 

litre of water for 10 minutes. Cool 

it and use the filtered decoction to 

wash hair. Do it daily for week 

and then twice a week or make a 

paste of powdered Acacia concinna 

(Shikakai) by adding water in 

it. Apply it over scalp and hair. 

Leave it for an hour. Wash hair 

with normal water. It also cleans 

your hair from roots to tips. 

Acacia concinna for age spots 

Make a fine paste of Acacia concinna 

fruit. Apply every day for 

15 minutes. Wash three times a 

day. 

Acacia concinna for gum diseases 

Take half table spoonAcacia concinna 

and boil it in two cups of 

water. Gargle with this lukewarm 

water three times a day. 

Acacia concinna for skin diseases 

Boil one table spoon Acacia concinna 

powder in one cup water. 

Cool it and apply on the skin. 

Acacia concinna (shikakai) pods for leprosy 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 491



 

Squeeze the juice of Acacia concinna 

(shikakai) pods. Apply it on 

effected parts twice a day. 

Acacia concinna for psoriasis: 

Take few pods of Acacia concinna. 

Boil them. Apply them over 

affected areas. 

Herbal treatment for constipation 

Make a paste of three leaf buds of 

Acacia concinna, two cloves of 

garlic and some black salt. Take it 

with cooked rice. 

Herbal treatment for fever 

Make a paste of three leaf buds of 

Acacia concinna, two cloves of 

garlic and some common salt. 

Take it with cooked rice. 

Herbal treatment for dandruff 

Grind three to four dried fruits of 

Acacia concinna (shikakai) without 

seeds. Add one to two teaspoons 

of fenugreek (methi in India) 

seeds, one teaspoon full wild 

turmeric (aamahaldi in India) root 

powder, one teaspoon of Indian 

Sarsaparilla (anantmool in India) 

root powder and one teaspoon of 

sandalwood powder. Mix well. 

Add water to form a thick paste. 

Massage the scalp with coconut 

oil, and then apply this paste. 

Leave it for half an hour. Wash off 

and shampoo. Do this once a 

week. 

Massage your scalp with lukewarm 

sesame oil. Boil four to five 

dried Acacia concinna (Shikakai 

in India) pods in two glass of water. 

Strain well. Let it cool. Use its 

water to rinse your scalp after an 

hour. It gives relief from dandruff 

and promotes hair growth. 

Acacia concinna (shikakai) and 

lemon both are beneficial herbs in 

treating dandruff. The mixture 

made up of these two, works 

wonder to make your hair free of 

Mall 

dandruff. Take 3 to 4 tablespoon 

of Acacia concinna (shikakai) 

powder. Make a paste by adding 

lemon (nimbu) juice. Apply it 

over scalp and leave it for an hour 

or two. Wash with water. Do it for 

a week every day and you may see 

half your dandruff has gone. 

Herbal treatment for head lice 

Grind ten dried fruits of Acacia 

concinna after discarding the 

seeds, half cup each of fenugreek 

seeds, wild turmeric roots, roots of 

Indian Sarsaparilla and Sandalwood 

chips. Massage the head 

with coconut oil and apply this 

powder. Rinse off after thirty 

minutes. 

Herbal treatment for jaundice 

Make a paste of one tea spoon 

tender Acacia concinna leaves, 

three pepper corns, one tea spoon 

tamarind pulp, half red chilli and 

some salt to add taste. Eat it with 

cooked rice. 

Herbal treatment for stomach ache 

Boil one cup Acacia concinna tree 

bark in one litre water and sieve it. 

Add powdered Indian Pennywort 

in it. Also add the powder of four 

black peppercorns, two cardamom, 

cinnamon, two cloves, one 

fourth nutmeg and half tablespoon 

long pepper. Mix it and leave it 

for a month before it is taken for 

medicinal use. Take one tablespoon 

twice a day. 

Herbal treatment for grey hair 

Soak Indian gooseberry, soap nut 

seeds (Ritha) and pots of Acacia 

concinna (Shikakai) in three cups 

of water for a night. Grind it. Use 

it as shampoo. 

Herbal treatment for frizzy hair 

Take following herbs in mentioned 

quantity, two hundred g In- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 492



Mall 

dian gooseberry (Amia), two hundred 

g Acacia concinna (Shikakai), 

twenty g Bacopa monnieri 

(Bhrahmi), one g Asphaltum 

(Shilajit), forty g dried pomegranate 

peel (Anar ka chilka), twenty 

g Eclipta alba (Bhringraaj), Soak 

them overnight in any iron bowl. 

Grind to make paste. Apply it on 

your hair and Scalp. Wash after 3 

hours with lukewarm water. Do 

not use shampoo. Shampoo may 

be used on next day. 

4.3. Aegle marmelos (L.) Corr 

It is a medium-sized, deciduous, 

armed tree. Leaves are trifoliolate. 

Flowers are yellowish. 

Fruits are large, globose. Phenology 

is April-May & March-July. In 

Katerniaghat Wildlife Sanctuary 

Aeglemarmelos is found only in 

teak plantation forest. 

Ethnobotanical potentiality 

Bael is one of the most important 

tree species used in various indigenous 

system of medicine in India, 

China, Burma, and Sri Lanka. Bael is 

used in all tridosa- vista (air), Pitta 

(phlegm) and kapha (cough). Out of 

more than 66 ethno-botanical uses of 

bael, 48 are exclusively for medicinal 

purposes. Almost all parts of bael are 

used in preparing medicine (Kala, 

2006). 

Leaf: Abscess, backache, eye complaints, 

abdominal disorders, vomiting, 

cut & wounds, ulcer, destroy, beriberi, 

weakness of heart, cholera, diarrhoea, 

cardio tonic, blood sugar, injuries 

caused by animals, nervous disorders, 

hair tonic, acute bronchitis, child 

birth, veterinary medicine for wounds, 

killing worms, fodder for sheep, goat 

and cattle, stimulation of respiration 

and contraction of de-nerved nictitating 

membrane in anaesthetized cats. 

Fruit: Astringent, diarrhoea, gastric 

troubles, constipation, laxative, tonic, 

digestive, stomachic, dysentery, brain 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 493 

 

 

 

 

 

 

 

 

 

 

 

 

& heart tonic, ulcer, antiviral, intestinal 

parasites, gonorrhoea, epilepsy. 

Root: Dog bite, gastric troubles, heart 

disorders, intermittent fevers, antamoebic, 

hypoglycemic, rheumatism. 

Bark: Stomach disorders, intermittent 

fevers, heart disorders. 

Seed: Febrifuge. 

Flower: Expectorant, epilepsy. 

Whole Plant: Abdominal pain, abscess, 

astringent back ache, dog bite, 

breast pain, cholera constipation, convulsions, 

cramp, diabetes, diarrhoea, 

dysentery, fevers, eye complaints, 

gastric trouble, abdominal disorders, 

jaundice, laxative, nausea night fever, 

heart disorders, snakebite, stomach 

disorder, vomiting tonic, cut & 

wounds. 

Root bark: Fish poison. 

Seed mucilage: Plaster for walls. 

Seed oil: Laxative. 

Wood: Beads worn by low caste, special 

couches for rheumatic patients. 

Gum around seed: To improves adhesive 

strength of water paints. 

Unripe fruit rind, Bark: Yellow dye. 

Stem: Pestles of oil and sugar mills. 

The medicine is prepared in the form 

of pills, powder and paste. Ayurvedic 

practitioners commonly use the roots 

of bael as an ingredient of dasmula 

(ten roots), which is useful in recovering 

the loss of appetite and use fruits 

in the preparation of chawanprash. 

Bael fruits regarded as an astringent 

are frequently used by various ethnic 

communties for the treatment of diarrhoea, 

dysentery, constipation, stomach 

ache, intestinal ulcer, diabetes, 

dyspepsia, heart diseases and cholera 

due to its digestive and carminative 

properties. 

Bael is highly valued in Ayurvedic 

medicine for the treatment of chronic 

diarrhoea and dysentery and as brain 

tonic. Bael possesses antiviral, antihelminthic 

anti-inflammatory, antibilious, 

anti-parasitical, anti-pyretic, 

anti-scorbutic, aromatic, astringent, digestive, 

febrifuge, haemostatic, anti-



diarrheal, laxative and nutritive properties. 

Ripe bael fruit is sweat, aromatic 

and nutritive, whereas fresh fruit is 

stringent and has laxative properties. 

Bael fruit powder exhibits anticancerous 

and anti-proliferative activities. 

The combinations of five parts of 

bael, such as, fruit, leaf, bark, root and 

flower is assumed to be effective for 

certain mental disorders. 

Unripe fruits pulp mixed with boiled 

rice water is taken twice a day to cure 

vomiting in pregnancy. Unripe fruits 

pulp mixed with sugar is taken with 

milk twice daily for curing urinogenital 

disorders. 

Half roasted unripe fruit pulp mixed 

with equal quantity of sugar is taken 

twice a day to cure dysentery. Unripe 

fruit pulp powder is taken twice daily 

to cure abscess. 

Bael leaf extract is taken twice a day to 

remove the intestinal worms. Leaf 

poultice is used as remedy in ophthalmic 

problems and ulcer. 

Leaf juice is reported to have multiple 

medicinal uses, including controls of 

diabetes. Cooling delicious drink prepared 

from fruit pulp along with sugar 

and tamarind diluted with water is useful 

for health. 

Baelroot decoction is given twice daily 

to cure fever and cold. Extract of bael 

root, pyaz (Allium cepa Linn.), and 

haldi (Curcuma domestica Valeton) 

mixed in equal proportion is put in the 

ears to relive earache and secretion 

from ears. Root decoction is used in 

the treatment of intermittent fevers and 

heart palpitation. 

Root and stem bark decoction is used 

in the treatment for fever and various 

types of heart disorders. Bael root is 

used in the treatment of abdominal 

pain, heart palpitation and urinary 

troubles. 

Bael tea is good for health and is used 

for flatulence, gastrointestinal problems, 

cough and chronic intestinal diseases 

in children. 

 

Mall 

For Hindus, the Bael is sacred tree, 

which they dedicate to the lord Shiva 

by offering of Bael leaves. Its three 

leaflets are assumed by the symbols of 

three gunas or attributes (e.g., satva, 

rajas and tamas, literally meaning morality, 

superiority and immorality, respectively); 

three Gods (Brahma, 

Vishnu and Mahesh); and three lives 

(past, present and future). Bael is considered 

to be extremely auspicious and 

cultivated around most of the Hindu 

temples. 

Phytochemicals of Aegle marmelos 

A. marmelos has been reported to contain 

several phyto-constituents mainly 

marmenol, marmin, marmelosin, marmelide, 

psoralen, alloimperatorin, rutaretin, 

scopoletin, aegelin, marmelin, fagarine, 

anhydromarmelin, limonene, a- 

phellandrene, betulinic acid, marmesin, 

impertorin, marmelosin, luvangentin and 

auropetene Rahman and Parvin . 

Due to the presence of various 

phyto-constituents the plant has anti-diarrhoeal, 

anti-microbial, anticancerous, 

anti-pyretic, antigenotoxic, 

anti-fertility, antiinflammatory 

anti-diabetic and diuretic 

activities. 

The essential oil isolated from the 

leaves of A. marmelos tree has proved 

to have antifungal activity against animal 

and human fungi like Trichophyton 

mentagrophytes, Trichophyton 

rubrum, Microsporum 

gypseum, Microsporum audounii, 

Microsporumcookie, Pidermophyton 

floccosum, Aspergillus niger, Aspergillus 

flavus and Histoplasma capsulatum. 

The leaf extracts and fractions have 

fungicidal activity against various 

clinical isolates of dermatophytic fungi. 

Various extracts of A. marmelos 

leaves, roots ad fruits have been reported 

to be active against many bacterial 

strains. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 494




Bael fruits are edible, contain high 

protein and are used in making tasty 

aromatic cold drinks and jam. Its fresh 

juice is better and pungent. In Myanmar, 

Bael fruits are used in making 

paints. Fruits are also used as a substitute 

for soap, as source of essential 

oils and perfumes. The mucilage of 

bael seed is good cementing material. 

Bael wood is used in building houses, 

making carts, agricultural implements, 

pestles, handles of tools and combs. A 

yellow dye is obtained from the rind 

of unripe fruits and is used in calico 

printing. An essential oil is also distilled 

from the rind. Dried fruit after 

removing the pulp are used as pill 

boxes for keeping valuable medicines 

and sacred ashes. Bael stem yields 

gum, which is used for improving the 

adhesive potency of water paints. Its 

wood is suitable for making charcoal. 

4.4. Albizzia lebbeck (Linn.) Benth. 

Synonym: Mimosa lebbeck Linn. 

It is a tall, unarmed, and deciduous 

tree distributed throughout India from 

the plains up to 900m in the Himalayas. 

It is tree growing to height of 18- 

30 m tall with a trunk 50 cm to 1 m in 

diameter. The leaves are bipinnate, 

7.5-15 cm long, with one to four pairs 

of pinnae, each pinna with 6-18 leaflets. 

The flowers are white, with numerous 

2.5-3.8 cm long stamens, and 

very fragrant. The fruit is a pod 15-30 

cm long and 2.5-5.0 cm broad, containing 

six to twelve seeds. In Katarniaghat 

Wildlife Sanctuary it is found 

only in miscellaneous forest with IVI 

0.9. 

 

Mall 

Ethnobotanical potential 

It uses included in environmental 

management, forage, medicine and 

wood. It is cultivated as a shade tree 

in North and South America. In India 

and Pakistan, the tree is used to produce 

timber. Wood from Albizia lebback 

has a density of 0.55-0.66g/cm 3 

or higher (Babu et al., 2009). 

Even a where it is not native, some 

indigenous herbivores are liable to 

utilize lebbeck as food resource. For 

example, the greater rhea (Rhea 

Americana) has been observed feeding 

on it in the cerrado of Brazil. 

Ethno-madicinal potential 

Lebbeck is an astringent, also used 

by some cultures to treat boils, 

cough, to treat the eue, flu, gingivitis, 

lung problems, pectoral problems, is 

used as a tonic, and is used to treat 

abdominal tumors. 

The bark is used medicinally to treat 

inflammation. 

In Sidha system of medicine the bark 

and flowers of this plant are used to 

treat arthritis (Mudaliar, 1936). 

The tribal people in Himachal Pradesh 

and Kashmir use this plant to 

treat inflammation (Srivastava et al., 

1986; Jain, 1991; Kapur, 1993). 

Balasubramaniam (1992) reported 

that the tribals point Calimere Wildlife 

Sanctuary, Tamilnadu use this 

plant to treat fractures. 

In Ayurvedic system of medicine, the 

stem bark of this plant is used to treat 

diarrhoea (Nadkarni, 1954), edema, 

poisoning, asthma and bronchitis 

(Gupta, 2004). 

Inflammation is complex pathophysiological 

process medicated by a 

variety of signalling molecules produced 

by leucocytes, macrophages 

and mast cells as well as by the activation 

of complement factors that 

bring about edema formation as a result 

of extravasation of fluid and proteins 

and accumulation of leucocytes 

at the inflammatory site (White, 

1999). All the steroidal and nonsteroidal 

anti-inflammatory drugs 

(NSAID’s), despite their great number, 

cause undesired and serious side 

effects. Therefore, development of 

new and more powerful drugs is still 

needed. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 495



 

 

 

 

Research in plants with medicinal 

properties and identification of the 

chemical components responsible for 

their activities have corroborated the 

traditional uses of ancient healing 

wisdom and lore and have proven the 

enduring healing potential of many 

plant medicines even in today’s hitech 

community. 

It is previously reported that the alcoholic 

extract of Albizia lebback 

protects the guinea pig against the antigen 

induced challenge (Tripathi et 

al., 1977; Barua et al., 1997). 

Further there it also reduced the level 

of histamine and raised the plasma 

cortisol in antigen challenged guinea 

pigs (Tripahti and Shukla, 1979) as 

well as in bronchial asthma patients 

(Tripathi et al., 1978). Das et al., 

(2003) and Pramanik et al., (2005) 

previously reported the antiinflammatory 

activity of the methanol 

extract of Albizia Lebback bark. 

Many saponins, such as lebbekanin 

A-H (Varshney and Khan, 1961; 

Varshney and Sharma, 1969; Varshney 

et al., 1973, 1976) and Albizziasaponin 

A-C (Pal et al., 1995), 

which contain oleanolic acid, echinocystic 

acid or acacic acid as sapogenins 

were reported from various parts 

of this plant. Further, melanoxetin 

okenin-3-one, (+) pinitol, (-) leucopelargonidin 

(Gupta et al., 1966). 

Alternative medicine for the treatment 

of various diseases is getting 

more popular. Many medicinal plants 

provide relief of symptoms comparable 

to that of conventional medicinal 

agents (Verpoorte, 1999). 

4.5. Albizia procera (Roxb.) Benth. 

Synonym: Mimosa procera Benth 

The habitat ranges from monsoon 

forest, mixed deciduous forest, savannah 

woodlands, pyrogenic grasslands, roadsides 

and dry gullies, to stunted, seasonal 

swamp forest. It is commonly found in 

open secondary forest. It is a large deciduous 

tree. Leaves bi-pinnate with a large 

Mall 

gland near the base of the petiole, leaflets 

rigidly sub coriaceous, grey beneth, glabrous 

obliquely truncate at the base. 

Flowers whitish in copiously panicled 

heads. Pods thin, brown, glabrous. White 

siris is a large, fast growing tree with an 

open canopy that is almost evergreen but 

becomes leafless for a short time in the 

dry season. It grows up to 30 meters tall. 

The bole can be straight or cooked; it can 

be branchless for up to nine meters and up 

to sixty cm in diameter. An ornamental 

tree, it is often planted along avenues and 

in gardens to beautify them. Phenology is 

in May-June and September-March. In 

Katarniaghat Wildlife Sanctuary it is 

found both in miscellaneous forest and 

Sal forest with IVI of 2.8 and 0.5, respectively. 


The tree is extensively harvested 

from the wild for its timber, many 

natural forests being managed on a 

forty year rotation. 

The tree is also grown as a plantation 

crop in Asia, Africa and the Americans. 

Fuel wood plantations are managed 

on a 20-year rotation. 

The cooked leaves are eaten as a 

vegetable. In times of scarcity the 

bark can be ground into a powder, 

mixed with flour and eaten. 

The tree is widely planted for its 

good soil binding capacity. 

It is occasionally cultivated as shade 

tree for tea and coffee plantations, 

where it also acts as a wind and firebreak. 

It is popular for the rehabilitation for 

seasonally dry, eroded and degraded 

soils. Its ability to grow on dry, 

sandy, stony and shallow soils makes 

it a useful species for reforestation 

for difficult sites. 

Good survival and rapid early growth 

have been reported in reforestation 

trials on both saline and alkaline 

soils, which are widely cultivated in 

agro-forestry system. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 496



Mall 

 

 

 

 

 

 

 

 

 

The bark can provide tanning material. 

It is used in India for tanning and 

dying. However, its low tanning content 

(12-17%), considerable weight 

loss in dying and difficult harvesting 

has limited its importance. 

When injured, the stem exudes large 

amounts of a reddish-brown gum that 

is chemically similar to, and used as a 

substitute for, gum arabic (obtained 

from Acacia senegal and other species). 

The leaves are known to have insecticidal 

and pesticidal properties. 

The branches (twigs) are used by tea 

planters as stakes for lying out tea 

gardens. These are found to split 

well. The species is popular along 

field borders. 

Pods and fallen leaves should be considered 

not as undesirable litter but as 

potential energy sources. It seems 

probable that if the pods of the related 

species A. lebbeck can yield ten 

barrels of ethanol per hectare, then 

this species could as well. 

The timber has large amount of nondurable, 

yellowish-white sapwood. 

The heartwood large and heavy, light 

or dark brown with light and dark 

bands. Due to the broadly interlocked 

nature of the grain, it is more suitable 

for use in large section where a bolder 

effect is desired, such as in largesized 

panels and tabletops. 

It seasons and polishes well. The 

wood is used chiefly for construction, 

furniture, veneer, cabinet work, flooring, 

agricultural implements, moulding, 

carts, carriages, cane crushers, 

carvings, boats, oars, oil presses and 

rice pounders. It is resistant to several 

 

species for termites. 

The chemical analysis of the wood 

indicates that it is a suitable material 

for paper pulp. Bleached pulp in satisfactory 

yields (50.3%) can be prepared 

from A. procera wood by the 

sulphate process. It is suitable for 

writing and printing paper (mean fibre 

length is 0.9 mm, mean fibre diameter 

is 0.021mm). 

The calorific value of dried sapwood 

is 4870 kcl/kg, and that of heartwood 

4865 kcl/kg. An excellent charcoal 

(39.6%) can be prepared from the 

wood, and it is widely used as a fuel. 


White siris is commonly used in traditional 

medicines. Some research 

has been carried out into the medicinal 

activities of the plant and a number 

of active compounds have been 

recorded. 

All parts of the plant are anticancerous. 

The roots contain alpha-spinasterol 

and a saponin that possess spermicidal 

activity at a dilution of 0.008%. 

A decoction of the bark is given for 

the treatment of rheumatism and 

haemorrhage. 

It is also considered useful in treating 

problems of pregnancy and for stomach-ache. 

The leaves are poultice on 

to ulcers. 

4.6. Alstonia scholaris (Linn.) R.Br. 

Synonym: Echites scholaris Linn. 

Alstonia scholaris found in India, 

Sri Lanka, Pakistan, Nepal, Thailand, 

Burma, South East Asia, Africa, Northern 

Australia, Solomon Islands, and Southern 

China. Alstonia scholaris is an evergreen 

tropical tree up to 80 ft in height, having 

greyish rough bark with lenticels, secreting 

white milky latex-rich in poisonous 

alkaloid, lateciferous which is bitter in 

taste. Leaves grow in clusters of seven, 

coriaceous, elliptic-oblong, 10 to 20 cm 

long, 3 to 4.5 cm wide, pointed at the 

base, rounded at the apex, glossy green on 

the upper surface, white or greyish on the 

underside. The tip of the leaf is rounded 

or shortly pointed, tapering towards the 

base. The blokes of larger trees are 

strongly fluted to 10 m. the outer blaze is 

cream to yellowish in colour with abundant, 

milky latex that flows rapidly when 

cut. The inflorescence is a much-branched 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 497



terminal panicle, up to 120 cm long; 

flowers 7-10 mm long white, cream or 

green, the tube hairy, lobes sparsely or 

densely pubescent, 1.5-4 mm long, the 

left margins overlapping, strongly perfumed. 

The fruits are thin pods that can 

grow up to 20 inches long. Fruit is made 

up two slender follicles which are pendulous 

and cylindrical follicles, 20 to 40 cm 

long, 4-5 mm in diameter. Seeds are 3 to 

4 mm long, with brown ciliate hairs on 

the ends. Phenology- December-June. In 

Katarnia Ghat Wildlife Sanctuary it is 

found only in miscellaneous forest with 

IVI 0.4. 


The wood is too soft for making 

anything- so it is usually used in making 

packing boxes, blackboards, etc. Alstonia 

scholaris tree has been used to make paper. 

Ethno-madicinal potential 

Alstonia scholaris has many medicinal 

properties like antimicrobial, antiamoebic, 

anti-diarrheal, antihypertensive, 

anti-malarial, febrifuge, stimulant, 

hepoprotective, immunemodulatory, 

anti-cancer, antiasthmatic, 

antioxidant, analgesic, antiinflammatory, 

anti-fertility, antidiabetic, 

etc. 

Alstonia scholaris used in the treatment 

of fevers, chronic diarrhea, dysentery, 

ulcers, rheumatic pains, cancer, 

malarial fever etc. 

The ripe fruit of the plant are used in 

syphilis and epilepsy. 

The milky juice of Alstonia Scholaris 

has been applied to treat ulcers. 

The bark of the Alstonia scholaris is 

used in Ayurvedic medicine to treat 

fever, malaria, troubles in digestion, 

tumors, ulcers, asthma and so forth. 

The leaves and the latex are applied 

externally to treat tumors. 

The dried leaves of the Alstonia 

scholaris are used as an expectorant. 

The leaves can be used to treat skin 

diseases. 

 

 

 

Mall 

The bark and roots are boiled with 

rice and eaten by girls daily for several 

weeks to treat excessive vaginal 

discharge. 

The roots and bark are used in traditional 

medicine as an anthelmintic, astringent 

tonic, alternative antidiarrhoeaticum, 

antiperiodicum, etc. 

The latex is used to clean wounds and 

can we used for chewing gum. 

4.7. Bombax ceiba DC. 

It is a large deciduous tree. Leaves are 

digitate, leaflets 5-7, flowers red or yellowish, 

capsules ovoid. Phenology is 

March-April. In Katarniaghat Wildlife 

Sanctuary is being found only in miscellaneous 

forest with IVI 11.00. 


The silk cotton tree is often referred 

to as the silent doctor for the 

host of medicinal benefits that is offers 

almost each part of the tree, including 

the bark, flowers, fruits, seed 

and leaves, gums, thorns have 

therapeutic potential. 

A herbal composition made from the 

bark of the tree, for example is administered 

for the treatment of male sexual 

and gastro-intestinal disorders like 

dysentery and diarrhoea. The pharmacological 

benefits are basically due to 

the presence of Glycosides and tannins 

in the root and stem. 

It has haemostatic properties and is 

administered during menorrhagia. 

Silk cotton extracts are used in eve 

care, tentax forte, acne pimple 

cream. The plant is also being used 

for general debility, diabetes, impotence, 

spermatorrhoea, urinary stones 

and liver disorders. 

Some of the diseases for example diarrhoea, 

dysentery, asthma, rheumatism, 

leprosy, leucorrhoea, body 

pain, wounds are included in antiinflammatory, 

analgesic, antimicrobial 

and oxytocic activities 

of plant as indirect evidence of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 498



scientific validation (Jain and Verma, 

2014). 

4.8. Diospyros cordifolia Linn. 

It is a large shrub or small tree. 

Leaves are ovate-oblong, ovate lanceolate, 

acute, base cordate or rounded and 

hirsute on both surface. Flowers pale 

white in axillary cymes. Fruits are globose 

yellow at maturity. Phenology- 

March-June and June-September, In 

Katarniaghat Wildlife Sanctuary is being 

found both in Sal forest and Teak Plantation 

forest with IVI of 3.2 and 3.0 respectively. 


It is of great use for human being. 

It has commercial value being used in 

Bidi industry as a raw material. 

Leaves are being used in stupefying 

fishes. 

It is being used in several ailments 

either as a cure or for the well-being, 

viz., used for lever disorders, whooping 

cough, leprosy, ulcers, gonorrhoea, 

fever as emetic and antihelminthic. 

Alcoholic extract are anti-inflammatory, 

antipyretic and analgesic. 

It is depressant, spasmolytic 

producing bradycardia and hypotension. 

Aqueous extract is being used 

in critical jaundicised condition. 

The fruits are consumed because of its 

juicy and sweet nature by local inhabitants 

(Mall, 2016). 

4.9. Ficus racemosa Linn 

Mall 

Synonym: Ficus glomerata Roxb. 

A large deciduous tree, buttressed 

at the base. Bark smooth reddish brown; 

blaze pink, fibrous with white latex turning 

yellow on exposing. Leaves are 5-15 

x 2.5-6.5 cm, alternate, ovate or ellipticlanceolate, 

entire, sub-acute, base rounded 

or acute, glabrous above, minutely dotted 

beneath. Receptacles 2-3.2 x 2-3.5 

cm, clustered on leafless branches, 

smooth or pubescent, red or pink at maturity. 

Plant is propagated by using cuttings 

of stem and root shakers or by seeds 

also. The flowers are pollinated by very 

small wasps. Phenology: April – July. In 

Katarniaghat Wildlife Sanctuary is being 

found both in miscellaneous forest and 

Teak Plantation forest with IVI of 9.7 and 

1.9, respectively. 


Traditionally it is used in Indian 

medicinal practice as astringent, 

carminative, stomachic, vermicide 

etc (Mall and Tripathi, 2017). 

The extract of fruit is used in leprosy, 

diarrhoea, menorrhagia. It is useful in 

the treatment of leucorrhoea, blood 

disorder, burning sensation, fatigue, 

urinary discharge, intestinal worms 

and as carminative. 

Leaves are astringent to bowels and 

good in case of bronchitis; leaves are 

used in dysentery young tender leaves 

are used for fair complexion. The decoction 

of leaves is used to wash the 

wounds and ulcers. 

Bark is useful in asthma and piles. 

The latex or milky juice is administered 

in chronic infected wounds, 

haemorrhoids, boils, traumatic swelling, 

toothache, vaginal disorder, 

wounds it promote healing very soon. 

The root sap is used for treating diabetes. 

Phytochemical properties: The leaf of 

this plant contains sterols, triterpenoides 

(lanosterol) and alkaloids, tannins 

and flavonoids. Stem bark gives 

gluanol acetate, β-sterol, lupenol, 

stigmasterol. Fruit contains gluanol 

acetate, glucose, tiglic acid, esters of 

taraxasterol, lupeol acetate and other 

phytosterols. 

4.10. Madhuca latifolia Roxb. 

Madhuca is a large deciduous tree 

reaching a height up to 20m. Leaves are 

large and broadly elliptic 12-20cm long. 

The bark is 1.2 cm thick. Flowers white to 

cream colour with tubular, fleshy and 

juicy corolla. Fruit berry, ovoid, green 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 499



at maturity and turn pinkish yellow 

when ripe. 


The madhuca contains protein, carbohydrate, 

fat, minerals, calcium, phosphorus, 

iron, carotenes, sugar. Vitamins 

and many other nutrimental 

chemical constituents. 

The bark of Madhuca is used to 

cure leprosy and to heal wound. 

The flower decoction is used for 

headache due to cough and cold. 

Paste of fresh bark is useful on joint 

and muscles pain. 

Whole plant decoction is taken orally 

which is useful in joint and muscles 

pain (Mall and Tripathi, 2017a). 

4.11. Syzygium cumini Skeels. 

It is a large evergreen tree with whitish 

brown bark. Every year the bark is 

shed off. Its leaves are simple pointed at 

the tip, somewhat leathery, oval to rectangular 

and somewhat shiny. Flowers are 

mostly white and appear in cluster from 

axil to leaves.The fruit is berry. 

Ethnobotanical potentiality: 

The fruit contains 88% moisture, 

0.7% protein, 0.1% fat, 19.7% carbohydrate 

and 0.4% minerals. Fresh 

fruit had the antioxidants 708 

mg/100g AEAC units. The ripe fruit 

contains anthocyanin pigment (Rao et 

al., 2006). Syzygium cumini is a wellknown 

anti-diabetic herb. 

It is a good immune modulator. It is 

also used in blood pressure, dysentery, 

diarrhoea and gingivitis. 

Mall 

4.12. Schleichera oleosa (Lour.) Oken. 

Synonims: Pistacia oleosa Lour. 

Schleichera trijuga Willd., Cussambium 

oleosum Kuntze., Melicocca trijuga Juss. 

It is a monotypic genus belonging 

to the same family to which the popular 

fruit ‘Litchi’ belongs. The generic name 

of kusum, Schleichera is derived after the 

Swiss botanist J. C. Schleicher who first 

descrived the tree. The species name oleosa 

is derived from the Latin word ‘oleosa’ 

meaning oil, as the seed kernels are 

rich in oil. Synonymously the tree is also 

reffered as Schleichera trijugaWilld.,the 

word trijuga stands for ‘three pairs’,based 

on thepresence of three pair of leaflets in 

a leaf. Kusum is a large forest tree with 

dense green foliage. Leaves pinnate with 

three pairs of leaf lets .Inflorescence raceme. 

Flowers are white and fruits small. 

The fruits are berry shaped, globose or 

ovoid with a hard skin. The seeds are 

brown, irregular elliptic, slightly compressed 

oily and enclosed in a succulent 

aril. The oil content of the seed is around 

59-72% with yellowish green color. Phenology 

is in October-November. 

It is locally known as kusum. The 

other common names are kusum, kusumb, 

kosumb, koshamara, Celon oak kosamara, 

lac tree, honey tree, gum lac tree, macassar 

oil tree, sukoshka, skrataka, jatudruma,koshamra, 

jantu vriksha and kshudra 

maukkuli, etc. It occurs in the Indian 

sub-continent and south East Asia. There 

are many trees that are grown for multiple 

products. They are known as multipurpose 

trees (MPT S ), a term widely used in 

agro-forestry. Kusum is also one among 

the multipurpose trees which has been 

proved to be useful in numerous ways 

from times immemorial. 

In Katarniaghat Wildlife Sanctuary 

(KWS) it occurs in all the three types 

of forests with different IVI values. In 

miscllaceous forest, Sal forest and Teak 

plantation the IVI value of the kusum 

plant is 1.5, 4.3 and 4.4, respectively. 

The available literature reveals 

that this multipurpose ethno-botanical, 

nutrimental, ethno-medicinal, ethnoveterinary 

and plant of environmental use 

in agro-forestry which provide shade, 

habitat for organisms, soil improvement, 

etc., many useful products are also obtained 

such as fruits, timber, fire wood 

and variety of metabolic chemicals which 

may be used in the form of home remedies 

and for traditional medicine. Considering 

the multipurpose importance of 

the tree, which is yet not popularise due 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 500



to one reason or the other despite providing 

an array of benefits (Mall and Tripathi, 

2017b). 

 

 

 

 

 

 

 

 

 

 

Mall 


The leaves, twigs and the seed- cake 

are used as fodder to feed cattle. 

The wood is suitable as firewood and 

makes excellent charcoal. 

Pressed oil cakes from kusum tree are 

rich source of crude protein, carbohydrate, 

fibre and other minerals 

and serves as nutritive cattle feed. 

The oil extracted from the seed, 

called as kusum oil is used for culinary 

and lighting purposes. 

The kusum oil is being used to cure 

itching, acne, burn and other skin 

problems. 

The oil is used in rheumatism by external 

massage. 

Kusum oil is used in hair dressing as 

well as for promoting hair growth. 

The pinkish-brown heart wood is 

very hard, durable and excellent to 

make pestles, cartwheels, axles, 

plows, tool handles, and rollers of 

sugar mills and oil presses. 

Kusum plant is known for lac cultivation. 

It is one of the major host plant 

commercially exploited for cultivation 

of the Indian lac insect (Kerria 

lacca). It supports the kusmi strain of 

lac insect, which produces good quality, 

natural, biodegradable and commercially 

important, light colored lac 

resin of demand by lac industry, thus 

fetching high remunerative prices to 

lac growers. The lac resins serves as 

a livelihood support to millions of 

poor farmers in states like Jharkhand, 

Chattisgarh, Orissa, Andhra Pradesh 

and West Bengal. Immature lac insects 

prefer semi-tender twig of 

kusum tree for sap sucking and start 

secreting resins surrounding their 

body. The resinous coatings of closely 

settled sessile insects eventually 

coalesce together to form an encrustation 

in five to six months. On 

kusum tree, two lac crops are produced 

in winter and summer season. 

About 34-38% of the total lac production 

of India is shared by the 

Kusum tree as lac host. There are also 

other lac hosts but the quality of 

kusum lac is far superior. The dence 

foliage of the mature kusum tree provides 

an additional advantage of supporting 

brood lac (inoculums stick 

lac with emerging larvae from the 

female resin cells) viability even during 

the very hot summer season, otherwise 

summer mortality of lac insects 

is a common problem with other 

host plants like Butea monosperma 

(palas) and Ziziphus mauritiana 

(ber). 

The seeds of kusum are a very rich 

source of oil (60-72%) for industrial 

implications. The seed oil called 

kusum oil is an important component 

of the Makassar oil used for hair 

dressing and cooling bath oil. 

Kusum oil is used in textile industry 

for batik applications and also for 

making soap. 

The bark of kusum tree produces tannins 

and dyes that are occasionally 

used in small-scale industries like 

tanning in leather industry. 

Young leaves and shoots-raw cooked 

in soups or steamed and served with 

rice. 

The ripe fruit is eaten raw which has 

a pleasant acid flavor. 

The unripe fruits are pickeled. 

Oil obtained from the seed called 

macassar oil, is sometimes used for 

culinary purposes. It contains cyanogenic 

compounds, which may cause 

giddiness and should be removed if 

the oil is used for human consumption. 

The kusum tree is also grown as an 

avenue tree or wayside tree. 

The tree is utilized for multifarious 

purposes and is a boon for a subsistence 

farmer. 

The extended foliage and canopy of 

the kusum tree provides good shade 

and is therefore, suitable for mixed 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 501



farming with other heat susceptible 

economic plants. 

Importance of S. oleosa as a biodiesel 

fuel 

The depletion of the conventional 

petroleum resources has become a problem 

of major concern in recent years. Extensive 

research is going on to find an alternative 

fuel. Since vegetable oils have 

properties similar with that of diesel, they 

are replacing diesel in the field of commercial 

transportation and agricultural 

machinery. But the direct use of vegetable 

oil is having adverse effects on the combustion 

engine. Therefore, these vegetable 

oils are converted to biodiesel. Blending, 

emulsification, thermal cracking, and 

trans-esterification are the few techniques 

used for the conversion of crude vegetable 

oil into biodiesel. At present, biodiesel 

is produced by sunflower oil, palm oil and 

soybean oil by trans-estrification process. 

These oil due to their non-toxic, biodegradable 

and renewable nature, have 

gained a lot of alteration by the researchers. 

Cetane number for biodiesel is higher 

than that of petroleum. Moreover, biodiesel 

does not contain aromatic components. 

The emission of carbon monoxide, 

hydrocarbon and particulate matter is also 

less as compared to that of diesel fuel. 

High cast of the above mentioned oil is 

the basic disadvantage associated with 

them. Hence, the non-edible type of oils 

yielded from tress such a mahua, sal, inseed, 

caster, karanji neem, rubber, 

jatropha, kusum, cashew, restaurants 

waste oils and greases slong with animal 

fats are best suited for the production of 

biodieses, for instance, S. oleosa seed oil, 

one of the many non-edible seed oil is 

found to have many cyanogenitic materials 

and free fatty acides (FFA) such as 

myristic acid, palmitic acid, palmitoleic 

acid, cis oleic acid, trans linolelaidic acid, 

cis linoleic acid, alpha linolenic acid, 

eicosadienoic, heneicosanoic, behenic 

acid, erucic acid, lingoceric acid, docosahexaenoic 

acid (Mikolajczek and 

Smith, 1971, Canacki and Gerpen, 2001 

Mall 

and Gandhi et al., 2011). Therefore, it is 

used in production of biodiesel. In a report 

by Gandhi et al.,2011 methyl ester 

was produced using S. oleosa seeds. 

Phytoremediation properties 

Callophyllum inophyllum L. and Bixa 

orellana L. (Chaturvedi et al., 2012). 

Mining, smelting of metalliferrous, 

dumping of waste, chemicals used in 

agriculture etc. Are the different 

source of soil pollution, but the waste 

rocks generated by mining is the main 

source of the metal pollution of soil. 

The direct consequences of the deposition 

of waste rocks on the surface 

are the loss of cultivatable lands, forest 

and grazing land (Clemente et al., 

2007, Rio et al., 2006 and Freitas et 

al., 2004). Activity such as grinding, 

crushing, washing and smelting, used 

to extract and concentrate metals, 

generate waste rocks and tailings. 

Most of the tailings exhibit acidic pH 

due to which the microbial activity 

decreases which in turn leads to the 

death of plants. Tailings fo not contain 

organic matter and are characterized 

by the high concentration of arsenic, 

cadmium, copper, manganese, 

lead, zinc and other heavy metals 

(Mukhopadhyay and Maiti, 2010). 

However some plants can exist in the 

region of high concentration of metals 

(Das and Maiti, 2008). Such plants 

can be used to restore the contaminated 

sites by the process of phytoremediation. 

Phytoremediation is an environmental 

friendly and cost efficient 

technique used to treat the contaminated 

soil, air or water through the use 

of plant without employing any soil 

excavation or mechanical clean up 

method. Although many physicchemical 

techniques are also available 

to extract metals such as acid leaching 

and electro-osmosis, but these techniques 

are quite costly and can decontaminate 

only small portions of land 

(Ramadhas and Murleedharan, 2005). 

Moreover, these techniques also dete- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 502



riorate biological activity of the soil 

and adversely affect its physical structure. 

Therefore, the phyto-remediation 

is the preferred technique to decontaminate 

the soil. This approach to 

remove the metal is called green mining 

because further extraction of metals 

can be done from the plant tissue 

(Clemente et al., 2007). 

Use as livestock feed 

A few non-conventional agroindustrial 

by-products including S. 

oleosa cake were checked for their effectiveness 

a livestock feed (Punj, 

1988). The presence of tannins adversely 

effects the utilization of various 

nutrients (Mc Leod, 1974). In addition, 

tannins are believed to create 

toxic effects by breaking down the alimentary 

canal tissues and the hydrolysable 

tannins make pathological 

changes in liver, kidney, heart, etc. 

When their concentration in blood increases 

further than the competence of 

the liver to deify them. The levels of 

tannins were determined using various 

chemical and biological methods. It 

was observed that in S. Oleosa, tannin 

levels in terms of total phenols (TP) 

and condensed phenols (CP) were 

low, and protein-precipitation capacity 

(PPC) could not be detected because 

of its very low level. Hence, it 

can be considered safe for incorporation 

in livestock feed since the harmful 

factors are absent (Makkar, 1990). 


Different plant parts (stem bark, seed, 

fruit and seed oil) of kusum are used 

in traditional medicines. 

The seed oil is used by the local vaids 

for curing skin diseases like scabies, 

itching, and acne. 

The bark decoction is also used 

against skin inflammation and ulcers. 

The bark decoction is also infused for 

curing malaria. 

The fine paste of the bark of Kusum is 

often used to control tissue swelling. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Mall 

The bark is known to contain medically 

important compounds like lupeol 

used in preparing analgesic and antitumerous 

agents like betulin and betulic 

acids. 

It balances kaph, useful in productive 

cough and asthema. 

It cleanses intestine. 

It is used in bleeding disorders like 

nasal bleeding and heavy periods. 

The unripe fruits are absortant, useful 

in diarrhea, neuralgia, paralysis, constipation 

and bloating. 

The fruit pulp improves hair strength 

and promotes hair growth. 

The ripe fruit improves digestion 

strength, improve taste and relieves 

anorexia. 

The leaf, seed, oil, and bark are used 

for treating rheumatoid arthritis, 

headache, myalgia,,skin disease, malarial 

fever and prophylactic against 

cholera. 

The bark is astringent and is used in 

fever as antipyratic, useful in pruritus. 

The kusum oil is bitter, sour, sweet 

which improves strength and immunity 

and can be taken regularly. It improves 

taste and relieves anorexia. 

The oil is digestive, induce mobility, 

causes diarrhea, purgative and relieves 

constipation. 

The fine paste of the bark which is 

astringent is mixed with oil is applied 

to cure itch and acne and other skin 

eruptions. 

The oil is useful in worm infection, 

skin diseases, in toxic conditions, poisoning, 

ulcer and wounds. 

The seed oil is also used for the cure 

of itch and acne. 

The seed oil is stimulating and has 

cleansing applications. 

The ripe fruit is often served with salt 

which improves digestion, useful in 

anorexia and nourishing. 

Ethno-veterinary potential 

The seed is grinded so as to make 

fine powder. It is mixed with wa- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 503



 

ter and given to cattle for removing 

worms from the stomach. 

The fine powder of the seeds is 

applied to wounds and ulcers of 

cattles to remove maggots. 

Phyto-chemical constituents 

Phytochemical studies have 

shown that its bark contains lupeol, lupeol 

acetate, betulin, betulinic acid, betasitosterol, 

and scopoletin (Dan and Dan, 

1986). A very recent report have also 

shown the existence of taraxerone and 

tricadenic acid A in the outer bark of the 

above plant (Ghosh et al., 2011). The 

bark also contains about 10% tannin and 

antitumor agents such as betulin and betulinic 

acid have also been isolated from it. 

Mall 

Anticancer activity 

Cancer is a term used for a disease 

in which abnormal cells trend to proliferate 

in an uncontrolled way and, in some 

cases metastasize. Extensive research has 

been done in order to find therapeutic 

drug for the treatment of cancer. Plant 

based products have been frequently examined 

as potential anticancer agents. 

The screening of various medicinal plants 

results in the isolation of bioactive compounds 

which have been reported as effective 

chemopreventive as well as chemo 

therapeutic agents (Kawamori et al., 

1999, Choi et al., 2001, Kirana et al., 

2003 and Sandhya et al., 2006). The phytochemical 

screening of S. oleosa revealed 

the presence of lupeol and butilinic 

acid type triterpene which have antineoplastic 

activity (Bhatia et al., 2013). This 

study provides a step toward the exploration 

of S. oleosa as a chemo preventive 

agent against cancer. A bulk of research 

revealed that the phyto-chemicals exhibit 

their anticancer properties either by suppressing 

the proliferation of tumor cells 

via suppression of various cell signalling 

pathways or by induction of apoptotic 

death in tumor cells by generation of free 

radical, such as reactive oxygen/nitrogen 

species (Bharti et al., 2003 and Pettit et 

al., 2000). A report involving the separation 

of an extract prepared from the bark 

and stem of Sri lankan tree S. oleosa results 

in the isolation of seven sterols, 

Scheicherastins (1-7) and two related 

sterols 8 and 9 designed as Schleicheols 1 

and 2. The isolated Scheicherastins exhibited 

cancer cell growth inhibitory properties 

(Pettit et al., 2000). 

Antioxidant activity 

Oxygen is used for generating 

metabolic energy in our body but it also 

produces reactive oxygen as by products 

during its various reactions in the body. 

Reactive oxygen species are usually atoms 

or a group of atoms having odd (unpaired) 

electrons, in aerobic cells these 

are produced during mitochondrial electron 

transport and several oxidation reactions 

(Forman and Torres, 2002). These 

reactive species acan, react with DNA 

and several other bio-molecules causing 

what is called ‘oxidative damage to DNA’ 

this damage causes changes in DNA such 

as stand breaks; changes at cross links 

between DNA and protein; changes as 

base tree sites among other changes (Dizdaroglu 

et al., 2002). Several medicinal 

plants, fruits, vegetable can decrease the 

risk of oxidative damage as they comprise 

of vitamins, carotenes, phenolic compounds, 

flavanoids, alkanoids, tannins, 

etc. which act as chemo-preventive agents 

(Dhir et al., 1993, Cozzi et al., 1997 and 

Thind et al., 2012). These phytochemicals 

can prevent damage by their 

radical scavenging ability. Thind et al. 

evaluated the hydroxyl radical scavenging 

potential of S. oleosa. Extracts of roots of 

S. oleosa with different solvents were 

tested for their anti-proliferative activity. 

Antioxidants are molecules which 

can safely interact with free radicals and 

terminate the chain reaction before vital 

molecules get damaged. The free radical 

damage can be prevented by several enzymes 

and the principle antioxidants such 

as vitamin E, beta-carotene, and vitamin 

C, present in the defence system of our 

body. Several studies have shown that 

plant phenolics also have antioxidant 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 504



properties (Velioglu et al., 1998, 

Maisuthisakul et al., 2007 and Li et al., 

2008). Natural polyphenols can have simple 

structure for example phenolic acid, 

phenylpropanoids, flavonoids can have 

simple structures like polymers, e.g., lignis, 

melanins, tannins (Bravo, 1998). Free 

radical scavenging property, metal chelating 

property, effects on cell signalling 

pathways and on gene expression contributes 

to the potential of phenolics as 

antioxidant therapeutic agents (Soobrattee 

et al., 2003). S.oleosa has been found as 

potent antioxidant due to the presence of 

phenolic compounds (Thind et al., 2011). 

Mall 

Antimicrobial activities 

In recent study two (Ghosh et al., 

2011) triterprnoids, namely taraxerone 

and tricadenic acid A were isolated from 

the outer bark and preliminary study on 

their antimicrobial activities were done 

against five different fungal pathogens 

namely Colletotrichum camelliae, 

Fusarium equisti, Altermaria alterata, 

Curvularia eragrostidis, Colletotrichum 

gloeosporioides by in vitro antifungal assay 

(Suleman et al., 2002 and Saha et al., 

2005) and against four bacterial pathogens 

namely. Escherichia coli, Bacillus 

subtilis, S aureus and Enterobacter by 

antibacterial assay. It was found that both 

taraxerone and tricardenic acid A had 

prominent activities against the fungal 

and bacterial pathogens. 

The enumerations collectively 

show the various pharmacological activities 

of S. oleosa. It has potential of anticancer, 

antioxidant and antimicrobial activities. 

It contains various poly phenolic 

compounds. The poly phenols scavenge 

free radicals and do not allow them to 

damage the cell. Due to its free radicals 

scavenging activity, S. oleosa is a potent 

antioxidant. Free radical scavenging activity 

can also be correlated to cytotoxicity. 

It exhibits toxicity against various 

cell lines and was found to be as effective 

anticancer agent. It, moreover, has 

a great scope of being an effective antimicrobial 

agent since it showed good activity 

against various microbes. It was 

also found that this plant has various environment 

aspects to it as well. The biodiesel 

produced from it. Is found to have 

many properties similar to that of diesel 

e.g. viscosity and volatility. Also its cetane 

number is higher than that of petroleum; 

therefore it can replace diesel for 

the combustion engine. On the basis of 

physic-chemical, growth and biochemical 

parameters C. inophyllum and 

B. orellana were found to be more capable 

for phyto-remediation of the contained 

soil compared to S. oleosa. Furthermore, 

it was observed that it contained 

low tannin levels, thus it can be 

considered safe to be used as a livestock 

feed. 

The major use of kusum tree is for 

cultivation of lac. However, other uses of 

kusum tree are currently underexploited 

but hold promise to benefit human life in 

many spheres. The plantation of kusum in 

suitable areas needs to be promoted in 

view of its advantages as MPT. There is a 

tendency to prefer quick growing trees in 

the forestation programmes, which may 

not always be advantageous or even ecofriendly 

in long run. 

The role of kusum plantation can 

mainly be envisaged in terms of economic 

benefits to the resource-constrained farmers 

dwelling around forest areas. 

4.13. Ziziphus mauritiana Lam. 

Ziziphus mauritiana is an extremely 

drought hardy and native fruit of 

India, found wild and cultivated. 


It is useful as food, fodder, nutrient, 

medicinal, construction material and fuel. 

Z. mauritiana is having tremendous 

medicinal properties, attributed by diverse 

group of secondary metabolites 

such as alkaloids, flavonoids terpenoids, 

saponin, pectin, triterpenoic acids and 

lipids. Jujubosides (saponin) isolated 

from Ziziphus reported to have haemolytic, 

sedative, anaxiolytic, and weetness 

inhibiting properties. Whereas, cyclopep- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 505



tide alkaloids, found to have sedative, 

antimicrobial, hypoglycaemic, antiplasmodial, 

anti-infectious, anti-diabetic, 

diuretic, analgesic, anticonvulsant and 

anti-inflammatory activities (Goyal et 

al.,2012). 

In spite of the fact that Ziziphus mauritiana 

having medicinal properties it is 

neither considered as important medicinal 

plant nor utilized for medicinal use in 

main stream therapeutic. 

From the present study, it is envisaged 

that the trees of Katarniaghat Wildlife 

Sanctuary has great socio-economic importance 

as they are being widely used for 

different purposes by the natives. Nature 

has provided a complete store house of 

remedies to cure ailments of mankind. 

Besides, traditional and commercial importance, 

they have tremendous ecological 

significance. Trees which are of leguminous 

nature and soil binding abilities, 

they all are suitable species for 

wasteland development. However, other 

uses of many trees are currently underexploited 

but hold promise to benefit human 

life in many spheres. The plantation of 

plants in suitable areas needs to be promoted 

in view of their advantages as 

malty purpose trees (MPT). There is a 

tendency to prefer quick growing trees in 

the forestation programmes, which may 

not always be advantageous or even ecofriendly 

in long run. The trees must be 

conserved and more plantations should be 

done either by utilisation of Biotechnology 

or through traditional methods. People 

conserve what they love. They love what 

they understand and they understand 

what they are taught. 

References 

Bajpai, O., Kumar, A., Mishra, A. K, 

Sahu, N., Behera, S. K. and 

Chaudhary, L. B. (2012a). Phenological 

study of two dominant tree 

species in tropical moist deciduous 

forest from the Northern India. International 

Journal of Botany 8, 66- 

72. 

Mall 

Bajpai, O., Kumar, A., Mishra, A. K., 

Sahu, N., Pandey, J., Behera, S. 

K. and Chaudhary, L. B. (2012b). 

Recongregation of tree species of 

Katarniaghat Wildlife Sanctuary, 

Uttar Pradesh, India. Journal of Biodiversity 

and Environmental Sciences 

2, 24-40. 

Babu, N. P., Pandikumar, P. and Ignacimuthu, 

S. W. (2009). Antiinflammatory 

activity of Albizia 

lebbeck Benth., an ethnomedicinal 

plant, in acute and chronicanimal 

models of inflammation. Journal of 

Ethnopharmacology. 125, 356-360. 

Balasubramaniam, P. (1992). Observation 

on the utilization of forest 

plants by the tribal’s of point Calimere 

wild life sanctuary, Tamil Nadu. 

Bulletin of Botanical survey of 

India 34, 100-111. 

Barua, C. C., Gupta, P. P., Patnaik, G. 

K., Kulsherestha, D. K. and Dhawan, 

B. N. (1997). Studies on the 

antianaphylactic activity of 

franctions of Albizzia lebbeck. Current 

science 25, 397-399. 

Bharti, A. C., Donato, N., Singh, S. and 

Aggrawal, B. B. (2003). Curucumin 

down regulates the constitutive 

activation of nuclear factor-kappa B 

and Ikappa B alpha kinase in human 

multiple myeloma cells, leading to 

suppression of proliferation and induction 

of apotosis. Blood 101, 

1053-1062. 

Bhaumik, S., Aanjum, R., Rangara, N., 

Pardhasaradhi, B. V. V. and 

Khar, A. (1999). Curicumin mediated 

apotosis in AK-5 tumor cells 

involves the production of reactive 

oxygen intermediates. FEBS Lett. 

456, 311-314. 

Bhatia, H., Kaur, J., Nandis, S., Gurnam, 

V., Chowdhary, A., Reddy, 

P. H., Vashishtha, A. and Rathi, 

B. (2013). A review on Schleichera 

oleosa: Pharmacological and Environmental 

aspects. Journal of 

Pharmacy Research 6(1),224-229. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 506



Bravo, L. (1998). Polyphenols: chemistry, 

dietery sources, metabolism and 

nutritional significance. Nutr Rev. 

56, 317-333. 

Celemente, R., Paredes, C. and Bernal, 

M. P. (2007). A field experiment 

investigating the effects of olive 

husk and cow manure of heavy 

metal availability in a contaminated 

calcareous soil from Murcia 

(Spain). Agric Ecosyst Environ. 

118, 319-326. 

Champion, H. G. and Seth, S. K. 

(1968). A Revised Survey of the 

Forest Types of India. Publication 

Division, Govt. of India, New Delhi. 

Chanacki, M. and Van Gerpen, J. 

(2001). Biodiesel production from 

oils and fats with high free fatty acids. 

Trans Am Soc Agric Engineers. 

44, 1429-1436. 

Chaturvedi, N., Dhal, N. K. and 

Ramareddy, P. S. (2012). Comparative 

phytoremediation potential of 

Callophyllum inophyllum L., Bixa 

orellana L. and Scheichera oleosa 

(Lour.) Oken on iron ore trailings. 

Int J Min Reclam Environ. 26,104- 

108. 

Chin, W.Y. (2005). Plants That Heal, 

Thrill and Kill SNP International, 

Singapore pp.172. 

Choi, J. A., Kim, J.Y. and Lee, J. Y. 

(2001). Induction of cell cycle arrest 

and apoptosis inhuman breast 

cancer cells by quercetin. Int J. Oncol. 

19, 837-844. 

Cozzi, R., Ricordy, R., Aglitti, T., Gatta, 

V., Perticone, P. and De salvia, 

R. (1997). Ascorbic acid and β- 

carotene as modulators off oxidative 

damage. Carcinogenesis.18, 

223-236. 

Dan, S. and Dan, S. 

(1986).Phytochemical study of 

Adensonia digitata, Psychotria adenophylla 

and S. oleosa. Fitoterapia 

57, 445-446. 

Das, M. and Maiti, S. K. (2008). Comparison 

between availability of 

heavy metals in dry and wetland 

Mall 

dealing of na abandoned coppertailing 

pond. Environ Monit Assess. 

137, 343-350. 

Das, A.K., Ahmad, F., Bachar, S.C., 

Kundu, J., Dev, S., 2003. Antiinflammatory 

effect of Albezia 

lebbeck (Benth.) Bark. Online 

Journal of Biological Science 3, 

685-687. 

Dhir, H., Kumar, A. and Sharma, A. 

(1993). Relative efficiency of Phyllanthus 

emblica fruit extract and 

ascorbic acid in modifying lead and 

aluminium-induced sister-chromatic 

exchanges in mouse bone marrow. 

Environ Mol Mutagen. 21, 229-236. 

Dizdarogula, M., Jaruga, P., Birincioglu, 

M. and Rodrigeuez, H. 

(2002). Free radical induced damge 

to DNA: mechanisms and measurement. 

Free Radicals Biol Med. 

166, 1102-111. 

Forman HJ and Torres M. (2002). Reactive 

oxygen species and cell signaling: 

burst in 

macrophage signaling. Am J. Respir 

Crit Care Med. 166, S4-S8. 

Freiteas, H., Prasad, M. N. V. and Pratas, 

J. (2004). Plant community tolerant 

to trace elements growing on 

the degraded soils of Sao Domingos 

mine in the south east of Portugal: 

enhvironmental implications. Environ 

Int. 30, 65-72. 

Gandhi, M., Ramu, N. and Raj, S. B. 

(2011). Methyl ester production 

from Schleichera oleosa. Int J 

Pharma Sci Res. 2, 1244-1250. 

Ghosh, P., Chakraborty, P., Mandal, 

A., Rasul, M. G., Chakraborty, 

M. and Saha, A. (2011). Triterpenoids 

from Schleichera oleosa of 

Darjeeling foothills and their antimicrobial 

ativity. Indian J. Pharm 

Sci. 73, 231-233. 

Goyal, M., Sasmal, D. and Nagori, B. P. 

(2012). Review on ethnomedicinal 

uses, pharmacological activity and 

phytochemical constituents of 

Ziziphus mauritiana (Z. jujuba 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 507



Lam., nonMill). Spatula DD2, 107- 

116. 

Gupta, A. K. (2004).Reviews on Indian 

Medicinal Plants.Vol I Indian 

Council of Medical Research, New 

Delhi, pp. 445-480. 

Gupta, A.K., Varalakshmi, P. (1998). 

Anitinflammatory activity of Lupeol 

linoleate in adjuvant induced 

arthritis. Fitoterapia 69, 13-19. 

Gupta, S. R., Malik, K. K. and Seshadri, 

T. R. (1966).Chemical components 

of Albizzia lebbek heart 

wood. Indian Journal of Chemistry 

4, 139-141. 

Iwasa S. (1997). Schleichera oleosa 

(Lour.) Oken. I. faridah Hanum. 

L.J.G. van der Maesen (Eds.). plant 

resources of South-east Asia No. 

11. Auxilary Plants, Prosea Foundation, 

Bogor Indonesia. pp. 227-229. 

Jhansi, P., Kalpana, P. and Ramanujan, 

G. K. (1994). Apidologie. 25, 

289-296. 

Jain, S.K., (1991). Dictionary of Indian 

folk medicine and Ethnobotany. 

Deep Publications, Lucknow, pp. 

17. 

Jain, V. and Verma, S. K. (2014). Assessment 

of credibility of some folk 

medicinal claims on Bombax ceiba 

Linn. Ind. J. Trad. Knowl. 13, 87- 

94. 

Kala, C. P. (2006). Etnhobotany and ethnoconservation 

of Aegle marmelos 

(L.)Correa.Indian Journal of Traditional 

Knowledge 5, 537-540. 

Kapur, S.K., 1993. Ethno-medico plants 

of Kangra valley (Himachal Pradesh). 

Journal of Economic and 

Taxonomic Botany 17, 395-408. 

Kaul, O. N. and Sharma, D. C. (1971). 

Forest type statistics. Indian Forester 

97, 432-436. 

Kirana, C., Mcintosh, G. H., Record, I. 

R. and Jones, G. P. (2003). Antitumor 

activity of extract of Zingiber 

aromaticum and its bioactive sesquiterpenoid 

Zerumbone. Nutr 

Cancer. 45, 218-225. 

Mall 

Kowamori, T., Lubet, R. and Steele, V. 

E. (1999). Chemopreventive effect 

of curcumin, a naturally accuring 

anti-inflammatory agent, during the 

promotion/progression stages of colon 

cancer. Cancer Res 59, 597- 

601. 

Lee, K. W., Hur, J. H. and Lee, C. Y. 

(2005). Antiproliferative effects of 

dietary phenolic substances and hydrogen 

peroxide. J. Agric food 

Chem. 54. pp. 7036-7040. 

Li, H. B., Wong, C., Cheng, K.W. and 

Chen, F. (2008). Antioxidant properties 

in vitro and total phenolkic 

contents in methanol extracts from 

medicinal plants. Lwt-Food Sci 

Technol. 41, 385-390. 

Mahaptma, S. P.and Sahoo, H. P. 

(2008). An ethno-medico botanical 

study of bolangi, Orissa, India: native 

plant remedies against gynaecological 

diseases. Ethanobot Leafl. 

12, 846 

Maisuthisakul, P., Suttajit, M. and 

Pongsawatmanit, R. (2007). Assessment 

of phenolic content and 

free radical-scavenging capacity of 

some Thai indigenous plants. Food 

Chem. 100, 1409-1418. 

Makkar, H. P. S. (1990). Tannin levels 

and their degree of polymerization 

and specific activity in soma agroindustrial 

by products. Biol Wastes 

31, 137-144. 

Mall, T.P. (2016). Diospyros cordifoia 

Roxb.- An under exploited potent 

ethnomedicinal feed- A review 

World J. Pharmaceut. Res. 5, 172- 

177. 

Mall, T. P. and Tripathi, S. C. (2017). 

Diversity of Wild Nutrimental 

Fruits of District Bahraich, Uttar 

Pradesh, India. Int. J. Curr. Biosci. 

Plant Biol.4, 65-77. 

Mall, T. P. and Tripathi, S. C. (2017). 

Kusam-A Multipurpose Plant from 

Katarniaghat Wildlife Sanctury of 

Bahraich (UP) India-A Review.World 

Journal of Pharmaceutical 

Research. 6, 463-477. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 508



Mall 

McLeod, M. N. (1974). Plant tannis-their 

role in forage quality. Nutr Abstr 

Rev.44, 803-815. 

Mikolajczak, K. L. and Smith, C. R. 

(1971). Cyanolipids kusum (Schleichera 

trijuga (seed oil). Northern 

Regional research Laboratory 

ARS/USDA Lipids. 6, 349-350. 

Moon, A., Khan, A. and Wadher, B. 

(2009). Biotherapeutic antibacterial 

of potential Schleichera oleosa 

against drug resident isolates. J 

Pure Apl Mirobiol.3, 181-186. 

Mukhopadhyay, S. and Maiti, S. K. 

(2010).Phytoremediation of metal 

mine waste.Appl Ecol Environ Res. 

8, 207-222. 

Mudaliar, K. S. M., (1936). Siddha Materia. 

Department of Indian medicine 

and Homeopathy. Chennai, pp. 

799-800. 

Nadkarni, A. K. (1954). Indian Materia 

Medica, Vol. 1. Popular Book Deparment, 

Bombay. 

Nemeikaite, C. A., Imbrasaite, A., 

Sergediene, E. and Cenes, N. 

(2005). Quantitative structureactivity 

relationships in pro-oxidant 

cytotoxicity of polyphenols: role of 

potential of phenoxyl radical/phenol 

redox couple. Arch Biochem Biophys. 

441, 182-190. 

Palanuvej, C. and Vipunngeun, N. 

(2008). Fatty acid constituents of 

Schleichera oleosaa (Lour) Oken 

seed oil. J. Health Res.22, 203-212. 

Pettit, G. R., Numata, A. and Cragg, G. 

M. (2000). Isolation and structures 

of Scheicherastins (1-7) and 

Schleicheols 1 and 2 from the teak 

forest medicinal tree Schleichera 

oleosa. J. Nat Prod.63, 72-78. 

Pitman, N. C. A., Terborgh, J. W., Silman, 

M. R., Percy, N. V., Neill, D. 

A., Ceron, C. E., Palacios, W. A. 

and Aulestia, M. (2002). A comparison 

of tree species diversity in 

two upper Amazonian Forests. 

Ecology 83, 3210-3224. 

Porra, R. J., Thompson, W. A. and 

Kriedmann, P. E. (1989). Determination 

of accurate extinction coefficients 

and simultaneous equations 

for assaying chlorophylls a 

and b extracted with four different 

solvents: verification of the concentration 

of cjlorophyll standards by 

atomic absorption spectroscopy. Biochem 

Biophys Acta. 975, 384-394. 

Pramanik, K. C., Bhattacharya, P., 

Chatterjee, T. K. and Mandal, S. 

C. (2005). Anti-inflammatory activity 

of methanol extracts of Albizzia 

lebbeck (Mimosaceae) bark. Europian 

Bulletin of Drug Research 13, 

71-75. 

Punj, M. L. (1988). Agricultural byproducts 

and industrial wastes for 

Livestock and poultry feeding. All 

India Co-ordinated research project 

India council of Agriculture Research, 

New Delhi. 

Ramadhas, S. and Murleedharan, C. J. 

S. (2005). Performance and emission 

evaluation of a diesel engine 

fueled with methyl esters of rubber 

seed oil. 

Rao, V. K., Shivshankara, S. and 

Prakas, G. S. (2006). Antioxidant 

properties of some underutilized 

fruits.National Symposium on Underutilized 

Horticultural Crops, 

IIHR, Bangalore.pp54. 

Rashid, A., Anand, V. K., Serwar, J. 

(2008). Less known wild edible 

plants used by Gujjar tribes of 

Dirtrict Ralouri, Jammu aand 

Kashmir State, India. Int. J. 

Bot.4, 219-224. 

Rio, M. D., Font, R., Moreno-Rojas, R. 

and De Haro-Bailon, A. (2006). 

Uptake of lead and Zinc of wild 

plants growing on contaminated 

soils. Ind Crop Prod.24, 230-237. 

Rodgers, W. A. and Panwar, H. S. 

(1988). Planning a Protected Area 

Network in India. Vol I & II. The 

Report of Wildlife Institute of India, 

Dehradun, India. 

Rout SD, Panda T and Mishra N. 

(2009). Ethno-medical plants used 

to cure different diseases by tribal 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 509



of Mayuribhanj district of North 

Orissa. Ethno-med.3, 27-36. 

Sandhya, T., Lathika, K. M, Panday, B. 

N. and Mishra, K. P. (2006). Potential 

of traditional ayurvedic formulation, 

Triphala, as a novel anticancer 

drug. Cancer Lett.231, 206- 

214. 

Saha, D., Dasgupta, S. and Saha, A. 

(2005). Antifungal activity of some 

plant extracts against fungal pathogen 

of tea (Camellia sinensis). 

Pharm Biol. 43, 87-95. 

Sakagami, H., Jiang, Y. and Kusama, 

K. (2000). Cytotoxic of hydrolysable 

against human tumor cell lines-a 

possible mechanism. Phytomedicine. 

1, 39-47. 

Singh, J. (2011). Sehat ke liya phal 

khayein, Phal-phool, Jan-Feb, IC- 

AR, New Delhi. pp.17-20. 

Singh, K. N. and Lal, B. (2006). Notes 

on Traditional Uses of Khair (Acacia 

catechu Willd.) by Inhabitants 

of Shivalik Range in Western 

Himalaya. Ethnobotanical Leaflets 

10, 109-112. 

Singleton, V. N. (1981). Naturally occurring 

food toxicants; phenolic substances 

of plant origin common in 

food. Adv Food. 27, 149-242. 

Sinha, A. K., Sharma, U. K., Abhisekh, 

S. and Nandini, S. (2010). A process 

for the preparation of crystalline 

and non-hygroscopic phenolic 

rich colored fractions from plants. 

Int Appl. WO Patent 

(2010)2010109286A1. 

Soobrattee, M. A., Neergheen, V. S., 

Luximon-Ramma, A., Aruoma, 

O. L. and Bahorun, T. (2005). 

Phenolics as potential antioxidant 

therapeutic agents: Mechanism and 

Actions. Mutation Research/Fundamental 

and Molecular 

Mechanism of Mutagenesis 579, 

200-213. 

Suleman, P., Al-Musallam, A. and 

Menezes, C. A. (2002). The effect 

of biofungicide Mycostop on 

Ceratosystis, redicicola, the casual 

Mall 

agent of black scorch of date palm. 

Biocontrol. 47, 207-216. 

Srivastava, T.N., Rajesekaran, S., 

Badola, D.P. and Shah, D.C. 

(1986). An indedx of the available 

medicinal plants used in Indian system 

of medicine from Jammu and 

Kashmir State. Ancient Science of 

Life 6, 49-63. 

Thind, T. S., Rampal, G., Agrawwal, S. 

K., Saxena, A. K. and Arora, S. 

(2010). Diminution of free raaadical 

induced DNA damage by extracts/fractions 

from bark of Schleichera 

oleosa (Lour.) Oken. Drug 

Chem Toxicol. 33, 329-336. 



(2011).In vitro antiradical properties 

and total phenolic contents in 

meth extracts/fractions from bark of 

Schleicheraoleosa (Lour.) Oken. 

Med Chem Res. 20, 254-260. 



(2012). Evaluation of cytotoxic and 

radical scavenging activities of root 

extracts of Schleichera oleosa 

(Lour.) Oken. Nat Prod Res. 26, 

1728-1731. 

Tripathi, K. P. and Singh, B. (2009). 

Species diversity and vegetation 

structure across various strata in 

natural and plantation forests in 

Katerniaghat Wildlife Sanctuary, 

North India. Tropical Ecology 50, 

191-200. 

Tripathi, S. N. and Shukla, P. (1979). 

Effect of Histamine and Albizzia 

lebbeck Benth, on guinea pig adrenal 

glands. Indian journal of experimental 

Biology 17, 915-917. 

Tripathi, S. N., Shukla, P., Mishra, A. 

K, and Udupa, K. N. (1978). Experimental 

and clinical studies on 

adrenal function in bronchial asthma 

with special reference to the 

treatment with Albizzia lebbeck. 

Quarterly Journal of Surgical Science 

14, 169-176. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 510



Varshney, I. P. and Khan, M. S. Y. 

(1961). Saponins and Sapogenins 

XI. On the presence of echinocystic 

acid and quercetin in flowers of the 

Albizzia lebbek Benth.Canidian 

Journal of Chemistry 39, 1721- 

1723. 

Varshney, I. P. and Sharma, S. P. 

(1969). Chemical Investigation of 

the Albizzia lebbek leaves and Albizzia 

lucida seeds and bark. Indian 

Journal of Applied Chemistry 32, 

10-11. 

Varshney, I. P., Geeta, H., Srivastava, 

H. C. and Krishnamurthy, T. N. 

(1973). Partial structure of 

Lebbekanin A, a new saponin from 

the seeds of Albizzia lebbek 

Benth.Indian Journal of Chemistry 

11, 1094-1096. 

Varshney, I. P., Pal, R., and Vyas, P. 

(1976). Studies on the Lebbekanin 

E, a new saponin from Albizzia 

Mall 

lebbek Benth. Journal of Indian 

Chemical Society LIII, 859-860. 

Velioglu, Y. S., Mazza, G., Gao, L. and 

Oomah, B. D. (1998). Antioxidant 

and total phenolics om selected 

fruits, vagetables and grain products. 

J Agric Food Chem. 46, 4113- 

4117. 

Verpoorte, R. (1999). Exploration of natures 

chemodiversity: the rolr of 

secondary metabolites as leads in 

drug development.Drug Discovery 

Today 3, 232-238. 

White, M. J. (1999). Mediators of inflammation 

of inflammatory process. 

Journal of Allergy and Clinical 

Immunology 103, S378-S381. 

Yu, L., Haley, S., Perret, J., Harris, M., 

Wilson, J. and Qian, M. (2002). 

Free radical scavenging properties 

of wheat extracts. J Agric Food 

Chem.. 50, 1619-1624. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 511



Free Radical Scavenging Potential and Anticancer 

Activity of Primula denticulata Sm. from North-Western 

Himalayas 

Bilal Ahmad Wani 1, *, Mohammed Latif Khan 1 and Bashir Ahmad Ganai 2 

1 Department of Botany, Dr. Hari Singh Gour Central University, Sagar, India- 470003 

2 Centre of Research for Development, University of Kashmir, Srinagar, 190006, India; 

*Correspondence: bilalenvsci@gmail.com; Tel: +91-9407579802 

Abstract: The present research work was aimed to evaluate the free radical scavenging potential 

and anticancer property of Primula denticulata ethanol extract (PDEE) against different 

human cancer cell lines. The antioxidant potential was determined by total phenolic 

and flavonoid content, DPPH free radical scavenging assay, hydroxyl radical scavenging 

assay and DNA damage assay. The anticancer activity was determined by SRB assay. 

Apoptotic induction in MiaPaca-2 cells was analysed by propidium iodide staining cell-cycle 

and DNA content analysis, and mitochondrial membrane potential loss was measured using 

rhodomine-123 as fluorescent dye through flow cytometry. The total phenolic and flavonoid 

content in PDEE was found to be 12.24±2.11 (mg GAE/g dry extract) and 7.06 ±1.31 

(mg catechin/g dry extract) respectively. The extract at 600 µg/ml concentration induces 

69.35% DPPH free radical inhibition. In hydroxyl radical scavenging, the extract showed 

54.51% inhibition at 120 μg/ml concentration. PDEE prevents DNA damage against oxidative 

stress in concentration dependent manner. Anticancer activity was evaluated against six 

human cancer cell lines (MiaPaca-2, A-549, PC-3, THP-1, HCT-116 and HOP-620). The 

extract showed significant anticancer activity in concentration dependent pattern. The highest 

activity was shown against MiaPaca-2 cell line. PDEE induces significant apoptotic induction 

in cells. Exposure of MiaPaca-2 cells to PDEE (0-100 μg/ml) caused dose dependent 

cell cycle arrest at G 0 /G 1 phase and induced apoptosis by increasing accumulation of cells at 

G 0 /G 1 phase. PDEE increased the apoptotic cell population from 11.4% in case of control to 

49.6% at 100 μg/ml. Further, PDEE induces loss of mitochondrial membrane potential (∆Ψm) 

to 99.5% at 100 μg/ml from 24.4% in control cells. These primary results depict the free radical 

scavenging potential and anticancer activity of P. denticulata extracts. These findings may 

serve as foundation to develop an anticancer drug from medically important P. denticulata. 

Keywords: Apoptosis; DNA damage; DPPH; MiaPaCa-2 cells; Primula denticulata 


Cancer after cardiovascular diseases 

is the second leading mortality 

cause and is rapidly becoming a global 

pandemic. The worldwide incidence and 

mortality of cancer in 2008 were 12.66 

and 7.56 million cases respectively. According 

to (World Health Organization, 

2010) report, the global cancer burden is 

expected to nearly double to 21.4 million 

cases and 13.5 million deaths by 2030. 

Pancreatic cancer is the fourth most 

common cause of cancer-related deaths 

across the world with incidence equalling 

mortality and continues to pose an enormous 

challenge to clinicians and cancer 

scientists (Hariharan et al., 2008). Among 

all pancreatic cancers, pancreatic ductal 

adenocarcinoma (PDAC) is the most 

common epithelial, exocrine pancreatic 

malignancy, representing more than 80% 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 512


Free Radical Scavenging Potential and Anticancer Activity of Primula sp. 

Wani et al. 

of the malignant neoplasms of the pancreas 

(Alexakis et al., 2004). 

Consumption of fruits and vegetables 

is known to impart reduction in the 

incidence of ischemic heart disease and 

some types of cancer, particularly stomach, 

oesophagus, lung, oral cavity and 

pharynx, endometrial, pancreas and colon 

cancers (Mathew et al., 2004). Similarly 

natural antioxidant supplements (ascorbic 

acid, tocopherols, anthocyanin, β-carotene 

and other polyphenols have been associated 

with lower incidences of cancers and 

cancer related diseases (Fleischauer et al., 

2003). During some pathophysiological 

conditions, excess amount of reactive oxygen 

species (ROS) is being generated by 

certain external agents such as UVradiations, 

drugs, pollution, other xenobiotics 

and as well as by endogenous chemicals, 

especially stress hormones (adrenalin 

and noradrenalin). The superoxide 

dismutase (SOD) and other defence 

mechanisms in living organisms are unable 

to scavenge excess of ROS completely, 

which causes damage to cellular molecules 

such as DNA, RNA, enzymes, lipids 

etc. that results in fluidity of biomembranes 

(Dean and David, 1993) and 

development of degenerative diseases including 

caners, cardiovascular, neurodegenerative, 

Alzheimer’s and inflammatory 

diseases (Shahidi et al., 1992; Gerber 

et al., 2002; Di Matteo and Esposito, 

2003; Sreejayan and Rao, 1996). Hence 

there is growing interest in natural polyphenolic 

compounds, present in medicinal 

and dietary plants that might help attenuate 

oxidative damage (Silva et al., 2005). 

The increased incidence of different 

types of cancers during the last few 

decades and the modern techniques for 

separation, structure elucidation, screening 

and combitorinial synthesis have led 

to the development of new anticancer 

drugs, drug combinations and chemotherapy 

strategies by exploration of enormous 

pool of biological, synthetic and natural 

products (Mukherjee et al., 2001). So far 

several potential anticancer lead molecules 

such as taxol, vincristine, vinblastine, 

podophyllotoxin, camptothecin, 

combretastatins, flavopiridol, bruceatin 

etc, with diverse chemical structures have 

been isolated from plants. Several biologically 

active analogues such as taxotere, 

isotaxel (taxol analogues) topotecan, irinotecan, 

rubitecan, lurtotecan, 9-Amino 

CPT (camptothecin analogues), etoposide, 

teniposide (podophyllotoxin analogues), 

vinorelbine, hydravin (Vinca alkaloid derivatives) 

have been synthesised from these 

front line anticancer lead molecules 

(Vandana et al., 2005). As a result, emphasis 

has now been shifted towards the 

screening of apoptotic inducers from natural 

sources particularly from plants in 

the form of extracts or as isolated compounds 

that specifically increase apoptotic 

cell death in cancerous cells. 

Primula denticulata Sm. (Primulaceae) 

is an important member of genus 

primula, which represent more than 400 

species (Richards, 1993). P. denticulata is 

commonly known as drumstick primula 

or tooth-leaved primula. P. denticulata is 

20-30 cm tall perennial rarely annual, deciduous, 

clump-forming plant with compact 

heads of many flowers. The plant is 

widely distributed from eastern Afghanistan 

and northern Pakistan, across the 

Himalaya to Yunnan, Sichuan and Guizhou 

in China. In Kashmir Himalaya, the 

species is widely distributed (Map-1). The 

species thrives best in moist, shady 

slopes, mostly near melting glaciers and 

moist meadows, ranging in altitude from 

2100 – 4050 meters. 


2.1. Chemicals 

Sulphorhodamine-B (SRB), 

RPMI-1640 medium, fetal bovine serum 

(FBS), streptomycin, sodium bicarbonate, 

5-Fluorouracil, paclitaxel, gentamycin 

sulphate, trypsin, 1,1-diphenyl-2- 

picrylhydrazyl (DPPH), folin–Ciocalteu 

reagent, catechin, gallic acid were procured 

from Sigma-Aldrich. Trichloroacetic 

acid (TCA), butylated hydroxytoluene 

(BHT), thiobarbituric acid (TBA), hydro- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 513



Wani et al. 

N 

Map 1: Distribution of Primulla denticulata Sm. in Kashmir Himalaya, J&K- India. 

gen peroxide (H 2 O 2 ), ferric chloride, dimethyl 

sulfoxide (DMSO), potassium ferricyanide 

were purchased from Merck. 

The other reagents used were all of analytical 

grade. 

2.2. Collection and identification of plant 

Primula denticulata Sm. at flowering 

stage was collected from Gulmarg 

region of Kashmir Himalaya (latitude 

34°3'27" N; longitude 74°23'9" E) at altitude 

of 2650 m. The healthy plant species 

were randomly collected by hand-picking 

and later identified by Dr. Anzar A. 

Khuroo at department of Botany, University 

of Kashmir. A specimen under 

voucher number KASH-1743 was preserved 

for future reference. 

2.3. Extract preparation 

Fresh and healthy leaves of P. 

denticulata (1Kg) were cleaned with dou- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 514



Wani et al. 

ble distilled water, dried under shade (25± 

2°C) for 5-6 days. The dried plant material 

was ground to powder form. The plant 

powder was extracted in soxhelt apparatus 

using ethanol as solvent at desirable temperature. 

The filtered extract was concentrated 

using Buchi rotavapour and stored 

in glass vials at 4 o C until used. 

2.4. Estimation of total phenolics 

The total phenolic content in leaf 

extract of P. denticulata was determined 

by Folin–Ciocalteu method as adopted by 

Slinkard and Singleton (1977) with slight 

modifications. To 0.2 ml of plant extract 

(1mg/ml) was added to 2.5 ml of 10% diluted 

Folin–Ciocalteu reagent and 2 ml of 

2.5% aqueous Na 2 CO 3. The reaction mixture 

was incubated at room temperature 

with intermittent shaking. The blue colour 

solution was read at 765 nm on UV– 

visible spectrophotometer. The absorbance 

of solution was compared against 

standard Gallic acid (50 mg %) calibration 

curve. 

2.5. Estimation of total flavonoids 

The aluminium chloride colorimetric 

method as described by Mcdonald 

et al., (2001) was used to determine the 

total flavonoid content of leaf extract. The 

principle of this method is based on flavonoid–aluminium 

complex formation, 

which shows absorbance maximum at 430 

nm. Briefly 0.5 ml (1mg/ml) of extract 

was mixed with 1.5 ml of ethanol, 0.1 ml 

of 10% AlCl 3 , 0.1 ml of 1M potassium 

acetate and 2.8 ml of distilled water. After 

5 min of incubation, the absorbance was 

read at 430 nm. Flavonoid concentration 

was expressed as milligrams of catechin 

equivalents per gram dry weight. 

2.6. DPPH assay 

DPPH assay is one of the most extensively 

used method for determining the 

antioxidant potential of any biological 

sample. DPPH is a purple stable free radical 

which is reduced to yellow colour 

complex 1,1-diphenyl-2-picrylhydrazine 

(DPPH-H) by compounds which are capable 

of donating hydrogen or electron. 

100 μl of different concentrations (100- 

600 μg/ml) of plant extract or standard 

antioxidant was added to 1 ml DPPH solution 

(0.5 mM). The solution was slightly 

shaken and kept stand for 30 min at 

room temperature under dark conditions. 

The yellow colour solution was read at 

517 nm against ethanol (Brand-Williams 

et al., 1995). The free radical inhibition 

was calculated as: 

Percentage inhibition = [(A c -A s )/A c ] x 

100 

Where, A c and A s are the absorbance of 

control and sample respectively 

Butylated hydroxytoluene and α- tocopherol 

were used as positive control. 

2.7. Hydroxyl radical (HO ● ) scavenging 

assay 

Deoxyribose assay was used to 

evaluate the hydroxyl radical scavenging 

potential of P. denticulata leaf extract 

(Halliwell et al., 1987). The HO ● generated 

in Fenton reaction attack deoxyribose 

to form products that upon heating with 

thiobarbituric acid at low pH yield a pink 

chromogen (TBARS). A reaction mixture 

containing deoxyribose (25 mM), FeCl 3 

(10 mM), ascorbic acid (100 mM), H 2 O 2 

(2.8 mM) in 10 mM KH 2 PO 4 (pH 7.4) 

with or without plant extract at various 

concentrations (20-120 µg/ml) and incubated 

at 37 0 C for 1h. Then 1 ml of TBA 

(1% w/v) and 1 ml of TCA (3% w/v) 

were added and heated at 100 0 C for 20 

min. Absorbance of TBARS was read at 

532 nm. Deoxyribose oxidation inhibition 

was calculated as: 

Percentage inhibition = [(A-B)/A] ×100 

Where, A is malonaldehyde produced 

when treated with extract and B is malonaldehyde 

produced without extract. Butylated 

hydroxytoluene and α- tocopherol 

were taken as the positive control. 

2.8. DNA damage assay 

The Prevention of oxidative DNA 

damage by PDEE was determined by 

method as previously described by Ghanta 

et al., (2007). Calf thymus DNA 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 515



Wani et al. 

(0.37μg) with and without plant extract 

(10, 30, 50, 80 and 100 μg) was incubated 

with 20 mM ferric nitrate 30 mM H 2 O 2 in 

20.0 mM phosphate buffer (pH 7.4) in a 

final reaction mixture volume of 20 μl for 

1h at 37 o C. Oxidative DNA damage was 

induced by hydroxyl radicals generated in 

Fenton reaction (Ani et al., 2006). Bromophenol 

blue (0.25%) and glycerol 

(30%) were added to terminate reaction 

mixture, followed by gel electrophoresis 

in 0.7% agarose. The gel was then visualized 

and photographed on gel doc. 

2.9. Cells culture 

Pancreatic cell line (MiaPaca-2), 

Lung cell line (A-549), Prostate cell line 

(PC-3), Leukaemia cell line (THP-1), Colon 

cell line (HCT-116) and Lung cell line 

(HOP-62) were purchased from National 

Cancer Institute, U.S.A and European 

collection of cell culture, UK. Cells were 

cultured in RPMI-1640 and MEM medium 

supplemented with nutrients and antibiotics. 

Cells were grown at 37 o C and 5% 

CO 2 level with relative humidity of 98%. 

2.10. Anticancer activity 

The anticancer activity of PDEE 

against different human cancer cell lines 

was evaluated by SRB assay as described 

by Monks et al., 1991. Briefly 100µl of 

cell suspension (1x10 5 cells/well) were 

cultured in 96-well plates and incubated 

overnight at 37 0 C and 5% CO 2 level. 20 

µl test material at various final concentrations 

(10-100 µg/ml) was added. Paclitaxel 

(1 µM) and 5-fluorouracil (20 µM) 

were used as standard anticancer drugs. 

Cell growth was stopped after 48 hrs of 

incubation by adding 50 μl of 50% TCA 

in each well and incubated further for 1h 

at 4 0 C. The plates were then washed, air 

dried and stained with 50 μl of 0.4% SRB 

dye in 1% acetic acid, followed by incubation 

for 30 min at room temperature. 

The unbound dye was then removed by 

washing with 1% acetic acid and kept 

overnight for drying. In each well, 100 μl 

of 10 mM tris-base was added to solubilise 

the dye followed by stirring for 5 min 

at room temperature. The optical density 

was read at 570 nm using ELISA reader. 

The experiments were done in triplicates. 

Percentage cell growth was calculated as: 

Percentage cell viability = [At/Ac] ×100 

Percentage cell growth inhibition = (100- 

percentage cell viability) 

Where At and Ac are absorbance of treated 

and control cells, respectively. 

2.11. DNA content and cell cycle phase 

distribution 

Human pancreatic (MiaPaca-2) 

cells were seeded in 6-well culture plates 

with cell density 2x10 5 cells/ml/well and 

incubated for 24 hrs. After incubation, the 

cells were treated with PDEE (0, 30, 50 

and 100 mg/ml) and again incubated for 

48 hrs. After 48 hrs treatment cells were 

collected by 5 min centrifugation at 1000 

rpm. The harvested cells were washed 

twice with phosphate buffer solution and 

fixed with 70 % ethanol at -20 ºC for 1h. 

The cells were then stained with DNA 

staining solution containing propidium 

iodide (20 mg/ml) and triton X-100 (1%) 

in PBS for 30 min in dark. FACScan was 

used to measure DNA content. For each 

data file, data was collected from 10,000 

cells. Cell Quest (Becton, USA) was used 

for analysis of histograms. 

2.12. Loss of Mitochondrial Membrane 

Potential (ΛΨm) 

Flow cytometry was used to measure 

the mitochondrial membrane potential 

loss (ΛΨm). Human pancreatic (MiaPaca- 

2) cells were plated in 6-well cultural 

plates with cell density of 1x10 6 

cells/ml/well and incubated for 24 hrs at 5 

% CO 2 level. The cells were then treated 

with PDEE (0, 30, 50 and 100 mg/ml) and 

again incubated for 48 hrs. Rhodamine- 

123, a cell permeable cationic dye was 

added one hour before termination of experiment 

and again incubated for 30 min. 

The cells were washed with PBS and pellets 

were collected by centrifugation. The 

collected pellets were re-suspended in 300 

ml of PBS. Florescence of Rh-123 in cells 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 516



Wani et al. 

were analysed at 485 nm by flow cytometer 

(Jung et al., 2006). 

2.13. Statistical analysis 

All of the experiments were done in 

triplicate. The data were recorded as 

means ± standard deviations and were 

analysed with SPSS software. 


3.1. Total phenolic and flavonoid content 

Polyphenolic compounds are very 

important plant bioactive constituents because 

of their scavenging potential due to 

the presence of hydroxyl groups (Hatano 

et al., 1989). Folin-Ciocalteu method is 

most widely used to measure the polyphenol 

contents, with the basic mechanism 

of electron transfer and reducing 

ability (Prior and Schaich, 2005). Using 

this quantitative assay, we found that the 

total phenolic content (TPC) of ethanolic 

leaf extract of P. denticulate was found 

to be 12.24 ± 2.11 (mg GAE/g dry extract) 

as depicted in Figure 1. Considerable 

attention has been received by polyphenols 

for their physiological role as antioxidant 

and anticancer agents (Othman 

et al., 2007). Polyphenolic compounds h- 

them to act as strong reducing agents, hydrogen 

donor’s metal chelaters and singlet 

oxygen quenchers (Miguel, 2010). The 

total flavonoid content in DPEE was 

found to be 7.06 ± 1.31 (mg catechin/g 

dry extract). Flavonoids are known exhibit 

many biological activities like antioxidant, 

anticancer, antimicrobial and antiinflammatory 

properties (Hodek et al., 

2002). 

3.2. DPPH free radical scavenging activity 

DPPH is a purple stable free radical 

at room temperature with characteristic 

absorbance at 517 nm. The nitrogen 

free radical of DPPH is easily quenched 

by an antioxidant to yellow coloured 

complex (1,1-diphenyl-2-picrylhydrazine). 

The decolourization of purple colour 

is stoichiometric depending on the 

number of electrons gained (Soares et al., 

1997; Mokbel et al., 2006; Singh et al., 

2002). DPPH radical scavenging potential 

of PDEE at different concentrations investigated 

in the present study was determined 

together with standard antioxidants 

(BHT and α-tocopherol) at the same concentrations 

(Figure 2). PDEE showed significant 

scavenging effect on DPPH free 

Figure 1: Represents the total phenolic 

and flavonoid content of PDEE. Each 

value represents the mean ± SD (n = 3). 

-ave been reported to possess strong antioxidant 

potential their by scavenging free 

radicals and protect cells against such oxidative 

damages (Kahkonen et al., 1999). 

The antioxidant potential of medicinal 

plants is due to the redox properties of 

polyphenolic compounds, which enables 

Figure 2: DPPH radical scavenging activity 

of PDEE and known antioxidant BHT 

and α-tocopherol. Values are means of 

triplicate experiments (n = 3) ± standard 

deviation. 

radical in concentration dependent manner. 

When compared with standard anti- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 517



Wani et al. 

oxidants used in the experiment, the extract 

showed relatively lower DPPH free 

radical scavenging potential. The extract 

at 600 µg/ml produced 69.35% inhibition, 

while as BHT and α-tocopherol produced 

85.95% and 89.65% inhibition at the 

same concentration. The DPPH radical 

scavenging activity of PDEE as such 

might prevent reactive radical species 

from damaging biomolecules such as 

DNA, protein, polyunsaturated fatty acids 

(PUFA) and sugars in susceptible biological 

and food systems. 

3.3. Hydroxyl (HO • ) radical scavenging 

activity 

Among different free radicals 

generated in biological systems, hydroxyl 

radical (HO • ) is one of the most reactive 

species, which is capable to damage almost 

all the molecule in living cells, 

which ultimately leads to carcinogenesis 

and mutagenesis (Manian et al., 2008; 

Hochestein and Atallah, 1988). This radical 

is considered to be one of the important 

initiators in the process of lipid 

peroxidation, abstracting hydrogen atoms 

from unsaturated fatty acids (Kappus et 

al., 1991). In the present study, hydroxyl 

radical scavenging ability was estimated 

by generating hydroxyl radicals using 

ascorbic acid–iron-H 2 O 2 (Fenton reaction). 

Antioxidant efficiency of PDEE 

was determined as the ability to scavenge 

the free radicals generated. The extract 

exhibited a concentration dependent scavenging 

of hydroxyl radicals which was 

comparable to the reference standards 

(BHT & α-tocopherol) at the same concentration. 

The percentage inhibition of 

hydroxyl radical scavenging is shown in 

Figure 3. The extract showed antioxidant 

activity in concentration dependent manner. 

A 120 μg/ml of PDEE, BHT and α- 

tocopherol exhibited 54.51%, 81.20% and 

88.25% inhibition, respectively. 

3.4. Prevention of oxidative DNA damage 

Oxidative damage by hydroxyl 

radicals make DNA susceptible by oxidation 

of guanosine or thymine to 8- 

hydroxyl-2-deoxyguanosine and thymine 

glycol which leads to mutagenesis and 

carcinogenesis (Ames et al., 1993). PDEE 

prevents calf thymus DNA from oxidative 

damage due to hydroxyl radicals generated 

by FeSO 4 and H 2 O 2 in Fenton reaction 

using agrose gel electrophoresis. Figure 4 

shows the protective effect of PDEE on 

calf thymus DNA. The Hydroxyl radicals 

induce DNA strand breaks and causes 

complete DNA damage (Lane 2). PDEE 

at different concentration (10–100 μg/ml) 

offered concentration dependent protection 

to DNA damage (Lane 3-7). Catechin 

(10μg/ml) was used as standard antioxidant 

(Lane 8). Thus, the results indicate 

that PDEE prevents DNA damage against 

oxidative stress. The hydroxyl radical 

quenching ability of polyphenolic compounds 

of P. denticulate could be responsible 

for the protection against oxidative 

damage. 

3.5. Anticancer activity 

The anticancer activity of PDEE was determined 

by SRB assay against six human 

cancer cell lines. The assay is based on 

measuring the content of cellular protein 

using SRB dye (Vanicha and Kanyawim, 

2006). Treatment of cells with PDEE (10- 

100 µg/ml) exhibited concentration dependent 

anti-proliferative effect (Table 1). 

The results of the present study reveal that 

the plant extract is very active against all 

the cell lines used. The highest percentage 

cell growth inhibition was observed in 

MiaPaca-2, HCT-116 and THP-1 with 

mean percentage value of 99.06 ± 0.30, 

98.45 ± 0.65 and 98.33 ± 1.25 respectively. 

The susceptibility of cells to the drug 

exposure was characterized by its IC 50 

values. Lower IC 50 value indicates the 

higher anticancer potential of plant extract. 

The results of our study reveal that 

PDEE inhibits proliferation of cancer cell 

lines. The cytotoxic activity may be due 

to individual polyphenolic phytochemicals 

that act synergistically with other 

compounds to display the anticancer activity, 

as has been suggested by Yang et 

al. (2009). 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 518



Wani et al. 

Figure 3: Effect of PDEE and known antioxidant BHT and α-tocopherol on hydroxyl radical 

scavenging potential. Values are means of triplicate experiments (n = 3) ± standard deviation. 

Figure 4: PDEE Protects calf thymus DNA f oxidative damage. Lane 1: Native calf thymus 

DNA; Lane 2: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid + 30mM H 2 O 2; Lane 

3: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid + 30mM H 2 O 2 + 10µg of extract; 

Lane 4: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid + 30mM H 2 O 2 + 30µg of 

extract; Lane 5: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid + 30mM H 2 O 2 + 

50µg of extract; Lane 6: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid + 30mM 

H 2 O 2 + 80µg of extract; Lane 7: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid + 

30mM H 2 O 2 + 100µg of extract; Lane 8: DNA + 20mM Ferric Nitrate + 100mM Ascorbic 

Acid + 30mM H 2 O 2 + 10µg of catechin. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 519



Wani et al. 

Table 1: Anticancer activity of ethanolic leaf extract of Primula denticulate # 

Sample 

Conc. 

(µg/ml) 

MiaPaca-2 

(Pancreatic) 

Percentage cell growth inhibition 

A-549 

(Lung) 

PC-3 

(Prostate) 

THP-1 

(Leukaemia) 

HCT- 

116 

(Colon) 

PDEE 100 99.06 ± 0.30 95.67 ± 

1.85 

97.09 ± 

2.09 

98.33 ± 1.25 98.45 ± 

0.65 

50 73.15 ± 2.76 39.18 ± 40.23 ± 93.27 ± 2.10 83.21 ± 

1.90 3.45 

1.67 

10 44.36 ± 2.09 17.05 ± 15.68 ± 38.35 ± 2.43 28.80 ± 

1.16 2.53 

1.56 

5-FU 20µM - - - 67.87 ± 1.56 67.00 ± 

1.87 

Paclitaxel 1µM - 70.78 ± 

2.45 

# The results represent mean ± S.D of three experiments. 

HOP- 

62 

(Lung) 

93.68 

± 3.20 

47.08 

± 2.34 

10.96 

± 3.05 

- 

- - - 72.43 

± 2.78 

3.6. Flow cytometric analysis 

To evaluate the action mechanism 

of cell growth inhibition by PDEE, further 

experiments were performed on human 

pancreatic (MiaPaca-2) cell line. In the 

present study, apoptotic induction was 

assessed by two assays; Cell-cycle phase 

distribution via propidium iodide (PI) 

staining flow cytometry and mitochondrial 

membrane potential loss using Rhodamine-123 

staining flow cytometry. MiaPaca-2 

cells stained with PI were treated 

with PDEE (0, 30, 50 and 100 µg/ml) for 

48 hrs showed that the percentage of 

apoptotic nuclei increased to 57.00% at 

100 µg/ml PDEE from 11.4% in control 

(Figure 5). 

Figure 5: PDEE induces apoptosis of human pancreatic cancer (MiaPaca-2) cells via cell 

cycle arrest. Flow cytometric analysis of MiaPaca-2 cells after propidium iodide staining. 

Cells were incubated for 48 h in presence of PDEE (0, 30, 50 and 100 µg/ml). Figures show 

the representative staining profile of one of two similar experiments. P1 is the population of 

apoptotic cells, which increases from 11.4% in case of control to 57.00% in case of 100 

µg/ml of PDEE. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 520



Wani et al. 

Figure 6: PDEE induced loss of mitochondrial membrane potential (∆Ψ m ) in human pancreatic 

cancer cell line (MiaPaca-2) incubated with extract at different concentrations (0, 30 

50 and 100 µg/ml) in 6 well plate for 48 h treatment. 

Apoptotic cells were characterised by degraded 

chromatin with high side-scatter 

(SSC) and low forward-scatter (FSC) 

properties (Bachir et al., 2012). The results 

indicate that PDEE blocks cell cycle 

progression, resulted in significant increase 

in population of cells in sub G 0 /G 1 

phase, which may be due to fragmentation 

of DNA, resulted in apoptotic cell death. 

Disruption of MMP (∆Ψ m ) is one of the 

earliest events that occur following apoptosis 

induction (Qi et al., 2010). MiaPaca- 

2 cells after treated with PDEE for 48 hrs 

resulted in loss of (∆Ψ m ) from 24.4% in 

control cells to a low of 99.5% at 100 

µg/ml of PDEE (Figure 6). MMP loss is 

mainly due to mitochondrial permeability 

transition pore, which causes Cytochrome-C 

release from mitochondria and 

ultimately triggers other apoptotic factors 

(Kroemer et al., 1997). The increase in 

inner mitochondrial membrane permeability 

is due to interaction of anticancer 

agents with mitochondria (Fulda et al., 

1998). 

4. Conclusion 

The P. denticulata ethanol extract 

exhibit potent antioxidant property as revealed 

by different antioxidant assays. 

PDEE showed strong anticancer activity 

and induces apoptosis in MiaPaca-2 cell 

line by arresting cells at G 0 /G 1 phase and 

inducing loss of MMP (∆Ψ m ). Further research 

is required to explore the potential 

of P. denticulata in developing an anticancer 

drug. 


Authors are thankful to UGC, 

New Delhi for providing financial support 

to Bilal Ahmad Wani in the form of Dr. 

D.S. Kothari Postdoc fellowship. 

References 

Alexakis, N., Halloran, C., Raraty, M., 

Ghaneh, P., Sutton, R., Neoptolemos, 

J.P. (2004). Current 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 521



Wani et al. 

standards of surgery for pancreatic 

cancer. British Journal of Surgery 

91, 1410-27. 

Ames, B.N., Shigenaga, M.K., Hagen, 

T.M. (1993). Oxidants, antioxidants, 

and the degenerative diseases 

of aging. Proceedings of the 

National Academy of Sciences 90, 

7915–7922. 

Dean, R.T., David, M.J. (1993). Reactive 

species and their accumulation 

on radical damaged proteins. 

Trends in Biochemical Sciences 

18, 437-441. 

Di Matteo, V., Esposito, E. (2003). Biochemical 

and therapeutic effects 

of antioxidants in the treatment of 

Alzheimer’s disease, Parkinson’s 

disease and amyotrophic lateral 

sclerosis. Current Drug target 

CNS neurological Disorders 2, 

95-107. 

Fleischauer, A.T., Simonsen, N., Arab, 

L. (2003). Antioxidant supplements 

and risk of breast cancer recurrence 

and breast cancer-related 

mortality among postmenopausal 

women. Nutrition and Cancer 46, 

15–22. 

Fulda, S., Susin, S.A, Kroemer G, Debatin 

KM. (1998). Molecular ordering 

of apoptosis induced by anticancer 

drugs in neuroblastoma 

cells. Cancer Research 58, 4453– 

4460. 

Gerber, M., Boutron-Ruault, M.C., 

Hercberg, S., Riboli, E., Scalbert, 

A., Siess, M.H. (2002). 

Food and cancer: state of the art 

about the protective effect of fruits 

and vegetables. Bulletin du Cancer 

89, 293-312. 

Ghanta, S., Banerjee, A., Poddar, A., 

Chattopadhyay, S. (2007). Oxidative 

DNA damage preventive 

activity and antioxidant potential 

of Stevia rebaudiana (Bertoni) 

Bertoni, a natural sweetener. 

Journal of Agricultural and Food 

Chemistry 55, 10962–10967. 

Halliwell, B., Gutteridge, J.M., Aruoma, 

O.I. (1987). The deoxyribose 

method: a simple “test tube” 

assay for determination of rate 

constants for reactions of hydroxyl 

radicals. Analytical Biochemistry 

165, 215-219. 

Hariharan, D., Saied, A., Kocher, H.M. 

(2008). Analysis of mortality rates 

for pancreatic cancer across the 

world. International Hepato- 

Pancreato-Biliary Association 10, 

58-62. 

Hochestein, P., Atallah, A.S. (1988). 

The nature of oxidant and antioxidant 

systems in the inhibition of 

mutation and cancer. Mutation Research 

202, 363-375. 

Hodek, P., Trefil, P., Stiborova, M. 

(2002). Flavonoids- Potent and 

versatile biologically active compounds 

interacting with cytochrome 

P450. Chemico-Biological 

Interactions 139, 1-21. 

Jung, G.R., Kim, K.J., Choi, C.H., Lee, 

T.B., Han, S.I., Han, H.K. 

(2007). Effect of betulinic acid on 

anticancer drug-resistant colon 

cancer cells. Basic and Clinical 

Pharmacology & Toxicology 101, 

277–85. 

Kahkonen, M.P., Hopia, A., Vuorela, 

H.J., Rauha, J.P., Pihlaja, K., 

Kujala, T.S., Heinonen, M. 

(1999). Antioxidant activity of 

plant extracts containing phenolic 

compounds. Journal of Agriculture 

and Food Chemistry 48, 

1485–1490. 

Kappus, H., (1991). Lipid peroxidation– 

Mechanism and biological relevance. 

In Aruoma, O.I., Halliwell, 

B., (Eds.), Free radicals and food 

additives (pp. 59-75).London, UK: 

Taylor and Francis. 

Kroemer, G., Zamzami, N., Susin, S.A. 

(1997). Mitochondrial control of 

apoptosis, Immunology Today 18, 

44–51. 

Manian, R., Anusuya, N., Siddhuraju, 

P., Manian, S., (2008). The anti- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 522



Wani et al. 

oxidant activity and free radical 

scavenging potential of two different 

solvent extracts of Camellia 

sinensis (L.) O. Kuntz, Ficus bengalensis 

L. and Ficus racemosa L. 

Food Chememisry 107, 1000– 

1007. 

Mathew, A., Peters, U., Chatterjee, N., 

Kulldroff, M., Sinha, R. (2004). 

Fat, fiber, fruits, vegetables, and 

risk of colorectal adenomas. International 

Journal of Clinical Nutrition 

78, 517-520. 

Mcdonald, S., Prenzler, P.D., Autolovich, 

M., Robards, K. (2001). 

Phenolic content and antioxidant 

activity of olive extracts. Food 

Chemistry 73, 73–84. 

Miguel, M.G. (2010). Antioxidant activity 

of medicinal and aromatic 

plants. A review. Flavour Fragrance 

Journal 25, 291- 312. 

Mokbel, M.S., Hashinaga, F. (2006). 

Evaluation of the antioxidant activity 

of extracts from buntan (Citrus 

grandis Osbeck) fruit tissues. 

Food Chemistry; 94,529−34. 

Monks, A., Scudiero, D., Skehan, P., 

Shoemaker, R., Paull, K., Vistica, 

D., Hose, C., Langley, J., 

Cronise, P., Vaigro-Wolff, A., 

Gray-Goodrich, M., Cambell, 

H., Mayo, J., Boyd, M. (1991). 

Feasibility of a high-flux anticancer 

drug screen using a diverse 

panel of cultured human tumour 

cell lines. Journal of the National 

Cancer Institute 83, 757. 

Othman, A., Ismail, A., Ghani, N.A., 

Adenan, I. (2007). Antioxidant 

capacity and phenolic content of 

cocoa beans. Food Chemistry. 

100,1523–1530. 

Prior, R.L., Wu, X., Schaich, K. (2005). 

Standardized methods for the determination 

of antioxidant capacity 

and phenolics in foods and dietary 

supplements. Journal of Agricultural 

and Food Chemistry. 53, 

4290–4303. 

Qi, F., Li, A., Zhao, L., Xu, H., Inagaki, 

H., Wang, D., Cui, X., Gao, B., 

Kokudo, N., Nakata, M., Tang, 

W. (2010). Cinobufacini, an aqueous 

extract from Bufo bufo Gargarizans 

cantor, induces apoptosis 

through a mitochondria-mediated 

pathway in human hepatocellular 

carcinoma cells. Journal of Ethnopharmacology. 

128, 654–661. 

Richards, J., Richards, A.J., Edwards, 

B. (1993). Primula timber (Botanical 

illustrations). Timber Press, 

Portland.Silva, B.A., Ferreres, 

F., Malva, J.O., Dias, A.C.P. 

(2005). Phytochemical and antioxidant 

characterization of Hypericum 

perforatum alcoholic extracts. 

Food Chemistry. 90, 157–167. 

Singh, R.P., Murthy, K.N.C., Jayaprakasha, 

G.K. (2002). Studies on 

the antioxidant activity of promegranate 

(Punica granatum) peel 

and seed extracts using in vitro 

models. Journal of Agricultural 

and Food Chemistry. 50, 81−6. 

Slinkard, K., Singleton, V.L. (1977). 

Total phenol analyses: automation 

and comparison with manual 

methods. American Journal of 

Enology and Viticulture. 28, 49– 

55. 

Soares, J.R., Dinis, T.C.P., Cunha, 

A.P., Almeida, L.M., (1997). Antioxidant 

activities of some extracts 

of Thymus zygi. Free Radical 

Research. 26, 469–478. 

Sofowora, A. (1993). Medicinal Plants 

and Traditional Medicine in Africa. 

2nd Edn. Spectrum Books Limited, 

Ibadan, Nigeria 1-153. 

Sreejayan, N., Rao, M.N.A. (1996). Free 

radical scavenging activity of curcuminoids. 

Arzneimittel forschung 

46, 169-171. 

Vandana, S., Arvind, S.N., Kumar, 

J.K., Gupta, M.M., Suman, 

P.S.K. (2005). Plant-based anticancer 

molecules: A chemical and 

biological profile of some im- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 523



Wani et al. 

portant leads. Bioorganic and Medicinal 

Chemistry 13, 5892–5908. 

Vanicha, V., Kanyawim, K. (2006). Sulforhodamine 

B colorimetric assay 

for cytotoxicity screening. Nature 

protocols 3, 1112-1116. 

Yang, J., Liu, R.H., Halim, L. (2009). 

Antioxidant and antiproliferative 

activities of common edible nut 

seeds. LWT– Food Science and 






ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 524



Panchakavya: Organic Fertilizer and Its Stimulatory 

Effect on the Seed Germination of Abelmoschus 

esculentus and Solanum melongena 

V. Ramya and S. Karpagam* 

Department of Botany, Queen Mary’s College, Chennai - 4, Tamil Nadu, India; 

*Correspondence: s.karpagam98@gmail.com; Tel.: +91 9444944835 

Abstract: Panchakavya is an incredible source of growth promoting substances. From ancient 

period cow‟s urine has been used as a medicine. In India, drinking of cow urine has 

been practiced for thousands of years. Panchakavya is a term used in Ayurveda to describe 

five important major substances, obtained from cow, which include cow‟s urine, milk, 

ghee, curd and dung all the five products posses medicinal properties against many disorders 

and are used for the medicinal purpose singly or in combination with some other 

herbs. This kind of treatment is called „panchakavya therapy‟ or „cowpathy‟. The indiscriminate 

use of chemical pesticides resulted in environmental problems. An alternative to the 

chemicals is the natural products, one such as the panchakavya. Panchakavya is the single 

organic input that acts as a fertilizer, pesticide, growth promoter and immunity booster. 

The effect of panchakavya (cow urine) on the seed germination was studied in two plants 

namely Abelmoschus esculentus L.Moench (Lady‟s finger) and Solanum melongena L. 

(brinjal). The germination percentage was calculated. After germination the shoot and root 

length was measured and seedling vigour index was calculated. Cytotoxic effect of panchakavya 

on cell growth and cell division was studied in onion bulbs. The panchakavya at 

1% concentration favoured the production of larger number of roots. This article highlights 

that panchakavya is more effective, easy to prepare, environmental friendly and could be 

used as a good fertilizer to boost the growth and productivity of agricultural crops. 

Keywords: Abelmoschus: mitotic index; panchakavya: Solanum melongena: seed germination 


Agriculture is means of livelihood 

for millions of people in India and 

worldwide with crops chiefly dependent 

on rainfall and fertilizers. India is not only 

self-sufficient in food production but 

also has a substantial reserve (Gupta and 

Gopal, 2001). The latest trend is turned to 

organic farming, since the side effects of 

chemical fertilizers and pesticides have 

been established. India is the third largest 

producer and consumer of chemical fertilizers 

in the world. Heavy use of chemicals 

in agriculture has weakened the ecological 

balance, in addition to the degradation 

of soil; water resources and quality 

of the food. At this juncture, a keen 

awareness has sprung on the adoption of 

organic farming as a remedy to cure the 

ills of modern chemical agricultural practice. 

It is very much essential to develop a 

strong workable and compatible package 

of nutrient management through organic 

resources for various crops, based on scientific 

facts, local conditions and economic 

viability (Nene, 1994; 1999). Cow 

is described as “Kamdhenu” (one which 

fulfills all the wishes) since vedic times in 

Indian civilization. According to ayurveda 

cow products are used to treat various 

disease conditions in human beings. Five 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 525


Use Organic Fertilizers for Sustainability 

Ramya and Karpagam 

products of cow called as panchakavya is 

an important component of many rituals 

and pooja in Hindus. (Gosavi and Jhon, 

2012; Sathasivam et al., 2010). In Sanskrit, 

panchakavya means the blend of 

five products obtained from cow (all these 

five products are individually called 

„Gavya‟ and collectively termed as „Panchakavya). 

When suitably mixed and 

used, has positive influence on living organisms. 

Panchakavya had got reverence 

in the scripts of Vedas (divine scripts of 

Indian wisdom), and Vrkshayurveda (vrksha 

means plant and ayurveda means 

health system). The texts on Vrkshayurveda 

are systematization of the practices 

that the farmers followed at field level, 

placed in a theoretical framework and it 

defined certain plant growth stimulants; 

among them panchakavya was an important 

one that enhanced the biological 

efficiency of crop plants and the quality 

of fruits and vegetables (Natarajan, 2002). 

Green revolution lead to intensified 

agriculture to meet the ever increasing 

demand for food and fiber, which is a 

practice at great cost to the environment 

resulting in continuous loss of natural 

ecosystems, ground water depletion, pollution 

and other environmental degradation 

(Gupta and Gopal, 2001). Alternative 

approaches to pest control is the concept 

of integrated pest management, 

where synthetic pesticides are only applied 

as a last resort and is now considered 

common practice in professional agriculture. 

The non-chemical alternatives 

include cultural practices, use of resistant 

varieties, creation of an environment favourable 

for natural enemies of pests and 

use of biological products and agents, including 

beneficial insects. 

The indiscriminate use of chemical 

pesticides in modern agriculture resulted 

in the development of several problems 

such as pesticide resistant insects, 

resurgences of target and non-target pest, 

destruction of beneficial organism like 

honey bee, pollinaters, parasites and 

predators and pesticide residues in food 

and fodder. The awareness about the 

health and environmental problems due to 

the continuous use of pesticides resulted 

in the development of integrated pest 

management and organic farming. 

Organic manure replaced chemical 

fertilizers, herbal extracts replaced 

pesticides and fungicides, but nothing was 

available to replace growth promoting 

hormones and immunity for plants. The 

organic system was imperfect and continued 

to be incomplete for want of an input 

to replace growth promoting hormones 

and immunity boosters, to maximize the 

efficiency of cultivated crops and coordinate 

the process leading to sustained 

higher productivity. 

Many alternatives are available to 

reduce the effects pesticides have on the 

environment. Alternatives include manual 

removal, applying heat, covering weeds 

with plastic, placing traps and lures, removing 

pest breeding sites, maintaining 

healthy soils that breed healthy, more resistant 

plants, cropping native species that 

are naturally more resistant to native pests 

and supporting biocontrol agents such as 

birds and other pest predators. 

Biological controls such as resistant 

plant varieties and the use 

of pheromones, have been successful and 

at times permanently resolve a pest problem. 

Integrated Pest Management (IPM) 

employs chemical use only when other 

alternatives are ineffective. IPM causes 

less harm to humans and the environment. 

The focus is broader than on a specific 

pest, considering a range of pest control 

alternatives. The use of phosphorous and 

nitrogen fertilizer in the global level has 

increased manifold, which effects the environment 

as run off into water bodies. 

The indiscriminate use of pesticide and 

fungicide has increased at global level, 

which is biomagnified at the tertiary level 

consumers. 

Panchagavya or panchakavyam is 

a concoction prepared by mixing five 

products of cow and used in traditional 

Indian rituals. The three direct constituents 

are cow dung, urine, and milk; the 

two derived products are curd and ghee. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 526




Figure 1: Global map of phosphorus fertilizer application rates (kg per ha of grid cell area) 

[source: https://ourworldindata.org/fertilizer-and-pesticides/#note-6]. 

Figure 2: Global map of nitrogen fertilizer application rates (kg per ha of grid cell area) 

[Source: https://ourworldindata.org/fertilizer-and-pesticides/#note-7]. 

These are mixed in proper ratio and then 

allowed to ferment. Panchamrita is a 

similar mixture that replaces dung and 

urine with honey and sugar. The mixture 

which is made using yeast as a fermenter, 

bananas, groundnut cake, and the water of 

tender coconut, is believed to be a potent 

organic pesticide and growth promoter 

- this is considered to 

be pseudoscience. 

The Sanskrit word Panchagavya m 

eans "mixture of five cow products". It is 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 527




also called cowpathy treatment based on 

products obtained from cows used 

in Ayurvedic medicine and of religious 

significance for Hindus. Panchgavya is 

also used as fertilizers and pesticides in 

agricultural operations, but has no scientific 

evidence to back its claims. 

Cow dung contained undigested 

fibre, epithelial cells, pigments and salts, 

rich in nitrogen, phosphorous, potassium, 

sulphur, micronutrients, intestinal bacteria 

and mucous, cow dung is also rich in bacteria, 

fungi and other microbial organisms 

(Nene, 1999). Singh (1996) recorded that 

cow dung had water 82% and solid matter 

18%. Reddy (1998) reported that cows 

urine is rich in urea and acted both as nutrient 

as well as hormone. Cow milk was 

used by farmers in ancient times and reported 

to be an excellent sticker, spreader, 

a good medium for saprophytic bacteria 

and a virus inhibitor (Nene, 1999). 

They are used as a Prasad in temples. 

A common usage is as a fertilizer 

and pesticide. Seeds can be treated with 

panchagavya. This was found useful in 

rhizome of turmeric, ginger and sugarcane 

and they yielded more, helps in plant 

growth and immunity. The medicinal usage 

of panchagavya, particularly cow 

urine, is practiced in Ayurveda. Proponents 

claim that cow urine therapy is capable 

of curing several diseases, including 

certain types of cancer, although these 

claims have no scientific backing. In fact, 

studies concerning ingesting individual 

components of Panchagavya, such as cow 

urine, have shown no positive benefit, and 

significant side effects, including convulsion, 

depressed respiration, and 

death. Cow's urine can also be a source of 

harmful bacteria and infectious diseases, 

including leptospirosis. Proponents claim 

it is an antibiotic growth promoter in the 

broiler diet, capable of increasing the 

growth of plankton for fish feed, the production 

of milk in cows, the weight of 

pigs, and the egg laying capacity of poultry 

chicken. 


2.1. Preparation of panchakavya 

The ingredients for panchakavya 

sample was collected from cow farm 

(Thiruvallur DT)) using sterile container. 

Based on the detailed review of literature 

panchakavya stock solution was prepared 

by using cow dung (2.5 Kg), cow‟s urine 

(1.5 L), cow‟s milk (1L), cow‟s curd (1 

L) and cow‟s ghee (0.5 kg). In addition, 

jaggery (1.5 Kg), tender coconut water 

(1.5 L) and ripe banana (6 Nos.) were also 

added as modification. All the materials 

were placed in a wide mouthed mud 

pot and kept open under shade. The contents 

were stirred twice a day for about 20 

mintues, both in the morning and evening 

to facilitate aerobic microbial activity. 

The panchakavya stock solution will be 

ready after 30 d (care should be taken not 

to mix buffalo products, local breeds of 

cow is said to have potency then exotic 

breeds) and is covered with a plastic 

mosquito net to prevent houseflies from 

laying eggs and the formation of maggots 

in the solution. Jaggery is dissolved in 

water and used while sugarcane juice is 

more suitable. 

2.2. Seed collection 

The seeds of Abelmoschus esculentus 

(ladies finger) and Solanum 

melongina (Brinjal) were bought from 

nursery Balaji traders, Chennai 601203, 

Tamil Nadu. Healthy Seeds of uniform 

size and shape were used for sowing. 

2.3. Treatments 

Treatments were given as: 1. Control 

(water); 2. Chemical (Cartap hydrochloride 

50% radon sp + tata tafgor dimethoate 

30% ec = 2:1 ratio and dissolved 

in 10 ml of water); 3. Panchakavya; 4. 

Panchakavya +Neem cake; 5. vermicompost 

were used. 

2.4. Germination of seeds 

The seeds (40 numbers) were 

soaked in sterile water in Petri plates with 

filter paper. After 24 hours, the seeds 

were observed for germination. The ger- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 528




minated seeds were counted, the radical 

and plumule length were measured (cm) 

and tabulated. 

2.5. Seedling length 

Two days after seed germination, 

ten normal seedlings were taken out carefully 

at random from each treatment and 

seedling length was measured from the tip 

of primary root to the tip of apical shoot. 

The average length (cm) of ten seedlings 

was calculated and expressed as mean 

seedling length. 

2.6. Seedling vigour index 

The seedling vigour index was 

calculated adopting the method suggested 

by Abdul-Baki and Anderson (1973) and 

expressed whole number treatment wise 

Vigour index = Germination percentage 

× Seedling length 

2.7. Effect of panchakavya on cell division 

and cell growth 

Cytological studies of root tip of 

Allium cepa was studied for 7 days. Onion 

bulbs were treated with diluted panchakavya 

(50%, 25%, 10% and 1%).and 

tap water as control. Allium cepa din 

(2n=16) (Rank and Nielsen 1994) were 

used as test system. The root tip from 

control and experimental setup were thoroughly 

washed in distilled water and 

fixed in Carnoy‟s fixative (ethanol, chloroform 

glacial acetic acid 6:3:1 v/v/v) and 

chromosome studies were done by acetocarmine 

staining technique. The fixed 

root tip were washed in distilled water 

and hydrolyzed in IN HCL for 10 min 

then treated in 45% acetic acid for 5 min 

and stained in acetocarmarine stain for 

10-15 minutes. After staining the root tips 

were squashed and observed under phase 

contrast microscope. 


The effect of panchakavya on 

seed germination of two plants namely 

Abelmoschus esculentus and Solanum 

melongena was studied in sterile Petri 

plates. Seeds of 40 numbers were soaked 

in 2ml of water (Control), in 2ml of 

Chemical control, (Cartap hydrochloride 

50% radon sp + tata tafgor dimethoate 

30% ec = 2:1 ratio and dissolved in 10 

ml of water), and 2 ml of panchakavya; 

2ml of Panchakavya + neem cake (2 

gm), 2ml of vermicompost as treatment, 

separately and was observed for 5 days. 

The germinated seeds were counted and 

the germination percentage was calculated 

in each of the treatment (Table 1 and 

2). The germination frequency was 100% 

in panchakavya and the adjuvant treatment 

in A. esculentus while it was only 

90% in S. melangena. The germination 

frequency was lesser in all the other 

treatments and it was only 50% in control 

for S. melangena. Among the panchakavya 

treated seeds, maximum germination 

was found in both A. esculentus 

and S. melongena. 

After germination the seedling 

vigour index was calculated by measuring 

the length of shoot and root. The data 

on shoot and root length of different 

treatment were measured by cm scale. 

The plants treated with 2ml panchakavya 

produced the longest shoot 

legnth (2.3) and root length (2) in Abelmochus 

esculentus. While in Solanum 

melongena longest shoot (1.2), root 

length (1.5) was in panchakavya treatment 

which was comparatively higher 

than control. The seedling vigour index 

was higher in panchakavya treated seedlings 

in both the plants which was 430 for 

A. esculentus and 243 for S. melangena 

which was higher than control and all 

other treatments (Table 1 and 2). 

Among the panchakavya and panchakavya 

+ neem cake treated seeds, 

maximum germination frequency was 

observed in Abelmoschus esculents and 

Solanum melongena. The shoot length 

was 2.5 and 1.2 in A. esculentus and S. 

melangena and root length was 2 and 

1.1in A. esculentus and S. melangena. 

There was a slight difference in 2ml of 

panchakavya + neem cake. The pan- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 529



chakavya and panchakavya + neem cake, 

is more effective, when compared to control 

as well as other treatments. The 100 

% germination was observed in 

A.esculentus in panchakavya and panchakavya 

+ neem cake and while only 90 

% germination was seen in Solanum 

melongena (Table 1 and 2). 

The growth of onion roots was observed 

on panchakavya at the concentrations 

of, (50%, 25%, 10%, and 1%). The 

highest number of onion roots, shoots 

length and root length was observed in 1 

% concentration of panchakavya, and it 


was measured in (cm) scale (Table 3 and 

4). 

The onion bulb developed more 

number of roots in 1% panchakavya 

which was 48, and the root length was 

7.5, and the shoot length was 7 which 

was higher when compared to other concentrations. 

At 1% concentration of panchakavya 

the number of cells in the microscopical 

field was 250 cells, of which 

48 of them was in metaphase stage. 

Whereas, in control it was lesser, 22 cells 

in metaphase stage out of the 250 cells in 

the microscopical field. The 1% concentration 

of panchakavya increased the 

Table 1: Pesticide consumption (in lakh tones) during, 1994-1997 (Heisey and Norton, 

2007) 

Country/Region Herbicides Insecticides Fungicides 

China NA NA NA 

India 6.8 37.2 9.4 

Other Asia 24.4 41 19.2 

Middle East/North Africa 9.7 19.5 14.1 

Sub-Saharan Africa 11.7 9.7 9 

Latin America/Caribbean 85.8 39.8 31.8 

All developing 138.4 147.2 83.6 

Transitional 35.6 7.9 23.2 

Industrialized 337.8 163.4 190.4 

World 511.8 318.4 297.2 

Table 2: Rate of seed germination in Abelmoschus esculentus 

No Treatment Time 

Duration 

(Hours) 

No of 

Seeds 

No of seed 

germination 

Germination 

% 

Vigour index 

Seedling 

Length 

1. Control 48 40 28 70 98 

2. Chemical 48 40 32 80 184 

3. Panchakavya 48 40 40 100 430 

4. Panchakavya 48 40 40 100 450 

+ Neem cake 

5. vermicompost 48 40 40 100 410 

Table 3: Rate of seed germination in Solanum melongena 

Time 

No of 

No Treatment Duration 

Seeds 

(Hours) 

No of seed 

germination 

Germination 

% 

Vigour index 

Seedling 

Length 

1. Control 48 40 20 50 70 

2. Chemical 48 40 28 70 133 

3. Panchakavya 48 40 36 90 243 

4. 

Panchakavya + 

Neem cake 

48 40 36 90 207 

5. vermicompost 48 40 32 80 168 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 530




Table 4: Effect of Panchakavya on growth of onion roots 

No. of roots 

Concentration 

S.No 

After four 

[%] 

days 

Root length 

[cm] 

Shoot length 

[cm] 

1. Control 26 5 3 

2. 50 37 5.5 5 

3. 25 37 6 6 

4. 10 37 7 6.5 

5. 1 48 7.5 7 

number of onion roots, when compared 

with control as well as other different 

concentration of panchakavya. 

The greatest challenge of the nation 

in the coming years is to provide safe 

food for the growing population in the 

country without degrading the environment. 

In this regard organic farming 

which is a holistic production management 

system for promoting and enhancing 

health of agro ecosystem, has gained wide 

recognition as a valid alternative to conventional 

food production and ensure 

safer food supply for human consumption. 

This farming system avoid large use 

of synthetic fertilizer, pesticides, growth 

regulators and livestock feed additives 

and relies on green manures, crop rotation, 

crop residues, animal manures, bio 

fertilizers, bio pesticides, different kinds 

of cow based liquid organic manures such 

as panchakavya, sanjibani, kunapajala, 

amrit pani etc. 

Many advanced countries mainly 

depend upon the dairy products because 

of their commercial, agricultural and nutritive 

properties. The dairy industries 

play a vital role in the development of the 

country. When a new house or building or 

even a temple constructed in India, the 

first to enter the premises would be the 

cow because this is considered to be auspicious. 

Cow‟s urine (Comiyam) is used 

in almost all the Hindu rituals. The potential 

of using panchakavya as growth promotor 

and biofertilizer is revealed in this 

work. The present study revealed the 

germination frequency of Abelmoschus 

esculentes, and Solanum melongena 

grown under the different treatments 

namely, Control, Chemical + fertilizer, 

panchakavya, adjuvated with neem cake, 

and vermicompost. 

The effect of adjuvants namely 

vermicompost and neem cake showed a 

slight difference, whereas the panchakavya 

alone was sufficient to enhance 

the seed germination. The panchakavaya 

favors cell growth, cell division (Table 5) 

which is known from the mitotic index. 

The onion bulbs produced longer roots 

and shoots when compare to control. The 

panchakavya at 1% concentration favoured 

the production of larger number of 

roots. The presence of growth promoting 

substance present in the panchakavya favored 

rooting. The present study reveals 

that panchakavya preparation is easy and 

it is cost effective and could be prepared 

easily by a farmer with his household expenses 

and availability. Rahul kumar et 

al. (2014) studied the effect of panchakavya 

fortified with Bauhinia plant 

extract which showed a positive response 

as an anthelminthic preparation. 

Table 5: Effect of Panchakavya on cell 

division [onion root tips] 

No. of cells 

Concentration 

No 

in 

[%] 

Metaphase 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 531 

No. 

of 

cells 

1 Control 22 250 

2 50 46 250 

3 25 44 250 

4 10 45 250 

5 1 48 250 

Sangeetha and Thevanathan 

(2010) studied the potential of panchagavya 

as biofertilizer against certain pulses 

by growing in soil amended with seaweed 

extract and panchagavya. Experimental




seedling recorded higher rates of linear 

growth of both shoots and roots as compared 

to controls. These seedlings produced 

264 to 390% more lateral roots 

than the control and maximum lateral root 

production was always observed in seedlings 

grown in soil amended with seaweed 

based panchagavya at low concentrations 

In recent years the people have 

recognized a number of commercial, medicinal 

and agricultural values from the 

various products of dairy forms. Tharun 

et al., (1983) carried out extensive works 

in this aspects and the environmental 

management in developing countries. 

The number of new methods of 

recycling and controlling measures of organic 

waste in urban and rural habits was 

proposed by (Furedy et al., 1989). The 

current global scenario firmly emphasizes 

the need to adopt eco-friendly agricultural 

practices for sustainable agriculture. 

Chemical input had an adverse impact on 

the health-care of not only soil but also 

the beneficial soil microbial communities 

and the crops. This eventually led to a 

high demand for organic products. Farmers 

all over the world realize the need to 

detoxify the land by switching over to 

organic farming dispensing with chemical 

fertilizers, pesticides, fungicides and 

herbicides. In India, organic farming was 

a well developed and systematized agricultural 

practice during the past and this 

ancient wisdom obtained through Indian 

knowledge is the use of „panchagavya‟ in 

agriculture for the health of soil, plants 

and humans. 

4. Conclusion 

Panchakavya is used in different 

forms. Such as foliar spray, soil application 

along with irrigation, seed treatment 

or seedling treatment etc. For foliar spray 

3% concentration is being used by organic 

farmers. Panchakavya was an important 

one that enhanced the biological efficiency 

of crop and the quality of fruits and 

vegetable production. Panchakavya not 

only enhances the plant yield and growth 

rate, it also reduces the insect invasion 

and fungal attack. The results of the present 

study clearly show that panchakavya 

is an organic fertilizer and growth promoter. 

It also increases the soil fertility. 

It is very much essential to develop a 

strong workable and compatible package 

of nutrient management through organic 

resources for various crops, based on scientific 

facts, local conditions, and economic 

viability for the sustainability. 


The authors would like to thank 

Dr. S.Karpagam, Head of the department 

of Botany, Queen Mary‟s College for 

providing the laboratory facilities. 

References 

Abdul-Barki, A.A, and Anderson, J.D, 

(1973). Vigour determination in soybean 

seed by multiple criteria. Crop 

Science 13, 630 – 63. 

Furedy,C, Bluemental,u.J, Strauss.M, 

Carnicross, L. (1980). Model for the 

effect of different control measures 

in reducing health risks from waste 

water. Sci.Tec., 21, 567 – 577. 

Gomathi, R., Isaivani Indrakumar and 

S. Karpagam (2014). Larvicidal activity 

of Monstera adansonii plant 

extracts against Culex quinequefaciatus. 

Journal of Pharmacognosy and 

Phytochemistry, 3(3), 160-162. 

Gosavi D.D. and Jhon, P.S. (2012). Effect 

of panchakavya Ghritra on some 

neurological parameters in albino 

rats. Asian journal of pharma ceutical 

science clinical Research, 5, 154 – 

156. 

Gupta and Gopal (2001). 

http:/www.epa.gov/oecaagct/ag101/c 

roppesticidesuse 

Natarajan, K. (2002). Panchakavya a 

manual. Other India press, Mapusa, 

Goa, India, pp, 33. 

Nene,Y.L. (1999). Seed health ancient 

and medicinal history and its rele- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 532



vance to present day agriculture In: 

Anicent and medical history of Indian 

agric S.L.Choudhary, G.S,Sharma 

and X.L, 

Nene (ed) (1911). Proc. of the summer 

school held from 28 th may, college of 

Agric, Jaipur, Rajasthan. 

Rank, J. and Nielsen, M.H. (1994). A 

modified Allium test as a tool in the 

screening of the genotoxicity of 

complex mixtures. Hereditas 118, 

49-53. 

Rahul Kumar, Amit Kumar, Kuldip 

Kumar, Vaishnavee Gupta, 

Triveni Shrivas, Kishu Tripathi. 

(2014). Synergistic anthelmintic activity 

of different compositions of 

panchagavya and Bauhinia variegata 

linn. International Journal of Phytopharmacology, 

5(2), 120-122. 


Sangeetha, V. and Thevanathan, R. 

(2010). Biofertilizer Potential of 

Traditional and Panchagavya 

Amended with Seaweed Extract. The 

Journal of American Science, 

6(2):61-67. 

Sathasivam, A., Muthuselvam, M., Rajendran, 

R. (2010). Antimicrobial 

Activities of Cow Urine Distillate 

against Some Clinical Pathogens. 

Global Journal of Pharmacology, 4, 

41-44. 

Tharun,G., N.,C., and Bidwelll, R. 

(1983). Environmental Management 

for Developing countries, vol 1: 

Waste and Water Pollution control – 

Review of Technical solutions. Asian 

institute of Technology, continuing 

Education centre, Bangokok, pp.48 – 

54. 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 533



Short Communication 

Increasing Human Interference in Katarniaghat 

Wildlife Sanctuary 

Shiv Pratap Singh* 

Department of Geography, Kisan P.G. College Bahraich, Affiliated to Dr. R.M.L. Awadh 

University, Faizabad, Uttar Pradesh, India;*Correspondence: principalkpgc@yahoo.co.in; 

Tel.: +919984146483 

Abstract: Katarniaghat Wildlife Sanctuary (KWS) is a part of Dudhwa Tiger Reserve. It is 

managed with the Dudhwa National Park and Kishanpur Wildlife Sanctuary. It is located 

near to the Indo-Nepal boarder. It is in the Bahraich district in Uttar Pradesh. The sanctuary 

comes under the tropical moist deciduous forest of the Himalayan Tarai-Bhabhar region. It 

is under laid on 31 may, 1976. It is situated between 27°41 – 27°56 N and 81°48 – 81°56 E 

covering an area of 551 km. The KWS is divided into six divisions. Four division (Katrniya 

,Nishangada, Murtiha, Dharampur) of it lies under the core zone and remaining two divisions 

are in buffer zone. Tharu is the main tribe of this area. Increasing human interference 

has been studied in the related research paper that outsteps the concept of Biosphere Reserve. 

. Due to growing tourism and farming in core zone, and human activities and habitats 

in buffer zone, it has be-come a critical situation to the bio-diversity of this wildlife sanctuary. 

In this article, the challenges of the Katarniyaghat Wildlife Sanctuary are highlighted. 

The highlighted challenges need to be addressed for the sustainable growth and development 

of the region and the associated communities. 

Keywords: Biodiversity; conservation; environment; human interference; sustainable development; 

wildlife 


In the 21 st century, due to growth in 

materialistic view of life and population 

explosion, the competition to make the 

life more and more comfortable by utilizing 

industrial products has become noticeable. 

To meet the consumers demand, 

industrial production is increased drastically; 

but unplanned and indiscriminate 

industrialization elevated the level of environment 

pollution and ecological imbalance. 

The growing number of animals 

and human, and pressure of agriculture 

and economic development destroyed the 

forests. Deforestation is sensitive in India; 

because not only it has a bad effect on the 

environmental activities but availability 

of fuel, food items, fodders and income 

also get affected. In this article, the example 

of Himalayan Tarai Ecosystem of 

Uttar Pradesh and the problems pervaded 

in biodiversity of Katarniyaghat Wildlife 

Division are highlighted. 

2. Introduction of the study field 

Katarniyaghat Wildlife division is 

situated in Nanpara Tahsil in Bahraich 

district of Uttar Pradesh in India. It lies 

along Indo-Nepal international border. It 

is situated between 27°41 - 27°56 N and 

81°48 - 81°56 E. This division is extended 

in about 551 km area; which is an im- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 534


Increasing Human Interference in Wildlife Sanctuary 

portant part of Himalayan Tarai - Bhabhar 

region. This wildlife sanctuary is included 

in the Project Tiger in 2003 for the conservation 

of tigers, Saryu (Kaudiyala and 

Girwa) river Dalphins, massive Crocodiles, 

Alligator and Turtles biodiversity. 

The forest of the sanctuary area has 

been classified into two major types: (1) 

The Sal forest and (2) the miscellaneous 

forest. This area is rich in biodiversity by 

including vast grasslands, dense forests of 

Sal, Sakhu and Teak trees and Aquatic 

areas. 

Pedagogically, the study area is made 

up of the alluvial soil of the Saryu River 

and its tributaries flowing adjoining to it. 

The study area has a tropical monsoon 

type climate with three distinct seasons 

i.e. summer (April to June) winter 

(November to February) and warm-rainy 

(July to September). March and October 

are considered as transition months between 

the seasons. The mean maximum 

temperature ranges from 22 degree Celsius 

in January to 40 degree Celsius in May 

and the mean minimum temperature 

ranges from 8 degree Celsius in January 

to 27 degree Celsius in June. The annual 

rainfall ranges from 36 to 142 cm in winter, 

34 to 662 cm in summer and 1294 to 

1689 cm in warm-rainy seasons. 

It is a natural aesthesis to see roaring 

Tigers, Leopard resting on tree, leaping 

Cheetal, Padha, Stage, Kakad and Boars 

digging the forest land with their long 

snout. 

To conserve the abundance of wild lives, 

U.P. government has divided it in 6 sub 

divisions. Four sub divisions (Katarnia, 

Nishangara, Murtiha, Bharthapur) are declared 

as a core zone and remaining two 

(Motipur, Kakraha) are declared as a 

buffer zone. North-east railway line passes 

through this division connecting the 

Bichhia-Katarniya tourist place. This line 

is parallel to the roadway that divides the 

zone into two parts (Figure 1). 

There is greatest biodiversity under 

the Katarniya zone. If we have a glimpse 

on biodiversity of biosphere reserve then 

we find endangered Ganga Dolphin, 

Singh 

Tiger, Cheetal, Stages, Rhinocerous, Elephant, 

Leopard, Boar, Padha etc. Under 

the endangered aquatic birds we find Lalsar, 

Surkhab, Nilsar, Gugral, Kaj kurchhia 

and little Mew. Some other birds like 

Peacock, Moorcock, Pheasant, Dhanesh, 

Log log, Hairns, Submarine bird, Crane, 

Wilture, Hawak, Kite, Owl, Caprimulgid, 

Magpia, Woodpecker, Khanjan, Mynah 

bird, Crow, Nightangle, Satbahin are also 

observed. 

Endangered reptiles namely, Iguanas, 

Pythans, Black Cobras and Cobras are 

found here. For conserving crocodiles and 

alligator a Crocodile Project has been established 

in the Katarniyaghat. Currently, 

this wild life division is being developed 

as a tourist place of Bahraich district. 

Tourists come here to see and watch wild 

animals and birds natural beauty as well 

from for-off places. The main tribe living 

here is ‘Tharu’ which lives in the buffer 

zone of the forest area. They are able to 

earn their livelihood by working in agricultural 

or non-agricultural sectors. 

3. Katarniaghat wildlife sanctuary and 

the concept of biosphere reserve 

Under the concept of bio-sphere reserve 

the conservation and enhancement 

of endangered environmental condition is 

implied. The entire zone is divided into 

three zones. First zone is the specific zone 

related to the internal part; where human 

entries are restricted. Second zone is 

known as transitional zone or mid-zone 

surrounded to the core zone; this zone is 

related to research centers, tourist places 

and utilization of fuels and non-living resources. 

Third zone which is situated 

round the mid zone is related to the habitats, 

agriculture, tourist places and utilization 

of non-living resources. But in the 

Bio-sphere reserve (Katarniyaghat Wildlife 

Sanctuary), the core zone (Katarnia, 

Nishangara, Murtiha, Bharthapur ) is being 

developed as Guest House (GH) and 

Tourist Centre (TC). Meanwhile, in the 

buffer zone, the activities of forestry and 

agriculture are growing for the earning of 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 535



livelihood for growing population density. 

Thus, the area of this zone is decreasing 

day by day. If these trends persist then 

Singh 

there is possibility of bio-diversity loss 

and imbalance of ecosystem. 

Figure 1: A diagrammatic sketch (map) showing the Katarniaghat Wildlife Sanctuary location 

in India. Katarniaghat Wildlife Sanctuary map shows the roads, railway lines and 

guest houses located within the sanctuary. 

4. Human activities in Katarniaghat 

wildlife sanctuary 

Approximately 20% of the area consisting 

mostly of the grassland is infested 

by Lantana sp. Over grazing and human 

activities including fire are disturbing. 

The Protected area also experiences pressure 

for fuel wood and fodder from the 

adjoining 25 villages and also from popu- 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 536



lation on the Nepal side. There are some 

forest villages (erstwhile Taungya villages) 

and a Central State Seed Research 

Farm near Girijapuri, situated inside the 

wildlife sanctuary covering 38.42 sq Km 

area that causes considerable disturbance 

to the wildlife, creating a break in the forest 

connectivity. 

Generally, we know that the harnessing 

of timber and fuel is right and to 

cut the forest for trade is illegal. It cannot 

be restricted without giving the employment 

to the effected people and maintaining 

facultative measures. In the studied 

area, the most of the human activities are 

done by rural people; because, the activities 

of cattle rearing and forestry are done 

directly. Along with these activities, illegal 

wood cutting and hunting is found in 

abundant measure. In the buffer zone of 

this sanctuary, the rural population and 

unplanned habitats are growing continuously. 

So the local people are overleaping 

the forest land and converting it into agricultural 

land by cutting the forest. In fact 

is a challenge of present time for the sanctuary. 

In addition to this, the fast running 

vehicles and north-east railway line cause 

the accident of wild lives of the forest area. 

The ecological system is widely disturbed 

by the noise pollution and savage 

pollution caused by the tourists. As the 

Sanctuary is situated at the border of Nepal; 

Nepalies from the border region are 

involved in lot of illegal activities related 

to forestry; which is also disturbing to the 

wildlife and forest of the Sanctuary. 

5. Response to cope with the changing 

theme (human activity) 

Though the management plant of the 

Protect Area Sanctuary (PAS) and working 

plant of Reserve Forest Sanctuary 

(RFS) have not been specifically oriented 

to cope with the identified Global Change 

Factors per se yet, Many initiatives are 

taken up to minimize the factors that contribute 

to habitat fragmentation and infestation 

of invasive species. Major factors 

Singh 

responsible for the fragmentation are; as 

follows: 

Burgeoning human population 

(and therefore the escalating pressure 

for resources) 

Changed socio-economic scenario 

(e.g. changed lifestyle of the Gujjars 

and Khatta holders, therefore 

overgrazing over cutting of firewood 

timber and fodder species) 

Encroachment of forest lands ( by 

agriculture monoculture plantation 

and other land use) 

Infrastructure development (like 

rail, road, hydroelectric and irrigation 

projects) 

Various illegal/ legal (timber harvesting, 

boulder mining in river 

beds etc.) 

approach of various departments 

 

 

Little/ no community participation 

Obsolete policies less amenable to 

adapt to changes, and 

Lack of adequate scientific 

knowledge and proper monitoring 

plants. 

The magnitude of the above stated problems 

leading to habitat fragmentation and 

biological invading varies. There are different 

responses from various stakeholders. 

The government forest department, 

local communities, and the main stakeholders 

that largely manage and use the 

natural resources of the landscape. There 

are some broad responses, which could be 

generalized across the sites for the betterment 

of the sanctuary. 


This paper highlighted the human activities 

in which are disturbing wildlife 

sanctuary. To protect the biosphere reserve 

people should be educated to inculcate 

the importance of conserving wildlife 

for the sustainability. The human interference 

in the buffer and core zone generated 

the danger to the bio-diversity. Forest 

department has recently started the plans 

to supply income to the local people but it 

is well know that only forest department 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 537



cannot do this. Other institution will have 

to take responsibility to make more 

chances to generate employments for the 

people in the vicinity of the sanctuary. 

Jawahar Rojgar Yojna, Samekit Gramin 

Vikas Karyakram and MGNAREGA will 

be helpful in providing proper jobs to locals 

to establish the right situation to conserve 

the wild lives. Task is challenging; 

but, it is essential for the sustainable 

growth and development of the people 

and the region. 

References 

Annual Report (2007). Government of 

India ministry of Environment and 

Forest. 

Census of India (2001). Table-D1005, 

Ministry of Home Affair, Office of 

the Registrar General Delhi; Kalyani 

Pub., 240-250. 

Forest Department Gajetiar (2009). Uttar 

Prdesh. 

Gosal, G.S. (1961). Internal Migration in 

India; A Reginal Analysis; Indian 

Geographical Joural 36(2), 106- 

121. 

Singh 

McComb, C. Benda (2007). Book: Wildlife 

Habitat Management Concepts 

and Application in Forestry, Secand 

Edition, pp-162-168. 

Verma, S. (2010). The Eco-Friendly 

Tharu Tribe: A Study in Socio- Cultural 

Dynamics; Joural Of Asia Pacific 

Studies 1, 177-187. 

Pandy,R.K. (IFS 2005-2008). Tiger Protection 

and Conservation in Indian 

Tarai: Project Work: New Delhi. 

Praveer, R. (2001). Book: Tharu Tribe; 

Bihar Hindi Granth Akadme, Patna. 

Sharma, P.B. (2007). Book: Ecology and 

Environment; Rastoge Publication, 

Meerut; pp 168-171. 

Semwal, R. L. (2005). Book: The Tarai 

Arc Landscap In India; Forests & 

Biodiversity Conservation Programme, 

New Delhi. 

Prakash, S. and Prasad, S. L. (2009). 

Some Aspects of Socio- Economic 

Study of the Tharu in Uttar Pradesh: 

An Asian Tribes Studies; 

Jounral Annals 28(1). 

Singh, Dr. savindra (2002). Book- The 

Environment Geography, Indian 

Press Publication, pp. 484-490 





ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 538

“Earth provides enough to satisfy every 

man's needs, but not every man's greed” 

--- Mahatma Gandhi

About Editors 

Subhash Bhore, PhD: Subhash completed his BSc (Botany) and MSc (Botany) degrees 

education at University of Pune, India. Immediately after completing his 

MSc (1996), he got an opportunity to work at ‘Biochemical Engineering 

Department’ and ‘Plant Tissue Culture Pilot Plant’ of the CSIR-National 

Chemical Laboratory, Pune, India. In June 2000, he received a Doctoral 

Fellowship (GRA) to pursue a PhD Degree in Molecular Genetics at the 

National University of Malaysia (UKM). In 2004, he was appointed as 

Senior Research Officer at Melaka Institute of Biotechnology (MIB), a 

research wing of Melaka Biotechnology Corporation, Malaysia. Based 

on his performance, in April 2005, he was promoted as ‘Principal Investigator & Head of 

R&D Department’ at MIB, Malaysia. In 2008, he was invited by the AIMST University as 

a ‘Visiting Faculty’ for their Department of Biotechnology and now serving as a Senior 

Associate Professor. In 2009, he was nominated for the AASIO (Association of 

Agricultural Scientists of Indian Origin) Young Scientist Award. He has published more 

than 50 peer-reviewed articles, 6 books and submitted more than 11,900 DNA sequences in 

Gene Bank; filed one patent, and received more than 10 awards/fellowships. As of June 

2017, he has supervised more than 72 students including postgraduates, undergraduates and 

industrial trainees. He is actively involved in research as well as teaching and advising of 

postgraduate and undergraduate students. You may contact him using email, 

subhash@aimst.edu.my or subhashbhore@gmail.com 

_________________________________________________________________________ 

Kasi Marimuthu, PhD: Marimuthu accomplished his BSc (Zoology); MSc 

(Environmental Biotechnology); PhD (Environmental Biotechnology/ 

Zoology Interdisciplinary) degree education at Manonmaniam 

Sundaranar University, Tamilnadu, India. In 2003 he joined as a Post- 

Doctoral Fellow at School of Biological Sciences, University Science 

Malaysia, Penang for 2 years. At present, he is working as a Professor 

in the Department of Biotechnology AIMST University, Malaysia for 

the last 12 years. He teaches Aquaculture, Biostatistics, Research 

Methodology, Biology of Invertebrates and Vertebrates courses for undergraduate 

biotechnology programme. His research interests include fish reproduction and breeding, 

larval rearing, hatchery management, fish immunology and aquatic toxicology related 

research. He has published 95 research papers in fisheries and aquaculture in various 

reputed and indexed journals. He has participated in more than 35 local and international 

conferences, seminars, and workshops. He has been appointed as an external examiner for 

six Indian Universities (Manonmaniam Sundaranar University, Annamalai University, 

Bharathiyar University, Bharathidasan University, Madras University, and Priest 

University) Tamilnadu, India. He is a life member in National Academy of Biological 

Sciences [NABS], India and Asian Fisheries Society. He is an editorial member in Indian 

Journal of Natural Products and Resources and Acta Ichthyologica Et Piscatoria. He is also 

presently serving as a Deputy Vice chancellor for Academic and International Affairs, 

AIMST University, Kedah Darul Aman, Malaysia. You may contact him at 

marimuthu@aimst.edu.my / aquamuthu2k@gmail.com. 

Manickam Ravichandran, PhD: Prof M. Ravichandran obtained his 

M.Sc Medical Microbiology from Christian Medical College, Vellore in 

1991 and gained PhD degree from Anna University, Chennai, India in 

1997. Currently his research interests include cholera vaccine, molecular 

diagnostics and phage therapy. He has constructed several cholera 

vaccine candidates for O139 Vibrio cholerae and developed several 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9

simple cold chain free molecular diagnostic kits. He has published 64 papers in 

international journals (Cumulative citations 584), supervised 40 postgraduate students, filed 

8 patents and commercialized 3 products on diagnostics. He has been awarded with 44 

national and international awards for the academic and research excellence including Ideas 

Inventions New Products (IENA), Nuremberg Germany; International Exhibition of 

Inventions: New Technologies and Products, Geneva and Anugerah Inovasi Negara. He is 

an associate member of Academy of Sciences Malaysia (ASM) and Expert panel member 

for R,D&C grants, Sciencefund, Technofund, Community Innovation Fund (CIF), 

Enterprise Innovation Fund (EIF), and Innovation (MOSTI); He was the Cluster Working 

Group (CWG) Committee member on Human Capital Development, Malaysian 

Biotechnology Corporation and the Committee member on ‘Top Research Scientists 

Malaysia’(TRSM) of Academy of Sciences Malaysia (ASM), Biotechnology Road map of 

the Kedah state was formulated under his supervision. He is currently the Chief Executive 

and Vice Chancellor of AIMST University, Kedah Darul Aman, Malaysia. He can be 

contacted at ravichandran@aimst.edu.my. 

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9



Published by AIMST University

Biotechnology for Sustainability: Achievements, Challenges and Perspectives

Create successful ePaper yourself

Delete template?

Save as template?