Environmental Biotechnology Biodegradation - Bioremediation - and Bioconversion of Xenobiotics For Sustainable Development

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9781771883627
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ENVIRONMENTAL
BIOTECHNOLOGY
9781771883627
Biodegradation, Bioremediation, and Bioconversion
of Xenobiotics for Sustainable Development
AUTHOR COPY
9781771883627
AUTHOR COPY
ENVIRONMENTAL
BIOTECHNOLOGY
9781771883627
Biodegradation, Bioremediation, and Bioconversion
of Xenobiotics for Sustainable Development
Edited by
Jeyabalan Sangeetha, PhD
Devarajan Thangadurai, PhD
Muniswamy David, PhD
Mohd Azmuddin Abdullah, PhD
AUTHOR COPY
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Library and Archives Canada Cataloguing in Publication

Environmental biotechnology : biodegradation, bioremediation, and bioconversion of
xenobiotics for sustainable development / edited by Jeyabalan Sangeetha, PhD, Devarajan
Thangadurai, PhD, Muniswamy David, PhD, Mohd Azmuddin Abdullah, PhD.
Includes bibliographical references and index.
Issued in print and electronic formats.
ISBN 978-1-77188-362-7 (hardcover).--ISBN 978-1-77188-363-4 (pdf)
1. Bioremediation. 2. Biodegradation. 3. Xenobiotics. 4. Sustainable development.
I. Thangadurai, D., author, editor II. Sangeetha, Jeyabalan, author, editor III. David,
Muniswamy, author, editor IV. Abdullah, Mohd zmuddin, author, editor
TD192.5.E58 2016 628.5 C2016-904275-8 C2016-904276-6
Library of Congress Cataloging-in-Publication Data

Names: Sangeetha, Jeyabalan, editor. | Thangadurai, D. editor. | David,
Muniswamy, editor. | Abdullah, Mohd Azmuddin, editor.
Title: Environmental biotechnology : biodegradation, bioremediation, and bioconversion
of xenobiotics for sustainable development / editors, Jeyabalan Sangeetha, PhD, Devarajan
Thangadurai, PhD, Muniswamy David, PhD, Mohd Azmuddin Abdullah, PhD.
Other titles: Environmental biotechnology (Apple Academic Press)
Description: 1st ed. | Toronto ; New Jersey : Apple Academic Press, 2015. |
Includes bibliographical references and index.
Identifiers: LCCN 2016027712 (print) | LCCN 2016028891 (ebook) | ISBN 9781771883627
(hardcover : acid-free paper) | ISBN 9781771883634 (eBook) | ISBN 9781771883634 ()
Subjects: LCSH: Bioremediation. | Biodegradation. | Green technology.
Classification: LCC TD192.5 .E59 2015 (print) | LCC TD192.5 (ebook) | DDC 628.5--dc23
LC record available at https://lccn.loc.gov/2016027712
Apple Academic Press also publishes its books in a variety of electronic formats. Some content that appears
in print may not be available in electronic format. For information about Apple Academic Press products,
visit our website at www.appleacademicpress.com and the CRC Press website at www.crcpress.com
AUTHOR COPY
ABOUT THE EDITORS
Jeyabalan Sangeetha, PhD, is an Assistant Professor in Central University
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of Kerala at Kasaragod, South India. She earned her BSc in Microbiology
and PhD in Environmental Science from Bharathidasan University,
Tiruchirappalli, Tamil Nadu, India. She holds also an MSc in Environmental
Science from Bharathiar University, Coimbatore, Tamil Nadu, India.
She is the recipient of the Tamil Nadu Government Scholarship and the
Rajiv Gandhi National Fellowship of the University Grants Commission,
Government of India, for her doctoral studies. She served as Dr. D.S. Kothari
Postdoctoral Fellow and UGC Postdoctoral Fellow at Karnatak University,
Dharwad, South India during 2012-2016 with funding from University
Grants Commission, Government of India, New Delhi. Her main research
interests are in environmental toxicology, environmental microbiology and
environmental biotechnology and her scientific/community leadership have
included serving as editor of an international journal, Acta Biololgica Indica.
Devarajan Thangadurai, PhD, is Senior Assistant Professor at Karnatak

University in South India; President of the Society for Applied Biotechnology;
and General Secretary for the Association for the Advancement of
Biodiversity Science. In addition, Dr. Thangadurai is Editor-in-Chief of sev-
eral journals, including Biotechnology, Bioinformatics and Bioengineering;
Acta Biologica Indica; Biodiversity Research International; and the
Asian Journal of Microbiology. He received his PhD in Botany from Sri
Krishnadevaraya University in South India. During 20022004, he worked
as CSIR Senior Research Fellow with funding from the Ministry of Science
and Technology, Government of India. He served as a Postdoctoral Fellow
at the University of Madeira, Portugal; University of Delhi, India; and
ICAR National Research Centre for Banana, India. He is the recipient of
the Best Young Scientist Award with a Gold Medal from Acharya Nagarjuna
University and the VGST-SMYSR Young Scientist Award of the Government
of Karnataka, Republic of India. He has edited/authored fifteen books in-
cluding Genetic Resources and Biotechnology (3 vols.), Genes, Genomes
and Genomics (2 vols.), and Mycorrhizal Biotechnology with publishers of
national and international reputation.
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vi About the Editors
Muniswamy David, PhD, joined Karnatak University, Dharwad, South

India, in 1995 as Assistant Professor and advanced to the rank of full
Professor of Zoology in 2012. He has vast teaching and research experi-
ence in the fields of ecotoxicology, molecular toxicology, microbial reme-
diation of xenobiotics, and fishery biology. He has published more than 80
papers in national and internationally reputed journals. Dr. David obtained
his PhD in Zoology from Sri Krishnadevaraya University, Anantapur, South
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India, and was awarded a postdoctoral fellowship by the University Grant
Commission, New Delhi, India. He was also named Scientist of the Year in
2013 from the National Environmental Science Academy, New Delhi, India.
He has successfully handled three major research projects funded by UGC,
DST-SERB, and Hutti Gold Mines Pvt. Ltd., Karnataka.
Mohd Azmuddin Abdullah, PhD, is an Associate Professor of Bioprocess

and Environmental Engineering at the Institute of Marine Biotechnology,
Universiti Malaysia Terengganu. He was a visiting scientist (1997) at Kinki
University, Japan, under the JSPS Fellowship and a postdoctoral scientist at
the Massachusetts Institute of Technology, USA, under the Malaysia-MIT
Biotechnology Partnership Program. He was a lecturer in the Department
of Biotechnology, Universiti Putra Malaysia and a Senior Lecturer and
Associate Professor in the Department of Chemical Engineering, Universiti
Teknologi PETRONAS, Malaysia. He has published more than 120 articles
in peer-reviewed journals and conference proceedings, and has published
many book chapters and technical reports on cell culture engineering, sec-
ondary metabolites, natural products, biomaterials, bioenergy, environmen-
tal remediation, drug delivery, photocatalysts, and chemical sensors. Dr.
Abdullah obtained an MEng degree in chemical engineering and biotechnol-
ogy from the University of Manchester Institute of Science and Technology,
United Kingdom, under a PETRONAS Scholarship, and a PhD in Bioprocess
Engineering from Universiti Putra Malaysia under a UPM Scholarship.
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CONTENTS
List of Contributors............................................................................. ix
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List of Abbreviations.........................................................................xiii
Preface.............................................................................................. xix
1. The Role of General Methods and Mathematical Models on Microbial

Evolution, Ecological Interactions, and Population Dynamics....................1
Subir Kumar Nandy
2. Identification of Microorganisms Carried by Aeolian Dust to

Other Continents and Their Impact on Public Health...............................17
Paraskevi A. Farazi
3. Chlorinated Compounds in Natural and Biotechnological

Processes: Merits, Risks, and Uses...............................................................41
Sndor T. Forczek, Josef Holk, Ludk Rederer, and Martin Ferenk
4. Environmental Impact of Pesticide Use on Microbial

Communities and Soil Bioprocesses: A Physiological,
Biochemical, and Molecular Perspective.....................................................67
Jeyabalan Sangeetha, Muniswamy David, Devarajan Thangadurai,
Etigemane Ramappa Harish, Jadhav Shrinivas, Prathima Purushotham,
and Kartheek Rajendra Malowade
5. Recent Advances in Applications of Nanomaterials for

Water Remediation........................................................................................97
Kaliyaperumal Rani and Barindra Sana
6. Fungal Dehalogenation: An Overview....................................................... 119

Raghunath Satpathy, Venkata Sai Badireenath Konkimalla, and Jagnyeswar Ratha
7. Insight of Biofuel Prospects from Microalgae as Renewable

Energy Source for Environmental Sustainability.....................................133
Ganapathi Sibi
8. Integrated Algal Industrial Waste Treatment and

Bioenergy Co-Generation............................................................................153
Mohd Azmuddin Abdullah and Ashfaq Ahmad
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viii Contents
9. Bioambiant Preservation of Raw Hides Using Plant-Based

MaterialsA Green Technology to Reduce Tannery
Waste Water Pollution.................................................................................225
Prafulla Namdeo Shede, Ashish Vasantrao Polkade, Pradnya Pralhad Kanekar,
Prashant Kamalakar Dhakephalkar, and Seema Shreepad Sarnaik
10. Phytoremediation of Organic Chemopollutants.......................................257
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Pradnya Pralhad Kanekar, Seema Shreepad Sarnaik, Parag Avinash Vaishampayan,
and Prafulla Namdeo Shede
11. Bioconversion of Palm Oil and Sugar Industry Wastes into

Value-Added Polyhydroxyalkanoate..........................................................287
Noor Fazielawanie Mohd Rashid, Ain Farhana Mohd Yatim, Al-Ashraf Amirul,
and Kesaven Bhubalan
12. Influence of Environmental Factors on the Prevalence of

Postharvest Deterioration of Raphia and Shea Fruits in Nigeria............307
Okungbowa Francisca Iziegbe and Esiegbuya Ofeoritse Daniel
13. Soil Remediation and Ecological Restoration from

Heavy Metal Pollution and Radioactive Waste Materials
using Fungal Genetic and Genomic Resources.........................................327
Jeyabalan Sangeetha, Devarajan Thangadurai, Muniswamy David, Jadhav Shrinivas,
Abhishek Channayya Mundaragi, Paidi Murali Krishna, Etigemane Ramappa Harish,
Prathima Purushotham, and Swapna Kishor Deshpande
14. A Multifaceted Bioremediation of Xenobiotics Using Fungi....................363

Devipriya Rabin Majumder
Index......................................................................................................................395
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LIST OF CONTRIBUTORS
Mohd Azmuddin Abdullah
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Institute of Marine Biotechnology, Universiti Malaysia Terengganu, Kuala Terengganu 21030,
Terengganu, Malaysia
Ashfaq Ahmad
Department of Chemical Engineering, Universiti Teknologi PETRONAS, Bandar Seri Iskandar, Tronoh
31750, Perak, Malaysia
Al-Ashraf Amirul
School of Biological Sciences, Universiti Sains Malaysia, Pulau Pinang 11800, Malaysia
Kesaven Bhubalan
Malaysian Institute of Pharmaceuticals and Nutraceuticals, MOSTI, Bayan Lepas 11700, Pulau Pinang,
Malaysia
Esiegbuya Ofeoritse Daniel

Plant Pathology Division, Nigerian Institute for Oil Palm Research (NIFOR), Benin City, Nigeria
Muniswamy David
Department of Zoology, Karnatak University, Dharwad 580003, Karnataka, India
Swapna Kishor Deshpande

Department of Botany, Karnatak University, Dharwad 580003, Karnataka, India
Prashant Kamalakar Dhakephalkar

Bioenergy, MACS Agharkar Research Institute, G.G. Agarkar Road, Pune 411004, India
Paraskevi A. Farazi
Mediterranean Center for Cancer Research, Department of Life and Health Sciences, University of
Nicosia, 46 Makedonitissas Avenue, P.O. Box 24005, Nicosia 1700, Cyprus
Martin Ferenk
Institute of Environmental and Chemical Engineering, Faculty of Chemical Technology, University of
Pardubice, Studentsk 573, Pardubice CZ-53210, Czech Republic
Sndor T. Forczek
Isotope Laboratory, Institute of Experimental Botany, Academy of Sciences of the Czech Republic,
Vdesk 1083, Prague CZ-14220, Czech Republic
Etigemane Ramappa Harish

Josef Holk
Isotope Laboratory, Institute of Experimental Botany, Academy of Sciences of the Czech Republic,
Vdesk 1083, Prague CZ-14220, Czech Republic
Okungbowa Francisca Iziegbe

Department of Plant Biology and Biotechnology, University of Benin, Benin City, Nigeria
Pradnya Pralhad Kanekar

Department of Biotechnology, Modern College of Arts, Science and Commerce, Shivajinagar, Pune
411005, India
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x List of Contributors
Venkata Sai Badireenath Konkimalla

School of Biological Sciences, National Institute of Science Education and Research (NISER),
Bhubaneswar, Odisha 751005, India
Paidi Murali Krishna

Devipriya Rabin Majumder

Department of Microbiology, Abeda Inamdar Senior College, 2390-KB Hidayatullah Road, Azam
9781771883627
Campus, Camp, Pune 411001, Maharashtra, India
Kartheek Rajendra Malowade
Abhishek Channayya Mundaragi

Subir Kumar Nandy

Technical University of Denmark, SltoftsPlads, 2800 Kgs, Lyngby, Denmark
Ashish Vasantrao Polkade

Microbial Culture Collection, National Centre for Cell Science, First floor, Central Tower, Sai Trinity
Building Garware Circle, Sutarwadi, Pashan, Pune 411021, India
Prathima Purushotham
Kaliyaperumal Rani
Nanyang Technological University, 62 Nanyang Drive 637459, Singapore
Jagnyeswar Ratha
School of Life Sciences, Sambalpur University, Jyoti Vihar, Burla, Odisha 768019, India
Noor Fazielawanie Mohd Rashid

School of Marine and Environmental Sciences, Universiti Malaysia Terengganu, Kuala Terengganu
21030, Terengganu, Malaysia
Ludk Rederer
Povod Labe, State Enterprise, Vta Nejedlho 951, Hradec Krlov CZ-50003, Czech Republic
Barindra Sana
Agency for Science, Technology and Research (A*STAR), 1 Fusionopolis Way, #20-10 Connexis North
Tower 138632, Singapore
Jeyabalan Sangeetha
Department of Environmental Science, Central University of Kerala, Kasaragod, Kerala 671316, India
Seema Shreepad Sarnaik

Microbial Sciences Division, MACS Agharkar Research Institute, G.G. Agarkar Road, Pune 411004, India
Raghunath Satpathy
School of Life Sciences, Sambalpur University, Jyoti Vihar, Burla, Odisha 768019, India
Prafulla Namdeo Shede

Department of Microbiology, MES Abasaheb Garware College, Karve Road, Pune 411004, India
Jadhav Shrinivas
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List of Contributors xi
Ganapathi Sibi
Department of Biotechnology, Centre for Research and Post Graduate Studies, Indian Academy Degree
College, Bangalore 560043, Karnataka, India
Devarajan Thangadurai
Parag Avinash Vaishampayan

Biotechnology and Planetary Protection Group, Jet Propulsion Laboratory, California Institute of
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Technology, Pasadena, CA 91109, USA
Ain Farhana Mohd Yatim
School of Marine and Environmental Sciences, Universiti Malaysia Terengganu, Kuala Terengganu
21030, Terengganu, Malaysia
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LIST OF ABBREVIATIONS
OOH Peroxide radicals
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3CP 3-chloropropionic acid
3HB 3-hydroxybutyrate
3HB-co-3HV Poly(3-hydroxybutyrate-co-hydroxyvaletare)
3HD 3-hydroxydecanoate
3HHx 3-hydroxyhexanoate
3HV 3-hydroxyvalerate
AgNPs Silver nanoparticles
AM Arbuscular mycorrhiza
AO7 Acid orange 7
AOX Adsorbable organic halogens
APS Ammonium persulfate
APX Ascorbate peroxidase
ARDRA Amplified ribosomal DNA restriction analysis
ASO Ascorbate oxidase
BMP Biochemical methane potential
BNF Biological nitrogen fixation
BOD Biological oxygen demand
C/N Carbon/nitrogen
C2Cl4 Tetrachloroethylene
C2HCl3 Trichloroethylene
Ca Calcium
CA Clofibric acid
CCA Chromated copper arsenic
cDCE Cis-1,2-dichoroethene
CDM Clean development mechanism
CER Carbon emission reduction
CF Concentration factor
CFCs Chlorofluorocarbons
CFG Cement flue gas
CFU Colony forming units
CHBr2Cl Chlorodibromomethane
CHCl3 Chloroform
CHIP Chromatin immunoprecipitation
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xiv List of Abbreviations
Cl Chlorium
CLE Council for Leather Export
CO2 Carbon dioxide
COD Chemical oxygen demand
Co-PCBs Coplanar polychlorinated biphenyls
CSL Corn steep liquor
Cu Copper
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CW Constructed wetland
Da Daltons
DAF DNA amplification fingerprinting
DART Dust at altitude recovery technology
DAS Days after sowing
DCA [UL-14C]-3,4-dichloroaniline
DCP Dichlorophenol
DCW Dry cell weight
DDT Dichloro-diphenyl-trichloroethane
DGGE Denaturing gradient gel electrophoresis
DHA Docosahexaenoic acid
DMSP Dimethylsulphoniopropionate
DNAPLs Dense non-aqueous phase liquids
DNT Dinitrotoluene
DNX Hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine
DOC Dissolved organic carbon
DOE Department of Energy
DSE Dark septate endophyte
EDC Endocrine disrupting chemical
EFB Empty fruit bunches
EPA Environmental Protection Agency
EST Expressed sequence tag
ETC Electron transport chain
eZVI Emulsified ZVI
FAME Fatty acid methyl ester
FBB Fresh fruit bunch
FDA Food and Drug Administration
FFA Free fatty acids
FFB Fresh fruit bunches
FGP Fungal Genome Project
FIIRO Federal Institute of Industrial Research Oshodi
FLT Fluoranthene
FTIR Fourier transform infrared spectroscopy
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List of Abbreviations xv
g m2 d1 Gram per meter square per day

GC-MS Gas chromatography-mass spectrometry
GHG Greenhouse gases
GHS Glutathione
GPX Guaiacal peroxidase
GSTs Glutathione S-transferases
H2CO3 Bicarbonate
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HDTMA Hexadecyltrimethylammonium
HE Harvesting efficiency
HFCW Horizontal flow constructed wetland
HFMD Hand, foot and mouth disease
HMX Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine
HPLC High performance liquid chromatography
HRT Hydraulic retention time
IMG Integrated microbial genomes
JGI Joint Genome Institute
Kg Kilogram
L Liter
LAS Linear alkylbenzene sulfonates
LCA Life cycle assessment
LCFA Long chain fatty acid
lcl-PHA Long-chain-length PHA
LH-PCR Length heterogeneity PCR
LiP Lignin peroxidases
LMW Low molecular weight
MBRT Methylene blue dye reduction test
mcl-PHA Medium-chain-length PHA
MDA Malondialdehyde
MeP Methyl parathion
MF Membrane filtration
Mg Magnesium
MIC Minimum inhibitory concentration
MM Minimal medium
MnP Manganese peroxidase
MNX Hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine
MPBR Membrane photobioreactor
MPOC Malaysian Palm Oil Council
MTs Metallothioneins
MUFA Monounsaturated fatty acids
NaCl Sodium chloride
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xvi List of Abbreviations
NH4Cl Ammonium chloride

NHGRI National Human Genome Research Institute
NIFOR Nigerian Institute for Oil Palm Research
nZVI Nanoscale zero-valent iron
O2. Superoxide ions
OH
Hydroxide ions
OLR Organic loading rate
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OMSW Olive mill solid waste
P(3HB) Poly(3-hydroxybutyrate)
PAHs Polycyclic aromatic hydrocarbons
PBR Photobioreactor
PCBs Polychlorinated biphenyls
PCDDs Polychlorinated dibenzo-r-dioxins
PCE Perchloroethylene
PCP Pentachlorophenol
PCR Polymerase chain reaction
PCs Phytochelatin synthase
PDB Protein Data Bank
PEI Polyethylenimine
PGPR Plant growth promoting rhizobacteria
PHA Polyhydroxyalkanoate
PKC Palm kernel cake
PM10 Particulate matter <10mm
POME Palm oil mill effluent
POMS Palm oil mill sludge
PPi Pyrophosphate
ppm Parts per million
PUFA Polyunsaturated fatty acids
PV Peroxide value
RAPD Random amplified polymorphic DNA
RAS Rennin angiotensin system
RDX Hexahydro-1,3,5-trinitro-1,3,5-triazine
RFLP Restriction fragment length polymorphism
RISA Ribosomal intergenic spacer analysis
RKF Raw kapok fibers
RMX Hexahydro-1,3,5-triaza-1,3,5-trinitrocyclohexane
RO Reverse osmosis
scl-PHA Short-chain-length PHA
SDS Sodium dodecyl sulphate
SEM Scanning electron microscopy
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List of Abbreviations xvii
SFA Saturated fatty acids

SHF Separate hydrolysis and fermentation
SKF Structurally modified kapok
SMKF Surface-modified kapok fiber
SOD Super oxide dismutase
SPC Solid-phase cytometry
SPM Suspended particulate matter
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SRT Solid retention time
SSCP Single-strand conformation polymorphism
SSF Simultaneous saccharification and fermentation
TAG Triacyl glycerols
TBA Terbuthylazine
TCA Tricarboxylic acid
TCDD 2,3,7,8-tetrachloro dibenzo-r-dioxin
TCE Trichloroethane
TDS Total dissolved solids
THM Trihalogenated methanes
TiO2 Titanium dioxide
TMPA Trimethylphenylammonium
TN Total nitrogen
TNT 2,4,6-trinitrotoluene
TOC Total organic carbon
TPH Total petroleum hydrocarbon
T-RFLP Terminal restriction fragment length polymorphism
UASB Up-flow anaerobic sludge blanket reactor
UASFF Upflow anaerobic sludge fixed film
UV-VIS Ultraviolet-visible spectroscopy
VAM Vesicular-arbuscular mycorrhiza
VBNC Viable but non culturable
VFAs Volatile fatty acids
VFCW Vertical flow constructed wetland
VOCl Volatile organochlorines
VP Versatile peroxidase
WAS Waste activated sludge
WHO World Health Organization
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PREFACE
Every fraction in our close proximities harbors inestimable living organisms
9781771883627
with untold scientific mysteries. Given our widespread environment, this
number would increase by leaps and bounds. This emphatically describes
the plethora of information which could be derived if this surreptitious en-
vironment is premeditated. The ability to probe the environment at the mo-
lecular level to create a new awareness of fundamental biological processes
therein has created an important new paradigm. This has been made possible
by the successful integration of biotechnology into the many facets of our
environment. Biotechnology is an emerging discipline that holds promise
to offer long-range solutions to our developmental and environmental con-
cerns. The future of our calling toward environment may in fact be in bio-
technology. Modern biotechnology is considered to be one of the potential
technologies of the current century to support sustainable development. That
being the case, we can expect the merger to follow, giving rise to environ-
mental biotechnology that involves specific application of biotechnology to
the management of environment and environment related socio-economic
developmental issues.
The present plight of the world as a victim to a surfeit of environmen-
tal setbacks ranging from global warming and ozone layer depletion to an
alarming increase in world pollution levels is threatening the existence of
the most intelligent species on earth. This has been enough for both en-
vironmentalists and laymen to wake up to the indisputable importance of
environmental protection and ecofriendly alternatives. Environmental bio-
technology is mainly the first alternative, with fields of application reaching
out to every quarter. Environmental biotechnology utilizes the biochemical
potential of microorganisms and plants for the preservation and restoration
of the environment. The primary role of environmental biotechnology is to
develop better approaches for sustainable development.
The purpose of this book is to present the reasonable manifestation of
the realistic biological approaches currently employed in environmental
biotechnology to address environmental problems. It covers the latest re-
search by prominent scientists and researchers in this fast growing field. This
most recent and updated book deals with various aspects of recent advances,
current trends and practical applications on the understanding of microbial
AUTHOR COPY
xx Preface
interactions, exploration of microbes in extreme environments, applications

of nanomaterials for water remediation, impact of toxicants on microbial
communities and soil bioprocesses, production of biofuel from microalgae,
algal industrial waste treatment, reduction of tannery waste water pollution,
phytoremediation of organic chemopollutants, bioconversion of palm oil
and sugar industry wastes, environmental influence on postharvest deterio-
ration of fruits, soil remediation, ecological restoration, heavy metal pol-
9781771883627
lution, radioactive waste materials, dehalogenation, and bioremediation of
xenobiotics using fungi.
In Chapter 1, S.K. Nandy gives an overview on the microbial evolution,
ecological interactions, and the population dynamics with general methods
and mathematical models. This is followed by the identification of micro-
organisms carried by Aeolian dust to other continents and their impact on
public health by P.A. Farazi and in Chapter 3, S.T. Forczek, J. Holk, L.
Rederer, and M. Ferenk discuss the merits, risks, and uses of chlorinated
compounds in natural and biotechnological processes. In Chapter 4, envi-
ronmental impact of pesticide use on microbial communities and soil bio-
processes using molecular approaches is highlighted by J. Sangeetha and her
colleagues. Discussion on the advances and applications of nanomaterials
for water remediation is highlighted by K. Rani and B. Sana in Chapter 5,
and fungal dehalogenation in Chapter 6 by R. Satpathy, V.B. Konkimalla,
and J. Ratha. A comprehensive review on the prospect of microalgal utiliza-
tion as renewable energy sources is made in Chapter 7 by G. Sibi, and on the
integrated industrial waste treatment and bioenergy co-generation in Chapter
8 by M.A. Abdullah and his team have been presented with recent informa-
tion. The use of green technology to reduce tannery waste water pollution
with the help of plant-based bioambiant preservatives is elucidated by P.N.
Shede, A.V. Polkade, P.P. Kanekar, P.K. Dhakephalkar, and S.S. Sarnaik in
Chapter 9. P.P. Kanekar and her collaborators interpret on the phytoremedia-
tion of organic chemopollutants in Chapter 10, followed by the discussion
on bioconversion of palm oil and sugar industrial wastes into polyhydroxy-
alkanoate by N.M.R. Fazielawanie, A.F.M. Yatim, K. Bhubalan, and A.A.
Amirul. Environmental influence on the prevalence of postharvest deterio-
ration of Raphia and Shea fruits is covered in Chapter 12 by O.F. Iziegbe
and E.O. Daniel. Soil remediation and ecological restoration from heavy
metal pollution and radioactive waste materials using fungi is discussed by
J. Sangeetha and her group in Chapter 13. Finally, the discussion on the
multifaceted bioremediation of xenobiotics using fungi is wrapped up in the
last chapter by D.R. Majumder.
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Preface xxi
The mission of editing and publishing this book has received immense
support of our foremost experts. We are deeply indebted to them for their
devoted consideration, valuable guidance, constant assistance, support, en-
durance, and constructive suggestions for the successful completion of the
book. We also wish to thank Sandy Jones Sickels, Vice President, and Ashish
Kumar, Publisher and President, at Apple Academic Press, Inc., for quality
production and humble attempts to publish this book. We owe our gratitude
9781771883627
to the people who were directly or indirectly involved in preparing this book,
for their co-operation and useful suggestions. We express thanks to our fami-
lies for their unrelenting support and constant encouragement.
Jeyabalan Sangeetha, PhD

Devarajan Thangadurai, PhD
Muniswamy David, PhD
Mohd Azmuddin Abdullah, PhD
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CHAPTER 1
THE ROLE OF GENERAL METHODS

AND MATHEMATICAL MODELS
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ON MICROBIAL EVOLUTION,
ECOLOGICAL INTERACTIONS, AND
POPULATION DYNAMICS
SUBIR KUMAR NANDY
Department of Systems Biology,Technical University of Denmark,
Lyngby, Denmark
CONTENTS
1.1 Introduction..........................................................................................2
1.2 Survival Strategy..................................................................................2
1.3 Cannibalism.........................................................................................3
1.4 Sporulation...........................................................................................5
1.5 Mathematical Models Related to Ecological Interactions...................9
1.6 Cannibalism vs Predation................................................................... 11
1.7 Conclusion and Future Perspectives..................................................12
Keywords....................................................................................................13
References...................................................................................................14
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2 Environmental Biotechnology
1.1INTRODUCTION
Bacillus subtilis is a spore-forming bacterium that can persist for years

in a resistant state. When faced with starvation, bacteria instead enter the
complex developmental pathway of spore formation. Gonzlez-Pastor et
al. (2003) and other researchers (Engelberg-Kulka and Hazan, 2003) have
found that bacteria en route to sporulation produce a toxin (sporulation-kill-
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ing factor) similar to peptide antibiotics that lyses sibling cells not commit-
ted to sporulation. The killing operon also produces its own export pump
and confers resistance to the killing peptide. The nutrient boost from the
lysed cells allows the surviving cells to postpone sporulation, to escape its
energetic costs, and to continue replication. A signaling factor (sporulation-
delaying protein) mediates this escape from sporulation via a transcription
factor that stimulates lipid oxidation and adenosine 5-triphosphate produc-
tion to restore energy reserves. The above studies of mixed culture require
an easy, effective, and fast methodology to quantify the metabolic active
cell count of different types of microorganisms. Both the survival strategy
and the microbial interactions including stress response in pure and mixed
culture are discussed in this chapter.
1.2 SURVIVAL STRATEGY
Microorganisms have evolved strategies to adapt in various stress environ-

ments such as change in temperature, pH, nutrient concentration, osmotic
pressure, and media. Therefore, the obvious question that arises is that what
are the mechanisms that underlie the evolution of these survival strategies?
Several studies in molecular biology report the signaling pathways that es-
tablish the phenotypic response. However, in the ecosystem, organisms need
to adapt in the presence of other microorganisms. The interactions of an
organism with its environment are fundamental to the survival of that organ-
ism and also the functioning of the ecosystem as a whole. The interrelation-
ship between various microorganisms under stress is relevant and has not
been studied sufficiently. Different types of bacterial interactions exist in
a mixed population. These are categorized as neutralism, mutualism, com-
mensalism, ammensalism, antagonism, symbiosis, and competition.
Limited resources of nutrients or environment create competition in a
mixed culture. The competition among the same species is termed as intra-
specific competition, while that in different species is termed as interspe-
cific competition. Cannibalism is an example for intraspecific competition.
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Microbial Evolution, Ecological Interactions and Population Dynamics 3
Froelich et al. (1979) have shown in anaerobic marine sediments that a

sulfate-reducing zone develops near sedimentwater interface and is also
underlined by a methanogenic zone. Studies have shown that this zone sepa-
ration reflects the competition between sulfate reduces and methanogens for
the same substrates (Lovely and Klug, 1983, 1986).
Under starvation condition, a majority of bacterial population die; how-
ever, some survive the nutrient-scarce environment by exploiting the nutri-
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ents made available from the dying cells. These surviving cells are highly
dynamic in nature and the interaction is termed as cannibalism. The can-
nibalistic interaction has been studied by Gonzlez-Pastor et al. (2003) and
Nandy et al. (2007). It is also proposed that instead of surviving from the nu-
trients coming out of the dying cells, survivors become fitter mutants and si-
multaneously survival strategy becomes an evolutionary strategy. Gonzlez-
Pastor et al. (2003) have found that B. subtilis can produce killing factor
under no nutrient medium to kill their own sister cells or siblings and survive
on the released nutrients to delay sporulation. Table 1.1 shows the different
existing ecological interactions.
TABLE 1.1 Different Ecological Interaction Processes are Summarized with Their System
Name and Responses.
Interaction Process Bacterial Response (Benefit: Y or N)
First Second
Neutralism N N
Mutualism Y Y
Commensalism Y N
Ammensalism N
Antagonism Y N
Y: yes; N: no.
1.3CANNIBALISM
B. subtilis is a Gram-positive soil-living bacterium, which adapts to differ-

ent phenotypes depending upon the nature of the surrounding environment.
These phenotypes are regulated in both cell density and environment-depen-
dent fashion. As bacteria move toward late log phase, they undergo sequen-
tially competence development, cannibalism, and sporulation.
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Cannibalism is a process in which bacteria produce a killing factor to kill

their own sister cells to survive under no nutritional conditions. In B. subti-
lis, a regulatory protein Spo0A is responsible for cannibalism in the stress
environment. Various interlinked signaling pathways regulate the activity of
Spo0A and these signaling pathways are also known to be involved in the
regulation of other physiological processes such as sporulation, synthesis
of degradative enzymes, and secondary metabolites. Under the severity of
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nutrient limitation, cells start to secrete several digestive enzymes, such as
proteases, which enable the bacteria to take food from several alternative
nutrient sources. Cells also start to secrete several antibiotics, which kill the
competitors, making the availability of nutrients for survival. If all these
adaptive ways are closed to survive, B. subtilis maintains its integrity by
undergoing sporulation.
skfA gene in skf operon is shown to be involved in the production of an
extracellular killing factor. Skf operon also produces the factors skfE and
skfF; these two factors confer resistance to killing factor. skfE behaves as
a ATP-binding cassette and skfF acts as a transporter. Together, they offer
resistance to mother cells by pumping out the killing factor. Sdp is another
operon that is regulated by nutrient limitation through spo0A. sdpC synthe-
sizes a 5kDa protein, which regulates the expression of yvbA. Transcription
factor yvbA upregulates the operon atp which is involved in ATP produc-
tion and also operon yusLKG which synthesize the enzymes involved in
lipid catabolism. Thus, higher expression of sdp operon generates energy
under no nutrient condition to make cells metabolically active, which delays
the sporulation in the cells expressing spo0A (Gonzlez-Pastor et al., 2003;
Westers et al., 2005).
Gonzlez-Pastor and colleagues have shown that during the nutrient
limitation, a subpopulation of bacteria become spo0A active cells, while re-
maining fraction of cells become spo0A inactive (Ellermeier et al., 2006). In
spo0A active cells, spo0A protein upregulates expression of skfA, skfE, skfF,
and sdpC. skfE and skfF together form killing factor transport protein com-
plex, conferring resistance to the parent cells. Since some cells are spo0A
inactive, skf operon remains inactive, making them sensitive to the killing
factor. sdp operon is also switched on in spoA active cells, which synthe-
sizes the signaling protein sdpC. sdpC upregulates yvbA which induce the
expression of operon for ATP synthase (atp) and lipid catabolism enzymes
(yusLKG), leading to energy production and delaying of sporulation. SdpC
signaling molecule also induces the yvbA gene in spo0A-negative cells.
yvbA represses the gene for sigma factor synthesis (Ellermeier et al., 2006).
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It has been suggested that sigma factor confers resistance to the cells by
making cells antibiotic resistant and also by detoxification of killing factor
(Gonzlez-Pastor et al., 2003; Ellermeier et al., 2006).
1.4SPORULATION
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Sporulation mechanism of Gram-positive bacterium involves a complex reg-
ulatory cascade which controls the expression of over 100 genes. This regula-
tory process is complex due to the temporal and spatial constraints. One of
the best examples is B. subtilis, which forms spores in the environment under
different stresses. At low nutrient condition, spores of B. subtilis are formed.
These spores are metabolically dormant for years and are resistant to several
factors such as heat and radiation. Metabolically and morphologically B. sub-
tilis spores are different from the normal vegetative cells. Several genes are
also identified to be involved in sporulation such as spo0A, spo0B, spo0E,
spo0F, spo0H, spo0J, and spo0K (Westers et al., 2005). In the absence of
either of these, the sporulation mechanism is blocked.
1.4.1 SPORULATION STAGES
This process involves a number of steps which can be monitored by light

and electron microscopy and completes within 8h (Driks, 1999). The whole
process is divided into six parts and these are as follows. (A) During sporula-
tion, morphology will change with sequential appearance of a series of tran-
scription factors, called sigma factors binding to core polymerase. Initially,
the mother cell was divided asymmetrically into two compartments. The
mother cell which is the larger compartment will serve the spore until the
complete development. This phenomenon will happen within 2h. E and F
become active in mother cell and forespore, respectively. (B) In the next lev-
el forespore engulfs into one membrane bound protoplast. G becomes active
in forespore whereas K express gene in the mother cell. (C and D) Then
one cortex forms in between forespore membranes, while the formation of
the coat from synthesis of protein in mother cell that assembles around the
forespore is visible by electron microscopy. (E) Finally the mother cell lyses
and the mature spore will come out in the environment. (F) Under nutrient
again spore can germinate and the nascent cell comes out in the environment
(Driks, 1999). Our main objective is to explain the sporulation process only,
and the whole process is described in Figure 1.1.
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FIGURE 1.1 Sporulation steps: (A) When a cell commits sporulation, the cell divides into
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mother cell and forespore in asymmetrically, H activity increases. (B) The forespore covers
with a membrane where G becomes active and K directs a final phase of gene expression. (C)
One cortex forms between the forespore membranes where final phase of gene expression has
been taken place with the help of GerE. (D,E) The dark coat has been visible by microscope
and after cell lysis spore comes out to the environment. (F) Spore can again germinate and
cell can resume vegetative growth (Driks et al., 1999).
1.4.2 METHODOLOGY TO STUDY SPORULATION
1.4.2.1 STAINING METHOD
The spores are differentially stained by using dyes that penetrate the spore
wall. An aqueous primary stain (malachite green) is applied and steamed
to enhance the penetration of the impermeable spore coats. Once stained,
the endospore do not readily decolorize and appear green. After fixing, the
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smears flood the malachite green over the slide and heat it for 5min, adding
more stain with time to time. Finally, wash the slides slowly with tap water
and counter stain with safranin for 30s. For example, the endospore of B.
subtilis shows green color and vegetative cells are in red (Rao et al., 1965).
1.4.2.2 AGAR METHOD
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Sporulation agar is employed to detect spore in the culture. The sample was
boiled at 90100C for 10min in a water bath and then spread on the sporu-
lation agar to count the spores after incubation for 24h (Driks, 1999).
1.4.3 REGULATION OF SPORULATION KILLING FACTOR
B. subtilis prefers cannibalism in the no nutrient condition. B. subtilis kills

its own sister cells by producing and exports a peptide sporulation factor
(skfA) where, skfA is a part of skf operon (skfA-H). This skf operon is re-
sponsible for immunity to SkfA and also for the production and export of
SkfA. Allenby et al. (2006) reported that the transcription of skfA induced
under phosphate starvation.
This phosphate starvation is responsible for the expression of genes in
PhoP and SigB (B) regulons of B. subtilis (Eymann et al., 1996; Hecker and
Volker, 1998; Antelmann et al., 2000; Prgai and Harwood, 2002; Allenby
et al., 2005). Spo0A is responsible for activation of PhoPregulon, and more-
over activated Spo0A (Spo0A~P) is responsible for sporulation induction,
and gene repression is induced by transition phase regulator AbrB. Thus,
spo0A mutant should not show cannibalism. PhoPregulon is upregulated
in spo0A null mutant and that is unable to initiate sporulation (Prgai et
al., 2004). Researchers have also reported that Spo0A regulates skf operon
which encodes sporulation killing factor (SkfA) (Fawcett et al., 2000; Molle
et al., 2003). The siblings of B. subtilis cells are lysed by induced SkfA and
they have not entered the sporulation pathway (i.e. Spo0A inactive), provid-
ing a source of nutrients to support this key differentiation process.
More than 125 genes are responsible for sporulation of B. subtilis that
is governed by a program of gene transcription (Stragier and Losick, 1996)
whereas in the early stages of development of DNA-binding Spo0A protein,
500 genes are expressed (Molle et al., 2003). Molle et al. (2003) demon-
strated the direct control of Spo0A using chromatin immunoprecipitation
in combination with gene microarray analysis to identify regions of the
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chromosomes at which an activated form of Spo0A binds in vivo. Therefore,

Spo0A is a master regulator for sporulation, whereas many of its effects on
the global pattern of gene transcription are likely to be mediated indirectly
by regulatory genes under its control. Similarly, the gene expression during
spore formation have very little information to describe properly.
Sporulation of B. subtilis is a developmental process that is most re-
sponsible for conversion of growing cell into a dormant cell type known
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as spore or endospore (Stragier and Losick, 1996). Normally sporulation
is governed by DNA-binding protein, Spo0A acting like both activator and
repressor of transcription. One of the members of response regulator family
of proteins, Spo0A and its activity controlled through phosphorylation by
a phosphorelay which consists of five histidine autokinases (KinA, KinB,
KinC, KinD and KinE) and two phosphorelay proteins (Spo0F and Spo0B)
(Jiang et al., 2000) that integrate environmental and physiological signals
in the decision to sporulate (Burbulys et al., 1991). Spo0F is phosphorylat-
ed to Spo0F~P, while kinases feed phosphoryl group and in turn, the phos-
phoryl groups to Spo0B which in the final step of the relay, phosphorylates
Spo0A to create Spo0A~P (Burbulys et al., 1991), target of phosphory-
lated form, involved in the formation of polar septum which divides cell
into forespores and mother cell compartments (Levin and Losick, 1996).
The transcription of genes and operons directed by Spo0A~P activates
the compartment-specific regulatory proteins F and E which direct tran-
scription in forespore and mother cell, respectively (Stragier and Losick,
1996). Recently it is also shown that Spo0A~P continues to function after
formation of the polar septum when it accumulates to high levels and di-
rects transcription in the mother cell (Fujita and Losick, 2003). Spo0A also
activates transcription of certain genes indirectly by repressing the gene
for the transition-state regulator AbrB (Strauch et al., 1990). Among the
targets of AbrB, stationary phase sigma factor H is the gene influenced
by Spo0A, where asymmetric cell divides. Genome-wide analysis of H
regulon (Britton et al., 2002) has confirmed that both mutant and Spo0A
mutant have similar transcriptional profile. Recently Stragier and Losick
(1996) showed that F and E are replaced by forespore G and mother
cell K, respectively. Whereas computational searches for binding sites for
each regulator in sequences upstream of H- and Spo0A-regulated genes
indicated that the list of presumed direct targets of each regulatory protein
is largely different (Fawcett et al., 2000; Britton et al., 2002). Therefore,
researchers used a recently developed procedure for carrying out chro-
matin immunoprecipitation (CHIP) in conjunction with DNA microarray
analysis (chip) to identify chromosome regions where Spo0A binds in vivo
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(Ren et al., 2000; Iyer et al., 2001; Molle et al., 2003). In addition, some
analysis also enabled to discover many additional genes that are likely to
be part of the sporulation program (Fawcett et al., 2000).
1.5 MATHEMATICAL MODELS RELATED TO ECOLOGICAL

INTERACTIONS
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1.5.1 GENERAL METHODS
In this chapter, competition between different species is discussed.

Competition refers to dependence of two factors on several factors such as
nutrient, light, and temperature. It is interesting to check the natural advan-
tage of one species over another. One of the best examples of competition
is cannibalism. Several models for the dynamics of a cannibalistic popula-
tion are derived under the assumption that cannibals attack only weaker and
smaller victims. Vito Volterra has developed a model for the growth of two
competing species, which is an attractive model in mathematical ecology
(Lotka, 1925; Volterra, 1926). The following equation represents a simple
model for predatorprey interaction (Bailey and Ollis, 1986):
dH
= rH aHP
dt
and
dP
= bHP mP
dt
It has two variables (P, H) and several parameters: H is the density of

prey, P is the density of predators, r is the intrinsic rate of prey population
increase, a is the predation rate coefficient, b is the reproduction rate of
predators per one prey eaten, and m is the predator mortality rate.
Many mathematical modeling and theoretical analyses have been con-
ducted for the decision-making of an individual microorganism so that to
maximize the currency of the individuals fitness. Several models have been
developed on the population dynamics of cannibalistic population (Cushing,
1991; Hansen, 1993). Structured and discrete models are available to explain
cannibalism for both continuous models (Diekmann, 1986) and discrete
models (Cushing, 1991). Cells are shown to adapt to the fluctuating envi-
ronments by bringing diversity within the population stochastic phenotype
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switching mechanism. The idea of randomization of phenotype has been

analyzed theoretically and computationally (Diekmann, 1986).
1.5.2 POPULATION DYNAMICS
On the one hand, cannibalism can be an effective mechanism for the regu-
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lation and equilibration of population density. On the other, it can result in
population oscillations too. Thus, cannibalism can promote either equilibra-
tion or oscillations depending upon the exact circumstances under which it is
practiced. Again in some cases cannibalism can be a crucial mechanism for
population survival. The so-called lifeboat strategy asserts that the resources
obtained from cannibalism can permit a population to survive during periods
of non-cannibalistic resource scarcity or in other circumstances under which
it could not otherwise survive (Cushing, 1991). For example, the cannibal-
ism of the young by adults may provide access to resources available to
young individuals that are otherwise unavailable to adults. Therefore, the
interplay between the negative and positive feedbacks loops in regulatory
network which are inherent in cannibalism can result in multiple steady
states and hysteresis. This can lead to catastrophic crashes to lower equi-
librium levels, as population parameters are changed below critical values
that cannot be reversed by increasing the parameters back above their criti-
cal values. This fact has been used by researchers, for example, to explain
the collapse of certain harvested fish populations and their failure to return
to pre-harvested levels after harvesting is ceased (although other explana-
tions are possible, such as evolutionary effects as studied). Only a handful
of dynamical models for populations practicing cannibalism appear in the
literature. There have been some studies of the extreme case of egg cannibal-
ism that address the destabilization effect of cannibalism. The possibility of
oscillations in a discrete age-structured cannibalism model was studied by
Cushing (1991). The effects of cannibalism can be developed using a simple
discrete age-structured model, one that is both analytically and numerically
very tractable. The simple model suggests that the phenomena are very like-
ly to be common in model cannibalistic populations and consequently might
also be expected to occur in more sophisticated models. Cannibalism, like
interspecies predation, is most commonly practiced by larger individuals on
smaller individuals. If we assume that age correlates with body size, then a
simple model could be built by distinguishing just two age classes, a juve-
nile class and an adult class, as is done in the simple model for intraspecific
competition introduced by Cushing (1991).
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1.6CANNIBALISM VS PREDATION
There are very few organisms that exhibit the rare phenomena called canni-
balism and predation in our ecosystem. In our previous study, both the prop-
erties have been shown in B. subtilis and Escherichia coli in a mixed culture
system (Nandy et al., 2007, 2008; Nandy and Venkatesh, 2008, 2014).
B. subtilis resorts to cannibalism to delay sporulation under nutrition-
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al stress. However, in ecosystem B. subtilis responds in presence of other
microorganisms. Methylene blue dye reduction test (MBRT) (Bapat et al.,
2006) was developed to quantify viable cell count of different microorgan-
isms in pure and mixed cultures. MBRT was also described to check the bac-
terial contamination in the industrial reactor to save batch time. This method
was further used to study the survivability of B. subtilis and E. coli as a
mixed culture in phosphate buffered saline (PBS)-lacking nutrients. Pure
culture of B. subtilis demonstrated cannibalism under stress condition or
limiting nutrient condition. A regulatory protein, Spo0A, present in fraction
of the culture is responsible for delaying sporulation that produces a killing
factor by activating skf operon and an associated pump to export the factor.
Other cells not containing spo0A are lysed. Therefore, there is a competition
started in the culture to survive in the nutritional limitation condition which
demonstrates cannibalism. Our extended study has also shown the predatory
behavior of B. subtilis in the presence of E. coli under severe nutritional
limitation (Nandy et al., 2007).
Further, the model developed in the previous study was empirical and
phenomenological, and structured model based on the molecular understand-
ing of the process of Spo0A regulation can be carried out. Environmental
changes are known to have an effect on the cannibalistic behavior of species.
Abiotic factors like temperature alter the interaction between species in ecol-
ogy and affect the population dynamics. It is important to study the effect of
temperature on the cannibalistic behavior of B. subtilis. It has been demon-
strated that the cannibalistic property of B. subtilis under extreme nutrient
deficiency is dependent on the medium temperature. The data were analyzed
by proposing a model using a delay differential equation. The dynamics of
growth and death in medium lacking nutrition result in an oscillatory behav-
ior of cell count. This was captured by simulating the dynamics using a delay
differential equation. The key parameters that determined the oscillatory be-
havior were the kinetic constants for growth and death. Since these param-
eters were dependent on temperature, the dynamics of cannibalism were also
strongly related to the medium temperature. At high temperatures (beyond
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40C) due to high kinetic constant of death and the absence of killing factor,
the dynamics demonstrated a first-order death kinetics. At low temperatures,
the growth rate was low which resulted in a very low amplitude oscillation.
The presence of the killing factor which indicates the process of cannibalism
was essential for observing the oscillating behavior. The presence of the kill-
ing factor was not observed at low initial cell counts (less than 1000 cells)
and at high temperatures, resulting in the absence of cannibalism (Nandy et
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al., 2008). The study is also extended to predatory behavior and the dynam-
ics show very satisfactory result.
Our previous results on the effect of external carbon and nitrogen on the
cannibalistic behavior of B. subtilis have already been described (Nandy et
al., 2008). It has been found that when carbon was introduced into a medium
in the absence of any other nutrients, the cannibalistic tendency was delayed.
This delay increased with the increase in the amount of glucose which is a
major source of carbon. Hence, the cells could not totally consume glucose.
Further, the cannibalism occurred at a later stage after the maximum utiliza-
tion of carbon in the medium. Thus, the cannibalism was observed to be very
sensitive to the amount of carbon present in the medium. However, when
the medium contained only nitrogen and was devoid of carbon, the effect
on cannibalism was minimal. Therefore, cannibalism was more sensitive
to carbon than to nitrogen, indicating that the phenomenon of cannibalism
was more energy dependent than nitrogen assimilation (Nandy et al., 2008).
Further, predatory behavior, another interesting property associated with
ecosystem, was also rare to observe in the microbial world. In the extended
work, B. subtilis maintains its viable cells under different combinations of
carbon and nitrogen effects with E. coli cells. In this work, as the concentra-
tion of carbon sources increases, a number of B. subtilis cells maintain more
than normal predation, whereas there is no effect in the presence of nitrogen.
Cannibalistic tendency was delayed with the increase in the concentration of
carbon source. For higher concentration of carbon, maximum glucose was
consumed, with some of the carbon sources remaining unused. Therefore,
predation was also sensitive to carbon and then nitrogen, which proves the
dependency on carbon in both cannibalism and predation.
1.7 CONCLUSION AND FUTURE PERSPECTIVES
Microorganisms under stress usually form endospores for their survival.

The endospores transform into vegetative cells under suitable conditions
for growth. Suitable conditions may be biotic in nature, such as media
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constituents or abiotic, such as temperature and pH. For example, B. subtilis,

a Gram-positive soil microorganism, can form endospores under nutritional
stress (Gonzlez-Pastor et al., 2003). However, the formation of endospore
is energy intensive and the organism resorts to spore formation only under
sustained stress conditions. Competence is a distinct phase from vegetative
growth and sporulation that takes place during the onset of late log phase
and under specific nutritional conditions. During competence, cells acquire
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extracellular DNA and get transformed. Since spore formation is a last re-
sort for survival under extreme condition, cells have evolved strategies to
delay spore formation. The organism uses strategies such as cannibalism
and competence to delay spore formation. In the nature, microorganisms
have to deal with competition from other microorganisms present in the en-
vironment. Thus, the response to stress has to be analyzed in the presence of
other microorganisms. This requires study of mixed culture under condition
of stress. Mixed culture can respond in various ways in an ecosystem. The
response varies from competition, symbiosis, predation, cannibalism, neu-
tralism, commensalism, and ammensalism.
In this chapter, a review of our previous study on cannibalistic and preda-
tory behavior of B. subtilis is reported. The organism is metabolically ac-
tive as demonstrated in previous studies. An extension of this study involves
quantifying metabolic state during cannibalism using molar balance or
metabolic flux analysis. Further, the model developed in this study was em-
pirical and phenomenological, and structured model based on the molecular
understanding of the process of Spo0A regulation can be carried out. These
studies can also be extended to other species and also be used to study other
interactions such as competition and mutualism.
KEYWORDS
Bacillus subtilis
Cannibalism
Ecological interactions
Endospore
Escherichia coli
Forespore
Gram-positive bacterium
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Killing factor
Mathematical models
Metabolites
Methylene blue dye reduction test
Microbial interaction
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Mixed culture
Phosphorylation
Population dynamics
Predation
Spore
Sporulation
Survival strategy
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CHAPTER 2
IDENTIFICATION OF
MICROORGANISMS CARRIED
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BY AEOLIAN DUST TO OTHER
CONTINENTS AND THEIR IMPACT ON
PUBLIC HEALTH
PARASKEVI A. FARAZI
Mediterranean Center for Cancer Research, Department of Life and
Health Sciences, University of Nicosia, 46 Makedonitissas Avenue,
P.O. Box 24005, Nicosia 1700, Cyprus
CONTENTS
2.1 Aeolian Dust......................................................................................18

2.2 Methods in Aeolian Dust Research....................................................21
2.3 Future Perspectives in Aeolian Dust Research Methods...................31
2.4 Conclusion.........................................................................................34
Keywords....................................................................................................35
References...................................................................................................35
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2.1 AEOLIAN DUST
2.1.1 CHARACTERISTICS, SOURCES, AND TRAVEL OF AEOLIAN

DUST
Aeolian dust originates from wind erosion of the regolith - the loose rock
and dust layer of bedrock - and consists of soil particles found in deserts
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or arid regions. The term Aeolian comes from the Greek word Aeolus,
who was the God of winds in ancient Greece. Aeolian dust events arise from
deserts of different continents such as the Sahara and Sahel deserts in Africa,
Australian deserts (the Simpson and Strzelecki deserts) as well as Lake Eyre
Basin and the western sector of the MurrayDarling Basin in Australia, the
Taklamakan, Gobi and Badain Jaran deserts as well as the Loess plateau in
Asia. The desert dust can actually be transported over long distances by air
currents that are able to carry it even to other continents (Revel-Rolland
et al., 2006; Yamaguchi et al., 2012). For example, more than one million
tons of Asian dust particles, which travel a distance of 30005000km, reach
Japan every year (http://www.nies.go.jp/index-e.html). Furthermore, dust
from North Africa has been reported in many European countries including
Greece (Crete), Spain, Italy, UK, France (Alps), and Scandinavia (Stevenson,
1969; Ricq de Bouard and Thomas, 1972; Bergametti et al., 1989; Nihln and
Mattsson, 1989; Rod et al., 1993; Franzn et al., 1994). Similarly, dust from
Africa reaches the United States and Caribbean (Prospero, 1999; Prospero
and Lamb, 2003). At the global level, estimates have shown that 0.55 bil-
lion tons of desert dust migrates by air annually (Perkins, 2001). The major-
ity of the dust (5075%) comes from the North African deserts, even though
in the last few decades there has been an increased migration of dust from
Asia, due to changes in the climate and desertification (Moulin et al., 1997;
Goudie and Middleton, 2001; Prospero and Lamb, 2003; Zhang et al., 2003).
In order for dust to enter the atmosphere, there needs to be a large supply
of it as well as strong surface winds, such as the low-level jet described in
the Bodele region in Africa (Washington and Todd, 2005). In addition, the
velocity of the wind matters on whether the dust will be raised in the atmo-
sphere and winds in the range of 6.513m/s have been reported to raise dust
in the air in Western Sahara region (Helgren and Prospero, 1987). Strong
vertical transport is needed to carry the dust into the troposphere, which oc-
curs under conditions of unstable thermal stratification as well as large-scale
synoptic upward motions (Estoque et al., 1986). A large supply of dust is
usually found in the most arid settings where there is no vegetation (New
et al., 2002). Atmospheric humidity has also been found to be extremely
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Aeolian Dust-Associated Microorganisms and Disease 19
important in the generation and transport of dust. Interestingly, dust concen-

tration increases with relative humidity (Csavina et al., 2014).
Aeolian dust is rich in many mineral and pollutant elements such as zinc
(Zn), chlorium (Cl), copper (Cu), lead (Pb), and sulfur (S) (Zhang et al.,
2010). In addition, the dust has been shown to carry persistent organic pol-
lutants such as pesticides, polychlorinated biphenyls, and polycyclic aro-
matic hydrocarbons (Garrison et al., 2006). Nitrate and sulfate ions have
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also been shown to be transported along with Aeolian dust (Moria et al.,
2003). Finally, quartz, crystalline aluminosilicates, iron oxides, and hydrox-
ides have been identified on travelling dust particles (Tondera et al., 2007).
2.1.2 AEOLIAN DUST AS A CARRIER OF MICROORGANISMS
Bacteria have been shown to attach on Aeolian dust with the potential to
migrate globally. Bacteria species such as actinobacteria, bacilli, and sphin-
gobacteria were found to be the predominant species in a study of Asian dust
(Yamaguchi et al., 2012). Furthermore, bacilli and sphingobacteria were ac-
tually able to grow on media suggesting that these species are not merely
transported by the dust but are also physiologically active (Yamaguchi et
al., 2012). Analysis of African dust reaching the Eastern Mediterranean has
revealed several bacteria such as firmicutes, actinobacteria, gammaproteo-
bacteria, betaproteobacteria, and cyanobacteria being carried by the dust.
Within spore-forming bacteria (such as firmicutes), bacilli and clostridia
have been identified. About 24% of sequenced clones were actually closely
related to human, plant, and animal pathogens, which have been linked to
diseases such as pneumonia, meningitis, bacteremia, and pathologic reac-
tions like endocarditis (Polymenakou et al., 2008). Bacteria carried by dust
have been identified in the Himalayas at a 6000m altitude and interestingly
these bacteria had good viability and growth potential. The most abundant
bacteria phyla were acidobacteria, proteobacteria, verrucomicrobia, and ac-
tinobacteria (Stres et al., 2013). Similarly, viable bacteria were identified in
tropospheric samples from the Gulf of Mexico (DeLeon-Rodriguez et al.,
2013), Asian dust traveling to Japan (Hara and Zhang, 2012; Yamaguchi
et al., 2012) and North America (Smith et al., 2012). These findings have
important implications as bacteria carried by the dust can potentially reach
their destinations alive and thus affect the host ecosystem. Bacteria genera
such as Microbacterium, Sphingomonas, Bacillus, and Streptomyces, as well
as the opportunistic pathogen Pseudomonas aeruginosa were isolated in the
Caribbean during African dust events (Griffin et al., 2001; Griffin et al.,
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2003). In addition, bacteria such as Bacillus, Gordonia, and Staphylococcus

have been found in African dust reaching the mid-Atlantic (Griffin et al.,
2006).
Fungi have also been shown to be carried by Aeolian dust. In California, an
outbreak of coccidioidomycosis caused by the fungus Coccidioides immitis
was reported after a severe dust storm (Williams et al., 1979). More recently,
the events of yellow fever associated with coccidioidomycosis were stud-
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ied in Phoenix Arizona, and associations between yellow fever events and
dust storm days were found (Sprigg et al., 2014). Many other types of fungi
such as Acremonium, Alternaria, Arthrinium, Aspergillus, Cladosporium,
Curvularia, Emericella, Fusarium, Nigrospora, Paecilomyces, Pithomyces,
Phoma, Penicillium, Torula, Trichophyton, and Ulocladium have been iden-
tified to be carried by dust (Griffin, 2007). These fungi are pathogenic and
therefore could have important implications in human health and disease
outbreaks following dust storms.
Viruses have not been investigated as extensively in terms of their ability
to be carried by dust during storms, however, there have been some timing
associations between disease outbreaks (such as foot and mouth disease in
livestock populations) and dust storms. These studies failed to show the pres-
ence of the relevant viruses on dust, however, the occurrence of these disease
outbreaks right after dust storms is strongly suggestive of the ability of dust
to carry viruses (Ozawa et al., 2001; Joo et al., 2002; Sakamoto and Yoshida,
2002). A more recent study has shown higher concentrations of ambient influ-
enza A virus during Asian dust storms, suggesting that the dust is able to carry
viruses during its travel (Chen et al., 2010). Another study has shown the abil-
ity of dust particles to induce inflammatory response of macrophages, further
suggesting that dust could contain microorganisms such as viruses and bac-
teria (Higashisaka et al., 2014). In the United States, bioaerosols (which can
include bacteria, fungi, viruses, pollen, cell debris, and bio-films), ranging in
size between 10nm and 100m have been identified to be transported via
dust storms, further implicating dust acting as a carrier of different types of
microorganisms, including viruses (Hallar et al., 2011).
2.1.3 THE IMPACT OF AEOLIAN DUST ON PUBLIC HEALTH
Considering that Aeolian dust carries all the aforementioned elements, pol-
lutants, and microorganisms, it comes as no surprise that the dust has a huge
impact on public health throughout its travel. For example, in Cyprus, an
island in Southeastern Mediterranean, increased hospitalization, as well as
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increased mortality linked to cardiovascular causes was reported during

sand storm days (Middleton et al., 2008; Neophytou et al., 2013). More spe-
cifically, a 2.43% increase in cardiovascular mortality was reported for each
10g/m3 increase in PM10 concentration (Neophytou et al., 2013). Ambient
concentrations of particulate matter consisting of particles smaller than
10m (PM10) have been shown to increase during Aeolian dust events (Pey
et al., 2012). Interestingly, in New Zealand all-cause mortality increased
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by 7% per 10g/m3 increase in PM10 exposure (Hales et al., 2012). Lung
cancer and respiratory-associated deaths showed higher associations. One
could assume that this effect would also apply to the increased concentra-
tions of PM10 associated with Aeolian dust. In fact, a study in Spain revealed
an increase of 8.4% in mortality per 10g/m3 increase in PM10 exposure
during Sahara dust storm days and only 1.4% increase in mortality during
non-Saharan dust days (Perez et al., 2008). A similar effect was reported in a
study in Italy, where a 22% increase in respiratory mortality of elderly peo-
ple over the whole year was associated with Saharan dust events and 33.9%
increase in respiratory mortality during the hot season (Sajani et al., 2011).
In Athens, Greece, a 0.71% increase in overall mortality was reported to be
associated with 10g/m3 increase in PM10 exposure during dust storm days,
with greater effects of people older than 75 years of age than younger age
groups (Samoli et al., 2011). In Japan, increased hospitalization of children
due to asthma was reported during days of Aeolian dust events (Kanatani
et al., 2010). Similar results were obtained in a study in the United States
(Texas), where increased number of hospital admissions due to asthma and
acute bronchitis were observed after dust events (Grineski et al., 2011). In
a two-year study in the Caribbean, scientists failed to show an association
between asthma-related hospital admissions and dust storm days, however,
it is unclear whether this has to do with the environmental setting of the
study (i.e., lower pollution levels in Barbados to start with) (Prospero et al.,
2008). Finally, allergic skin reactions in Japan during Asian dust events were
suggested to be linked to metals bound to and carried by the dust during its
travel (Otani et al., 2012).
2.2 METHODS IN AEOLIAN DUST RESEARCH
To reach the conclusion that the Aeolian dust is a carrier of microorganisms

and has an impact in public health, several studies have been conducted in
different regions of the world. Although these studies have the same overall
goal, their methodology differs especially in the following three aspects: (1)
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the choice of method of dust collection, (2) the geographic location of dust
collection, and (3) the method of microorganism characterization and iden-
tification. The next sections summarize these different ways of studying the
problem of microorganism transfer by the Aeolian dust to other destinations.
9781771883627
FIGURE 2.1 The important issues a scientist has to consider in the study of the
microorganism contents of Aeolian dust. The figure summarizes the considerations
a scientist has to make in regards to methods of dust sampling as well as the options
for sampling location. In addition, the methods used thus far in order to characterize
and identify microorganisms carried by the dust are shown.
2.2.1 METHODS OF AEOLIAN DUST COLLECTION
2.2.1.1SAMPLING
An interesting method of dust sampling involves use of a fabricated dust

sampler with wet beads, which are able to absorb dust particles. Such a
method was used by Yamaguchi et al. (2012) who studied the dispersion of
bacterial cells on Asian dust (Yamaguchi et al., 2012). The wet beads were
packed inside a stainless can and the air was collected through a sterilized
Teflon inlet tube. The sampling device can easily be attached on a small
airplane to collect air and dust at a higher altitude, as was done in the afore-
mentioned study. The beads are suspended in a certain volume of particle-
free water and filtered onto a sterilized polycarbonate membrane filter of
small pore size (such as 0.4m) to collect the dust particles. The dust is thus
trapped on these filters, which can be used for DNA extraction or microor-
ganism growth studies by placing the filters on agar media.
Air samples can also be collected using multi-stage impactors. These
impactors are attached to high-volume air samplers and can fractionate
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particulate matter into different fractions that differ in size and then they are
captured on filters. They can be set to collect air at a constant flow rate. Such
a sampler was used in the Greek study and was set to collect air particles at
constant flow rate of 740L/min. The dust was trapped on glass fiber filters
(Polymenakou et al., 2008). Such impactors are easy to attach on air sam-
plers and use in different land-settings. They are usually put on a platform to
avoid contamination from nearby surfaces. For example, Polymenakou et al.
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(2008) put the impactor on a 5m high platform.
Other types of collectors can be used for the collection of air samples
containing dust. For example, in-house designed portable sampling systems
have been used, which consist of a vacuum pump attached to a manifold
(PVC-pipe) secured inside a carrying case. Cellulose nitrate membrane fil-
ters are placed on the manifold in order to capture dust particles from the air
drawn. Such a sampling system was used in a study in Mali in West Africa
(Kellogg et al., 2004).
When sampling is performed at higher altitudes (e.g., mountains), where
the level of air pollution is minimal compared to urban settings, it can be
done in much simpler ways. For example, even a simple collection of air
for a longer period of time (e.g., weeks to a month) in tubes might work in
a clean environment setting. Such a study was performed to study micro-
organism diversity in the Himalayas (Stres et al., 2013). In this study, the
method of collection involved setting 50 ml sterile falcon tubes prefilled
with a layer of 2cm tall 100% polyester wool (pre-treated with a solution of
10% formaldehyde in 70% glycerol to prevent in situ microbial growth). The
tubes were left open for 30 days at an altitude of 50006000m every 200m
intervals. The tubes were then rinsed with sterile water and the water was
passed through a filter (0.22m size) to capture any microorganisms and
subsequently isolate DNA for additional analyses (DNA and microorganism
count).
At high altitudes in mountainous areas, another form of sample is snow.
It is easy to collect and transport for further analysis. For example, Stres et
al. (2013) collected snow samples in 50ml sterile falcon tubes immediately
after snowfall to ensure it was clean and fresh, then allowed the snow to
thaw and fixed it using formaldehyde to avoid microbial growth other than
what was in the snow sample initially. The thawed and fixed snow sample
was then filtered on a membrane and processed for further analysis as de-
scribed earlier (Stres et al., 2013). An alternative method to collecting snow
in a sterile way is to remove the top layer of snow and collect snow from
below. This was actually done in a study investigating the microbial diver-
sity carried by Aeolian dust to the Alps (Chucochina et al., 2011). In this
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study, to ensure sterile collection of snow, 25cm of snow in contact with

air was carefully removed with a clean shovel and clean snow was collected
underneath using sterile plastic containers. To further ensure sterility, the re-
searchers were wearing single-use Tyvek coveralls and vinyl gloves to avoid
any bacterial contamination other than the bacteria contained in the snow.
In this case, the snow was allowed to thaw and was concentrated using cen-
trifugal filter devices which allow concentration and desalting of solutions.
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Concentration is achieved by ultrafiltering the sample solution through an
anisotropic membrane. The centrifugal force essentially directs solvents and
low molecular weight solutes through the membrane into the filtrate vial,
whereas macrosolutes are retained above the membrane inside the sample
reservoir.
An interesting new approach to collect dust samples for investigation
of potential microorganisms carried by the dust involves the use of an air-
sampling device that is placed under the wing of an F-104 Starfighter jet
and can capture particles directly from clouds at a maximum altitude of
25000 ft (http://uk.mobile.reuters.com/article/environmentNews/idUK-
BRE9B510F20131206). The device (called DART - Dust at Altitude
Recovery Technology) is currently being used by researchers at the
University of Florida and the results should come out in the coming years
(http://www.insidescience.org/content/tracking-dust-around-world/1643;
http://phys.org/news/2013-12-high-altitude-device-airborne-pathogens.
html). The fact that collection of microorganisms can be performed right be-
fore the entry of the dust into the destination route allows the identification
of microorganisms carried by the dust, which would have not mixed with
local microorganisms, thus making the results much clearer.
All the methods of Aeolian dust collection described thus far involve
collection of the dust from air or snow samples. Dust can also be collected
from water samples, for example water from lakes. A study like this was
conducted in Spain, where the method of collection involved taking wa-
ter samples from the surface of lakes in the Pyrenees ranging in altitude
from 16202240m above sea level. The water samples were filtered twice
through 0.2m polycarbonate membranes to exclude local contaminating
microbes (Hervs et al., 2009). The filtered water sample can then be pro-
cessed further through a filter as described above to capture microorganisms
or directly as a source of the microorganisms for further characterization and
identification studies.
Aeolian dust originates from the deserts and thus a sample of it can be
collected directly from the source that is the desert sand. In fact, many studies
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have collected desert sand samples in search of microorganisms. Typically,

a certain amount of sand is collected and used for further analysis. For ex-
ample, in a study aimed at identifying gamma radiation resistant bacteria
in the Sahara desert, sand samples were collected, subjected to a dose of
15kGy gamma radiation, and subsequently plated on agar plates to iden-
tify colony-forming bacteria. Such bacteria would be good candidates for
travelling to far-away destinations and surviving harsh conditions (de Groot
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et al., 2005). Another study used sand samples from the Republic of Chad
in Africa, which were collected aseptically in tubes and used for microbial
characterization and DNA isolation analyses (Giongo et al., 2013).
2.2.1.2 GEOGRAPHIC LOCATION
Aeolian dust can be collected in different environmental settings. For ex-

ample, it can be collected at the source (i.e., the desert sand itself or air
samples from the desert of dust origin) (Kellogg et al., 2004; de Groot et al.,
2005; Giongo et al., 2013). Since the impact of Aeolian dust on destinations
far from the source is huge, it is also important to collect dust samples from
far away destinations of Aeolian dust travel. The latter can be achieved in
different ways. Some investigators choose to collect air samples on land
(just a few feet from the ground) (Polymenakou et al., 2008). For example,
in a Greek study sampling was done on a 5m high platform (located at the
University of Crete Campus) with the aim to reduce possible effects from
near-surface sources (Polymenakou et al., 2008). The problem with such
studies is that their urban setting increases the probability that microorgan-
isms of local origin (due to local air pollution) will be collected along with
the Aeolian dust sample. Thus, in such a setting it would be important to
compare dust collection on days with Aeolian dust events and days with
pollution from other sources. Of course, this increases the expenses as more
sequencing will need to be undertaken.
Other investigators try to avoid the local contamination problem by set-
ting up air collection at high altitudes (e.g., in the Alps or the Himalayas)
(Chucochina et al., 2011; Stres et al., 2013). In the Himalayas for example,
air sampling has been performed at an altitude of 50006000m (Stres et al.,
2013). At high altitudes, sample collection can also be achieved by collect-
ing snow samples (Chucochina et al., 2011; Stres et al., 2013). For example,
in the Alps in France snow was sampled at an altitude of 4250m from days
with Saharan dust events (during which the snow had a reddish-brown color)
and clean days (during which the snow had a clean color) (Chucochina et
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al., 2011). Even at high altitudes investigators still choose to sample at other
points for comparison. For example, in the study of dust in the Alps samples
from a nearby city of Grenoble (at 200m altitude) during rain deposition
were collected as well as samples from the Sahara desert for comparisons of
microorganism contents.
An interesting way to collect air samples containing Aeolian dust is by
collecting air near the destination route at a high altitude using an airplane.
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For example, a study in Japan was conducted by taking samples at an alti-
tude of 900m about 10km away from the coasts, which ensures that the
dust collected is not mixed with local pollution and that the microorgan-
isms identified were carried by the dust (Yamaguchi et al., 2012). Similarly,
an impactor device was mounted within a collector housing located on the
underside of a Lockheed Martin ER-2 to collect air samples at 20000m in
two different locations in the United States (Griffin, 2004). Samples col-
lected at such high altitudes minimize the chance of picking up local micro-
organisms, thus making the identification of microorganisms carried by the
Aeolian dust easier. Of course they require a more refined set up, however,
once the set up is in place the study of microorganism identification is more
straight-forward.
2.2.2 METHODS OF CHARACTERIZATION OF

MICROORGANISMS CARRIED BY AEOLIAN DUST
Once dust samples are collected from various sources as described above,
it is important to characterize their contents. Techniques such as scanning
electron microscopy (SEM) with energy dispersive X-ray analysis have been
used previously to study the chemical composition of the dust in elements
such as hydrogen, metals, and so forth as well as the size of the dust particles
(Matsuyama et al., 2008; Yamaguchi et al., 2012). SEM focuses an electron
beam across the surface and detects secondary or backscattered electron sig-
nals that allow the researcher to obtain detailed high-resolution images of
the sample. An energy dispersive X-ray analyzer is used along with the SEM
and it provides identification of elements and information on the quantitative
composition of the sample. This technique was used to study dust in Japan
for example (Yamaguchi et al., 2012).
To visualize bacteria on the surface of dust particles (in order to quantify
bacterial abundance) scientists can use a laser-scanning microscope attached
to a microspectrophotometer. This technique was used in the aforementioned
study of dust in Japan (Yamaguchi et al., 2012). Nucleic acids were stained
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with a fluorescent dye to distinguish bacteria from dust. Essentially, dust

particles are first suspended in water, fixed, and filtered on polycarbonate
membrane filters for staining. The technique can be quite tricky because
of high fluorescence background from soil itself. Efforts to reduce sample
high auto-fluorescence have been successful by using SYBR Green-I (which
stains the nucleic acids) and treating the specimens with hydrofluoric acid
(Morono et al., 2009). Bacterial cell concentrations in dust samples can also
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be measured using flow cytometry as was done in a study of dust in the
Alps (Chucochina et al., 2011). In this technique, samples are fixed (e.g., in
glutaraldehyde), mixed with a dye (e.g., SYBR Green I) and put through a
flow cytometer. Bacterial abundance has also been successfully determined
using 16S rRNA gene quantification by real-time PCR (Yamaguchi et al.,
2012). The colony-forming units of bacteria present per gram (g) of sand
are calculated by collecting the sand sample and then cultivating bacteria
on agar by streaking a specific amount of sand (suspended in saline) on agar
(Giongo et al., 2013).
2.2.3 METHODS OF IDENTIFICATION OF MICROORGANISMS

CARRIED BY AEOLIAN DUST
A popular method for identifying microorganisms involves sequencing of

the 16S rRNA gene which is shared among all bacteria but bears some se-
quence differences since it has nine variable regions (Weisburg et al., 1991;
Cox et al., 2013). Sequencing of this gene allows the identification of bacte-
ria on the dust. The 16S rRNA has been described as a molecular chronom-
eter (Woese, 1987). It is highly conserved due to its critical function in the
cell as a component of the ribosomes, which play an essential role in protein
translation. The 16S rRNA gene is about 1550bp in length and is com-
posed of both conserved and variable regions (Cox et al., 2013). Universal
primers, which are complementary to the conserved regions, are used to
amplify the gene in any bacterium and subsequently sequencing allows the
identification of the slight sequence differences between bacteria. Genbank,
which contains the sequences of all sequenced bacteria thus far, and other
databases such as the ribosomal database project (http://rdp.cme.msu.edu/),
Greengenes (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi), and SILVA
(http://www.arb-silva.de/) are used to make comparisons of the sequences
retrieved from experimental samples with the database sequences so as to
enable matching and identification of bacteria (Clarridge, 2004). Various
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software products have been developed to assist in these comparisons such

as QIIME (http://qiime.org/) and Mothur (http://www.mothur.org/).
Typically the sample DNA is amplified by PCR using universal prim-
ers for 16S rRNA, cloned into a vector and then a certain number of clones
are sequenced using the same primers for bacteria identification (Kellogg
et al., 2004; Polymenakou et al., 2008; Chucochina et al., 2011). The same
technique can be used for 18S rRNA in order to identify fungi, which can
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also be identified by microscopy (Kellogg et al., 2004). When DNA is iso-
lated from bacterial colonies on agar plates then cloning is not necessary
and bacterial DNA is PCR-amplified and sequenced immediately (de Groot
et al., 2005). Sequencing can also be coupled to restriction fragment length
polymorphism (RFLP) analysis, which allows classification of bacteria into
operational taxonomic units based on their restriction pattern (Wei et al.,
2007; Polymenakou et al., 2008; Chucochina et al., 2011). In addition to
standard sequencing, high throughput sequencing can be used for bacteria
identification, which does not require cloning of the 16S rRNA PCR prod-
ucts. In fact, pyrosequencing has been successfully used for this type of
studies (Stres et al., 2013). Pyrosequencing is a sequencing method based
on the generation of light every time a nucleotide is added in the sequencing
reaction. Essentially pyrophosphate (PPi) is released after the addition of a
nucleotide to the growing chain. The PPi is then converted into adenosine
triphosphate (ATP) by the enzyme ATP sulfurylase in the presence of am-
monium persulfate (APS). ATP then drives the conversion of luciferin to
oxyluciferin with the help of the enzyme luciferase and ultimately light is
generated and detected to allow for reading the sequence.
A study of sand samples from the desert attempted to use three differ-
ent molecular approaches to identify bacteria in order to assess their effi-
ciency. To this end, the scientists: (1) amplified the 16S rRNA gene by PCR,
cloned the PCR product and then sequenced, (2) amplified the 16S rRNA
gene and then performed high throughput sequencing, and (3) performed
high throughput sequencing directly on the DNA sample without amplifying
it. Cloning the PCR products reduced the number of bacteria the scientists
were able to identify to 13 genera. High throughput sequencing was more ef-
ficient. A total of 270 genera were identified using high throughput sequenc-
ing of amplified DNA and 260 genera were identified using high throughput
sequencing of non-amplified DNA. Of note, high throughput sequencing of
non-amplified DNA allowed the identification of Archaea, which was not
possible with the other two methods (Giongo et al., 2013). This development
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is important, as sequencing a DNA sample directly without having to am-

plify it first is more efficient.
2.2.4 METHODS OF IDENTIFICATION OF LIVE

MICROORGANISMS CARRIED BY AEOLIAN DUST
9781771883627
Identifying the microorganisms carried by the dust from the deserts to far-
away destinations is important, however, it is even more important to deter-
mine which microorganisms actually make it to the destination alive. The
aforementioned methods allow for the identification of bacteria, however,
this could be meaningless if the bacteria are not alive and therefore would
have no impact on health. Along these lines several researchers tried to in-
vestigate the growth potential of the bacterial cells on the dust particles by
incubating dust particles in different types of media (nutrient-rich and nu-
trient-poor liquid media as well as on agar media). To this end, Hervs et
al. (2009) tried to identify microbes that are actually viable in lakes in the
Pyrenees in Spain ranging in altitude from 16202240m above sea level
(after a major dust storm event) as well as sandy soil from Mauritania in
the Sahel region. The samples were filtered twice to exclude local contami-
nating microbes and then supplemented with different nutrients to enrich
for any bacteria carried by the dust that are actually viable. The supple-
ments included: (1) organic carbon (sodium acetate 1mM, final concentra-
tion), (2) nitrogen (casamino acids 0.04% w/v, final concentration), and (3)
phosphorous (KH2PO4 10mM, final concentration) (Hervs et al., 2009).
Identification of the viable bacteria was then carried out by 16S rRNA gene
sequencing as already described.
Several studies have attempted to obtain counts of viable microbes from
air samples of small volumes (<200L) and have shown an order of hun-
dreds of bacteria and fungi that are capable of surviving dust transport over
long distances. Live microorganisms identified in the Virgin Islands included
plant pathogens (25%) and opportunistic human pathogens (10%) (Griffin et
al., 2001). In this study, filters onto which dust from air was collected were
plated and grown on R2A agar, which is a low-nutrient medium suitable
for culturing stressed microbes (Reasoner and Geldreich, 1985). Similar in-
vestigations in Africa have shown live animal pathogens (10% of bacteria),
plant pathogens (5% of bacteria), and opportunistic human pathogens (27%
of bacteria) (Kellogg et al., 2004). An in-house designed portable sampling
system containing a pump and a manifold was used to collect air samples.
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The samples were collected on a cellulose nitrate membrane filter of 0.2m

size. The filters were plated on R2A agar and grown as in the previous study.
2.2.5 METHODS OF INVESTIGATION OF THE IMPACT OF

AEOLIAN DUST ON PUBLIC HEALTH
9781771883627
To investigate the impact of microorganisms carried by the Aeolian dust on hu-
man health, scientists try to make associations of dust episodes, identification
of microorganisms, and increases in the incidence of particular diseases, infec-
tions, and so forth. One example is the investigation of the link between Asian
dust in Japan and hand, foot, and mouth disease (HFMD). To achieve this, the
scientists collected bioaerosol samples (after dust events) from rooftops in an
attempt to investigate whether the enterovirus was present in these air samples.
At the same time, they analyzed hospital records and studied the hospitaliza-
tion rate for HFMD during dust days as a function of suspended particulate
matter (SPM, which increases following dust events). Indeed, hospitalization
rates increased in association with SPM, however, scientists failed to identify
the enterovirus (http://ehp.niehs.nih.gov/isee/p-2-02-13/). Studies of asthma
and allergies in children have identified dust as a predictor for increased in-
cidence of these conditions using logistic regression (Bener et al., 1996). In
addition, another hint that dust has implications in respiratory diseases and
public health comes from the fact that areas that are severely affected by desert
dust such as the Aral Sea and the Caribbean have the highest asthma rates in
the world (Howitt, 2000). In Barbados for example, asthma rates increased 17
times in just over two decades (highest rates in the 1823% range) (Howitt
et al., 1998). In Trinidad, a retrospective study has shown increased hospital
visits due to pediatric asthma during African dust events (Gyan et al., 2005). It
has been found that fungal and bacterial spores as well as molecules of the mi-
crobes such as endotoxins and mycotoxins can trigger respiratory stress. Thus,
one could infer from these observations that a potential explanation of higher
asthma rates after dust storms involves the contents of dust in microorganisms
and toxins (Braun-Fahrlander et al., 2002; Griffin and Kellogg, 2004).
Another way to study the impact of the dust on human health is to study
its effect in various biological processes. Dust for example, has been shown
to cause lipid peroxidation and DNA damage (Athar et al., 1998). In addi-
tion, study of the elements and other chemicals carried by dust can reveal
ways in which the dust can have an impact on public health. The finding
that dust carries radioactive elements, such as 90Sr and 137Cs (which can
be toxic and carcinogenic), suggests that Aeolian dust can have significant
AUTHOR COPY
long-term effects in public health, including an increase in the burden of

cancer (Igarashi et al., 2011). In addition, chromium which is carcinogenic
in some forms has been identified as one of the largest elemental compo-
nents of Aeolian dust, raising the possibility of an impact of the dust on
cancer (Liu et al., 2011; Aydin et al., 2012). The dust has also been shown to
carry persistent organic pollutants such as pesticides, polychlorinated biphe-
nyls, and polycyclic aromatic hydrocarbons (Garrison et al., 2006). There
9781771883627
has not been a demonstrated association between Aeolian dust and cancer
yet nor has there been direct evidence that dust has carcinogenic properties
at the molecular level. This represents an area open for investigation.
2.3 FUTURE PERSPECTIVES IN AEOLIAN DUST RESEARCH

METHODS
2.3.1 ALTERNATIVE METHODS TO CAPTURE AND

CHARACTERIZE MICROORGANISMS IN AEOLIAN DUST
2.3.1.1 MICROORGANISM COLLECTION
Bacteria and fungi have also been successfully captured and identified from
the stratosphere at heights up to 41km. The collection of air sample in this
case was achieved using the deployment of balloon-borne cryosamplers as
reported earlier (Lal et al., 1996; Brugger et al., 2012). The probes in these
cryosamplers were carefully sterilized to ensure that the microorganisms cap-
tured would be those from the stratosphere and no other contaminating micro-
organisms. The microorganisms in this study were identified and studied by
SEM. Even though this method has not been used on Aeolian dust samples, it
can easily be adopted for this sort of study. Of course, it needs to be adjusted
to the altitude that dust can be lifted to during dust storms. Aeolian dust might
only be able to reach lower levels of the stratosphere (Griffin, 2004).
2.3.1.2 MICROORGANISM QUANTIFICATION AND

CHARACTERIZATION
Quantifying bacteria on dust samples can be tricky as mentioned in a previ-

ous section due to high fluorescence background of the soil. However, if
bacteria are captured on agar plates, then quantifying them would be easier.
Techniques such as automated counting of bacterial colony-forming units
AUTHOR COPY
on agar plates have been developed which make use of an illuminator, a

camera, and colony-counter software algorithms. The application of such
techniques can help quantify the bacterial abundance in dust samples across
different conditions (Wainwright et al., 2003). Such techniques can be ap-
plied to quantify bacterial number on Aeolian dust samples.
Another interesting technique used to characterize bacteria and other
microorganisms such as fungi is solid-phase cytometry (SPC) which com-
9781771883627
bines epifluorescence microscopy with flow cytometry. In this technique the
sample containing suspect microorganisms is filtered, fluorescently labeled,
scanned through a laser which excites the fluorophore and analyzed through
computer software. The advantage of using SPC is that it is fast and works
well with low numbers of microorganisms. One limitation of the technique
is the limited availability of compatible stains and the requirement for filter-
able samples that are clear, aqueous solutions (Williamson et al., 2003).
2.3.1.3 ISOLATION OF VIRUSES FROM DUST
Bacteria are relatively easy to isolate from dust samples, however, viruses
present many challenges. Work on the extraction of viruses from soil has
shown that the success of the extraction depends on the elution buffer used
as well as the enumeration technique used. For the latter, plaque assay, epi-
fluorescence microscopy, and transmission electron microscopy were com-
pared. It was shown that the constitution of each soil affected the success of
each of these methods, thus requiring extensive testing to determine the most
efficient method of isolating viruses in a particular soil setting (Lies et al.,
2010). When it comes to dust investigations, dust from different deserts or
dust from the source versus the destination should be experimentally tested
to identify the best way to isolate viruses. This is essential in order to ensure
complete characterization of viruses in these samples. Viruses represent a
major public health concern, thus their efficient identification and character-
ization would be essential. These findings should be taken into consideration
in studies of Aeolian dust aimed at identifying viruses.
2.3.2 ALTERNATIVE METHODS TO CAPTURE LIVE

MICROORGANISMS FROM AEOLIAN DUST
There are various ways to capture live microorganisms from air samples and
grow them, which can be utilized in studies of Aeolian dust. The simplest
AUTHOR COPY
way is sedimentation, which basically involves exposing agar plates to

the air. The disadvantage of this method is that larger particles will settle
on the plate first and thus prohibit smaller particles from settling. In ad-
dition, agar can dry out, which reduces the chances of microbial growth.
Finally, the method is very crude as there is no way to measure the vol-
ume of air sampled and thus makes reproducing the sampling impossible
(http://www.foodquality.com/details/article/878155/Air_Sampling_101.
9781771883627
html?tzcheck=1). Another way to capture live microorganisms involves the
use of an impactor, which basically is a jet that draws air in the sampler
by vacuum and directs the air to the surface of a petri dish with agar. The
dish is on a turntable, thus allowing the dish to turn and collect different
microorganisms in different areas of the plate. One of the drawbacks of this
method is that it can potentially miss smaller particles. Media dehydration
in this case as well can limit the growth of microorganisms (http://www.
appliedphysicsusa.com/MicrobialAirSamplers.html; http://electroiq.com/
blog/1997/06/examining-ways-to-capture-airborne-microorganisms/).
Sieve samplers represent another way to sample air. The setup of these
samplers involves a perforated plate which is in front of an agar plate and
allows air to pass through. A sieve sampler can also be set up in a stacked
way, where several perforated plates and agar plates are set one after the
other with the size of the perforation reducing as the air goes through the
arrangement of plates. This allows for the capture of larger particles in the
first plates and smaller particles in the last plates. One of the problems with
the sieve samplers is that if the sampling time is not short or if there is not
high humidity the area around the perforation on the agar plate can dry out
consequently inhibiting the growth of certain microorganisms. In addition,
sieve samplers are not very good at capturing small particles, unless the air
flow rate is fast, which can affect the viability of the microorganisms.
Another way to obtain air samples is through centrifugal samplers. This
type of sampler basically uses an impeller inside an open shallow drum,
which draws air in by creating a vortex. A centrifugal force accelerates the
air into the drum, which is lined with a thin layer of agar, thus allowing
capture and growth of microorganisms. Once again, these samplers fail to
capture very small particles, for example, less than 10% of particles smaller
than 2m actually get deposited.
To overcome the difficulty of capture of small particles, glass imping-
ers can be used. These basically draw air through a curved suction tube and
accelerate the air toward the bottom through a jet. The particles are cap-
tured in a liquid at the bottom and then processed for microorganism growth
studies. As already mentioned, these impingers actually allow the capture of
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particles of all sizes including very small particles. Their major disadvantage
is that because they are made of glass they are not disposable and actually
need to be prepared every time for sampling. However, for the purpose of
collecting air samples during a dust event these impingers might work quite
well. Cooling of the sample here is essential to avoid evaporation of the liq-
uid and thus loss of microorganisms from the sample.
A very efficient sampling technology is gelatin membrane filtration. The
9781771883627
flow rate can be programmed and microbes are captured on a gelatin mem-
brane filter that is about 300m thick. The filter is porous allowing microor-
ganisms to be captured inside as well, not just at the surface and actually, it
allows for the capture of even the smallest microorganisms, such as viruses.
In addition, since the filter is made up of 50% water it does not present prob-
lems of drying out and thus the viability of microorganisms is unaffected,
contrary to all other air collection methods described. The filter is easy to
remove and place on a standard agar petri dish for microorganism growth.
In addition, the filter dissolves very easily in the agar. If microorganisms
are to be captured on filters instead of agar plates, then the choice of filters
when collecting air samples is crucial in ensuring the viability of captured
microorganisms. It was found that HEPA filters for example support the vi-
ability of captured microorganisms for even up to 210 days for some types
(Mittal et al., 2011).
Finally, to overcome the low capture efficiency and low viability prob-
lems encountered when sampling microorganisms on agar plates due to dry-
ing of the agar, a new instrument was recently developed, which rotates the
agar plates, thus allowing for continuous more efficient collection of micro-
organisms and water retention on the agar (essentially rotation prevents dry-
ing of the agar surface and improves microorganism viability) (http://www.
mbv.ch/documents/paper_continuos_monitoring_2012.pdf).
2.4CONCLUSION
The study of the microorganism contents of Aeolian dust is an exciting area

of research that is expected to benefit public health tremendously. If scien-
tists manage to put together a detailed catalogue of all the microorganisms
the dust is able to carry with it alive, this will pave the way for better under-
standing the dusts impact on human disease. In addition, by knowing what
microorganisms to expect with dust episodes, health officials can more effi-
ciently screen dust particles for these microorganisms, identify them quickly
and eradicate them in an attempt to prevent infectious disease outbreaks
AUTHOR COPY
following Aeolian dust episodes. The methods that are necessary to collect
dust particles and identify microorganisms carried by the dust are in place
as described in earlier sections (Fig. 2.1). Up to now research studies have
been performed on an individual group basis. It is now time to think about
creating a network of scientists that work together to study the microorgan-
ism contents of the dust in different continents using standard protocols and
collection methods. This will help better understand the global impact of
9781771883627
Aeolian dust and manage its negative health effects.
KEYWORDS
16S rRNA sequencing

Aeolian dust
Air sampling device
Asthma
Microorganisms
Multi-stage impactors
Public health
Respiratory diseases
Scanning electron microscopy
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CHAPTER 3
CHLORINATED COMPOUNDS IN
NATURAL AND BIOTECHNOLOGICAL
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PROCESSES: MERITS, RISKS, AND USES
SNDOR T. FORCZEK1, JOSEF HOLK1, LUDK REDERER2,
and MARTIN FERENK3
1
Isotope Laboratory, Institute of Experimental Botany, Academy of
Sciences of the Czech Republic, Vdesk 1083, Prague CZ-14220,
Czech Republic
2
Povod Labe, State Enterprise, Vta Nejedlho 951, Hradec Krlov
CZ-50003, Czech Republic
3
Institute of Environmental and Chemical Engineering, Faculty of
Chemical Technology, University of Pardubice, Studentsk 573,
Pardubice CZ-53210, Czech Republic
CONTENTS
3.1 Introduction........................................................................................42
3.2 Chlorinated Compounds in Natural Processes...................................42
3.3 Atmospheric Cycle of Chlorinated Compounds................................43
3.4 Formation of Organochlorine Compounds........................................44
3.5 Volatile Chlorinated Compounds in the Soil.....................................47
3.6 Formation of Chlorinated Compounds by Cyanobacteria.................48
3.7 Volatile Chlorinated Compounds of Anthropogenic Origin..............51
3.8 Chloroform.........................................................................................52
3.9 Adsorbable Organic Halogens...........................................................54
3.10 Sample Study.....................................................................................55
3.11 Conclusion.........................................................................................59
Acknowledgments.......................................................................................60
Keywords....................................................................................................61
References...................................................................................................61
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3.1INTRODUCTION
Chlorine, similarly to other elements, undergoes a complex biogeochemical

cycle that includes the formation, conversion, and degradation of different
inorganic and organic forms of chlorine. Chlorinated compounds participate
in natural processes, biological, and chemical processes forming volatile or-
ganochlorine compounds; human activities also change their presence in the
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environment. Chlorinated pollutants are in the center of interest due to their
reactive nature, causing degradation of ozone in the atmosphere, and due
to health concerns, as some chlorinated compounds are highly toxic. Some
volatile chlorinated hydrocarbons (VCHs) are both reactive and toxic, such
as chloroform. Moreover, chloroform has natural and anthropogenic sources
and can be formed in abiotic and biotic processes. In this chapter, we dis-
cuss the role of VCHs in the natural environment and their anthropogenic
impact. Furthermore, statutorily determinations of adsorbable organic halo-
gens (AOX) and chloroform will be evaluated in connection with a sample
study in a clean area Hamry, Czech Republic. The catchment has low human
activity and the collected water in the Hamry water reservoir is used as a
source of drinking water. The area contains many bogs and forests and a
high content of dissolved organic carbon has therefore been determined in
it. The high concentration of AOX and chloroform in the water source area
is caused by the natural biological activity of the soil. The compounds can-
not be found in the reservoir, as the AOX are diluted, while chloroform is
evaporated during on their course to the reservoir.
3.2 CHLORINATED COMPOUNDS IN NATURAL PROCESSES
Chlorine is present in every part of the geosphere, including atmosphere,

hydrosphere, lithosphere, and biosphere as well. Natural processes in-
cluding several abiotic and biogenic ways yield chlorinated compounds.
Organochlorines (Clorg), in which chlorine is covalently bonded to an organic
compound, participate in the complex biogeochemical cycle of chlorine in-
volving the formation, conversion, and degradation of different inorganic
and organic forms of chlorine. Natural sources, such as wild fires, volca-
noes, and other geothermal processes account for a wide range of volatile or-
ganochlorines (VOCl) and inorganic chlorine (Lobert et al., 1999). Chlorine
is present in volcanic gases not only as hydrogen chloride gas, but also as
organic forms of chlorine, such as chloromethane, chloroform, tetrachloro-
ethylene, carbon tetrachloride, and several chlorofluorocarbons (CFCs) such
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Chlorinated Compounds in Natural and Biotechnological Processes 43
as Freons, which are chemicals formerly thought only to result from the hu-
man activities (Gribble, 1999).
It has been suggested that chlorine contributes to the decay of soil organ-
ic matter (SOM) leading to the formation of large molecules of chlorohumus
(Asplund, 1995; Matucha et al., 2010). Degradation of chlorohumus leads
to smaller intermediates, such as chlorinated acetic acids, anisol-, orcinol-
and hydroquinone-based substances, which may be taken up by plants or
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fungi. In addition to insoluble compounds or smaller molecules present in
soil and dissolved in pore water, VOCl are also present in soil. Some of the
VOCls can also be found in hundreds of years old ice in Antarctica (Saito et
al., 2006), demonstrating that volatile VOCls have been emitted long before
anthropogenic activity altered the atmosphere of the Earth (Lovelock et al.,
1973).
Production of Clorg is widespread in every domain of living organisms,
including Archaea, Bacteria, and Eukarya as well. Examples of production
and emission are found in but not restricted to animals, almost all higher
and lower plants, fungi, algae, and microorganisms (Yokouchi et al., 2002;
Gribble, 2003). Many of the biogenic Clorg are volatile and so can be emitted
to the atmosphere, where relevant atmospheric reactions take place.
3.3 ATMOSPHERIC CYCLE OF CHLORINATED COMPOUNDS
Several reactions take place in the atmosphere between inorganic chloride

and chlorinated compounds. Most of them are notoriously well known, as
chlorinated compounds have long been considered as of anthropogenic ori-
gin only and studied in connection with ozone depletion (Gay et al., 1976;
Tuazon et al., 1988; Nelson et al., 1990; McCulloch and Midgley, 1996).
Chlorine and other halogens enter the atmosphere in the form of sea salt
spray as water particles are lofted into the atmosphere by the motion of
ocean waves. These particles can undergo reactions with trace atmospheric
gases and internal mixing with anthropogenic pollutants which are depos-
ited on the surface of the particles. Several studies have shown that NaCl
particles in the atmosphere are depleted in chloride and have attributed this
to reactions with inorganic acids. These reactions, which are unique for aero-
solized particles, were previously overlooked and are important in atmo-
spheric chemistry models (Laskin et al., 2012). The reactions in atmospheric
particles containing sea salt and organic acids liberate HCl (g) and promote
the formation of organic salts in the particle phase. In the atmospheric en-
vironment, the released HCl (g) may result in consecutive acidification of
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coexisting neighboring particles, and trigger additional acidbase reactions,

especially with alkaline components of mineral dust (Sullivan et al., 2007).
The uptake of chlorine species onto dust particles modifies the chlorine
chemistry budget in the marine boundary layer, thus altering the overall HCl
cycle and the cycles of important species involved in these heterogeneous
reactions (such as SOx, NOy, ClOx, and O3). These processes will therefore
affect the chemical budgets of ClOx, NOx, and SOx species in the marine
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boundary layer (Sullivan et al., 2007; Faxon and Allen, 2013). The chemi-
cally reactive gases ClOx, NOx, and SOx can form organically bound chlorine
in the atmosphere (Gay et al., 1976; Tuazon et al., 1988; Itoh et al., 1994;
Sidebottom and Franklin, 1996).
Precipitation washes out chloride and water-soluble chlorinated com-
pounds from the atmosphere. Chloride therefore does not stay in the atmo-
sphere, but it is transferred by wet and dry deposition. The halide concentra-
tions of real seawater are: Cl19000mg/L, Br65mg/L, I,IO30.060mg/L,
while in wet deposition, chloride concentration can be still significant for
terrestrial ecosystems; under continental climate it can reach values around
1mg/L. In the form of precipitation, Cl ends up on the soil surface or on
the vegetation, wherefrom it is taken up by plants or washed down into the
topsoil by tree throughfall and stem-flow. Chloride in aerosols is directly
captured by vegetation, ranging from several kg/ha/year in continental areas
(e.g., 250mg/m2/year in Central Europe) up to tens of kg/ha/year in coastal
areas (e.g., 4000mg/m2/year in Western France) depending on geographi-
cal situation and weather conditions (Delalieux et al., 2006). Estimates of
the yearly production of atmospheric marine salt range from 109 to 1010
metric tons, and a realistic value is most probably somewhere in between
(Blanchard, 1985; Erickson and Duce, 1988). Estimates of relative contribu-
tions of dry and wet removal processes to total sea-salt removal vary largely,
from an amount of 67% salt brought down by rain to 33% dry deposition
(Blanchard, 1985; Erickson and Duce, 1988).
3.4 FORMATION OF ORGANOCHLORINE COMPOUNDS
Chloride which enters the terrestrial ecosystems becomes dissolved in pre-

cipitation, running, or soil water, where it can react with soil organic sub-
stances, leading to the formation of halogenated compounds (Johansson et
al., 2000; Godwin et al., 2003; Matucha et al., 2010).
Formation of halogenated organic compounds can happen in soil in an
abiotic way (Keppler et al., 2000) or through the action of exoenzymes
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produced by microorganisms and plants. Myneni (2002) reported that sta-

ble, chlorinated hydrocarbons with aliphatic and aromatic groups are formed
rapidly at the expense of inorganic Cl during weathering and humification
of plant material. The magnitude of VOCl production in soil is difficult to
estimate, and the achieved emission rates have so far not been used to esti-
mate a global emission rate, but the results show that the terrestrial ecosys-
tem has an enormous potential to release CH3Cl, CH3Br, and CH3I into the
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atmosphere through an abiotic process.
In addition to the abiotic formation, the emission of VOCl by living or-
ganisms was also confirmed. The emissions of chloroform, carbon tetrachlo-
ride, or chloromethane in addition to numerous brominated and iodinated
organic compounds from some marine and terrestrial sources, such as ma-
rine macroalgae, coastal salt marshes, forest vegetation, and soil, are well
documented (Laturnus et al., 1996; Laturnus et al., 1998; Giese et al., 1999;
Rhew et al., 2000; Hoekstra et al., 2001; Svensson et al., 2007; Laturnus et
al., 2010). The pathways of formation of some chlorinated compounds have
been elucidated while others are still to be revealed. Formation of haloace-
tic acids and chloroform is closely interconnected in the soil, and both are
intensively studied. Both compounds are found to be produced during deg-
radation of resorcinolic structures, which are common structural elements
of humic material. In the course of their formation, the aromatic rings are
chlorinated and ring cleavage occurs, followed by haloform reaction with
aliphatic chains and final cleavage into trichloroacetic acid (TCA) and chlo-
roform. The former is in prevalence in acidic soils while the latter dominates
in basic soils with a pH over eight (Hoekstra et al., 1999a; Hoekstra et al.,
1999b). Haloacetic acids are readily taken up by vegetation (Forczek et al.,
2004), or can be formed from volatile halogenated hydrocarbons inside the
plants due to detoxification processes (Schrder et al., 2003; Weissflog et al.,
2007). Active movement and metabolism of halogen containing xenobiotics
have been intensively studied, and the role of glutathione in plants was elu-
cidated (Schrder and Wolf, 1996; Schrder et al., 2007). Plants possess an
elaborate enzyme-based detoxification system for organic xenobiotics and
agrochemicals, comprising a metabolic cascade proceeding in three phases.
In phase I, the xenobiotics are activated by P450 monooxygenases, peroxi-
dases, or similar enzymes that catalyze oxidation, reduction, or hydrolysis
of the foreign compound. Detoxification in the strict sense of the word pro-
ceeds in phase II, where the xenobiotic is conjugated to biomolecules, that is,
sugars via glycosyltransferases or to the tripeptide glutathione via glutathi-
one S-transferases. Glutathione conjugates are less toxic and lipophilic than
the parent xenobiotics, but they might still carry some unwanted properties
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rendering them problematic for the cell. It is therefore generally accepted

that xenobiotic conjugates are sequestered from the cytosol in higher plants
in phase III by the activity of tonoplast MRP transporters, which belong to
the wide family of ABC-transporters (Schrder et al., 2007).
VOCl emission by plants is theoretically possible: (1) during the detoxi-
fication of halogenated compounds; (2) due to the emission of already taken
up VOCl from the environment, or; (3) due to de novo synthesis. To answer
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the first possibility, chloroacetic acids were studied as model compounds
in plants by radiotracer methods (Blanchard, 1954; Forczek et al., 2004).
No degradation products were found in most cases, which points to total
mineralization by dechlorination and CO2 emission, whereas in some cases
trichloromethyl compounds were found (Mayer, 1957). The main degrada-
tion product during thermal decomposition of TCA is chloroform, which
was however never found in plants (Forczek et al., 2004). In the second
place, the re-emission of previously absorbed VOCl has also been studied,
and it is a completely reversible process in soils (Chen and Dural, 2002).
Plants are also capable of absorbing VOCl, and based on this capability,
some lichens are used as bio-indicators of air pollution (Conti and Cecchetti,
2001). Re-emission is therefore also possible in plants. In the third place,
the VOCl emission by plants was determined by field and laboratory experi-
ments (Yokouchi et al., 2000; Forczek et al., 2015). The emitted VOCl are
proved to be the products of de novo synthesis of plant enzymes.
The enzymes responsible for the formation of VOCl in plants can be heme
peroxidases (EC 1.11.1.X) such as chloroperoxidase (EC 1.11.1.10), or non-
heme peroxidases such as vanadate-dependent peroxidases (EC 1.11.1.7),
perhydrolases, and FADH2-dependent halogenases (Walter and Ballschmiter,
1992; Asplund et al., 1993; Wever and Hemrika, 2001; van Pe, 2003); most
of them ubiquitously present also in soil by microorganisms and fungi (e.g.,
Caldariomyces sp., Pseudomonas sp., Streptomyces sp.).
The halogenating enzymes containing the heme group or vanadium in
their molecule require hydrogen peroxide for their halogenating activity.
Perhydrolases and FADH2-dependent halogenases do not contain either a
heme group or any metal ions but the former also require hydrogen perox-
ide for their halogenating activity. The reaction mechanisms of halogenating
enzymes vary. Perhydrolases act in the presence of H2O2. Perhydrolysis of
the acyl-enzyme intermediate results in the formation of peracids, which
can oxidize halide ions to hypohalous acids which then act as the haloge-
nating agent. On the other hand, heme-type haloperoxidases and vanadate-
dependent peroxidases produce free hypohalous acid as the halogenat-
ing agent also in the presence of H2O2 and halide ions. Thus, heme-and
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vanadium-containing haloperoxidases and also perhydrolases are halogenat-

ing enzymes without any substrate specificity and regioselectivity. The ha-
logenation reactions in some biosynthetic pathways like 7-chlorotetracyclin
biosynthesis are catalyzed by enzymes with high substrate specificity and
regioselectivity (van Pe, 2003). FADH2-dependent halogenase enzymes
(EC 1.14.14.7) are therefore responsible for specific halometabolite synthe-
sis, whereas haloperoxidases and perhydrolases are probably involved in
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defense mechanisms in which unspecific halogenation occurs.
3.5 VOLATILE CHLORINATED COMPOUNDS IN THE SOIL
Sodium chloride, the most common compound of chlorine, primarily comes

to terrestrial ecosystems by atmospheric transport from the oceans and sec-
ondarily by weathering of bedrocks, which is negligible (Winterton, 2000;
berg, 2003). Some anthropogenic sources/activities such as irrigation, road
salting, or coal combustion locally increase the concentration of salt in soil,
and can cause immense damage. In terms of environmental impact, chloride
was considered a chemically inert substance and its role in the forest eco-
system has been considered negligible. After degradation of plant residues,
forest soil comprises mainly lignin, humic and fulvic acids, and a variety of
other substances resulting from their further degradation, altogether called
humus. Halogenation in soil by chlorine radicals can produce chlorinated
SOM (chlorohumus) containing a wide range of chlorinated compounds
such as large chlorinated humic and fulvic acids, and a number of low-mo-
lecular chlorinated compounds. The formation of chlorinated aromatic com-
pounds derived from lignin degradation is also anticipated. These substances
are present in the runoff from forest ecosystems. Low-molecular chlorinated
compounds, such as chloroacetic acids and chloroform are relatively well
detectable. The formation of chloroacetic acids has been demonstrated in
forest soil by investigations during long-term model experiments with the
help of chlorine 36 (36Cl) and carbon 14 (14C) isotopes (Matucha et al.,
2007a, 2007b) and formation of VCHs was proved (Rohlenov et al., 2009).
The chlorination achieved through abiotic formation and by living or-
ganisms, described in previous sections, produces a wide spectrum of vola-
tile and non-volatile halogenated compounds from inorganic halides. The
above-mentioned chlorinating enzymes are present in the forest ecosystem
not only as a product of plants, but also due to the action of fungi and mi-
croorganisms, and are located either inside the cells of living organisms, or
as extracellular enzymes (exoenzymes) emitted into the soil. Chlorination
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occurs fairly fast, chlorine is bound to organic molecules and the biogeo-
chemical cycle of chlorine continues in soil (Winterton, 2000; berg, 2003).
Through volatilization and mineralization, chlorine gets back into the atmo-
sphere. Most of the chlorine in the atmosphere in an organic form is chloro-
methane, chloroform, and carbon tetrachloride (Butler et al., 1999; Gribble,
1999; Montzka et al., 1999).
Formation of chloromethane by fungal methylation of chloride was re-
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ported by Harper (1985), and reaction of chloride with pectin (Hamilton et
al., 2003) leads to the same VOCls as those that cause damage to the ozone
layer. Myneni (2002) and Leri et al. (2006) showed also chlorination of plant
litter material.
Vinyl chloride found in the atmosphere also has natural sources, contrary
to previous considerations that dedicated all of it to anthropogenic sources
only. Soil air and ambient air from a rural area in Northern Germany were
investigated for volatile chlorinated halocarbons, and the concentrations of
vinyl chloride in the soil air were significantly enhanced as compared to
ambient air, indicating a natural formation of this compound in the soil. A
series of laboratory experiments using different soils and model compounds
clearly proved that vinyl chloride can be produced during soil processes
(Keppler et al., 2000; Keppler et al., 2002). The authors propose that this
highly reactive compound can be formed during the oxidative degradation of
organic matter in soil, for example in a reaction between humic substances,
chloride ions, and an oxidant (ferric ions or hydroxyl radicals). The redox-
sensitive aromatic compounds in soil such as catechols and -quinones can
be degraded to CO2, accompanied by the release of vinyl chloride and other
volatile chlorinated compounds (Keppler et al., 2000).
3.6 FORMATION OF CHLORINATED COMPOUNDS BY

CYANOBACTERIA
Cyanobacteria, very simple prokaryotic oxygenic phototroph microorgan-

isms found in nearly every conceivable habitat on earth, also produce chlori-
nated compounds. Some of these are simple compounds well known in plant
physiology, such as halogenated hydrocarbons (Gribble, 2003), whereas
others are specific secondary metabolites such as chlorinated cyanotoxins.
The blue-green algae Nostoc and Oscillatoria are two taxa rich in both types
of organohalogen compounds. For example, blue-green algae produce aeru-
ginosins, found predominantly in the genera Microcystis and Planktothrix.
These toxins can hold chlorine in their molecule in more positions (Fig. 3.1)
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Nostocarboline
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Aeruginosins, where X denotes different amino acids that can occupy the second position:
Leu, Ile, Phe, Tyr, and homotyrosine. Y denotes the C-terminal variable moieties as shown
to the right (from top): argininal (linear tautomer), argininol, agmatine, and 1-amino-2-
(N-amidino-3-pyrrolinyl)-ethyl (Ishida et al., 2009).
Cryptophycin A
Nostocyclophane B
FIGURE 3.1 Chlorinated natural compounds isolated from blue-green algae.

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(Ishida et al., 2009). The potent anticancer drug candidate cryptophycin A

(Fig. 3.1) was also isolated from cultures of a blue-green alga (Nostoc sp.),
and the structurally novel nostocyclophane (Fig. 3.1) is produced by Nostoc
linckia (Gribble, 2003). These special secondary metabolites are encoded in
specific gene clusters, aimed by the algae to produce highly specific com-
pounds. As these genes were identified, it will be possible to search for and
identify with the aid of genome-mining approaches organisms with similar
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genes, which can encode promising new natural products.
Although some cyanobacteria actually produce halogenated compounds,
their role in the global halocarbon production is not well established. Studies
on distributions and sea-to-air fluxes of VOCls were conducted only very
recently (Roy et al., 2011) and only poor correlations were found between
concentrations of halogenated compounds with various marker pigments.
The results support the existence of multiple sources and sinks of halogenat-
ed compounds, which might obscure the relationship between halocarbons
and phytoplankton composition. In another study, however, He et al. (2013a,
2013b) found a positive correlation between chlorophyll-a, salinity, and nu-
trients on the distributions of gaseous halocarbons in the coastal shelf of the
Yellow Sea and the East China Sea. Elevated levels of chloroform (CHCl3),
trichloroethylene (C2HCl3), tetrachloroethylene (C2Cl4), chlorodibromo-
methane (CHBr2Cl), and other simple halogenated hydrocarbons were found
in sea water, whereas a decreasing trend was found with the distance from
the coast, with low values found in the open sea, bringing evidence that phy-
toplankton is related with halocarbon emission. These results indicate that
the coastal shelf contributes significantly to the global oceanic emissions
of gaseous halocarbons, and especially phytoplankton is responsible for its
formation, but they do not state explicitly the role of cyanobacteria.
The formation of chlorinated compounds in blue-green algae is known
to have allelopathic effects to affect other organisms living in the same
aquatic environment. Active compounds are synthesized to fight the harm-
ful cyanobacterial water bloom. Some chemicals are derived from natural
compounds, such as those found and identified from decomposition of straw,
which show toxicity toward cyanobacteria. Several chemicals were derived
and are tested from anthraquinone (found in lignin) and stilbenes, which are
produced by plants in response to stress. Further compounds found to have
cyanocide properties are the alkaloids of carbolines such as nostocarboline
(Fig. 3.1), isolated from the cyanobacterium Nostoc (Jancula et al., 2011).
Nostocarboline, which is a novel neurochemical to fight Alzheimers dis-
ease, also contains chlorine in the molecule (Becher et al., 2005). Many of
the chemicals used as natural or synthetic herbicides are chlorine-containing
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compounds. Their metabolites and degradation products may differ in many

aspects from the original compounds. In addition to changes of physico-
chemical properties, their subsequent biological and toxicological proper-
ties are modified. The change of solubility and hydrophobicity defines new
hazards present in the novel compounds.
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3.7 VOLATILE CHLORINATED COMPOUNDS OF
ANTHROPOGENIC ORIGIN
In addition to naturally produced organohalogens, human activity also re-

leases halogenated compounds to the environment, which then pose risk on
human health. Halogenated organic compounds constitute the largest frac-
tion of priority pollutants as designated by the U.S. Environmental Protection
Agency (U.S. EPA), with over 50 % of the compounds containing chlorine
or another halogen. Quite often, the mentioned pollutants are volatile, and
that is why they reach the natural environment.
As a simple organochlorine, chloroform can serve for example to estimate
human activities. Anthropogenic sources of chloroform and other VOCls are
industrial production processes, chlorination of drinking, swimming pool,
and cooling water, pulp and paper bleaching (de Fouw, 1994), and waste
incineration (Jay and Stieglitz, 1995). Other sources of chloroform include
hazardous waste sites and sanitary landfills. Chloroform is one of the most
strictly controlled compounds in drinking water among other trihalogenated
methanes (THM) that were found to be carcinogenic in animal tests. U.S.
EPA has classified chloroform as a group B2, probable human carcinogen,
and the U.S. Food and Drug Administration has banned its use in consumer
products in 1976. Chloroform in the drinking water is therefore regulated.
To protect drinking water from pathogens, water suppliers often add a disin-
fectant, such as chlorine, to raw water. However, disinfectants can react with
naturally occurring materials present in the water to form halogenated by-
products, which may pose health risks. Hazardous disinfection byproducts
include THM and haloacetic acids formed during chlorination of drinking
water, wastewater, and swimming pools, where dissolved organic materials
react with chlorine, chloramine, and bromine.
Nowadays chloroform is primarily used as a solvent in pharmaceutical
industry and as an important raw material for producing dyes, pesticides,
and hydrochlorofluorocarbons (HCFCs). The U.S. EPA has posed a con-
trol on the maximum contaminant level of chloroform in drinking water
to 70 gL1, whereas in European countries the guideline for maximum
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acceptable concentration level is 200g L1. Several European countries set

a lower level for example, in the Czech Republic the maximum acceptable
concentration level of chloroform is 30gL1 or 50gL1 of THM.
Several methods are used to remove anthropogenic pollution from natu-
ral environment in situ. They are applied according to the nature of the pol-
lutant, such as air stripping, carbon adsorption, soil venting or other aeration
technologies, and biological remediation. Biotechnological methods based
9781771883627
on biological systems, living organisms, and their derivatives, though still
in the research stage can help to clean environmental pollutions by chlori-
nated products in natural ecosystems. In bioremediation, contamination or
hazardous substances are turned into less toxic or non-toxic form by re-
moval or breakdown of these substances. Some examples of bioremedia-
tion-related technologies are phytoremediation, bioventing, bioleaching,
landfarming, bioreactor, composting, bioaugmentation, rhizofiltration, and
biostimulation. Biodegradation of contaminants can be achieved by dechlo-
rinating microorganisms having enzymes which remove halogens by hydro-
lytic nucleophilic substitution (Thompson et al., 2005; Huang et al., 2014).
Some enzymes work in both directions, not only dehalogenating, but also
achieving halogenation whereas other enzymes degrade different parts of
the organic unit. Still other enzymes conjugate the compounds to xenobiotic
substrates. Cyanobacteria and other microorganisms utilized in biotechno-
logical applications therefore can produce VOCl, which can have adverse
effects on health and on the environment (Huang et al., 2014).
During bioremediation, chloroform can also be emitted as a degradation
product, for example in the course of bioaugmentation of carbon tetrachlo-
ride remediation (Wang et al., 2002). Bioremediation of chloroform-contam-
inated sites is considered a favorable alternative to physical and chemical
approaches such as air stripping or sorption onto activated carbon that trans-
fer contaminants from one medium to another rather than causing contami-
nant destruction (Cappelletti et al., 2012).
3.8CHLOROFORM
Although chloroform alone may play a minor role in the global chlorine
cycle, it may be worth considering more seriously when taken together with
other naturally produced chlorocarbons. To highlight chloroform among
the above-mentioned facts on VOCl, we can summarize that it is formed in
many different ways naturally and through anthropogenic action. The ma-
jor anthropogenic sources of chloroform are fossil-fuel combustion, waste
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incineration, and industrial releases primarily due to pulp and paper manu-
facturing and water treatment (Laturnus et al., 2002). Chloroform can be
transformed from other VCHs produced by human activity. Other uses of
chloroform, such as industrial solvent, surgical anesthetic or in cough syr-
ups, and toothpastes are banned or replaced by different solvents.
Chloroform is one of the most complex compounds in terms of its sourc-
es. According to the hypothesis of Hoekstra et al. (1999), chloroform can-
9781771883627
not be produced by decarboxylation of TCA in soil (Hoekstra et al., 1999a;
Hoekstra et al., 1999b), while Schler et al. (2003) claim that moderately
elevated temperature (5070C, 30min) already leads to thermal decom-
position of TCA to chloroform. In general, elevated temperature is not ex-
pected in soil, whereas it can happen in plant leaves, due to strong irradiance
from full sunlight (Dickey et al., 2005). Plant temperatures generally follow
the diurnal pattern of air temperatures, but they may be higher (sometimes
10C or more); this supports Schlers hypothesis (Schler et al., 2003) on
partial thermal degradation of TCA. Chloroform and methyl-chloroform are
released from biomass burning (Rudolph et al., 2000), rice fields (Khalil
et al., 1998), termite mounds (Khalil et al., 1990), fungi (Hoekstra et al.,
1998a, 1998b), and spruce forests (Haselmann et al., 2000a; Haselmann et
al., 2000b). Chloroform represents the most abundant halocarbon in the at-
mosphere (Harper, 2000; Cappelletti et al., 2012) but the anthropogenic flux
of chloroform into the environment is too much low to account for observed
background concentrations. Therefore, it is clear that natural sources must
make major contributions.
Natural abiotic ways of chloroform formation include, but are not limited
to, emission of chloroform from volcanic sites, wild fires, and geothermal
processes and abiotic formation in the soil. Biogenic formation of chloro-
form is also fairly diverse. The oceans have been found to be a major source
for naturally produced volatile halogenated compounds, including chloro-
form. Marine organisms, mainly phytoplankton and macroalgae have been
found to produce chloroform. Budget calculations indirectly indicate that
the terrestrial ecosystem may also be an important source of volatile halo-
carbons, thereby contributing to the global halogen cycle. Though terrestrial
natural sources of chloroform are still poorly investigated, some sources
have already been identified. Khalil et al. (1998) found a positive flux of
chloroform liberated into the atmosphere during the investigation of rice
fields. Dimmer et al. (2001) discovered a flux of chloroform from Irish peat-
land ecosystems in globally significant amounts. Chloroform in soil can be
formed from TCA, especially in alkaline media (Haiber et al., 1996; Schler
et al., 2003), through intracellular microbial activity and via the activity of
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exoenzymes in the soil. Metabolism of TCA in plants can also produce chlo-
roform, while in soil, chloroform is produced by fungi as the result of the
chlorination of natural organic matter by naturally produced hypochlorous
acid (Hoekstra et al., 1998a, 1998b). Concentration patterns of TCA and
chloroform in leaves suggest that TCA might be partly decarboxylated to
the intermediate trichloromethanide which forms chloroform, but this was
never confirmed in experiments (Schler et al., 2003; Forczek et al., 2004).
9781771883627
Due to its low tendency to sorb to soil organic carbon, chloroform has a high
mobility in aquifers (Cappelletti et al., 2012).
3.9 ADSORBABLE ORGANIC HALOGENS
Until the 1980s, halogenated compounds present in nature were considered

exclusively as industrial products. To detect these possibly harmful com-
pounds in natural environment, the AOX method was introduced, originally
developed to quantify anthropogenic pollution of surface waters such as var-
ious herbicides (e.g., 2,4-dichlorophenoxyacetic acid, atrazine, and TCA),
pesticides (DDT, lindane, etc.), chlorinated waste products (emitted e.g.,
during cellulose bleaching, or in PCB manufacturing) or chlorinated sol-
vents (perchloroethylene, chloroform). The method to determine adsorbable
water-soluble organohalogen compounds in water on charcoal was proposed
by Khn in 1976 (Mller, 2003) and has been included in 1985 into the
German DIN-Norm 38409 as a sum parameter. During AOX (where X stands
for chlorine, bromine and iodine) determination, halogenated compounds
are adsorbed on activated carbon in a slightly acidic aqueous solution, and
combusted thereafter along with the carbon, and then the halogen content is
determined by microcoulometric titration. AOX therefore are determined as
a group parameter, without further characterization of the compounds.
Investigations conducted since then have clearly shown that plants grow-
ing in marine, limnic, and terrestrial environments are a natural source of
organohalogens, which are also measured as AOX (Mller, 2003). During
the development of monitoring of environmental quality and pollution stud-
ies of anthropogenic activities, high levels of AOX have been revealed in
Scandinavia and elsewhere in the world in pristine natural areas (Silk et al.,
1997). In these vast forest areas, no herbicides or pesticides were ever ap-
plied or no industrial enterprises were present whereas in surface waters tens
to hundreds of g/L AOX were determined.
In natural terrestrial ecosystems, the most common halogen is chlorine
and halogenated substances are therefore also containing mostly chlorine.
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Further research proved that chlorinated substances of natural origin are

arising from chlorination of SOM. It has been shown that the chlorination is
carried out in the forest soil and mainly catalyzed by microbial exoenzymes,
specifically by chloroperoxidases (Asplund et al., 1993). Studies of Clorg in
forest ecosystems and watercourses were conducted primarily by a group
from Linkping University (berg, 2002; Johansson et al., 2003; berg,
2003; berg et al., 2005; berg and Sandn, 2005; Bastviken et al., 2007).
9781771883627
The resulting Clorg have not been characterized in detail. The results indicated
only the presence of chlorinated humic and fulvic acids, and additional chlo-
rinated substances of low molecular weight (LMW), possibly chloroacetic
acids, chloroform, chlorophenol, chloroanisole, and other chlorinated aro-
matic compounds, some of which could be toxic even at low concentrations.
The Clorg formed in the forest soil and determined as AOX usually fall
between 200 and 1000mgCl/kg soil and its amount is proportional to the
total carbon content of the soil and often exceeds its free inorganic chloride
(Cl) content. The formation of various types of chlorinated compounds in
forest soils leads to more easily degradable and better water-soluble soil
components. The dissolved Clorg in effluent waters from the forest ecosys-
tem can end up in reservoirs of drinking water, where these substances then
contribute to increased concentration of AOX. According to the AOX indi-
cator, the water quality is then deteriorated. It is important to understand the
biogeochemical cycle of chlorine and to separate natural processes from an-
thropogenic influences in forest ecosystems to identify the threats stemming
from human activities. Water catchments with high concentrations of total
organic carbon (TOC) and Clorg in the water are common worldwide; AOX
levels can pose health risk, which raises concerns on the consumers. One
such water source can be found in the catchment area of the Hamry water
reservoir (Czech Republic).
3.10 SAMPLE STUDY
The Povod Labe s.p. (Elbe River Basin, Czech State Enterprise) has exten-
sively monitored the level of AOX in the catchment area of the Hamry water
supply reservoir between 2006 and 2008 (Ferenk et al., 2008). Although
the reservoir is situated in a lightly populated and anthropogenically non-
loaded area, elevated concentrations of AOX have occasionally been found
in the surface waters. High concentration of TOC has also been found here,
because humic substances are leached from the forest and peatland areas in
the catchment. No xenobiotics were found during the detailed analyses of
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water samples, where 80 LMW anthropogenic organohalogen compounds

(especially herbicides and pesticides) were tested individually. Haloacetic
acids were not found in the water either. The absence of natural haloacetic
acids in the analyzed samples was not expected due to their rapid microbial
degradation in soil and soil water (Matucha et al., 2003). The chloroacetic
acids are supposed to be degraded very rapidly, without the formation of
VOCls, especially without chloroform and chlorinated methanes (Matucha
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et al., 2003, 2007a, 2007b). The findings gathered during the 20092010
monitoring indicate that the occasionally elevated AOX values, which reach
or exceed the specified national limit for sources of drinking water, were not
caused by human activity in the area but by natural soil processes. Similar
findings were reported in Sweden (berg, 2002; berg, 2003; Johansson et
al., 2003; berg and Sandn, 2005) showing that AOX measures both natu-
ral and anthropogenic Clorg, whereas in some cases AOX determination does
not include important compounds such as chloroform (which is evaporated
during the preparation of soil samples in the course of drying). This conclu-
sion was also confirmed in directed experiments performed by our team.
Therefore, the question arose whether substances of natural origin can pol-
lute surface waters like in this case, transporting compounds to the Hamry
reservoir from forest and peatlands in the catchment by streams.
The measured concentrations of AOX crossed the hygienic threshold for
sources of drinking water (30g/L) which, according to the present legisla-
tion in effect, indicated deterioration of drinking water. In this case, water
cannot be used as raw material for the preparation of drinking water, or spe-
cial precautions must be taken to remove the pollutants. This procedure can
be a financial burden and sometimes technically impossible or unavailable.
Recent publications in the literature are addressing the fate of chlorine in a
forest ecosystem, but monitoring studies including organochlorine are still
unique. The study also confirms the conclusions of Muller that AOX in the
original form cannot be simply used for indication of anthropogenic pollu-
tion by halogenated compounds (Mller, 2003). This is especially the case
when bogs are present in the watershed, where halogenated SOM, chlorinat-
ed humic acids, and other chlorinated compounds are being formed (Holk
et al., 2010).
The source area of one of the tributaries to Krejcarsk stream, bearing
large fields of peatlands with low drainage, contained higher concentration
of humic substances and many metabolites than running waters outside the
area. Humic substances leached from forest and peatland areas of the Hamry
water reservoir are responsible for higher TOC and AOX content of the sur-
face waters. The found values of AOX and TOC in the sample study showed
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a correlation between the concentrations of AOX and humic substances.

These findings are in agreement with the natural origin of the determined
AOX. This confirms the original assumption that Clorg are produced natu-
rally in the forest ecosystem during biotic and abiotic chlorination of com-
plex organic matter present in the soil, such as fulvic and humic substances,
lignin, and a wide variety of their chlorinated degradation products.
Although concentration of AOX in several cases exceeds the specified
9781771883627
national limit for drinking water in the Hamry catchment area, the water in
the reservoir itself, which is used to prepare drinking water, never reaches
the AOX hygienic limit.
During the monitoring, the study focused on the explanation of the pres-
ence and the origin of AOX in the catchment area of the Hamry water reser-
voir, which is an important source of drinking water in the area (Pardubice
region, Czech Republic) supplying approximately 20000 people with water.
Within the framework of cooperation in the years 20102011, the Institute
of Experimental Botany, Academy of Sciences of the Czech Republic; the
Pardubick kraj (Pardubice region, Czech Republic); the Povod Labe,
sttn podnik (Elbe River Basin, Czech State Enterprise); the Vodrensk
spolenost Chrudim, a.s. (Waterworks Co. Chrudim plc.) and the Vodovody
a kanalizace Chrudim, a.s. (Water Utilities Co. Chrudim plc.) participated in
studying the Hamry water catchment. The study revealed the primary impor-
tance of natural processes in the formation of AOX, and assessed the poten-
tial health risks to humans using water reservoirs such as Hamry as sources
of drinking water, which was found to be negligible. At the same time, the
study can endorse the most appropriate water treatment technologies for
preparation of drinking water. In this case, the removal of humic substances
and/or TOC would be sufficient. Regarding the presence of naturally oc-
curring chloroform, which was determined before the water treatment as a
model compound for the occurrence of volatile chlorinated compounds in
raw water, it can be treated as anthropogenic chloroform, also normally oc-
curring during water chlorination.
To further characterize dissolved macromolecules, we separated a LMW
fraction of 0500 daltons (Da) by ultrafiltration. This way, large molecules
are separated from smaller molecules which can usually pose a health risk.
Some of the low molecular compounds such as chloroacetic acids or chloro-
form are rapidly decomposed in water samples or evaporated during sample
preparation. Some of these low molecular compounds in the LMW fractions
such as chloroform, chloroacetic acids, chlorophenol, chloroanisole, chlo-
rinated dicarboxylic acid, and so forth can pose a health risk in increased
concentrations. For this reason, they are regulated in the drinking water by
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national laws in many countries. The AOX and TOC concentration were
determined in the original water samples and in LMW fractions. In order
to analyze and characterize compounds in the LMW fractions, we used a
pre-concentration method using a DEAE-cellulose packed column. The use
of DEAE-cellulose for the concentration of humic substances has been de-
scribed previously (Hiraide et al., 1994; Ibrahim et al., 2008). Fractions with
high absorbance (indicating high concentration of humic acids) were select-
9781771883627
ed for further concentration, acidified with hydrochloric acid to pH 1, NaCl
was added and then extracted with methyl t-butyl ether. The ether extracts
were concentrated by rotary evaporator, derivatized with diazomethane and
analyzed by GC-MS, as recommended for chlorinated phenols.
According to our expectations, UVVIS spectrum analysis of ultra-
filtrated fractions had a much lower overall extinction than the original
water samples. Moreover, the samples showed a distinct plateau in the
region of 260280nm, which can correspond to the presence of organic
molecules with aromatic ring or heterocycle in their molecules. It can be
assumed that these substances might arise from the degradation of humic
substances and lignin degradation. From these results, we can conclude
that abiotic and biotic chlorination enables high-molecular substances in
the forest ecosystem to facilitate their degradation, as their fractions are
more water-soluble.
Hydrolysis of large humic substances during their degradation, along
with chlorination, and chlorination of SOM can produce chlorinated organic
substances some of which are volatile. The chlorinated volatile substances
may partially increase the measured AOX level of water, but their presence
in the samples is not controlled and can change to various extents during
sample transport and preparation. Hence, volatile compounds have to be de-
termined separately from non-volatile compounds. During our monitoring
campaigns, we determined the levels of chloroform in water samples in ad-
dition to AOX determinations. The results indicate that chloroform produc-
tion is largely due to biotic chlorination of microorganisms in the soil and is
dependent on the season and temperature. The level of chloroform in surface
waters is also dependent on the weather as chloroform level shows a close
correlation with water temperature.
AOX and TOC analysis of original water samples and ultrafiltrated frac-
tions with molecular size under 500Da showed that the AOX to TOC ratio
in the LMW fractions is higher than in the original samples. This means
that LMW fraction is either more halogenated or faster degraded than large
molecules. By definition, compounds quantified by the AOX method are
dissolved in water and not well soluble in apolar solvents. Therefore, their
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lipophilicity is low, meaning that they have a low potential to bioaccumula-

tion or biomagnification.
Analyses of organochlorine compounds by gas chromatography-mass
spectrometry (GC-MS) in preconcentrated fractions of ultrafiltrates showed
that the achieved concentration factor does not yet allow detailed analysis
of the present compounds. The determinations led us to suspect that chlo-
rophenols and chloroanisoles might be present, but only in concentrations
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which are at the limit of detection of the method used. We are not yet able to
distinguish whether the traces of chloroanisole come from studied samples,
or if they are produced by methylation of chlorophenol, which theoretically
can happen by natural processes. In model experiments using radioactive
chlorine Cl-36, where forest soil was incubated with radiolabelled chloride,
traces of radioactive 4-chlorophenol and radioactive chloroacetic acid were
present in the water extracts (Matucha et al., 2007a, 2007b; Rohlenov et
al., 2009).
3.11CONCLUSION
Our study was based on a monitoring campaign which was carried out in
20062008 by the Povod Labe s.p., in the catchment of the Hamry wa-
ter reservoir. During the first monitoring campaign, elevated levels of AOX
were found in the catchments area, which only occasionally exceeded the
specified national limit (30g/L) for sources of drinking water. This limit
has not been exceeded in the water reservoir itself. The continued monitor-
ing in 20092010 was aimed to elucidate the AOX levels of water samples
and find out whether it is caused by anthropogenic contaminants. Our study
confirmed the deduction set out in the conclusions of monitoring conducted
by Povod Labe s.p., that is that substances collectively described by the
parameter AOX are in this case of natural origin. This was also found in a
study of the presence and origin of chlorinated organic substances in surface
waters in southeastern Sweden, which was originally also designed to detect
anthropogenic substances. From the research conducted in the Hamry water
reservoir it is evident that the observed AOX are of natural origin and that
the chlorinated compounds arise mainly during microbial chlorination of
SOM. SOM is part of humus formed from the biodegradation of dead plant
material.
The AOX concentration limit for sources of drinking water (i.e., 30g/L)
was exceeded in some cases and at some locations during the second moni-
toring campaign in 20092010, for example in stagnant peat waters and their
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draining stream waters. These waters were carrying compounds from peat-
land humic substances and many chlorinated metabolites of natural origin.
The chlorinated metabolites are measured by the AOX method, but are not
necessarily toxic. Running waters outside the area diluted these compounds,
and volatile compounds are evaporated in their course to the water reservoir.
The found AOX values were proportional to TOC values as well as to low-
molecular substances, indicating that LMW fraction is either more haloge-
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nated or faster degraded than larger molecules. Experiments on microbial
chlorination of SOM using radiotracer techniques confirmed the formation
of LMW substances, which have been identified as dichloro- and TCAs,
chloroform, and other chlorinated substances. These latter substances, sus-
pected to be present in trace amounts and are presumed to be chlorophenols
and chloroanisoles, are still waiting to be identified. Unidentified chlorophe-
nol compounds are of special interest because in low concentrations they
may have an impact on human health. The attempts to identify and deter-
mine the content of LMW substances in surface waters is still in the research
stage.
The concentrations of chloroform in natural waters were much lower
(0.10.8g/L) than the maximum permissible value (30g/L) for drinking
water since its volatility further reduces its concentration in raw water. Some
naturally produced organohalogens (such as chloroform) cannot be distin-
guished from anthropogenic counterparts, and have to be treated in the same
way, whereas chlorinated humic substances probably do not represent harm
to human health and can be easily removed from raw water.
ACKNOWLEDGMENTS
The results of the 20102011 monitoring were obtained within the frame-
work of cooperation of the Academy of Sciences of the Czech Republic with
the Pardubick kraj (Pardubice region, Czech Republic), the Povod Labe,
sttn podnik (Elbe River Basin, Czech State Enterprise), the Vodrensk
spolenost Chrudim, a.s. (Waterworks Co. Chrudim plc.), and the Vodovody
a kanalizace Chrudim, a.s. (Water Utilities Co. Chrudim plc.). The authors
also gratefully acknowledge financial support provided by the Grant Agency
of the Czech Republic (Grant Number 13-11101S).
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KEYWORDS
Adsorbable organic halogens

Biogeochemical cycle of chlorine
Bioremediation
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Chlorinated organic compounds
Chlorinated plant products
Chloroacetic acids
Chloroform
Chlorohumus
Chloroperoxidases
Chlorophenols
Cyanobacteria
Hygienic threshold levels
Natural bioactive compounds
Natural chlorination
Radiotracer studies
Sodium chloride
Soil organic matter
Trihalomethanes
Volatile organochlorines
Water disinfection byproducts
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CHAPTER 4
ENVIRONMENTAL IMPACT OF
PESTICIDE USE ON MICROBIAL
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COMMUNITIES AND SOIL
BIOPROCESSES: A PHYSIOLOGICAL,
BIOCHEMICAL, AND MOLECULAR
PERSPECTIVE
JEYABALAN SANGEETHA1, MUNISWAMY DAVID2,
DEVARAJAN THANGADURAI3, ETIGEMANE RAMAPPA HARISH2,
JADHAV SHRINIVAS2, PRATHIMA PURUSHOTHAM3, and
KARTHEEK RAJENDRA MALOWADE2
1
Department of Environmental Science, Central University of Kerala,
Kasaragod, Kerala 671316, India
2
Department of Zoology, Karnatak University, Dharwad 580003,
Karnataka, India
3
Department of Botany, Karnatak University, Dharwad 580003,
Karnataka, India
CONTENTS
4.1 Introduction........................................................................................68
4.2 Pesticides and Its Applications..........................................................70
4.3 Environmental Impact of Pesticides..................................................73
4.4 Agriculturally Important Microorganisms.........................................74
4.5 Pesticide Impact on Soil Microbial Diversity....................................78
4.6 Impact of Pesticides on Soil Bioprocesses........................................81
4.7 Molecular Assessment of Pesticide Impact on Microbial Diversity.....83
4.8 Conclusion and Future Perspectives..................................................86
Keywords....................................................................................................87
References...................................................................................................88
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4.1INTRODUCTION
Pesticides are agrochemicals which are in use to prevent pests infesting

crops. Usage of pesticides largely increased from the past few decades
which even started from pre-sowing stage. In agricultural fields, soil is
the most important component as it is the site for biological interactions.
Repeated application of pesticides ultimately contaminates the soil via spray
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drift during foliage treatment, ground water through seepage, and surface
water through runoff. Ultimately, pesticide contamination adversely affects
the soil ecosystem function by affecting microflora, macroflora, fauna, and
physicochemical properties of the soil. Finally, it leads to the degradation of
soil fertility. Nutrient cycle, roots, plants, and soil biological activities nor-
mally found in the top 2030 cm (812 in), known as the rhizosphere. The
most important microflora of the rhizosphere soil are nitrogen fixing bac-
teria, phosphate solubilizing bacteria, and mineral solubilizers. In addition,
many types of bacteria produce plant-growth promoting substances. These
microorganisms are responsible for the overall fertility of the soil (Asadu et
al., 2015). The excess and frequent application of pesticides may adversely
affect these kinds of agriculturally beneficial microorganisms and ultimately
spoil fertility of the soil.
The production and utilization of agro-based chemicals has been widely
undertaken by low to average income countries. Plants, soil matrix, and soil
organisms are having interrelationship as triad in the rhizosphere region,
where one compound in triad affected by external sources will have the
impact on other two compounds (Coleman et al., 2004). In general, broad
spectrum of pesticides acts against pests, insects, or weeds and kill them
but also affects the non-target beneficial organisms including microbes.
Thus, use of pesticides shows severe threat to microbial diversity of the
agricultural soil. Mode of action of the pesticides differs based on the ac-
tive chemical compounds. However, during their application majority of
the pesticides are affecting non-target organisms in addition to the pests.
Hence, the repeated application of the pesticides ultimately leads to loss of
biodiversity.
In particular, assessment of side effects of chemical or biological com-
pounds is a complex and problematic in environmental systems (Rebecchi
et al., 2000). Bioavailability of pesticides to soil organisms, including rhi-
zosphere microorganisms is of paramount importance to the expected effect
(Gevao et al., 2000). Bioavailability of the pesticide residues basically de-
pends on the percentage of pesticide used, soil type, leachate, and degradation
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Environmental Impact of Pesticide Use on Microbes and Soils 69
of the compound. A wide variety of pesticides are known to persist in soil

environment without degradation and are further found to pollute water table
as well as soil environment. Consequently, it influences micro- and macro-
organisms through bioaccumulation in food chain (Macdonald et al., 2000;
Gill and Garg, 2014). In general, continuous application of pesticides leads
to several negative impacts on the environment which cannot be ignored.
Directly or indirectly, pesticides may affect the fundamental biochemical
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reaction of soil ecosystem such as organic matter mineralization, nitrogen
fixation, nitrification, denitrification, ammonification, sulfur and phospho-
rous solubilization (Kinney et al., 2004; Menon et al., 2005; Hussain et al.,
2009). Many of the agriculturally important bacterial species are more sensi-
tive to pesticides. Filimon et al. (2015) proved the inhibition of nitrification
due to the sulfonylurea herbicides. Fungicides particularly, chlorothalonil
and dinitrophenyl have shown adverse effects on the rhizosphere micro-
organisms which are response for the nitrification and denitrification pro-
cesses (Niewiadomska and Klama, 2005; Lang and Cai, 2009). It has been
reported by earlier researchers that residual concentration of pesticides like
pentachlorophenol, DDT, and methyl parathion from soil are known to af-
fect signaling in leguminous plants such as alfalfa, peas, and soybeans with
symbiotic soil bacteria.
This phenomenon is comparable to endocrine disruption of pesticides
in human and animals, thereby significantly disrupting N2 fixation (Fox et
al., 2007; Mnif et al., 2011). Organochlorine pesticides are more persistent
and its metabolites are more toxic than other types of pesticides, hence it
has been banned to use in agriculture in many of the developed countries
(Ravikumar et al., 2013).
Pesticide residues are found in many agricultural products due to
continuous use of pesticides in agricultural fields. On the basis of global
concern and widespread criticism, certain pesticides like DDT, dieldrin,
endosulfan, and lindane have been banned citing the potential threat on
ecosystem. These pesticides are having long half life, for example, half
life of DDT in soil is from 22 to 33 years, toxaphene up to 14 years, mirex
about 12 years, dieldrin about 7 years, and chlordecone up to 30 years;
hence, it posses high persistence in the environment. Additionally, many
pesticides are potential to accumulate in the fatty tissues of living organ-
isms due to its water phobic or low water solubility nature (Cone, 2005).
Hence, pesticides are great threat to the biodiversity including macro- and
microorganisms.
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4.2 PESTICIDES AND ITS APPLICATIONS
The term pesticide is used to define certain group of chemicals that are syn-
thetically produced yet, are of biological origin, and are used to counter
pests of plants and animals that could harm the productivity, processing, and
transport of food and agricultural products (Arthur et al., 2000; Mushobozi
and Santacoloma, 2010; Saini et al., 2014). The term synthetic pesticide
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comprises the wide range of compounds which include organochlorine, or-
ganophosphate, pyrethroids, and carbamates having a property to act as an
insecticide, fungicide, herbicide, rodenticide, molluscicide, nematicide, and
plant growth regulators (Aktar et al., 2009). Pesticides are important because
of their tremendous benefits to the civilized people, through integral part
of the process by reducing the losses of yield from the weeds, disease and
insect pests that can markedly reduce risk in forestry, public health and the
domestic sphere and, of course, in agriculture (Vinita and Veena, 2015).
The present scenario of agriculture is dependent on the wide range of
pesticides which include insecticides, fungicides, and herbicides (Lopez et
al., 2002). An ideal pesticide should be toxic to only target species and non-
toxic to other organisms including humans. Additionally, higher crop yield
by the sage of pesticides should bring additional revenue to the people that
lead to wealth of the country (Aktar et al., 2009).
About 38% of global land are terrestrial biomes which have been utilized
for agricultural practices such as the cultivation of food, pasture, feed, and
fodder crops (Tilman et al., 2001; Foley, 2011). During the past six decades,
expansion and intensification of agricultural practices led to the rapid in-
crease in pesticide production (>75%) with the worldwide market of about
US $50 billion (Tilman et al., 2001; Stehle and Schulz, 2015). Traditional
agriculture has witnessed a considerable crop and economic losses in several
parts of third world countries, wherein the industrial production of pesti-
cides and their steady use as part of green revolution in the past have shown
potential increase in crop productivity and plant protection (Warren, 1998;
Webster et al., 1999).
Since pesticides play a significant role in an agricultural production and
as such farmers invest significant sums in management of pests. Therefore,
it is imperative that the quality of pesticides is assured for which the system
of collection of samples requires improvement, infrastructure for analysis of
pesticides samples particularly in the synthetic pesticides and plants growth
regulators. List of common pesticides used for different crops, and its target
pests are given in Table 4.1.
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TABLE 4.1 List of Common Pesticides Used in Different Crops and Its Target Organisms.
Pesticide Target and Crops Used References
Chemical Class
Phorate Insect, Organo- Bajra, barley, maize, paddy, sor- Rajendran (2003);
phosphate ghum, wheat, black gram, green Indiradevi (2010);
(OP) gram, pigeon pea Bhushan et al. (2013)
Mancozeb Fungicide, Potato, tomato, wheat, maize, Bhushan et al.
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Dithiocarbamate paddy, jowar, chilies, onions, tapi- (2013)
oca, ginger, sugarbeet, cauliflower,
groundnut, grapes, guava, banana,
apple, cumin, tobacco, mustard,
black pepper, pearlmillet, cucumber
Methyl Insect, Paddy, cotton, black gram, green Rajendran (2003);
Parathion Acaricide (OP) gram, soybean, mustard, groundnut Bhushan et al.
(2013)
Cypermethrin Insect, Brinjal, cotton, cabbage okra, sug- Rajendran (2003);
Pyrethroid arcane, wheat, sunflower, rice Bhushan et al.
(2013)
Carbendazim Fungicide, Paddy, wheat, barley, tapioca, Bhushan et al.
Carbamates cotton, jute, groundnut, sugarbeet, (2013)
beans, cucurbits, brinjal, apples,
grapes, walnut, rose, ber, mango
Monocroto- Insect, OP Paddy, maize, bengal gram, green- Bhushan et al.
phos gram, pea, red gram, sugarcane, (2013)
cotton, castor, mustard, citrus fruits,
mango, coffee, cardamom
Malathion Insect, OP Paddy, sorghum, soybean, cot- Rajendran (2003);
ton, castor, groundnut, mustard, Indiradevi (2010);
sunflower, okra, cauliflower, radish, Bhushan et al.
turnip, tomato, apple, grape, mango (2013)
Quinalphos Insect, Organo- Chilies, paddy, sugarcane, sorghum, Bhushan et al.
thiophosphate okra, cotton, brinjal, tomato, tea, (2013)
tur, groundnut, wheat, bengal gram,
blackgram, red gram, french bean,
soybean, jute, mustard, sesame,
cabbage, cauliflower, onion, apple,
banana, citrus fruits, mango, pome-
granate, cardamom, coffee, gram,
safflower
Carbofuran Insect, Barley, bajra, sorghum, jute, Bhushan et al.
Carbomates groundnut, french bean, potato, (2013)
tomato, apple, citrus fruits, maize,
paddy, mustard, soybean, sugarcane,
bhindi, chilies, cabbage, wheat,
brinjal, banana, peach, mandarins,
cotton, pea, tea, sweet pepper
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TABLE 4.1 (Continued)

Pesticide Target and Crops Used References
Chemical Class
Phosalone Insect, OP Barley, paddy, sorghum, cotton, Bhushan et al.
jute, groundnut, bhindi, brinjal, cab- (2013)
bage, chilies, tomato, tea, mustard
Chlorpyrifos Insect, OP Rice, beans, gram, sugarcane, cotton, Rajendran (2003);
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groundnut, mustard, brinjal, cabbage, Bhushan et al.
onion, apple, ber, citrus fruits (2013)
Dimethoate Insect, OP Bajra, maize, sorghum, red gram, Bhushan et al.
cotton, castor, groundnut, mustard, (2013)
safflower, bhindi, brinjal, cabbage,
cauliflower, chilies, onion, potato,
tomato, apple, apricot, banana,
citrus fruits, fig, mango, rose
Dichlorvos Insect, OP Paddy, wheat, soybean, sugarcane, Bhushan et al.
castor, groundnut, mustard, sun- (2013)
flower, cucurbits, cashew
Paraquat Weed, Tea, cotton, potato, rubber, rice, Rajendran (2003);
Dichloride Herbicides wheat, maize, grapes, apple, aquatic Bhushan et al.
weeds (2013)
Zineb Fungicide, Jowar, paddy, wheat, ragi, tobacco, Bhushan et al.
Organosulfur onion, potato, tomato, chilies, brin- (2013)
compound jal, cucurbits, cauliflower, cumin,
apple, citrus fruits, cherries, grapes,
guava
Captan Fungicide, Chilies, potato, apple, cherry, Rajendran (2003);
Organochlorides grapes, cabbage, cauliflower, Bhushan et al.
(OC) brinjal, beans, tomato, citrus fruits, (2013)
rose, paddy, tobacco
2,4D Weed, Chloro- Paddy, maize, wheat, sorghum, po- Rajendran (2003);
phenoxy acid tato, sugarcane, citrus fruits, grapes Bhushan et al.
(2013)
Fenvalerate Insecticide, Cotton, cauliflower, brinjal, okra Bhushan et al.
Organochlorides (2013)
Triazophos Sucking and Cotton, rice, soybean, brinjal Bhushan et al.
chewing (2013)
insects, OP
Acephate Insect, OP Cotton, safflower, rice Bhushan et al.
(2013)
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4.3 ENVIRONMENTAL IMPACT OF PESTICIDES
Throughout the world, farmers are following a number of pest controlling

measures by using different chemical pesticides to control pest infestation
and minimize crop yield losses. Pesticides are highly biologically active
substances that can threaten the ecological integrity of aquatic and terrestrial
ecosystem; hence, there is a need to incorporate good agricultural practices
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(De Leo and Levin, 1997). In most parts of old world, pesticides were simply
applied without proper understanding on ecology of pests, cost of pesticides,
cost-benefit analysis, mode of application, type of pesticide to be used, crop
specificity and different developmental stages of the crop plants, which are
all potential attributes for heavy crop as well as economic losses (Ombe,
2014). The level of pesticide consumption is comparatively less (Indiradevi,
2010) in several developing and poorly developed countries like Sri Lanka,
Lebanon, India, China, Bangladesh, Philippines, Mali, Ecuador, Zimbabwe,
and Vietnam than that of many other developed and industrialized nations
(Rola and Pingali, 1993; Van Der Hoek et al., 1998; Dung and Dung, 1999;
Wilson, 2000; Huang et al., 2001; Ajayi, 2002; Maumbe and Swinton, 2003;
Rahman, 2003; Yanggen et al., 2003; Gupta, 2004; Salameh et al., 2004).
Recently, Stehle and Scuze (2015) for the first time reported at the glob-
al scale that, more than 50% of detected insecticide concentrations exceed
regulatory threshold levels, thereby posing threat to aquatic biodiversity be-
cause of surface water pollution due to modern farming practices. Angelini
et al. (2013) and Anzuay et al. (2015) indicates that the pesticide application
caused a decrease on the number of cultivated nitrogen fixing population
of peanut soils of Cordoba as well on the nitrogen fixing ability of these
soil. According to World Health Organization (WHO), one million cases
of pesticide poisoning occur every year and consequently there are 20000
deaths globally. The most damaging ecological disturbance of injudicious
use of pesticides is the existence of high concentration of pesticide residues
in food chain, including cereals, pulses, vegetables, fruits, milk and milk
products (including mothers milk), fishes, poultry, meat products, and water
(Jeyanthi and Kombairaju, 2005).
The use of pesticides and production of synthetic pesticides doubles in
future due to increased cash-crop and plantation-style farming in the mod-
ern agricultural activity. Also, the rate of individual risk cases of intentional
and unintentional acute poisoning may increase over the next decade de-
spite a decrease in the proportion of the overall population directly involved
in agricultural production (Aktar et al., 2009). Hence, revisions regarding
the present regulatory methods and process of pesticide usage are necessary
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to minimize the damage to environment due to exploitation of synthetic

chemicals for agricultural purpose. To prevent any harmful effects on the
environment, the level of pesticide residues in air, soil, and water should be
monitored regularly. The monitoring of pesticide residues in food is one of
the most important approaches in minimizing the potential hazard to human
health. When unacceptable levels of pesticides are found, appropriate steps
should be taken to identify the cause and to prevent recurrence.
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4.4 AGRICULTURALLY IMPORTANT MICROORGANISMS
The era of green revolution in the 20th century was the result of intense
agricultural activity tagged with modern scientific technologies. This was
accompanied with surprising cost to the ecological system which further
led to environmental unsustainability and extensive threat to biodiversity
(Vance, 1998). As a result, immense global response was received toward
the deteriorating environment by suggesting certain measures which could
support the development of sustainable agriculture and increased productiv-
ity without compromising the environmental health (Vance, 2001; Noble
and Ruaysoongnern, 2010).
One of the feasible technologies to achieve this condition was to avoid
the application of hazardous agrochemicals which included synthetic fertil-
izers and toxic pesticides, instead to proceed with eco-friendly methodolo-
gies like employing symbiotic microorganisms which could help in attain-
ing good growth of crops and better health of livestock in addition to their
protection from pests and additionally impart resistance to environmental
stresses (Higa and Parr, 1994; Yang et al., 2009). The interaction of microbes
with plants is a well-established process due to the habitat availability shown
by plants to different microbes. The wide range of habitats for microbial as-
sociation include, phyllosphere which refers to aerial plant part, endosphere
referring to internal transport system and rhizosphere; zone of influence of
root system (Lynch, 1990; Lindow et al., 2002). Certain groups of free-liv-
ing bacteria that are present in soil are known to promote the growth of plant
by colonizing the plant roots, and are hence known as plant growth promot-
ing rhizobacteria (PGPR) (Kloepper et al., 1989; Cleyet-Marcel et al., 2001).
The plant microbe interaction is of primary importance in order to car-
ry out transformation and solubilization of nutrients from soil which is in
limited fashion under the absence of microbes. This in turn is in crucial
proposition for plants to realize their genetic ability to utilize nutrients and
grow. Considering biological aid for better production of crops is becoming
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more acceptable by wide range of farmers around the globe for its ability to
induce higher growth rate and in addition avoid the hazardous conditions
which would arise upon making use of synthetic chemical fertilizers. With
this scenario, PGPR has attained a pivotal role for its potential to increase
crop yield along with sustainable environment (Sturz et al., 2000; Shoebitz
et al., 2009). The occurrence of agriculturally important microbes is avail-
able under a cluster of symbiotic (Rhizobium sp.) as well as non-symbiotic
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(Azotobacter, Azospirullum, Bacillus and Klebsiella sp.) bacterial species
which are being used to ensure maximum productivity of agricultural crops
(Burd et al., 2000; Cocking, 2003).
Environmental factors are known to play a crucial role in determining the
composition of bacterial communities that are known to exist with constant
interaction with plant species. One among these factors is identified to be
the nature of soil which can determine the microbial existence in associa-
tion with the plant roots (Lundberg et al., 2012; Bulgarelli et al., 2013). At
the same time, reports have suggested that the host plant is known to play a
crucial role in determining the microbiota of its root habitat (Marschner et
al., 2005; Doornbos et al., 2011) especially endophytic bacterial communi-
ties (Haichar et al., 2008). The enormous contribution could be seen by the
important microbes that are known to establish their association with plants
and are often known to play a critical role in determining environmental
stability. Analysis of these microbes under molecular levels have revealed
that more than 4000 species are known to be present in one gram of soil.
However, many of these microorganisms have known to be non-cultivable
which include a plant symbiotic vesicular-arbuscular mycorrhiza (VAM) en-
domycorrhizae which are often seen in angiosperms and gymnosperms or
are viable yet in non-cultivable conditions (Colwell and Grimes, 2000).
The accessibility of nutrient pool in an ecosystem is highly dependent on
ability of microbes existing in that very environment and their capacity to up-
take the nutrients from soil. Retention of nitrogen by microbes has been iden-
tified as a temporary supply of nitrogen in few terrestrial ecosystem (Zogg
et al., 2000; Bardgett et al., 2003) thereby potentially limiting the export of
nitrogen to adjacent ecosystems and groundwater (Brooks et al., 1998).
Understanding the localization of nitrogen due to seasonal variations is
important for acquiring nitrogen by plants that are more particular to eco-
systems with poor nitrogen availability. This is where microbial species tend
to localize maximum amounts of nitrogen thereby helping the plants during
autumn and preserve it throughout winter until the arrival of spring dur-
ing which it could be released for utilization of plants (Zogg et al., 2000;
Bardgett et al., 2005).
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The genus Frankia constitutes an important group of microorganisms

that have immense agricultural importance. They are filamentous, sporulat-
ing, and Gram-positive bacteria that are capable of fixing atmospheric nitro-
gen through establishment of symbiotic nodules in different dicotyledonous
plants (Susamma et al., 2002). The presence of nif genes in Frankia makes
it possible to carry out nitrogen fixation (Lois et al., 1999; Joel et al., 2002).
One of the important and well-known relationships between legume-Rhizo-
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bium is seen between few plants and microbes. The rapid growing nature of
Rhizobium makes it easy to be isolated it in pure culture. Frankia on other
end is also a strong symbiotic organism that is known for its alliance with
plants like Alnus and Casurina. As compared with the other species of sym-
biotic groups, Frankia constitute highly active strains (Ganesh et al., 1994).
It is known to give rise to root nodules in which nitrogen is actively con-
verted to ammonia. They are highly diverse and they differ in morphology
when compared to actinomycete genera. They are known to form vegetative
hyphae, sporangia, and vesicles (Tjepkema et al., 1980). Vesicles are char-
acterized to protect nitrogenase, as it is highly reactive to O2. The establish-
ment of Frankia in to plants is known to be gained by root hair infection.
After gaining the entry through root hair infection, Frankia is known to form
nodules on lateral roots with cortical cylinder of vascular tissue (Ganesh et
al., 1994). Vegetative mycelia are the active state of Frankia that is seen in
root nodules. Thus, most of the nitrogen requirement that a plant is in need
of is supplied by Frankia species. The process of fixation of nitrogen begs
the need of large amount of energy and this is in turn provided by the host
plants in the form of organic carbon. The primary significant role is played
by the soil condition which is more or less a deciding factor for establish-
ment of Frankia with the plants in a symbiotic relationship (Reddel et al.,
1986; Smolander et al., 1988).
The identification of Rhizobium as source of fixing nitrogen in root nod-
ules is considered as not less than a milestone in the field of agricultural
microbiology (Fisher and Newton, 2004). The process of nitrogen fixation
is carried out in the root nodules wherein the free nitrogen reacts with H
molecules resulting in the formation of NH3 (Zahran, 1999). This process is
carried out by Rhizobium symbiotically during which it is gaining the energy
from host plant (Kondorosi et al., 2013). Rhizobium is identified as a Gram-
negative organism which is known to live freely in soil (Trinick, 1973). The
uniqueness of Rhizobium is due to its potential to fix atmospheric nitrogen
thereby helping the host plant for maintenance of its normal physiology
(Lancelle and Torrey, 1984). However, certain strains of rhizobia-legume
relations are constrained and not similar as above. Few species are known to
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exist as an endosymbiotic microorganism which is known to gain entry in to

root of legumes through root hair thereby forming the root nodule. One of
the prominent natures of rhizobia is that it has been found to be associated
with development of shoot and root growth in rice plants (Yanni et al., 1997;
Yanni and Abd-El-Fattah, 1999).
Azotobacter is a genus which is known to exist in symbiotic association
with plants under aerobic soil conditions. It is a Gram-negative organism
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and is known for fixing the nitrogen (Lakshminarayana, 1993). They are
known to be either free living or in association with plants (Gandora et al.,
1998; Martyniuk and Martyniuk, 2003). The distribution of Azotobacter sp.
is complex and is mainly dependent on critical environmental factors in-
cluding soil condition and its constituents. The species of Azotobacter has
been used as a crucial biofertilizer in association with cereals and rice crop
by employing seedling dip method and seed dipping technique (Singh et
al., 1999; Rttimann et al., 2003). Species of Azotobacter are known to be
highly specialized toward the nitrogen fixation process. Since the nitrogen
fixation is known to be highly sensitive to O2, the specialty of Azotobacter to
counter the O2 inside cells perhaps makes it feasible to fix the nitrogen com-
fortably (Yu et al., 2005). O2 is known to have antagonistic approach toward
nitrogenase enzyme, however, the high rate of respiration in Azotobacter is
known for utilizing the free O2 thereby protecting the nitrogenase enzyme
as well. One of the crucial factors involved in nitrogen fixation process is
the participation of homocitrate ions and C source is the chief requirement
to supply energy to microbes (Kanungo et al., 1997). A. vinelandis is known
for its high nitrogenase activity under in vitro conditions (Schubert et al.,
1976; Smith et al., 1976). Few reports indicating its involvement in growth
of wheat crop are also evident (Kader et al., 2002).
Azolla is a fern that is known to be free-floating. It is capable of fixing
atmospheric nitrogen under the collaborative approach with nitrogen fixing
cyanobacterium Anabaena azollae. Azolla encompasses seven species and
is a critical symbiotic complex in the aquatic ecosystem (Mian, 2002). This
organism is known to reside in dorsal lobes of Azolla leaves endophytically
and known for supplying nitrogen to rice crop. The lone plant cyanobacterial
complexity exists between Azolla-Anabaena that is used as a biofertilizer in
agricultural scenario. The remarkable ability of Azolla to retrieve soils there-
by dejecting harmful invasions by weed species is known to be well evident
in rice crops. In addition, its use in waste-water treatment and degradation of
certain heavy metals has also been well documented. Besides these, its use
in the form of aquaculture feed has also been practiced and the terminology
Azobiofer has been used to describe this (Hove and Lejeune, 1996).
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Cyanobacteria, commonly known as blue green algae are an important

class of bacteria that are known to derive energy through the process of pho-
tosynthesis (Stewart, 1980). The bacteria are known to be widely distributed
with its characteristic presence seen as phototrophic biofilms that are seen in
fresh water and marine waters as well. The Cyanobacteria are found to be
highly resistant to climatic conditions and under unfavorable environmental
conditions are known to pose special structures called as heterocysts. The
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heterocyst is known to contain nitrogenase which has a pivotal role to play in
the process of nitrogen fixation. The heterocyst forming cyanobacteria are
given special importance for their ability to fix the atmospheric nitrogen and
efficiently converting it to ammonia, nitrites, and nitrate which the plants can
readily absorb for fulfilling their energy requirement. Ample studies indicat-
ing the use of Cyanobacteria have been presented under rice field conditions
and is known to reduce the usage of chemical urea (Uma and Kannaiyan,
1999). Their contribution to the field of soil chemistry is also immense,
as they are known to prevent the process of soil erosion. Cyanobacteria is
found to highly benefit the rice plants by the production of certain growth
promoters, that is followed by elevating the phosphorus availability by the
process of excreting organic acids which is further speculated to contribute
in preventing soil erosion ultimately.
Rhizosphere microorganisms are playing a vital role to supply nutrients
to the plants, providing growth promoting substances to enhance the plant
growth, protecting plants from pathogens and also to maintain the soil fertil-
ity (Ahmad et al., 2008; Ahemad and Khan, 2011). The plant-microbe inter-
actions are important for the solubilization, mobilization, and transformation
of minerals to the plants (Shoebitz et al., 2009; Hayat et al., 2010). Many
researchers have studied the effect of pesticides on root colonizing microor-
ganisms in various crop fields (Anderson et al., 2004; Fox et al., 2007; Datta
et al., 2011).
4.5 PESTICIDE IMPACT ON SOIL MICROBIAL DIVERSITY
Pesticides greatly influence soil bioprocesses carried out by microorgan-

isms. These also affects the bioavailability of organic compounds and even
affect the process by which microbes convey organic compounds in soil to
their mineralized forms and thus biotransformation (Demanou et al., 2004;
Kinney et al., 2005). However, recent advances has lead to the develop-
ment of molecular tools and techniques which can be used in understanding
the impact of pesticides on microbial community structures and functions
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(Widenfalk et al., 2008). Various environmental factors and properties of

soil play a major role in determining the effect of pesticide on soil micro-
flora (Ecobichon, 1991). Due to indiscriminate use of pesticides, a large
deviation in standard quality of organic matter in soil affecting diverse soil
microbial community in it. Since microbial biomass one or the other way is
linked with various nutrient recycling and biotransformation, any alteration
in their population could seriously alter soil fertility. Many pesticides are
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intended to ward off certain insect pests. However, more than 90% of foliar
application reach soil and thereby affect non-target microorganisms altering
their vital biochemical processes, thus cell division and molecular composi-
tion (De Lorenzo et al., 2001). Initial pesticide exposure decreases the mi-
crobial diversity, but prolonged persistency leads to resistance or tolerance.
Ryan (1999) reported that routine agricultural practices involving chemical
pesticide may alter some groups of soil organisms, but the overall integrity
of community structure would remain constant. However on the contrary,
Kalia and Gosal (2011) studied overall soil microbial community structure
from rice and wheat cultivating lands under pesticide stress and concluded
that average population in all soil microbes considered for study decreased
drastically with pesticide application.
Soil properties and chemical nature of a pesticide decides the fate of
pesticide in soil; as transformations like degradation, transit, and adsorption/
desorption. According to Singh and Walker (2006), soil microorganism and
their metabolites often react with pesticides and thus alter the physiological
and biochemical properties of soil microbes. However, available microbial
activity and biomass in a given soil determines soil health and fertility, as
the major nutrient transformations are linked with microorganisms in soil.
Many recent studies shows nugatory effect of some pesticides on soil mi-
crobial population thus soil fertility (Pampulha and Oliveira, 2006; Zhou
et al., 2006). More precisely, decrease in soil fertility implies reduction in
microbial activity and increase implies induction in soil microbial activity.
On the contrary, some consortia of microorganisms are capable of utiliz-
ing administered pesticides for their energy requirements to grow, develop,
and multiply. There are reports stating administered pesticides reduced the
microbial diversity but helped to increase the functional diversity of micro-
bial community (Pampulha and Oliveira, 2006; Wang et al., 2006). However,
pesticides may outnumber some group of microorganisms by removing and
outnumbering them from competition. For instance, fungicide application as
reported by Chen et al. (2001) removed the activity of certain fungi leading
to rapid elevation in bacterial population. Similar activity was reported by
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Lpez et al. (2006) with increase in heterotrophic mesophilic and psychro-

philic aquatic bacteria when applied with herbicide simazine.
Despite several other parameters, numerous environmental factors deter-
mine the effect of pesticide on soil microorganisms. One of such major fac-
tor is the bioavailability of pesticide in soil environment. Several adsorption/
desorption reactions modulate the concentration of pollutant in the vicinity
of the soil and hence its bioavailability (Katagi, 2008). Further, Menon et al.
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(2004) have reported inhibitory activity of chlorpyrifos and quinalphos are
differed with sandy loam, and loamy sand as the bioavailability differed with
respect to different soil environments. Gundi et al. (2005) observed syner-
getic effects of three insecticides (monocrotophos, quinalphos, and cyper-
methrin) at lower level in black clay soil and harmful effect at higher level
of concentration. On the contrary, Widenfalk et al. (2004) reported toxic ef-
fect of pesticides on fresh water sediment microbial community even under
predicted environmentally safe concentrations (Table 4.2).
TABLE 4.2 Effect of Some Pesticides on Soil Microorganisms.

Pesticide Target Microbe and Effects References
Methamidophos Soil bacteria (decreased Wang et al. (2006)
biomass)
Metalaxyl Soil microflora (decreased Sukul and Spiteller (2001)
biomass)
Mefenoxam, Bensulfuron Inhibition in N-fixing bacteria Monkiedje et al. (2002)
methyl
Isoproturon Actinomycetes and fungi (sup- Nowak et al. (2004)
press growth)
Captan Rhizobium ciceri (decrease in Kyei-Boahen et al. (2001)
viable count)
Atrazine, Metribuzin Bradyrhizobium sp. (adverse Khan et al. (2006)
effect)
Agroxone, Atranex, 2,4-D Rhizobium phaseoli, Azoto- Das and Mukherjee (1998)
amine bacter vinelandii (most toxic)
Further, apart from bioavailability, certain other parameters like soil

texture, organic matter, and available vegetation may also influences pes-
ticide toxicity toward microbial biomass. The resistance to pesticides may
get enhanced by the additional supplementation of carbon sources (glucose,
acetate, amino acids) (Mishra and Pandey, 1989). This effect could be well
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documented in the soil which has been well tilled. Agronomically impor-
tant microorganisms such as bacteria as well as arbuscular mycorrhiza (AM)
form a symbiosis together. This interaction is often affected by pesticide
application resulting in stress and deviation from normal conditions (Sainz
et al., 2006). Fungi are the most affected at this function as they are prone
to growth, development, nitrogen fixation, and other metabolic activities.
However, further work is needed in understanding comparative sensitivity
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of fungi to many pesticides (Ma et al., 2004).
4.6 IMPACT OF PESTICIDES ON SOIL BIOPROCESSES
Biotransformation is one of the key aspects in microbial biochemical pro-

cesses. They have the ability to transform various biomolecules like nitrogen
(N), phosphorus (P), sulfur (S) and carbon (C). Biochemical reaction such as
mineralization of organic matter, nitrogen fixation, and ammonification are
usually affected by pesticides directly or indirectly either by acting upon mi-
crobes alone or by acting on enzymes from microbial metabolism (Kinney
et al., 2005; Menon et al., 2005). Biological nitrogen fixation (BNF) is one
of the major components of nitrogen fixation which is estimated about twice
(about 175 million tons) the amount of nitrogen fixation from non-biological
sources (Tate, 1995). However, pesticides are known to affect the root nodu-
lation and BNF in legumes. Niewiadomska (2004) and Niewiadomska and
Klama (2005) have studied and reported the adverse effects of carbendaz-
ime, thiram (fungicides), and imazetapir (herbicide) on nitrogenase activity
of Rhizobium leguminosarum, Sinorhizobium meliloti, and Bradyrhizobium
sp., and clover, lucerne, and serradella plants.
Biological activities are more pronounced in black soil than red soil. This
is because black soil contains more organic matter than red soil (Shrinivas
and David, 2015). Hence, organic matter is the most crucial part of the soil
which determines fertility of soil. Quality of organic matter and dynamics is
often controlled by biological activities of the soil and rate at which nutrient
recycling takes place. Some researchers have thrown light on how pesticides
deteriorates nutrient recycling and biological decomposition of organic
matter under grassland area, forest ecosystem and desert place (Weary and
Merriam, 1978; Perfect et al., 1981; Santos and Whitford, 1981). Whereas,
others have documented beneficiary effect of pesticide on mineralization
processes. Sukul (2006) reported significant decrease in C and N content of
the soil with 30-day exposure to metalaxyl.
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Pesticides when applied can interfere with bacterial communities af-

fecting many biological processes such as nitrification, denitrification, and
ammonification. Kinney et al. (2005) studied the effect of fungicides and
herbicides (mancozeb, chlorothalonil, prosulfuron) on nitrifying bacteria
and concluded toxic potentials of these pollutants on nitrification (N2O) and
denitrification (NO) which are supposed to be environmentally significant
trace gases. Similar observation was made by Ogunseitan and Odeyemi
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(1985) in tropical soil with lindane, captan, and malathion on nitrification,
phosphate solubilization and sulfur oxidation. They observed nitrification
and phosphate solubilization were deprived in 30 days exposure period with
all three pesticides. It was even reported that malathion increased sulfur oxi-
dation while lindane and capta affected the reaction adversely.
Soil also contains certain enzymes in free form, immobilized and within
microbial cells and represent normal/abnormal health of soil. Certain foliar
applied pesticides however reach soil system thereby interacting with soil
microbes and may affect enzymatic activities (Shrinivas and David, 2015).
Monkiedje and Spiteller (2002) have reported negative impact of pesticides
on hydrolases, oxidoreductases, and dehydrogenase activities in the soil.
Whereas, there are reports advocating the increased enzyme activities and
ATP contents in the soil due to certain pesticide applications (Shukla, 1997).
Malkomes (1997) attributed such differences to the dual behavior of pesti-
cides (both harmful and beneficial for soil enzymes), diversity and various
stages of the processes taking place in soil that are frequently overlapped.
Enzyme activity in soils reflects not only enzymes in soil solution and liv-
ing tissue, but also enzymes bound to soil colloids and humic substances
(Nannipieri et al., 1990).
However, on a lighter note there is no comprehensive information to un-
derstand the role of pesticides on soil microbes as well bioprocesses despite
of extensive researches conducted all over. This is because some pesticides
act as energy sources by providing carbon and nitrogen to microbes and
are easily get degraded in return. Whereas, some act as recalcitrant and ad-
versely affect soil microflora and bioprocesses. Soil enzymatic activity acts
as a biological index to measure the soil fertility and biological process in
soil and is also reduced by the pesticides (Monkiedje et al., 2002; Antonious,
2003). The mineralization of organic compounds and biotransformation of
nutrients by soil microorganisms are also adversely affected by the pesticides
(Demanou et al., 2004; Niewiadomska, 2004; Kinney et al., 2005; Mahia et
al., 2008). Therefore, it is naive to give a definite conclusion on pesticide
effect on soil micropopulation. The same is true in case of soil biochemical
processes and soil enzymatic activities.
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4.7 MOLECULAR ASSESSMENT OF PESTICIDE IMPACT ON

MICROBIAL DIVERSITY
In this scientific era, molecular techniques are most widely used to study the
effect of agrochemicals on the function and structure of microbial communi-
ties, as this technique is culture independent. However, the impact of agro-
chemicals on the genetic structure and degradation ability of soil microbial
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communities have been less investigated (Hussain et al., 2009). Reports by
Schloss and Handelsman (2004) have indicated the availability of not less
than 50 bacterial phyla of which 50% of bacterial communities have been
represented on the basis of molecular sequencing. Culturable microorgan-
isms constitute <1% of all microbial species (Hugenholtz, 2002). For ex-
ample, from soil samples most of the microbial species belong to one of four
phyla (Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria) due to
their ease of cultivation in laboratory conditions. Conversely, Acidobacteria
comprise approximately 20% of soil bacterial communities which are repre-
sented by few genera; such bacterial species are difficult to cultivate in labo-
ratory conditions (Schloss and Handelsman, 2004). These findings suggest
that in depth characterization of environmental microbial population needs
molecular techniques for isolation, cultivation, and characterization.
The introduction of culture independent methods such as 16S rRNA and
DNA based methods to assess the total microbial diversity has found the
solution for the limitations of culture dependent methods. Assessment of
microbial diversity in environmental samples is possible through molecu-
lar tools (Sharma et al., 2014). Since last two decades, amplification and
sequencing of 16S rRNA has been in use for the assessment and identity
of abundance and taxonomic variation of microbial species in the environ-
ment (Pace, 2009). Gradually, 16S rRNA observed shows variation among
the strains of same species (Acinas et al., 2004). Thus, molecular tools have
been developed as a new approach for revealing the microbial diversity in
different environments.
There are apparent improvements in the microbial diversity study
through several scientific and technological developments in nucleic acid
sequencing methods (Rastogi and Sani, 2011). The advent and effect of se-
quencing techniques has changed the assessment of microbial diversity and
its functional pattern in an ecosystem (Chistoserdova, 2010; Manickam et
al., 2010; Morales and Holben, 2011). For the assessment of environmental
risk, environmental impact, and public health, culture independent methods
would be used rather than culture dependent methods, so as to infer the sig-
nificance of a specific taxonomic group in a community (Vaz-Moreira et al.,
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2011). There are two types of molecular tools that are available to assess the
microbial diversity such as whole community analysis and partial commu-
nity analysis (Fig. 4.1).
9781771883627
FIGURE 4.1 Molecular methods to characterize the diversity of microorganisms from
pesticide contaminated soil samples (Rastogi and Sani, 2011).
The strategy of partial community analysis is based on polymerase chain

reaction (PCR) methods, where the characterization of the microbial species
can be achieved by the total RNA/DNA extracted from the environmental
samples. The PCR product might have the mixture of genetic sequences of all
microorganisms including viable but non-culturable (VBNC) microorgan-
isms present in the sample (Rastogi and Sani, 2011). PCR amplification of
16S rRNA from an environmental sample has been used for all types of sam-
ple due to its ubiquitous nature, structurally and functionally conserved, hav-
ing variable, and highly conserved regions (Hugenholtz, 2002). In microbial
diversity studies, 16S rRNA technology is a prime choice through its suitable
gene size and increasing number of 16S rRNA sequences available in data-
base for comparison. Using 16S rRNA sequencing methods the phylogenetic
relatedness for the known microorganism can be estimated, in which the
AUTHOR COPY
closest affiliation of a new isolate is assigned. According to Ghebremedhin

et al. (2008), RNA polymerase beta subunit (rpoB), gyrase beta subunit
(gyrB), recombinase A (recA) and heat shock protein (hsp60) have also been
used in microbial ecology to differentiate the microbial species. These am-
plified PCR products further analyzed either by (i) genetic finger printing
such as Denaturing- or Temperature Gradient Gel Electrophoresis (DGGE/
TTGE), Single-Strand Conformation Polymorphism (SSCP), Random
9781771883627
Amplified Polymorphic DNA (RAPD), DNA Amplification Fingerprinting
(DAF), Amplified Ribosomal DNA Restriction Analysis (ARDRA),
Terminal Restriction Fragment Length Polymorphism (T-RFLP), Length
Heterogeneity PCR (LH-PCR), and Ribosomal Intergenic Spacer Analysis
(RISA), (ii) clone library method, or (iii) DNA microarray or using a combi-
nation of these three techniques (Fromin et al., 2002; Haack et al., 2004; de
Figueiredo et al., 2007; Rastogi and Sani, 2011).
Sequence analysis of 16S rRNA is generally used by researchers in
microbial ecology using conserved gene sequence regions. Though, this
technique does not provide adequate resolution at species and strain level
(Konstantinidis et al., 2006). In this view, whole community analysis pro-
vides an inclusive outlook of genetic variability and diversity of microbial
population compared to partial community analysis. In principle, complete
genetic information present in the total DNA extracted from the environ-
mental sample is thoroughly analyzed by whole community analysis and
GC content (Rastogi and Sani, 2011). DNA-DNA hybridization (DDH)
offers promising, accurate, efficient, and reliable results for the compari-
son among the whole genome of different organisms. Moreover, Guanine-
Cytosine (G+C) content of DNA is varying within different prokaryotes at
35%, which can also be used to compare the genomic composition of phy-
logenetically related bacterial community present in samples from extreme
environments (Nsslein and Tiedje, 1999).
Shotgun cloning method is also used to sequence the whole microbial
genome. Assessing the microbial systems through whole-genome analysis is
a wide-ranging and incorporated approach to understand the structural and
functional pattern of microbial ecology (Huson et al., 2007). Through the
genome sequencing technique, massive amount of information gathered and
deposited in the searchable databases that could be incorporated with other
bioinformatics tools which available in the Integrated Microbial Genomes
(IMG) web server (Markowitz et al., 2010). In addition, metagenomics is
commonly used to analyze the collective microbial genomes from an envi-
ronmental samples (Riesenfeld et al., 2004). Metagenomics is also function-
ing based on the principle of sequencing whole genome to sequencing and
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analyzing the entire genetic composition of environmental microbial com-

munities (Rastogi and Sani, 2011; Imfeld and Vuilleumier, 2012).
The unscientific and uncontrolled excessive and indiscriminate use of pes-
9781771883627
ticides in the agricultural sector results in many human health problems and
environmental pollution. Hazards arising during the application of pesticides
are mainly due to lack of information, knowledge, awareness, poor super-
vision during spraying, absence of proper legislation or of enforcement of
legislation, and sale on the open market of highly toxic pesticides. Hence, it
is necessary to monitor the occupational health of workers in the agricultural
sector, with appropriate surveillance and record-keeping (Roy, 2015). Food
and Agricultural Organization (FAO) introduce the word for safe use of pes-
ticide in the environment and it define it as Good Agricultural Practice
(GAP) in the use of pesticide as the officially recommended or authorized
usage of a pesticide under practical conditions at any stage of production,
storage, transport, distribution and processing of food and other agricultural
commodities, bearing in mind the variations in requirements within and be-
tween regions, and which takes into account the minimum quantities neces-
sary to achieve adequate control, the pesticides being applied in a manner
so as to leave a residue which is the smallest amount practicable and which
is toxicologically acceptable (FAO, 1977). In developing countries, it is
necessary to train the general public regarding health problems in the ag-
ricultural sector with respect to quantities of pesticides to be used for the
respected crops, scientific way of pesticide application, the current state of
health of workers in relation to pesticide exposure, coordination between
hospital physicians, occupational health specialists, the number, sex, and age
of the workers exposed, and so forth. Comprehensive occupational health
histories should be obtained from all workers adversely affected by pesticide
exposure (Roy, 2015).
The use of pesticide is beneficial for plant growth by protecting it from
the damage by the pests and insects, but decreasing the overall soil fertil-
ity and contaminating the environment. Soil microorganisms are playing a
crucial role in biogeochemical cycle, which maintains soil fertility for plant
growth and productivity. According to many research findings, continuous
application of pesticides poses adverse effect on soil especially rhizosphere
microorganisms (Sethi and Gupta, 2013). The effects of pesticides on mi-
crobial community and soil bioprocesses are based on the nature of the
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chemical compound in pesticide, its metabolites, and quantity of the pesti-

cide application. Elucidating the risk of pesticides on microbial diversity and
soil biological processes can be achieved only through the molecular assess-
ment of whole microbial community in pesticide contaminated-environment
(Hussain et al., 2009). The field of microbial ecology is enduring the ex-
ceptional desirable changes along with the development and application of
molecular genomic tools.
9781771883627
Our knowledge and understanding on microbial composition and nutri-
ent dynamics in agricultural soil ecosystem is still insufficient. In future,
several issues related to the impact of pesticides on microbial diversity is
need to be addressed and widely acceptable risk assessment frame work in
agro-ecosystems with microbial indicators should be prepared. Currently,
research focus is shifting toward the exploration of the soil microbial re-
sponses to pesticide contamination. A complete analysis is necessary for the
global patterns of microbial species diversity which can be obtained from
sequencing methods and its alteration by the pesticide chemical exposure.
KEYWORDS
Agrochemicals
Azobiofer
Biological index
Biologically active substances
Biotransformation
Endosphere
Environmental impact
Frankia
Microbial indicators
Nitrogen fixation
Pesticides
Plant growth regulators
Rhizobium
Rhizosphere
Soil microbial diversity
Synthetic pesticide
AUTHOR COPY
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CHAPTER 5
RECENT ADVANCES IN APPLICATIONS

OF NANOMATERIALS FOR WATER
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REMEDIATION
KALIYAPERUMAL RANI1 and BARINDRA SANA2
1
Division of Bioengineering, School of Chemical and Biomedical
Engineering, Nanyang Technological University, 62 Nanyang Drive
637459, Singapore
2
p53 Laboratory, Agency for Science, Technology and Research
(A*STAR), 1 Fusionopolis Way, #20-10 Connexis North Tower
138632, Singapore
CONTENTS
5.1 Introduction........................................................................................98
5.2 Nanomaterials for Water Remediation.............................................100
5.3 Nano-Based Technologies vs Conventional Technologies
for Water Treatment.........................................................................109
5.4 Conclusion and Perspectives........................................................... 111
Keywords.................................................................................................. 112
References................................................................................................. 112
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5.1INTRODUCTION
According to National Nanotechnology Initiative, nanotechnology (NT) re-

fers to engineering functional systems at the nanoscale (about 1100 nm size)
where unique phenomena facilitate innovative applications. This emerging
transdisciplinary field opens novel perspectives in all sectors including med-
icine, biology, cosmetics, optical science, semi conductor, memory and stor-
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age technologies and electronic and automotive industry. Nanotechnology
plays an inevitable role in improving the environmental sustainability by
means of detecting, preventing, and removing pollutants and also by devel-
oping environmental friendly products (Figs. 5.1 and 5.2).
FIGURE 5.1 Applications of nanotechnology in various sectors.
FIGURE 5.2 Environmental applications of nanotechnology.

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Applications of Nanomaterials for Water Remediation 99
Nanoremediation refers to the application of nanomaterials for remedia-

tion. These nanomaterials are highly reactive and effectively transform and
extenuate the pollutants through oxidation or reduction. The standout fea-
tures of nanomaterials such as large surface area, well-defined structure, and
easy dispersability make them superior to conventional remediation technol-
ogies. They could be readily tailored according to the pollutants of concern.
Further, nanoremediation technologies eliminate the necessities of ground
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water being drawn out above ground level and soil being shipped to different
sites for treatments (Fig. 5.3) (Otto et al., 2008).
Well
defined
structure
High High
reactivity surface area
ADVANTAGES OF
NANOMATERIALS
Easily
High tailored for
specificity specific
applications
Easy
dispersability
FIGURE 5.3 Salient features of nanomaterials.

Figure 3
Various nanotechnological platforms have been developed that could

offer cost-effective solutions to numerous environmental concerns. For
instance, nanoparticles containing zero-valent iron hold great promise for
remediation of contaminated groundwater. Nanotitanium dioxide potential-
ly degrades organic pollutants, which are deleterious to the environment.
Carbon nanotube membranes are of great use in the process of desalination.
Nanofilters are employed to clean up ground or surface water contaminated
with chemicals and hazardous substances. Nanosensors are availed to detect
waterborne contaminants. Nanosilver is applied to disinfect water owing to
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its antimicrobial potency. Overall, there is a multitude of promising envi-

ronmental applications for nanotechnology; this chapter highlights some
of the fascinating developments in the applications of following nanoma-
terials such as nanoscale zero-valent iron (nZVI), titanium dioxide (TiO2),
silver nanoparticles (AgNPs), nanomembranes, nanotubes, nanoclays, and
nanocomposites.
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5.2 NANOMATERIALS FOR WATER REMEDIATION
5.2.1 NANOSCALE ZERO-VALENT IRON (nZVI)
Numerous nanoparticles have been widely applied for remediation of aque-

ous contaminants; however, nanoscale iron (nZVI) gained a great deal of
attention in the recent years and symbolizes a latest generation of environ-
mental remediation technology. Nano zero-valent iron is a nanoscale materi-
al of 10 to 100nm in diameter and is considered as a novel nanomaterial for
soil and ground water remediation. They exhibit a typical core shell struc-
ture containing metallic iron core surrounded by a delicate amorphous oxide
shell (Yan et al., 2010). In bimetallic nanoparticle, nZVI is often mixed with
another metal such as palladium, silver, or copper, which acts as a catalyst.
The inclusion of noble metal for instance palladium makes the remediation
more effective as it catalyzes the dechlorination and hydrogenation process-
es more effectively (Dhakras, 2011).
nZVI has numerous advantages such as abundant surface area, which
renders enhanced surface reactivity, exorbitant flexibility for in situ applica-
tions, efficiency to extenuate widespread environmental pollutants. In spite
of these merits, nZVI bears certain site-specific requisites, which need to be
fulfilled for efficient remediation. For instance, site characterization such
as location, geologic conditions, concentration and types of contaminants,
geochemical features, ground-water flow velocity, and depth are mandatory
before nZVI is being applied. The most frequently used method of applica-
tion of nZVI is injection of an nZVI suspension directly into an aquifer.
Injection techniques include infiltration wells, sleeve pipe, push infiltration,
or gravity infiltration. Another mode of application is by using a permeable
reactive barrier, where the trench is loaded with nZVI particles (Mishra and
Patel, 2009).
The nZVI appears to be very potent for degrading a wide a spectrum of
organic pollutants including polychlorinated biphenyls (PCBs), chlorinated
hydrocarbon (organochlorine) pesticides, and chlorinated organic solvents
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(Zhang, 2003). The mechanisms through which chlorinated solvents such as

trichloro ethane (TCE) are degraded by nZVI involve two pathways name-
ly reductive dechlorination and -elimination (Arnold and Roberts, 2000).
The dechlorination reactions take place at the surface of nZVI and utilize
the excess electrons formed from iron corrosion in water. This results in
the production of nonchlorinated hydrocarbons such as ethane and ethene.
However, the degradation of TCE by -elimination pathway results in the
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formation of chloroacetylene, which is then converted to acetylene with
the elimination of chlorine. Acetylene is further converted into ethane and
ehthene (Arnold and Roberts, 2000). It has been demonstrated that nZVI
is efficient in reducing inorganic anions such as nitrate, arsenate, arsenite,
perchlorate, selenite, and chromate. When compared to granular iron, nZVI
bears high catalytic rate and sorption potency. Further, nZVI is effective in
eliminating heavy metals such as Pb and Ni present in aqueous solution by
reducing them to zero-valent metals (USEPA, 2013).
nZVI particles have shown the potential to reduce perchlorate present in
aqueous solution to chloride completely without forming any intermediates
(Cao et al., 2005). It has been demonstrated that iron oxide nanoparticles
are efficient in removing arsenic (As) through irreversible binding. Further,
nZVI has been shown to catalyze the rapid transformation of As (V) to As
(III) at neutral pH. However, for this reduction, nZVI was needed at larger
quantity due to presence of phosphate, sulfate, and dissolved organic carbon
(DOC) present in the solution (Kanel et al., 2006).
Chlorinated dense non-aqueous phase liquids (DNAPLs) remain as one
the major constituents of groundwater contamination. nZVI acts as a re-
mediation agent for DNAPL sources. As nZVI reduces the concentration
of contaminants, it favors the formation of concentration gradient between
DNAPLs and the aqueous phase, resulting in the translocation of DNAPLs to
the dissolved aqueous phase. This in turn creates the necessity for DNAPLs
to be treated again (Watlington, 2005). To overcome this issue, emulsified
ZVI (eZVI) has been introduced. This contains iron particles in water sur-
rounded by an oil-liquid membrane. The properties of eZVIs membrane
(hydrophobicity) are similar to that of DNAPLs and hence are miscible with
DNAPLs. Owing to the diffusion of halogenated hydrocarbons in DNAPL
through the membrane into the interior aqueous phase where zero-valent
iron presents, abiotic reductive dechlorination occurs. Moreover, the pres-
ence of hydrophobic membrane in eZVI protects the nZVI from other water
constituents, thereby enhancing its availability for treating target contami-
nants (Hara et al., 2006).
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5.2.2 TITANIUM DIOXIDE (TiO2)
Nanocatalyst based photocatalysis is considered as a promising approach for

treating contaminated water. The catalysts harness the UV radiation from
sunlight and utilize the energy to degrade the pollutants. Photocatalysis
involve in the degradation of wide spectrum of pollutants such as organic
compounds and acids, inorganic compounds, pesticides, and dyes. There
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are several potential photocatalysts, among which titanium dioxide (TiO2) is
emerging as a promising option for water remediation owing to its semicon-
ducting, energy converting, and gas sensing natures. They are very versatile
and serve both as oxidative and reductive catalysts hence have been widely
explored for oxidative transformation of organic and inorganic pollutants
(Mayo et al., 2007). TiO2 catalyzes the oxidation process through the forma-
tion of hydroxyl-free radicals and the reduction process through peroxide
formation. The photocatalytic process catalyzed by TiO2 is clearly demon-
strated in Figure 5.4. In TiO2, the electrons are confined to energy bands
namely valence band and conduction band. When TiO2 is illuminated by UV
light, electrons are excited from the valence band to the conduction band.
As a result, electron vacancies known as holes are produced in the valence
band (Mills and Davies, 1993). As the holes are of positively charged, they
form OH and H+ ions upon binding with water molecules. Electrons reduce
the dissolved oxygen to superoxide ions (O2.). As these superoxide ions are
highly reactive in nature, they bind with water molecules to produce perox-
ide radicals (OOH) and hydroxide ions (OH). Upon binding with H+ ions,
peroxide radicals produce OH and OH. The hydroxide ions are oxidized
by the holes to OH. Hence, OH ions formed during the photocatalysis di-
rectly attack the contaminants present in the aqueous mixture (Linsebigler
et al., 1995) (Fig. 5.4).
TiO2 nanoparticles are cost effective, photocatalytically active, chemi-
cally inert and are highly resistant to corrosion. The larger surface area of
TiO2 nanoparticles provides huge catalytic surface (Theron et al., 2008).
These salient features make them to use as semiconductor photocatalyst in
water purification systems. In the presence of UV light, TiO2 nanoparticles
are highly efficient in the removal of carbon from water contaminated with
organic wastes (Chitose et al., 2003). However, they are unable to absorb
visible light. Doping of transition metals and anionic nonmetals such as ni-
trogen, carbon, sulfur, or fluorine into TiO2 enhances its optical absorbance
to the visible region resulting in enhanced photocatalytic potency (Asahi et
al., 2001; Noworyta and Augustynski, 2004; Livraghi et al., 2005; Ni et al.,
2007). For instance, surface modification of TiO2 nanoparticles with metals
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such as gold and platinum elevates the photocatalytic activity as the metals
keep the electrons and holes from recombining. Further surface modifica-
tion with chelating agents such as lauryl sulfate and arginine provides high
surface area and thereby increases the active sites resulting in enhanced pho-
tocatalytic activity (Makarova et al., 2000). Similarly, nitrogen-doped TiO2
nanoparticles were efficient in degrading azo dyes and Fe(III)-doped TiO2
nanoparticles were efficient in the degradation of phenol (Liu et al., 2005;
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Nahar et al., 2006).
FIGURE 5.4 Mechanism of TiO2 photocatalysis.
Microbial contaminants constitute a major part of water contamination.

It has been demonstrated that nitrogen-doped TiO2 nanoparticles are effi-
cient in degrading microbial contaminants of water (Shalini et al., 2012).
UVvisible light activated TiO2 nanoparticles demonstrated enhanced bac-
tericidal activity against E. coli (Ireland et al., 1993). Chemical oxygen de-
mand (COD) (Kemnetz and Cody, 1996) test is often used to determine the
concentration of organic compounds in water and hence acts as a useful
measure of water quality. TiO2 electrodes are of great use in detecting chemi-
cal oxygen demand of water, hence are employed as sensors for measuring
the degree of water contamination (Kim et al., 2000).
Owing to the stable nature of TiO2 in water, it could be immobilized onto
thin films or membranes. The main goal of immobilizing TiO2 is to eliminate
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the post separation predicaments related to the amorphous TiO2. Apart from
this, TiO2 immobilization offers greater catalytic area, increased surface
hydroxyl groups and enhanced adsorption efficiency (Zhu and Zou, 2009;
Esparza et al., 2010; Jin and Dai, 2012). TiO2 films are employed for filtra-
tion and bacterial inactivation purposes. TiO2 films can be fabricated using
different deposition techniques such as reactive sputtering, chemical vapor
deposition and solgel process. The solgel method is the most popular ap-
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proach because of its cost effectiveness and simplicity. The characteristics
and the potential applications of TiO2 films strongly rely on their synthesis
procedures and relevant operational parameters (Sobczyk-Guzenda et al.,
2013). It has been demonstrated that Ag/TiO2 slurry solution and TiO2 films
fabricated by sol-gel method showed exceptional efficiency in the photo-
catalytic degradation of wastewater (Domnguez-Espndola et al., 2013).
5.2.3 SILVER NANOPARTICLES
Silver nanoparticles (Ag NPs) possess unique optical, thermal and electrical
properties and have been widely used as anti-bacterial agents. Ag NPs are
mainly derived from silver nitrate and silver chloride. It has been reported
that the antibacterial activities of Ag NPs mainly depend on the size and
shape of the particles. Smaller Ag NPs exhibits enhanced bactericidal activ-
ity when compared to large size silver particles (Makhluf et al., 2005) and
truncated triangular silver demonstrated exalted antibacterial potency than
spherical and rod-shaped nanoparticles (Pal et al., 2007).
The exact mechanism through which Ag NP exert its antibacterial effect
has not been clearly elucidated yet, however, various modes of actions have
been proposed. It has been demonstrated that Ag NPs bind with the bacte-
rial cell wall and cause structural changes in the membrane, which results in
increased cell permeability and ultimately death (Sondi and Salopek-Sondi,
2004). It has also been suggested that silver ions released by the nanopar-
ticles inactivate various enzymes of the bacterial cells by effectively binding
with the SH functional groups of enzymes (Feng et al., 2000; Matsumura et
al., 2003). Spectroscopy studies demonstrated that Ag NPs produce highly
reactive free radicals, which in turn disrupt cell membrane resulting in cell
death. These free radicals also attack the enzymes of the electron transport
chain and inhibit electron transport system (Danilczuk et al., 2006; Kim et
al., 2007). The potency of Ag NPs to bind with the sulfur and phospho-
rus components of DNA molecules provokes DNA damage and thus inter-
feres with DNA replication (Hatchett and White, 1996). In gram-negative
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bacteria, Ag NPs cause dephosphorylation of tyrosine residues resulting in

altered phosphotyrosine profile of bacterial peptides, which in turn interfere
with signal transduction pathways and inhibit bacterial growth (Shrivastava
et al., 2007) (Fig. 5.5).
AgNPsbindwith
AgNPs
bacterial
bacterialcellwall
cell wall
9781771883627
ProteinOxidation
Inhibitssignal
ENZYMES transduction
Dephosphorylation
InhibitsDNA
DNA replication
ROS
ELECTRONTRANSPORT
CHAIN
CELLDESTRUCTION
h ll
Enhancescell
permeability
Cellmembrane
Cell membrane
disruption
FIGURE 5.5 Antibacterial mechanisms of Ag NPs.
Immobilized Ag NPs showed greater antimicrobial activity, for instance,

Ag NPs embedded with cellulose acetate fibers exhibited enhanced antimi-
crobial activity against both gram negative and gram-positive bacteria (Son
et al., 2004). Further, Ag nanoparticles were incorporated into polysulfone
membranes and used as nanofilters for water purification. The Ag NPs im-
pregnated polysulfone membranes exhibited greater biofouling resistance
(Zodrow et al., 2009). It has also been reported that these membranes pos-
sess excellent antimicrobial activities against E. coli, Pseudomonas, and so
forth (Chou et al., 2005; Lee et al., 2007). Further, Ag NPs immobilized
on amine-functionalized silica surface has been proven to possess enhanced
bactericidal activity (Agnihotri et al., 2013).
5.2.4 NANOFILTERS, NANOMEMBRANES, AND NANOTUBES
Membrane technology plays an instrumental role in wastewater treatment

and desalination of seawater. Membrane filtration (MF) involves the use
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of selective permeable membrane for the separation of dissolved solutes in

aqueous mixture. One of the recent promising approaches in MF is the use
of nanomembranes for filtration processes. NF membranes bear pore sizes
between 0.2 and 4 nm. These pores are smaller than those of microfiltration
and ultrafiltration membranes and are larger than those of membranes used
in reverse osmosis process. Hence these membranes possess characteristics
between those of ultra filtration (Makhluf et al., 2005) and reverse osmosis
9781771883627
(RO) membranes. NF membranes are extensively employed in wastewater
and potable water treatments. They are effective in removing turbidity, in-
organic species such as Ca and Na and microbes. These membranes hold
great promise in reducing water hardness and also in removing dissolved
organic pollutants. NF-RO systems are employed in seawater desalination
which primarily consisting of two steps where NF membranes are used for
filtration prior to RO.
Recently carbon nanotube filters gained a great deal of attention in the
field of water remediation. These filters posses numerous advantages such as
hydrophobicity and high porosity. Despite the salient feature of CNT mem-
branes, which could effectively filter water and retain Na+/Cl ions, they are
capable of desalinating seawater. The efficiency of CNT membrane in the
removal of sodium chloride from seawater has been demonstrated recently
by Srivastava et al. (2004). In addition, these membranes are of great use in
removing bacteria such as Escherichia coli or nanometer sized poliovirus
from water (Srivastava et al., 2004). In another approach, a novel carbona-
ceous nanofilter was synthesized using continuous spray pyrolysis process.
For this, n-hexane was used as a carbon source and ferrocene served as a
catalyst source. It has been demonstrated that at pressures of 811 bar, these
filters efficiently remove viruses from contaminated water (Mostafavi et al.,
2009). The added advantages of carbon nanotube filters are that they are eas-
ily cleaned up either by ultrasonication or autoclaving.
Carbon nanotubes are known to be effective against chemical contami-
nants including heavy metals such as Cr3+, Zn2+ and Pb2+, metalloids such
as arsenic compounds, organics such as polycyclic aromatic organic com-
pounds (PAH), and atrazine (Li et al., 2005; Peng et al., 2005; Di et al.,
2006; Hedderman et al., 2006; Yang et al., 2006; Gotovac et al., 2007; Rao
et al., 2007; Yan et al., 2008). Biological contaminants form a major portion
of potable water contaminants and are mainly comprised of three catego-
ries namely microbes, biological toxins and natural organic matters (NOM).
Carbon nanotubes possess exceptionally high bacterial adsorption capacities
and serve as adsorbent media for eliminating biological contaminants. The
standout features of carbon nanotubes such as high and selective adsorption
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of pathogens make them superior to conventional adsorbents (Upadhyayula

et al., 2009).
The evolution of nanotechnology in the realm of ceramics has introduced
innovative opportunities for the establishment of nanoceramic filters, which
are of great use in removing viruses from blood stream. These filters possess
exceptional chemical and thermal stability. Currently, nanoceramic mem-
branes and filters are commercialized and used mainly for the removal of
9781771883627
virus from blood. However, it only eliminates free viruses present in the
blood stream (Zhao et al., 2008).
5.2.5 NANOCLAYS AND NANOCOMPOSITES
Nanoclays are one of the most affordable natural nanomaterials found in the
clay fraction of soil. Owing to their outspread occurrence in nature, clays
have long been availed as low cost flocculants and sorbents of environmen-
tal contaminants such as suspended particles, disease-bearing organisms,
and other harmful pollutants (Churchman et al., 2006). The suitability of
various nanoclays has been explored for water remediation over the past
years. It has been reported that clays have a myriad of potential applications,
which include the removal of oil and grease, heavy metals, chemicals and
also reclaiming nitrogen from nitrogen-rich effluents (Kemnetz and Cody,
1996; Lo, 1996; Churchman, 2002a, 2002b; Yuan, 2004; Theng et al., 2006;
Srinivasan, 2011).
As clays are layered minerals with space in between the layers, they
readily adsorb water molecules and ions. They possess superior adsorp-
tion capacity, which make them attractive for applications in photocatalysis
process (Chong et al., 2010). Clays also exchange adsorbed ions with their
immediate environment. However, clays are catalytically inactive and lack
the porosity. Hence, various researches have been carried out to manipulate
them. The addition of materials such as metals, polymers with clays result
in the formation of nanocomposites (clay composites). The salient features
of montmorillonite clays such as excellent hydration and swelling capac-
ity, durability, enlarged interlayer space, and immense reactivity make them
excellent targets for manipulation. Modification of montmorillonite through
pillaring with various polyoxy cations of Zr4+, Si4+, Al3+, Fe3+, Ti4+, Ga3+ or
Cr3+ and so forth resulting in enhanced metal adsorption capacity suggesting
its potential in the elimination of heavy metals like Cd, Cu, Mn, Cr, Co, Ni,
Fe, Pb, and Zn (Bhattacharyya and Gupta, 2008).
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Recently, polymer-clay nanocomposites are emerging as promising op-

tions for remediation owing to their enhanced thermal, mechanical, and
barrier performance features resulting from the large contact area between
clay particles and polymer on a nanoscale. These nanocomposites have tre-
mendous potential in removing organic pollutants, anionic pollutants, and
herbicides especially atrazine, a well-known long-lived persistent herbi-
cide (Breen, 1999; Churchman, 2002; Zadaka et al., 2009). For instance,
9781771883627
chitosan-montmorillonite composites efficiently remove anionic pollutants
(An et al., 2007; Li et al., 2009). Its efficiency in adsorbing selenium from
water was exceptional when compared to commercially available adsorbents
(Bleiman and Mishael, 2010). Chitosan-coated montmorillonite clay has
been reported to be more efficacious compared to natural clay in the purge
of tungsten (Gecol et al., 2006). In a batch test performed by Yuan et al.
(2009), montmorillonite-supported magnetite nanoparticles were proven to
be effective in removing hexavalent chromium. Ti-pillared montmorillonite
has tremendous potential in the elimination of arsenite and arsenate from
aqueous mixture (McNally et al., 2010).
Nitrates remain as the major pollutants of groundwater contamination in
many parts. Hydrochloric acid activated montmorillonite exhibited greater
nitrate adsorption competency. Recently, binary clay composites have been
prepared and investigated for the removal of metals and other common wa-
ter pollutants. For instance, the combination of carbon and sodium montmo-
rillonite binary clay demonstrated increased sorption of lead compared to
carbon alone (Ake et al., 2001).
Bentonite is well known for its competency to adsorb and remove tox-
ins, impurities, heavy metals, and chemicals. Bentonite clay bears a strong
negative charge while toxins bear positive charge; hence, the clay particles
adsorb toxins easily resulting in the efficient removal of heavy metals such
as lead and zinc from water (Mishra and Patel, 2009). Addition of iron ox-
ide to bentonite clay has been shown to enhance competency of bentonite
in adsorbing metal ions such as Ni2+, Cu2+, Cd2+, and Zn2+ (Oliveira et al.,
2003). Thermally activated bentonite possesses highest adsorptive capac-
ity for U(VI) from aqueous solutions (Aytas et al., 2009). It has been dem-
onstrated that bentonite-polyacrylamide composite effectively adsorb Cu2+
ions from the ground and surface waters (Zhao et al., 2010). Magnesium in-
corporated bentonite (MB) showed enhanced capacity for fluoride removal
and therefore serve as an potent adsorbent for defluorinating water (Zhao et
al., 2010). Besides magnesium, manganese and lanthanum (La) incorporated
bentonite were also explored for fluoride adsorption and both were showed
to be potent in deflourinating water, however, the efficiency of La-bentonite
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was much higher as compared to Mg-bentonite, Mn-bentonite (Kamble et

al., 2009).
Another type of clay that gained much popularity in the recent years is
allophane nanoclay. Allophane nanoclays are inexpensive, eco-friendly and
most importantly, recoverable after use. It contains greater concentration
of active aluminum, hence competent in adsorbing and precipitating phos-
phates. Hence, this clay was reported to be effective in treating eutrophic
9781771883627
water, meatworks effluent and sewage water (Yuan and Wu, 2007). In batch
experiments carried out by Etci et al. (2010) demonstrated that beidellite, an
inexpensive natural mineral, holds excellent adsorption capacity for lead and
cadmium ions present in aqueous solutions.
Organically modified clays known as organoclays are often applied in
the upstream sector of petrochemical industry for eliminating hydrocarbons
from water produced during refining processes (Pereira et al., 2005). Various
organoclays have also been explored for treating toxic organic chemicals
released by pharmaceuticals and pesticides industries. Carbamazepine
is most widely prescribed anticonvulsant and mood stabilizer. Owing
to its low biodegradability, it is highly persistent in the water systems.
Trimethylphenylammonium (TMPA), and hexadecyltrimethylammonium
(HDTMA) smectites showed greater sorption for Carbamazepine from water
(Zhang et al., 2010). Also, poly(4-vinylpyridine-co-styrene)-montmorillon-
ite has been reported to remediate atrazine from water (Zadaka et al., 2009).
5.3 NANO-BASED TECHNOLOGIES VS CONVENTIONAL

TECHNOLOGIES FOR WATER TREATMENT
Conventional technologies employed for water treatment include filtration,

UV radiation, chemical treatment, and desalination. Even though they have
been extensively used for many decades, they have certain drawbacks too.
For instance, chemical treatment such as chlorination not only destroys
disease-causing microbes, but also gives a possibility of producing toxic
chlorinated fragments. In addition, chlorination gives the undesirable odor
and taste to the treated water. UV radiation is considered as effective means
of disinfection; however, it is inefficient in treating organic contaminants of
water. Even though, filtration techniques such as reverse osmosis (RO) and
ultrafiltration play inevitable part in water treatment, these techniques are
not competent in treating water contaminated with chemicals, drugs, person-
al care products, industrial additives and surfactants, and chemicals. Taken
together, the conventional technologies are not efficient enough to address
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all potential issues of water contamination. Moreover, new microcontami-

nants released into the aquatic environment also cause water contamination
and potential threat to aquatic life.
Nanotechnology has been considered holding tremendous potential in
solving problems related to water contamination. Nanotechnological ap-
proaches hold promises in treating a wide spectrum of existing and emerging
water contaminants. Further, it aids to improve the performance of existing
9781771883627
technologies of water treatment. In recent years, various nanomaterials have
been extensively studied in eliminating various organic, inorganic pollut-
ants from water. Nanomaterials such as nano zero-valent iron, TiO2, silver
nanoparticles, carbon nanotubes, nanomembranes, and nanoclays act as ma-
jor contributors for the development of more efficient water treatment strate-
gies. Figure 5.6 displays various conventional and nano-based technologies
available to treat contaminated water.
Conventional technologies for Nano-based technologies for

water treatment water treatment
Nanocatalyst based
Heat and UV radiation technologies
Eg.nZVI,TiO2
Chemical treatment
1. Coagulation-Flocculation
Magnetic nanoparticles
2. Chemical disinfection Conventional
3. Flocculant-Disinfection and Nano-
based
techniques for
Filtration water Nanoadsorbents
1. Ceramic filters treatment Nanoclays and Nanocomposites
2. Sand filters
Eg. Montmorillonite and Chitosan-
3. Charcoal and activated
coated montmorillonite
carbon filters
Desalination Nanofiltration
1. Reverse Osmosis
Nanomembranes and Nanotubes
2. Distillation
FIGURE 5.6 Conventional and nano-based technologies for water treatment.

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5.4 CONCLUSION AND PERSPECTIVES
Nanotechnology is an innovative emerging technology and its potential envi-

ronmental applications include pollution detection, monitoring, remediation
and control. In this chapter, we presented an overview of recent advances in
the applications of selected nanomaterials in water remediation. Table 5.1
displays a list of nanomaterials used in the remediation of various inorganic,
9781771883627
organic, and microbial contaminants of water. This clearly reveals that nano-
materials have already provided a significant impact on water remediation,
which is expected to exponentially grow during the next years. However,
most of the nanomaterials have been examined only in the lab-scale so far.
Owing to the nanoscale nature and potentially high reactivity resulting from
the large surface to volume ratio of nanomaterials, the potential risks associ-
ated with all new nanosystems need to be evaluated. However, at this point
of time, not much information is perceived about the fate, transport, and
toxicity of nanomaterials in the environment. Hence, it is of immense sig-
nificance to carry out systematic studies to explore the toxicological aspects
of nanomaterials with regard to the environment. Nevertheless, the current
scenario evidently demonstrates that nanotechnology holds a great promise
in the realm of water remediation. Further, the knowledge gained in this field
so far, paves the way to establish novel strategies for applying nanomaterials
for practical and large-scale water remediation.
TABLE 5.1 Summary of Nanomaterials Used in the Remediation of Various Inorganic,

Organic, and Microbial Contaminants of Water.
Nanomaterials Examples Target Pollutants
TiO2 based nanoparticles TiO2 NPs, mesoporous Organic pollutants, phenol, rhoda-
(NPs) and nanopowders TiO2 nanosized powder mine B dye
Iron based nanoparticles Nano zero valent iron chlorinated solvents, polychlori-
nated biphenyls, arsenic, nitrates,
humic acid
Silver nanoparticles Ag NPs Antibacterial activity (against E. coli)
Metal oxide based ZnO NPs, CUO NPs Antimicrobial activity (against E.
nanoparticles coli, S. aureus)
Bimetallic nanoparticles Pd/Au, Fe/Pd, Cu/Fe Chlorinated compounds, lindane,
atrazine, nitrate
Magnetic nanoparticles MnFe2O4, MgFe2O4, Zn- Cr(VI), As(III), As(VI), organic
Fe2O4, CuFe2O4 pollutants
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Nanomaterials Examples Target Pollutants
Nanoclays and Montmorillonite and Nitrates, lead, zinc, Cd, Cu2+, U(VI),
nanocomposites composites, bentonite and phosphates, herbicide such as
composites, allophane, atrazine
organoclays
Nanotubes Carbon nanotubes Heavy metals such as Pb, Cu, Co,
9781771883627
Mn, Zn, lead, fluoride, organic
pollutants
Nanomembranes Nanofiltration membranes Nitrates, natural organic matters
with TiO2 such as fulvic acid and humic acid
Dentrimers Polyamindoamine, diami- Copper, lead, polycyclic aromatic
nobutane poly dendrimers hydrocarbons
KEYWORDS
Antimicrobial activity
Heavy metals
Inorganic ions
Nano zero-valent iron
Nanoclays
Nanomaterials
Nanomembranes
Nanotechnology
Nanotubes
Organic pollutants
Silver nanoparticles
Titanium dioxide
Water remediation
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CHAPTER 6
FUNGAL DEHALOGENATION:
AN OVERVIEW
9781771883627
RAGHUNATH SATPATHY1, VENKATA SAI BADIREENATH
KONKIMALLA2, and JAGNYESWAR RATHA1
1
School of Life Sciences, Sambalpur University, Jyoti Vihar, Burla,
Odisha 768019, India
2
School of Biological Sciences, National Institute of Science Education
and Research (NISER), Bhubaneswar, Odisha 751005, India
CONTENTS
6.1 Introduction......................................................................................120
6.2 Available Enzyme Systems and Bioreactor
Consideration in Fungi.....................................................................121
6.3 Interdisciplinary Approach toward Fungal Dehalogenation............126
6.4 Conclusion and Future Perspectives................................................128
Keywords..................................................................................................129
References.................................................................................................129
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6.1INTRODUCTION
Microbes, such as fungi play a vital role in preserving the balance of global
bio-geochemical cycling of organic components in our environment. The
mechanism includes both synthesis and degradation of organic compounds
in conjunction with their derivatives and intermediates (Madsen, 2011).
Some of the fungi adapt to these organic chemicals in the environment and
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utilize them as a substrate for metabolism whereas some secrete extracel-
lular enzymes to degrade the organic chemicals. This mechanism has been
established by identification and characterization of specialized enzyme sys-
tems and metabolic pathways in different microorganisms of diverse habitats
(Van Der Meer, 1997; Gbel et al., 2004). The genetic architecture of these
microbes is established based on the gene sequence and their divergence pat-
tern has been studied earlier. The study infers that due to presence of gene
clusters, the microbes have developed different metabolic pathways for deg-
radation of varied toxic organo-chemicals (Van Der Meer, 1992; Ding et al.,
2012). This mechanism, by which the toxic organic compound is converted
to non-toxic metabolite by a biological organism is referred to as bioreme-
diation. One amongst the potential bioremediation technique is dehalogena-
tion, where the microbe has the ability to degrade halogenated toxic organic
compounds (Singh et al., 2004). The organo-halide compounds are among
the largest group of environmental chemicals and also important intermedi-
ate substances of the global halogen cycle occurring in nature. Thus, the
dehalogenation phenomena are grabbing attention for its great environmen-
tal significance (Gribble, 2003). Many types of microorganisms like algae,
fungi, bacteria, and archea have been studied previously for the dehalogena-
tion process and currently attempts are made to replace halogenated toxic
chemicals by using the suitable one. Out of about 4000 natural halogenated
compounds, the most common biological origin is generally chlorinated
phenols and phenolic ethers, halogenated terpenes, chlorinated amino acids
and peptides, halogenated alkaloids, bromo- and chloro-substituted pyrroles,
chlorinated insole, halogenated thiophenes, chlorinated prostaglandins, and
varied antibiotics (Hardman, 1991). However, the toxic effect exhibited by
different organo-halide compounds, and their capability for bioaccumulation
in the food chain, food web, and the creation of environmental contamina-
tion is of great concern in the current scenario. Several type of microor-
ganism posses the dehalogenation potential by diverging their metabolism.
Thus, it is all important to study and understand the diverse microbial im-
pact (in molecular level) on the dehalogenation process, which would open
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Fungal Dehalogenation 121
the door to understanding more about the carbonhalogen cleaving process

(Erable et al., 2006).
6.2 AVAILABLE ENZYME SYSTEMS AND BIOREACTOR

CONSIDERATION IN FUNGI
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Until now, little is understood about the role of yeasts and fungi in the deha-
logenation process of the toxic pesticides as compared especially to bacteria.
Basically the white rot fungi grow by hyphal extension through the soil and
having an advantage for gaining better access to some of the pollutant chem-
icals especially halogenated pesticides and herbicides (Atagana, 2004; D
Baldrian, 2008). Being aerobic in nature in case of fungi, the complete deg-
radation of halogenated substance occurs through the Krebs cycle, but the
best-known transformations take place through co-metabolism. However,
different factors like soil type, pH, organic matter, fungal biomass, moisture,
and aeration are also important equally that affect the process. The fungi
induce specific dehalogenating enzymes, which is based on the adaptation to
the halogenated pollutant of interest. The important biochemical reactions in
the fungal degradation of organohalogens are alkylation, dealkylation, am-
ide/ester hydrolysis, dehalogenation, dehydrogenation, hydroxylation, ether
cleavage, ring cleavage, oxidation, reduction, condensation, and conjugate
formation. There are four broad categories of enzymes and their response
mechanism available in fungi as presented in Figure 6.1.
Halogenated compound Biotransformation Experimental evidence for

types mechanism dehalogenation
Chlorinated heterocyclic, Oxidation, Methylation Spectral changes, Organic-X X- ,

Halogenated and glycosylation, % of 14CCO2, Reductive
organophosphorous, Intermediate reduction dehalogenation, Biotransformation
Herbicides, Halogenated Hydroxylation, by oligomerization, Elimination,
Benzenes, Chlorinated Reductive X Benzoquinone, FTIR analysis,
bis (phenyl) ethane dehalogenation Toxicity detection by Microtox test
FIGURE 6.1 Different biotransformation strategy and methods used to detect halogenated
metabolites during fungal dehalogenation.
6.2.1PEROXIDASES
Peroxidases enzymes are equally well known as oxido-reductases, which uti-

lizes the hydrogen peroxide to catalyze corresponding oxidation reactions.
Many numbers of peroxide enzymes have been identied in fungal sources
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and characterized at the molecular level (Ana et al., 2000). The extracellular
peroxidases of white rot fungi include three basic enzymes, such as lignin
peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase
(VP). The general mechanism of the enzyme is given below:
Step 1: Enzyme (LiP, MnP, VP) + H2O2

Intermediate Cationic Radical (Oxidized) + H2O
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Step 2: Intermediate Cationic Radical (Oxidized) + Substrate Oxidized
Intermediate (neutral) + substrate (reduced)
Step 3: Oxidized intermediate (neutral) + Substrate
Enzyme (LiP, MnP, VP) + Oxidized Product
These enzymes have been classified to the Class II fungal heme per-
oxidases and both LiPs and MnPs belong to a family of multiple isozymes
coded by multiple genes (Cameron et al., 2000).
6.2.2LACCASES
Laccase enzymes are widely distributed in fungi, higher plants, bacteria,

and insects. More than 60 fungal strains, belonging to various classes, such
as Ascomycetes, Basidiomycetes, and Deuteromycetes have been demon-
strated to produce laccase (Higson, 1991). Laccase enzyme catalyzes the
substrate by a specialized oxido-reductase activity via a mediator, schemati-
cally explained below (Fig. 6.2a).
FIGURE 6.2 (a) Showing the general mechanism of laccase enzyme action; (b) Catalytic
mechanism of halo acid dehalogenase.
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Laccase activity was also noticed in the cultures of a wide range of fungi,
from ascomycetes to basidiomycetes, and from wood and litter decomposing
fungi to ectomycorrhizal fungi. The white rot fungus Trametes pubescence
MB 89 is a source of the laccase production at the industrial level (Gianfreda
et al., 1999; Muhammad et al., 2012).
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6.2.3 HALO ACID DEHALOGENASE FROM FUNGI
The haloacid dehalogenase are the group of enzymes, which removes the
halide group from haloacid. The general mechanism of this enzyme is repre-
sented in Figure 6.2b. Various works on the characterization and identifica-
tion of bacterial haloacid dehalogenase enzyme have been performed and
from some of their report a possible haloacid dehalogenase activity in fungi
has been proposed (Ridder et al., 1997; Loubna et al., 2005; Papajak et al.,
2006). Evidence of dehalogenation reaction from the experiment is normally
obtained by radioactive labeling procedure of CO2, O2 in the microbial me-
tabolism (Liu et al., 1995). As per Expasy data base records (www.expasy.
org), currently there are about 228 numbers of predicted sequences of halo
acid dehalogenase from different fungal sources.
6.2.4 P450 SYSTEM IN FUNGAL DEHALOGENATION
The cytochrome P450 consists of a large and diverse group of enzymes

belongs to the super family of monooxygenases. These enzymes catalyze
diverse reactions the organic substances include oxidation of primary and
secondary metabolites also in xenobiotic detoxification (Chigu et al., 2010;
Nazir et al., 2011). The enzymatic properties and substrate specificity are
influenced by their redox partners. Filamentous fungi contain many numbers
of cytochrome P450 often possess cytochrome P450 reductases as redox
partners (Lah et al., 2011). Many studies have been done to distinguish the
potential function of cytochrome P450-catalyzed dehalogenation of organo-
halide compounds. Recently the dehalogenation process of Polaromonas
sp. strain JS666 has been conformed on the substrate cis-1,2-dichoroethene
(cDCE) and in the mechanism for cDCE degradation involvement of P450
monooxygenase has been established (Luke et al., 2001). The general mecha-
nism of fungal cytochrome 450 enzyme system includes epoxidation of C=C
double bonds followed by hydroxylation of the halo organic compounds.
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Many toxic halogenated compounds like perchlorobenzenes and perfluoro-

benzenes are converted to their non-toxic phenols and keto-forms.
Epoxidation Hydroxylation
R-X R-O-OX R-O-H
(Halogenated toxic substance) (Epoxide derivatives) (Hydroxylated non-toxic derivatives)
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Much considerable success has been accomplished by the applying
the fungal cells (cultures) in several ways, such as immobilization method
and utilizing them in different bioreactors. Different types of fungal biore-
actors are established for the treatment of various halogenated pollutants
(Table6.1). The potential of certain fungi in the eld of bioremediation can
be raised by a number of elements, such as media composition, static and
agitated culture condition, pH and temperature, C and N sources and salt
concentration, initial concentration of halogenated pollutant, and so on.
TABLE 6.1 Showing Some Common Type of Bioreactor Systems Involving Fungal Species
for Dehalogenation Purpose.
Bioreactor Types Fungal Organism Utility References
Upow column Penicillium Treatment of paper and Taeli et al. (2004)
bioreactor camemberti pulp effluent
Fixed lm bioreactor P. chrysosporium Chlorinated low-molec- Paszczynski et al.
ular-weight phenols (1985)
(Immobilized) Cell Trametes AOX reduction of pulp Pallerla and Chambers
entrapment versicolor and paper wastes (1995)
(Immobilized) Coriolus Monoaromatic chloro- Roy-Arcand and
Adsorption versicolor phenolics removal Archibald (1991)
Two-step sequential Paecilomyces sp. AOX reduction Singh et al. (2005)
bioreactor
Static flask culture P. chrysosporium Pentachlorophenol Udayasooriyan et al.
dehalogenation (2007)
Unlike fungi, bacteria are often unable to degrade substituted molecules

due to low water solubility and availability. Fungi possess a wide range of
enzyme systems and most of them being extracellular and non-specific,
hence capable for degradation of a large group of chemicals. Fungi are cos-
mopolitan in nature and they are capable of degrading a list of commonly
used halo-organic pollutants individually or in mixtures (Table 6.2).
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TABLE 6.2 Example of Fungal Sources that Degrade Halogenated Pollutants with Proposed
Mechanism.
Fungal Sources Act on Halogenated Proposed References
Substances Mechanism
Fusarium solani AM203, Halolacetones Hydrolytic Johnson (2009)
Botrytis cinerea AM235, dehalogenation
Beauveria bassiana AM278
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Phanerochaete chrysospo- Pentachlorophenol Oxidation-reduction Liu and Zhao
rium, Trametes versicolor, (2004), Krupp
Inonotus dryophilus et al. (2006)
Pycnoporus cinnabarinus 2-hydroxy-5-chloro- Laccase mediated Bansal (2005)
biphenyl dehalogenation
Candida maltose Monochlorophenol Cycloisomerization Moore et al.
of the cis, cis chlo- (2010)
romuconic acid
Pleurotus ostreatus, Polychlorinated Laccase mediated Zhang et al.
Trametes versicolor biphenyls dehalogenation (2006)
Mucor sp., Trichoderma sp. 3-chloropropionic Haloacid Barr and Aust
acid (3CP) dehalogenation (1994)
Trametes elegans, Phlebia Pulp and textile in- Partial Grabarczyk
radiata, Panus crinitus, dustry waste waters demethylation (2012)
Trametes villosa
Phanerochaete sordid, Industrial waste wa- Laccase and lignin Mileski et al.
Trametes versicolor, ter with halogenated peroxidases (1988)
Bjerkandera sp. BOS55 substances
Mucor plumbeus pentachlorophenol Oxidation followed Alleman et al.
(PCP) by dehalogenation (1995)
Pleurotus pulmonarious, Atrazine Hydroxylation Schultz et al.
Phanerochaete (2001)
chrysosporium
Phelbia tramellosa Alachlor Oxidation Elke et al.
(1992)
Dichomitus squalens 2,4-dichlorophen- Mn2+ mediated Zeddel et al.
oxyacetic acid hydration (1993)
Stachybotrys sp. strain 2,6-dichloroaniline Laccase and lignin Sepideh et al.
DABAC 3 peroxidises activity (2013)
Penicillium camemberti Lindane - Peralta-Zamora
et al. (1999)
Penicillium frequentans 3,4-dichlorophenol, Oxidative Ed de and Jim
Strain Bi 7/2 2,4-dichlorophenol dehalogenation (1997)
Trametes versicolor Perchloroethylene Cytochrome P-450 Carvalho et al.
(PCE) system (2011)
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Fungal Sources Act on Halogenated Proposed References
Substances Mechanism
Phanerochaete TCE Lignin peroxidase Mougin et al.
chrysosporium enzyme (1997)
Trichoderma sp. Gc1 and DDD - Ferrey et al.
Penicillium miczynskii Gc5 (1994)
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Caldariomyces fumago 4-Fluorophenol Chloroperoxidases Reddy et al.
enzyme system (1997)
Phlebia brevispora Coplanar polychlo- - Annibale et al.
TMIC33929 rinated biphenyls (2006)
(Co-PCBs)
6.3 INTERDISCIPLINARY APPROACH TOWARD FUNGAL

DEHALOGENATION
Along with traditional molecular level analysis, various modern and novel
methodologies have been implemented in the recent studies on the micro-
bial dehalogenation mechanisms and the same can be utilized for in-depth
study about fungi. Starting from establishment of fungal culture to charac-
terize it as an dehalogenating agent have provided key knowledge for un-
derstanding the catalytic mechanism and engineering of bio-dehalogenation
process (Fig. 6.3). Especially, the structural study of the enzymes provides
the path to trace out the specificities and activities involved in this type
of bio-catalytic mechanism (Hlavica, 2013). There are several aspects, in
which the study about dehalogenation couples the approaches including
Sourceofhalogenatedsub
bstances
Selectionofsuitablefungalstrainfromthepollutionsite CollectionandisolationoftotalDNAfromthenaturalpollutionSite
Optimization of the culture condition

Optimizationoftheculturecondition 18S rRNA geneamplification
18SrRNA gene amplification
Supplyofsuitablelabelledpollutantstotheculture Phylogeneticanalysisandidentificationoffungalgenes
PrimaryscreeningforbiotransformationbyTLC/GC rDNA preparation,cloning,expressionandscreeningfor

biodegradation
Identification of all products from 1HNMRand

Identificationofallproductsfrom H NMR and 13CNMR
C NMR
FIGURE 6.3 Approaches from identification to characterization of a known (left) or

unknown (right) fungal species as an acceptable dehalogenating microbe.
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metagenomics, which is a culture-independent technique for analysis of the

genetic and metabolic potential of natural and model microbial communities
that degrade organic pollutants (Nishino et al., 2013). In addition to this,
the proteomics has also having significant contribution to understand the
individual organisms at the molecular level thereby finding various protein
functions with respect to the responses (Alcalde et al., 2006).
During the last few years, the field of dehalogenation has been seen in a
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rapid progress in the identification of novel fungi as well as gene, sequencing
of whole genomes, and the genetic adaptability potential. Hence, combined
effort of microbiology, biotechnology, and bioinformatics is required to ana-
lyze the fungal dehalogenation systems. Although a large number of fungi as
well as their enzymes have been isolated, identified, and studied, however,
there is a little knowledge available about the genes encoding these enzymes.
From the advance sequencing platforms, there is a substantial amount of se-
quence information is available in the public database, that include the gene
sequences, EST, whole genomes, proteome, metagenome, and so on. This
mass of data provides a bigger opportunity in bioinformatics to annotate
and evaluate the novel information associated with them (Peralta-Zamora
et al., 1999; Pritz et al., 2013). The development of a large number of da-
tabases, software tools to analyze data in the past few years has facilitated
the researcher for a wide range of applications to understand the microbial
dehalogenation process (Fig. 6.4). Hence the interdisciplinary methodology
is truly essential to determine the metabolic potential as well as the diversity
in fungal systems to understand better the complex molecular regulation and
control in them (Field, 2003; Seifert, 2009; Carvalho et al., 2011).
Whole genome sequencing

PCR, 2D gel electrophoresis,
Phylogenetic analysis Vs 3D structure modelling and
sequencer, assembly of small gene
Microbial evolution fragments, prediction of promoters
docking
Gene duplications, gene insertion, XRAY crystallography, NMR,
gene deletion, gene-fusion events, Theoretical modelling, docking
horizontal gene transfer and domain (protein-ligand and protein-
level restructuring protein)
Methods in microbial
dehalogenation
Reconstruction of metabolic
pathways
Identification of gene and protein Global network of reactions catalyzed
ORF prediction, gene prediction by QSBR analysis for dehalogenation by enzymes, network of gene-groups
statistical or artificial intelligence methods, Theoretical biodegradation analysis, connected through the reactions,
pairwise and multiple sequence analysis, Identification of mechanism, and catalyzed by enzymes embedded in the
protein domain and motif prediction degradation feasibility gene-groups, global modelling of
, chemical reactions in the microbial cells
FIGURE 6.4 Various interdisciplinary strategies applied in molecular data to explore

dehalogenation process.
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Biological treatment strategies for the bioremediation of organo-halide pol-

lutants are widely utilized as it is eco-friendly and also linked with low-cost
treatment methods. Therefore, several researchers are curious about the de-
velopment of suitable fungal technology for the biodegradation of haloge-
nated pollutants. Filamentous fungi, including zygomycetes, ascomycetes,
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and basidiomycetes (especially white rot fungi) have been shown to be po-
tentially useful to degrade recalcitrant xenobiotics (Kordon et al., 2010). In
the bioremediation process like dehalogenation, the bacterial consortia are
mostly come into focus and fungi are much less studied. In actual there is a
greater potential of dehalogenation mechanism exists in fungi in comparison
to bacteria, due to their rapid growth, capable of more biomass production
and aggressive hyphal growth in soil. Hence, more research should be fo-
cused on fungal flora for dehalogenation purpose.
The preliminary and advanced knowledge about established molecular
structures of a particular enzyme and its products are essential to study the
carbonhalogen bond making and bond breaking catalyzing process. To date
there is no three-dimensional (3D) experimental structure available for halo
acid dehalogenase enzyme from fungal source in protein data bank (PDB,
www.rcsb.org/pdb). Hence conducting research in elucidating of structure
of novel proteins and understanding their function and mechanism would be
of prime importance.
Genetic engineering technology is another approach normally practiced
to produce recombinant genes coding for suitable enzymes with a strategy of
their large-scale production. This strategy could be accustomed to produce
dehalogenating enzymes from fungi to study the synthesis and degradation
kinetics of the enzymes (Pieper et al., 2004).
Identification of new fungal strain from microbial community, discovery
of specific genetic and metabolic pathways followed by protein and meta-
bolic engineering approach will generate enzymes with altered biological
properties for biodegradation of harmful halogenic substances (Schneider et
al., 2010; Yin et al., 2011).
Halogenated products are used in the synthesis of many pesticides and
pharmaceuticals. These substances also create a risk to the health of the living
organism; can be degradable by a process called dehalogenation. The fungi
are considered as a suitable organism for degradation of halogenated organic
substances. Many new fungal strains with efficient potential are isolated and
their metabolic pathways in the process have been elucidated. Starting from
the discovery, development of the novel environmental biocatalysis attracts
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several scientists to pursue research on this. Hence, a thorough interdisci-

plinary analysis about the genetic, enzyme system, and the mechanism of
induction of the enzymes would make fungi as a great chunk of resource
material for the future research and industry.
KEYWORDS
9781771883627
Bioinformatics
Bioreactor
Bioremediation
Dehalogenase
Dehalogenation
Enzyme system
Fungal source
Halogenated substances
Metabolism
Pollutants
Xenobiotics
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CHAPTER 7
INSIGHT OF BIOFUEL PROSPECTS

FROM MICROALGAE AS
9781771883627
RENEWABLE ENERGY SOURCE FOR
ENVIRONMENTAL SUSTAINABILITY
GANAPATHI SIBI
Department of Biotechnology, Centre for Research and Post Graduate
Studies, Indian Academy Degree College, Bangalore 560043,
Karnataka, India
CONTENTS
7.1 Introduction......................................................................................134
7.2 Open and Closed Culture Systems...................................................134
7.3 Batch and Continuous Culture.........................................................135
7.4 Cultivation Strategies.......................................................................136
7.5 Selection of Microalgal Species......................................................137
7.6 Environmental Parameters Affecting Lipid Production...................138
7.7 Alternate Substrates for Algal Cultivation.......................................142
7.8 Extraction Methods..........................................................................143
7.9 Conclusion.......................................................................................144
Keywords..................................................................................................144
References.................................................................................................145
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7.1INTRODUCTION
Around 70% of the total global energy requirement is represented by fu-

els, and finding sufficient supplies of energy for the future is one of the
most daunting challenges. Fluctuating oil prices, increasing gaseous emis-
sions and their effect on green house, climatic change, and global warming
stresses the urgent need to find new feedstocks for fuels. Biofuels promote
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environmental sustainability in terms of non-toxic, clean energy with a con-
sequent decrease in greenhouse effect. Microalgae offer great promise to
contribute a significant portion of the renewable fuels due to their higher
photosynthetic activity, biomass productivity, CO2 fixation, O2 production,
faster growth rate than higher plants, and ability to grow in non-arable land
unsuitable for agricultural purposes. The properties of biodiesel from mi-
croalgal oil in terms of density, viscosity, acid value, and heating value are
comparable to those of fossil fuels. However, prior to industrial scale ap-
plication, a series of key challenges have to be resolved. The capital costs to
transform microalgal biomass into biofuel are high and face a broad range
of grand challenges to become technologically and economically viable. A
systematic approach and process integration are critical factors in a success-
ful future for algal bio-refineries. This chapter presents views and opinions
on key technical challenges associated with microalgal culture system, cul-
tivation conditions, growth medium, strain selection for highest growth rate,
increased biomass productivity integrated with CO2capture, lipid accumula-
tion with adequate composition, and extraction methods.
7.2 OPEN AND CLOSED CULTURE SYSTEMS
The selection of culture system for microalgal biofuel production can be

in either open or closed systems. Under open system, microalgae are mass
cultured in artificial circular, open ponds, and cascades from long time
(Richmond, 1986; Becker, 1994). Open ponds can be further categorized
as raceway, circular, inclined, and unmixed ponds. Raceway ponds are the
most applicable for both the pilot-study level and commercial scale because
of their easy and higher productivity (Borowitzka, 2005; Putt et al., 2011).
Circular ponds and inclined ponds are capable of achieving algal growth
rates as high as 21 g m2 d1 (Benemann and Oswa, 1993) and 31 g m2 d1
(Doucha et al., 2006), respectively. Unmixed open algal ponds are generally
avoided due to the low productivities and suitable for only selected algal
species.
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Microalgal Biofuels for Environmental Sustainability 135
Higher biomass production and lesser energy requirement are the advan-
tages of open system (Hase et al., 2000; Jorquera et al., 2010). At the same
time, open systems require large area of land and are continuously threatened
by invading species, such as undesired algae and bacteria. Monocultures of
algae under open culture system are achieved by maintaining an extreme
culture environment, such as high pH, salinity, and nutritional status (Lee,
1986). However, such approaches do not necessarily exclude bacteria, zoo-
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plankton, and other biological contaminants. Another disadvantage of open
system is that local weather conditions influence the cultivation conditions,
which is further hardly controlled and makes the production seasonal (Perez-
Garcia et al., 2011). Further, lower atmospheric CO2 concentration could
slow down the cell growth of microalgae, as there is a poor mass transfer
limitation in open systems. Due to these constraints, it is difficult to maintain
monocultures of desired algae in open culture systems (Lee, 2001), which
have led to the development of enclosed tubular (Gudin and Chaumont,
1983; Pirt et al., 1983; Robinson, 1987; Tredici and Materassi, 1992; Lee et
al., 1995; Borowitzka, 1999) and flat bed (Tredici et al., 1991; Pulz, 1994;
Hu et al., 1996) photo bioreactors.
The design of a photobioreactor is more complicated compared to an
open pond and one should consider the fundamental principles, such as light
regime, gas liquid mass transfer, nutrient supply, and oxygen removal sys-
tem. Irradiance supply influences the cell growth, CO2 fixation, and bio-
chemical composition of microalgae (Chrismadha and Borowitzka, 1994).
Reducing the light path increases the light available to each cell and increas-
es growth rate. Poor mixing of gas liquid mass transfer leads to increased
O2 concentration and CO2 stripping in the photobioreactor, which inhibits
the growth of the microalgae (Hoekema et al., 2002). However, cultivation
in closed systems is costlier compared with open ponds, which include light
illumination, CO2 feed, cultivation medium, and circulator system. In return,
microalgal productivity in a photobioreactor is higher and has less contami-
nation (Wu et al., 2005).
7.3 BATCH AND CONTINUOUS CULTURE
Batch culture of microalgae is defined as a culture period, where the cul-

tivated microalgal cells are harvested at once and continuous culture is
conducted based on the dilution rate. Growth rates and biomass can be regu-
lated and maintained for extended time periods by varying the dilution rate.
Higher microalgal production can be achieved in the continuous culture
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mode compared with batch culture. McGinn et al. (2012) found that the
biomass productivity of Scenedesmus sp. was two times greater when grow-
ing in a continuous chemostat than in batch cultivation. Similar findings
were obtained by Wen et al. (2014) by cultivating Chlorella pyrenoidosa
in the continuous culture under varying nitrate conditions. Further, maxi-
mum lipid productivity of 144.93 mg L1 d1 when compared to 96.28 mg
L1 d1 in batch culture during the study. Under semi-continuous cultivation
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with nitrogen limitation and pH regulation, 3.64-fold higher lipid produc-
tivity was obtained in C. pyrenoidosa (Han et al., 2013). In another study,
excess light combined with a growth-limiting nitrogen supply resulted in
up to 12.4% w/w triacyl glycerol (TAG) accumulation in turbidostat cul-
tures of Neochloris oleoabundans (Klok et al., 2013). In contrast, Tang et
al. (2012) reported that varying dilution rates has increased biomass produc-
tivity of Chlorella minutissima and Dunaliella tertiolecta but had no effect
on lipid productivity. Sobczuk and Chisti (2010) found that lipid content in
Choricystis minor did not change significantly with various dilution rates in
chemostat culture.
7.4 CULTIVATION STRATEGIES
Under optimal growth conditions, microalgae synthesize fatty acids for

esterification into glycerol based membrane lipids, which make up about
520% of their dry cell weight. Under stress conditions, algae have been
found to produce significantly higher concentrations of neutral lipids. The
ability of microalgae to adapt their metabolism to varying cultural condi-
tions provides opportunities to modify or control the formation of targeted
compounds. Various systems are used for production of microalgae, which
can be autotrophic, mixotrophic, and heterotrophic. Heterotrophic cultiva-
tion has been known for decades, as it is regarded as the most practical and
promising way to promote productivity of biomass and high levels of lipids
(Grima et al., 2003; Olaizola, 2003; Miao and Wu, 2006). Under autotrophic
and heterotrophic cultivation, light penetration is inversely proportional to
the cell concentration and algal biomass hence, light limitation is the ma-
jor factor during microalgal cultivation (Chen, 1996). Another method of
microalgal cultivation for higher biomass and lipid production is two-stage
cultivation. In this strategy, microalgal cells first grow rapidly under growth-
optimized conditions, and then are transferred to conditions where light irra-
diance (Zhang et al., 2009), nutrition (Su et al., 2011), culture pH (Han et al.,
2013), as well as other factors (Liu et al., 2008; Das et al., 2011) are adjusted
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to promote lipid accumulation at the expense of cell growth. It is possible to

increase both biomass and lipid production in microalgae by choosing op-
timum cultural conditions and nutrient composition of the growth medium
(Sibi, 2015a).
7.5 SELECTION OF MICROALGAL SPECIES
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Microalgae are good option for biodiesel production than terrestrial crops.
However, selection of the most adequate species needs to take into account
for productivity and economic viability. Factors, such as the ability of mi-
croalgae to develop using the nutrients available or under specific environ-
mental conditions, fatty acid composition of the different microalgae species
should be considered simultaneously in the selection of the most adequate
species or strains for biodiesel production. Chlorella, Crypthecodinium,
Cylindrotheca, Dunaliella, Isochrysis, Nannochloris, Nannochloropsis,
Neochloris, Nitzschia, Phaeodactylum, Porphyridium, Scenedesmus,
Schizochytriu, and Tetraselmis have oil levels between 20 and 50% and
should be considered for biodiesel production (Table 7.1).
TABLE 7.1 Lipid Content of Microalgal Species.

Microalgal Species Lipid Content Lipid Productivity
(% dry weight biomass) (mg L1 day1)
Ankistrodesmus sp. 24.031.0
Botryococcus braunii 25.075.0
Chaetoceros muelleri 33.6 21.8
Chaetoceros calcitrans 14.616.4/39.8 17.6
Chlorella emersonii 25.063.0 10.350.0
Chlorella minutissima 57
Chlorella protothecoides 14.657.8 1214
Chlorella sorokiniana 19.022.0 44.7
Chlorella vulgaris 1440/56 11.240.0
Chlorella pyrenoidosa 2.0
Chlorococcum sp. 19.3 53.7
Crypthecodinium cohnii 20.051.1
Dunaliella salina 6.025.0 116.0
Dunaliella primolecta 23.1
Dunaliella tertiolecta 16.771.0
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Microalgal Species Lipid Content Lipid Productivity

(% dry weight biomass) (mg L1 day1)
Dunaliella bioculata 8.0
Dunaliella salina 1420 33.5
Ellipsoidion sp. 27.4 47.3
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Euglena gracilis 14.020.0
Haematococcus pluvialis 25.0
Isochrysis galbana 7.040.0
Monodus subterraneus 16.0 30.4
Monallanthus salina 20.022.0
Nannochloropsis oculata 22.729.7 84.0142.0
Nannochloropsis sp. 12.053.0 37.690.0
Neochloris oleoabundans 29.065.0 90.0134.0
Nitzschia sp. 16.047.0
Oocystis pusilla 10.5
Pavlova salina 30.9 49.4
Pavlova lutheri 35.5 40.2
Phaeodactylum tricornutum 18.057.0 44.8
Porphyridium cruentum 9.018.8/60.7 34.8
Scenedesmus dimorphus 640
Scenedesmus obliquus 11.055.0
Scenedesmus quadricauda 1.918.4 35.1
Skeletonema costatum 13.551.3 17.4
Spirulina platensis 4.016.6
Spirulina maxima 4.09.0
Thalassiosira pseudonana 20.6 17.4
Tetraselmis suecica 8.523.0 27.036.4
Adapted from Becker (1994), Illman et al. (2000), Miao and Wu (2006), Spolaore et al.
(2006), Liu et al. (2008), Natrah et al. (2008), Xiong et al. (2008), and Mata et al. (2010).
7.6 ENVIRONMENTAL PARAMETERS AFFECTING LIPID

PRODUCTION
Different nutritional and environmental factors, cultivation conditions, and

growth phases may affect the fatty acid composition. Lipids act as a second-
ary metabolite in microalgae, maintaining specific membrane functions, and
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cell signaling pathways while responding to the environment changes. The

quantity and quality of oils produced by algal cells are directly proportional
to the stimulus received from the surroundings. Stressful environmental con-
ditions change the use of carbon uptake by algae for proliferation to energy
storage in the form of oil. The lipid content, composition, and the propor-
tions of various fatty acids of microalgae vary according to the environmen-
tal or culturing variables, such as light intensity, growth phase, photoperiod,
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temperature, salinity, CO2 concentration, nitrogen, and phosphorous concen-
tration. Further, both the quantity and quality of lipids produced will vary
with the identity of the algal species.
7.6.1 NUTRIENT CONTENT
The growth and lipid accumulation of microalgae are affected by nutrition

concentration of the growth medium. Cellular lipid levels of microalgae will
increase under nutrient stress with triacyl glycerols as the dominant propor-
tions (Illman et al., 2000). Under nitrogen limitation or starvation conditions,
excess carbon from photosynthesis is channeled into storage molecules,
such as triglyceride or starch (Scott et al., 2010). Further transferring mi-
croalgal cells from normal nutrient to nitrogen-depleted media will gradu-
ally change the lipid composition from free fatty acid-rich lipid to mostly
triglyceride-containing lipid (Takagi et al., 2000). The deprivation of nitro-
gen enhances the lipid production in microalgae (Chen et al., 2008; Li et al.,
2008; Li et al., 2013) and produces more favorable triacyl glycerols by in-
ducing changes in fatty acid chain length and saturation for biofuel conver-
sion. Phosphate limitation caused significant changes in the fatty acid and
lipid composition ofMonodus subterraneus (Khozin-Goldberg and Cohen,
2006). However, nitrogen deficiency was more effective than phosphate de-
ficiency in C. zofingiensis (Feng et al., 2012). Significant increase in lipid
content of Desmodesmus sp., Nannochloropsis oculata, C. minutissima and
Botryococcus spp. were observed under nitrogen starvation conditions (Liu
et al., 2008; Yeesang and Cheirsilp, 2011; Cao et al., 2014; Surendhiran and
Vijay, 2014; Rios et al., 2015).
7.6.2 CARBON DIOXIDE
Microalgae are capable of taking zero-energy form of carbon and synthesiz-

ing it into a high-density liquid form of energy and are capable of storing
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carbon in the form of natural oils or as a polymer of carbohydrates. CO2 is

known to influence the lipid content of algae and alterations in the com-
position of the fatty acids are dependent on the CO2 concentration during
the algal growth. CO2 concentration in the range of 1040 ml min1 was
reported to increase the lipid productivity of microalgae (Chiu et al., 2009;
Ho et al., 2010; Lv et al., 2010; Ortiz Montoya et al., 2014). However, under
high concentrations of CO2, the algal growth was affected as unutilized CO2
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will be converted to H2CO3 thereby reducing the pH of the medium. Hence,
optimum CO2 levels are required to obtain maximum biomass and enhanced
lipid production using microalgae.
7.6.3TEMPERATURE
Temperature is known to influence the lipid accumulation in microalgal

cells. Many microalgae have displayed increasing growth and lipid con-
tent with increasing temperature. At low temperature, microalgae synthe-
size higher ratio of saturated fatty acids and increased temperature resulted
in decreased neutral lipid and polyunsaturated fatty acids. Total lipid con-
tent was increased at lower temperature in fresh water and marine micro-
algae (Hoffmann et al., 2010; Bohnenberger and Crossetti, 2014). At the
same time, increasing temperature has resulted in higher lipid production
in Nannochloropsis oculata and Ettlia oleoabundans (Converti et al., 2009;
Yang et al., 2013; Subhash et al., 2014).
7.6.4SALINITY
Salinity influences physiological and biochemical mechanisms of microal-

gae and can lead to increment in the lipid content due to changes in the fatty
acid metabolism. Restoration of turgor pressure, regulation of the uptake
and export of ions through the cell membrane, and accumulation of osmo-
protecting solutes and stress proteins get activated when cells are exposed
to salinity. This leads to stress generation inside the algal cells causing in-
creased total lipid accumulation, which act as a reserve energy material until
favorable conditions arise (Alkayal et al., 2011; Talebi et al., 2013). Further,
increase in unsaturated fatty acids proportion was observed under salt stress
on the other hand higher levels of saturated fatty acids has also been reported
under high salt conditions (Kan et al., 2012). Another advantage of cultivat-
ing microalgae under high alkaline salt conditions is to limit contaminants
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and competing microorganisms. Salinity acts as growth-limiting factor with-

in natural biotic communities in open pond systems by inhibiting invasive
organisms.
Higher lipid contents were observed in Scenedesmus species that were
subjected to salt stress (Walsby, 1982; Kirrolia et al., 2011; Kaewkannetra et
al., 2012). Duan et al. (2012) have reported a 21.1% increase of lipid yield
in C. vulgaris under salt induced osmotic stress. Increased salt concentra-
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tions resulted in a higher intracellular lipid content to 70% in Dunaliella
cells (Takagi et al., 2006). Salinity stress triggered both biomass growth and
lipid synthesis in microalgae significantly with total and neutral lipid content
of 23.4 and 9.2% along with higher amounts of saturated fatty acid methyl
esters (Mohan and Devi, 2014). However, higher salt conditions inhibited
the cell growth at the same time. Rao et al. (2007) reported increase in the
relative proportions of palmitic acid and oleic acid in Botryococcus braunii
at different levels of salinity. Similar results were obtained by Ruangsombon
(2012) along with varying light and nutrient conditions. Lipid accumulation
of Nannochloropsis salina under salinity stress has significantly increased
at a concentration of 36%. Elevated salinity conditions leads to increased
non-polar lipid content and decrease in membrane lipid content (Bartley et
al., 2013).
7.6.5 METAL IONS
Metal ions influence the algal biomass and lipid production. Heavy metals
like cadmium, copper, and zinc are known to increase the total lipid content
of Euglena gracilis (Einicker-Lamas et al., 2002). The totallipidcontent
andlipidproductivity of Scenedesmus increased 28.2% and 29.7% in the
presence of iron, magnesium, and calcium with the addition of EDTA during
cultivation (Ren et al., 2014). Liu et al. (2008) reported the effect of iron on
C. vulgaris and the total lipid content was raised up to 56.6%. Lipid accumu-
lation in C. protothecoides by copper stressed lipid biosynthesis was studied
by Li et al. (2013) where optimized biomass andlipidyield were achieved
by 6.47 g L1 and 5.78 g L1. Copper stress has influenced the lipid produc-
tion in Chlorella at qualitative and quantitative manner. Higher concentra-
tions of fatty acids were observed in C. vulgaris, C. protothecoides, and C.
pyrenoidosa at copper levels of 4 mg L1 (Sibi et al., 2014). The effect of
hexavalent chromium on fatty acid composition and lipid peroxidation was
studied in Euglena gracilis by Rocchetta et al. (2006).
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7.6.6 OXIDATIVE STRESS
Environmental stresses trigger the excessive formation and accumulation

of intracellular reactive oxygen species (ROS) in algae, which cause dam-
age through the oxidation of cellular components. However, algal cells are
able to mediate anti-oxidative defense and under oxidative stress, the lip-
id profile of many microalgae is reported to be altered. Kang et al. (2014)
9781771883627
have used oxidative stress to induce lipid production in Chlorella vulgaris.
Yilanciooglu et al. (2014) have determined the oxidative stress-mediated
increased cellular lipid content up to 44% by application of exogenous H2O2.
Nitrogen depletion results in the co-occurrence of ROS and lipid accumu-
lation in diatoms (Liu et al., 2012). Association of increased ROS levels
and cellular lipid accumulation under different environmental stress con-
ditions was also shown in green microalgae. Osundeko et al. (2013) have
reported that lipid content of Chlorella luteoviridis and Parachlorella hussii
were increased under oxidative stress, which could be used for biofuel feed
stock production. However, a mechanistic understanding of the connection
between oxidative stress and increased algal lipid accumulation requires fur-
ther investigation (Hong et al., 2008).
7.7 ALTERNATE SUBSTRATES FOR ALGAL CULTIVATION
Growth medium provides necessary nutrient sources for algal growth and
under heterotrophic conditions, the cost of growth medium is high, therefore
economic considerations demand much cheaper and easily available resourc-
es. Cellulosic biomasses are abundant renewable resources for the produc-
tion of biofuel. A large amount of valuable compounds are present in crop
residues after harvesting, which could be used as nutrient sources. Utilizing
lignocellulosic biomass offers the possibility of renewable source of carbon
and nitrogen that can be used to cultivated microalgae. Heterotrophic micro-
algae are capable of converting organic carbon sources to intracellular oil
that could be used to produce biodiesel efficiently. Organic carbon sourc-
es like starch hydrolysates from Jerusalem artichoke (Cheng et al., 2009),
sweet sorghum (Gao et al., 2010), cassava (Lu et al., 2010), waste molasses
(Yan et al., 2011), rice straw (Li et al., 2011), wheat bran (El-Sheekh et al.,
2012), sweet sorghum, and rice straw (Sibi, 2015b) were utilized to cultivate
microalgae as cost-effective approach to displace glucose.
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7.8 EXTRACTION METHODS
Lipids due to their high energy density are a very attractive feedstock for
biofuel production. The biodiesel product ion process from microalgae con-
sists of two major steps. First step includes biomass cultivation, harvesting,
drying, and lipid extraction. This is followed by the conversion of extracted
lipids to biodiesel and final purification. One of the main challenges in the
9781771883627
biodiesel from microalgae is extraction of lipids from the harvested bio-
mass. Lipid extraction continues to be a significant challenge toward the
commercial production of microalgal oil, even though a multitude of ex-
traction methods have been followed. The method of Folch et al. (1957)
that uses chloroform and methanol (2:1) was optimized for isolation and
purification of lipids from animal tissues. The method of Bligh and Dyer
(1959) that uses chloroform and methanol (1:2) followed by chloroform ex-
traction was originally optimized for extraction of phospholipids from fish
muscles. It should be noted that microalgae contain unusual lipid classes and
fatty acids differing from higher animal and plant organisms (Guschina and
Harwood, 2006). Further, following different lipid extraction methods can
result in widely varying estimations. Hence, careful choice and validation
of analytical methodology in microalgal lipid extraction is needed. The ef-
ficient extraction of lipids is highly dependent on the polarity of the organic
solvents used (Hamilton et al., 1992; Lewis et al., 2000). In general, solvent
mixtures containing a polar and a non-polar solvent could extract a greater
amount of lipids.
Various solvent systems were performed for extraction of lipids from
microalgae. Li et al. (2014) have compared the extraction methods and
found supercritical CO2 technique for lipid extraction in Tetraselmis sp.
Ryckebosch et al. (2012) found chloroform-methanol (1:1) was best solvent
mixture for total lipid extraction from lyophilized microalgae. Lipid extrac-
tion from Botryococcus braunii was carried out using chloroform-methanol
(2:1), hexane-isopropanol (3:2), dichloroethane-ethanol (1:1), and acetone-
dichloromethane (1:1) by Lee et al. (1998) and chloroform-methanol (2:1)
has produced highest lipid content. Grima et al. (1994) have used seven sol-
vent mixtures to extract lipids from Isochrysis galbana and obtained 93.8%
of lipid using chloroform-methanol-water (1:2:0.8).
Chloroform known for its carcinogenicity and its decomposition yields
phosgene and hydrochloric acid inflicts chemical modification of lipids
(Schmid et al., 1973). Matyash et al. (2008) have used methyl tert-butyl ether
(MTBE) extraction to avoid carcinogenic chloroform for lipid recovery.
Hexane can be considered for lipid extraction considering its low toxicity
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and removal of non-polar lipids from crude lipids efficiency (Prommuak

et al., 2012). Chen et al. (2011) have produced 88% total lipid recovery by
using hexane-ethanol (3:1). Yang et al. (2014) indicated ethanol extraction
of lipids from wet biomass of Picochlorum sp. and obtained 33.04% yield,
whereas, Fajardo et al. (2007) used ethanol and hexane to extract and purify
lipids from dried microalga Phaeodactylum tricornutum.
9781771883627
7.9CONCLUSION
All of the elements for the production of lipid-based fuels from algae have
been demonstrated in this chapter. It is clear that algal lipids can be extracted
and converted to biodiesel or other transportation fuels but the relevant ques-
tion is not whether biofuels from algae are possible, but rather whether they
can be made economically and at a scale sufficient to help contribute to
global fuel demand. A number of major technical challenges are needed to
achieve this goal. Significant attention and support should be given to both
basic and applied research on algae for biofuels. Photosynthetic microalgae
are technically viable and attractive alternatives for terrestrial crops and lig-
nocellulosic biomass. The promise of algal biofuels comes with the vision of
a novel form of large-scale production with economic viability of alternate
fuels for sustainable environment.
KEYWORDS
Algae
Autotrophic
Biodiesel
Biofuel
Biomass
Chlorella
Environmental sustainability
Fatty acid
Heterotrophic
Lipid production
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Microalgae
Open pond
Photobioreactor
Photosynthetic
Renewable energy
9781771883627
Scenedesmus
Triacyl glycerols
Tubular bioreactor
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Tredici, M. R.; Materassi, R. From open ponds to vertical alveolar panels: The Italian experi-
ence in the development of reactors for the mass cultivation of phototrophic microorgan-
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Walsby, A. Cell-water and cell-solute relations. In The biology of cyanobacteria, Carr, N.,
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Wen, X.; Geng, Y.; Li, Y. Enhanced lipid production in Chlorella pyrenoidosa by continuous
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Yeesang, C.; Cheirsilp, B. Effect of nitrogen, salt and iron content in the growth medium
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CHAPTER 8
INTEGRATED ALGAL INDUSTRIAL

WASTE TREATMENT AND BIOENERGY
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CO-GENERATION
MOHD AZMUDDIN ABDULLAH1 and ASHFAQ AHMAD2
1
Institute of Marine Biotechnology, Universiti Malaysia Terengganu,
21030, Kuala Terengganu, Terengganu, Malaysia
2
Department of Chemical Engineering, Universiti Teknologi
PETRONAS, 32610, Seri Iskandar, Perak, Malaysia
CONTENTS
8.1 Introduction......................................................................................154
8.2 Industrial Waste Remediation..........................................................156
8.3 Bioenergy.........................................................................................163
8.4 Large-Scale Application..................................................................170
8.5 Case Study Palm Oil Mill Wastes.................................................190
8.6 Conclusion and Future Outlook.......................................................203
Keywords..................................................................................................204
References.................................................................................................204
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8.1INTRODUCTION
Algae are a group of photosynthetic prokaryotes and eukaryotes, divided into

several divisions and kingdoms (Borowitzka et al., 2012). These are look-like
unicellular or simple multicellular organisms, living as distinct individuals,
in pairs, in clusters, colonies, or in sheets of individuals. Microalgae do not
form roots, stems, or leaves, but with high surface area to volume ratio, may
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reach sizes visible to the naked eye as minute green particles. They differ from
macroalgae in size and exist not only in aquatic but also in terrestrial eco-
system and in wide ranging environmental conditions, from icy North Pole to
the humid tropics and volcanic area. Prokaryotic algae include cyanobacteria
(Cyanophyceae), and eukaryotic microalgae are green algae (Chlorophyta)
and diatoms (Bacillariophyta) (Li et al., 2008; Lim et al., 2010). With an esti-
mate of more than 50,000 species, only a limited number of around 3000, have
been studied and identified (Richmond, 2004). The most plentiful microalgae
are single-cell drifters in plankton called phytoplankton which are competent
for rapid uptake of nutrients and carbon dioxide, have fast cell growth and
much higher photosynthetic efficiency than the land-based plants, although
the photosynthetic process is similar (Bajhaiya et al., 2010). As the energy de-
mand increases, the solution may lie in the application of microorganisms and
algae as the source of bioenergy (Rittmann et al., 2008; Laurens et al., 2012).
Among issues to be addressed in developing energy-based crops for biofuels
is the competition between fuels and food production, the effect of which has
been an increase in food prices (Somerville, 2007; Rude and Schimar, 2009).
The photosynthetic and heterotrophic natures of algae and the reported higher
oil productivity than the best producing oil crops make them highly potential
as alternatives to energy crops (Converti et al., 2009; Bajhaiya et al., 2010).
Algae are the main synthesizers of organic matter in aquatic habitats.
Since early time, microalgae have been used in human health food prod-
ucts, feeds for fish and livestock, and cultured for high-value of oils (Molina
et al., 1999; Spolaore et al., 2006), high-value chemicals for pharma- and
nutraceuticals and pigments such as carotenoids (Spolaore et al., 2006;
Borowitzka, 2010), and long-chain polyunsaturated fatty acids (PUFAs)
and phycobilins (Mendes et al., 2009). Industrial, municipal, and agricul-
tural wastewater treatments by algal culture systems enhance degradation
and improve CO2 balance with lower energy demand for oxygen supply in
aerobic treatment stages (Samori et al., 2013; Zhang et al., 2013). Among
the species for biofuel production includes Nannochloropsis oculata and
Tetraselmis suecica (Fig.8.1) and those suitable for wastewater treatment
include Scenedesmus sp., Chlorella sp., and Chlamydomonas reinhardtii
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Algal Industrial Waste Treatment and Bioenergy Co-Generation 155
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FIGURE 8.1 Productive microalgae species for biofuel production: a) Nannochloropsis

salina, b) Dunaliella salina, c) Tetraselmis suecica (Burton et al., 2009; Greenwell et al.,
2009).
(Oswald, 2003; Leadbeater, 2006). Nannochloropsis belongs to the class

of Eustigmatophyceae and is the most widely studied species owing to
its relatively high growth rate and lipid content with high nutritional val-
ues and resistance to mixing and contamination, which fit the needs of the
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biofuel industry and aquaculture (Roncarati et al., 2004; Bentley et al., 2008).
Tetraselmis suecica, a marine green agellate belonging to Chlorophyceae,
has good nutritional properties and contains C16:0 and C18:1 as predomi-
nant fatty acids for biofuel and as feeds for bivalve molluscs, penaeid
shrimp larvae, and rotifers (Harwood et al., 2009). Isochrysis galbana, a
Haptophyceae also has good nutritive values for aquaculture, principally
to feed mollusk larvae, as well as fish and crustaceans in the early stages of
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growth (Wikfors, 1994). Cyanobacteria and Chlorella sp. are efficient for
the treatment of organic pollutants (Kirkwood et al., 2003). Chlorella sp. can
be found in freshwater-bodies and grows well in municipal and agricultural
wastewaters and sludge (Kong et al., 2010).
In many Chinese lakes such as Lake Chaohu, Lake Taihu, and Lake
Dianchi, algal blooms have caused major water pollution, resulting in the
death of fish and illnesses. At the same time, these actually provide large
amount of biomass for value-added utilization (Miao et al., 2004). The role
of algae in waste treatment is both to incorporate nutrients and to provide
oxygen to bacteria, which in turn involve in the bacterial degradation of
organic materials in the wastewater, the same process utilized in activated
sludge. The presence of microalgae infact reduces the chemical oxygen de-
mand (COD) and biological oxygen demand (BOD), phosphorus, nitrogen,
and pathogens in a more cost effective way than activated sludge (Singh and
Dhar, 2011). The major challenge, however, is in determining a way that
allows downstream processing of algae to make it suitable for producing
biofuel and other bioproducts (Christenson et al., 2011). The integrated pro-
cesses that combine algae cultivation and wastewater treatment system for
biomethane production can reduce the cost and especially when combined
with CO2 mitigation. This versatility makes algae among the most interest-
ing organisms under research to solve global problem related to greenhouse
gases (GHG) emission, waste remediation, and bioenergy generation.
8.2 INDUSTRIAL WASTE REMEDIATION
8.2.1 LIQUID WASTES
Organic and inorganic substances which are freed into the environment as
a result of domestic, agricultural, and industrial wastewater can lead to pol-
lution. Most are hazardous and must be treated prior to disposal into the
waterways and land surfaces. Conventional primary and secondary treat-
ments of the wastewaters remove the easily settled materials and oxidize the
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organic materials. However, secondary treatment still releases large amount

of phosphorus and nitrogen which are directly responsible for eutrophication
of rivers, lakes, and seas (Lau et al., 1997; Trepanier et al., 2002). Secondary
effluents are also loaded with refractory organics and heavy metals. This
is where algal may become elegant solution in the tertiary and quaternary
treatments harnessing the ability of algae to use inorganic nitrogen, phos-
phorus, and amino acids for their growth and the capacity to remove heavy
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metals, as well as toxic organic compounds to prevent secondary pollution
(Abdel-Raouf, 2012; Guiry and Guiry, 2014). Algae cultivation actually
requires high amount of nutrients rendering the process economically and
environmentally not competitive (Halleux et al., 2008; Sialve et al., 2009).
Wastewaters can be a cost-effective alternative to synthetic culture media,
especially those derived from agro-industrial facilities which usually contain
high nutrient concentration (Markou and Georgakakis, 2011). Algalbacte-
rial systems for agro-industrial wastewater treatment have gained attention
where algae assimilate nutrients and through photosynthesis, produce dis-
solved oxygen that is immediately available to bacteria for the oxidization
of wastes whilst releasing CO2 needed for algal growth (De-Bashan et al.,
2004; Shilton et al., 2008). These avoid external oxygen supplementations
as required in the conventional processes, allowing nutrients recovery into
the biomass and reducing CO2 emissions (Molinuevo-Salces et al., 2010).
The tertiary algal biotreatment can then be coupled with the production of
bioenergy and extraction of lipids or biocompounds.
Heavy metals are other major pollutants as a result of industrial and min-
ing activities, use of fertilizers and pesticides, and release from fuels and
microelectronic products. Although toxic level of heavy metals may hinder
photosynthesis and kill the cells algae readily take up heavy metals from the
environment and induce heavy metal stress responses in the form of bind-
ing factors and proteins. The removal rate of metal ions such as aluminum,
calcium, ferum, manganese, and magnesium from plastic manufacturing
and electroplating waste water vary among the algae species, ranging from
50 to 99% (Wang et al., 2009a; Woertz et al., 2011). Macroalgal species
such as Lamiaria, Sargassum, Macrocystis, Ecklonia, Ulva, Lessonia, and
Durvillaea have been reportedly efficient for binding copper, nickel, lead,
zinc, and cadmium (McHugh, 2003). Combination of these algal nutrient
uptake, elevated pH, and high dissolved oxygen concentration avoids any
chemical additives and eutrophication leading to improved, safer, less ex-
pensive and more efficient approach to wastewater treatment and heavy met-
als removal (Munoz and Guieysse, 2006).
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8.2.2HYDROCARBONS
Leaks and accidental spills of petroleum-based products occur frequent-

ly from oil exploration, production, refining, transport, and storage. The
amount of natural crude oil seepage estimated at 600,000 metric tons per
year (Kvenvolden and Cooper, 2003). These have become major contribu-
tors to water and soil pollution (Holliger et al., 1997) which cause serious
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damage on eco-system with the accumulation in animals and plant tissues,
resulting in deaths or mutations (Alvarez and Vogel, 1991). Bioremediation
by microbial consortia can detoxify or remove pollutants owing to their di-
verse metabolic capabilities. It is an evolving, non-invasive and relatively
inexpensive method for the removal and degradation of many environmen-
tal pollutants (Leahy and Colwell, 1990; April et al., 2000; Ulrici, 2000;
Medina-Bellver, 2005). Algae and protozoa are the essential members of the
microbial community in both aquatic and terrestrial eco-systems, but reports
are scanty on their capabilities for hydrocarbon biodegradation. Isolated alga
Prototheca zopfi could utilize crude oil, a mixed hydrocarbon substrate and
exhibit extensive degradation of n-alkanes and isoalkanes as well as aromat-
ic hydrocarbons (Walker et al., 1957). Nine cyanobacteria, five green algae,
one red alga, one brown alga, and two diatoms have been reportedly capable
of oxidizing naphthalene (Cerniglia et al., 1980), but protozoa has not been
shown to utilize hydrocarbons. Diatoms species Skeletonema costatum and
Nitzschia sp. could degrade hydrocarbons simultaneously though the latter
is more efficient. Microalgae species show comparable or greater efficiency
in removing the mixture of hydrocarbons suggesting that the presence of
polyaromatic hydrocarbon may stimulate the degradation of the other hydro-
carbon (Hong et al., 2008).
Phenolic compounds such as nitrophenols and chlorophenols are the tox-
ic industrial pollutants (Khan et al., 1981a; Shigeoka et al., 1988; Wang et
al., 2001b; Aravindhan et al., 2009). The halophenols can be found in pulp
mill effluents (Xie et al., 1986), agricultural and residential runoff (Ahlborg
and Thunberg, 1980), and sewage and wastewater discharges (Stasinakis et
al., 2008) from wood pulp bleaching, water chlorination, textile dyes, oil re-
fineries, chemical, agro-chemical, and pharmaceutical industries (Rodriguez
et al., 1996; Perez et al., 1997; Fahr et al., 1999). Some of the phenolic com-
pounds are suspected to be endocrine disruptors and have deleterious effects
on humans and other organisms in the natural eco-system at concentrations
lower than the discharge standard for phenols (Schafer et al., 1999). Their
abatement in STPs is often insufficient because phenolics are highly toxic
to anaerobic and aerobic bacteria (Capasso et al., 1995). The detoxification
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potential of phenol by different microrganisms, mainly bacteria and fungi,

has been extensively studied (Kahru et al., 1998). Microalgae that are ca-
pable of phenol degradation include Chlorella sp., Scenedesmus obliquus
and S. maxima (Kleckner and Kosaric, 1992). Four species of freshwater
microalgae have been shown to mineralise phenol (Ellis, 1977) and green
Ankistrodesmus degrade various phenols (Pinto et al., 2002). After three
months of selective enrichment with -chlorophenol and -nitrophenol, two
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microalgal species, Chlorella vulgaris and Coenochloris pyrenoidosa, have
been isolated from the aquatic communities recovered from the waste dis-
charge container fed with several aromatic pollutants. As an axenic culture,
the consortia grown under 24h light regime are capable of biodegrading
50mgL1 of -chlorophenol within 5 days. Addition of zeolite as an ad-
sorbing material does not improve -chlorophenol removal. However, with
-chlorophenol at 150mgL1 introduced to the culture supplemented with
zeolite, the growth rate of the consortia improves, but with longer lag phase
(16 against 14 days without zeolite) (Lima et al., 2004). A golden brown
unicellular chrysophyte Ochromonas danica, investigated for the degrada-
tion of phenols in the dark and in aerobic conditions, has been found to affect
the metacleavage of exogenous phenol by utilizing the reactions for its en-
ergetic requirements (Semple et al., 1999). O. danica metabolize phenol and
shows heterotrophic growth at 96mgL1 phenol (Semple and Cain, 1997).
However, Ochromonas may not be completely suitable for wastewater treat-
ment as far as mass cultivation is concerned. As with other chrysophytes,
it possesses endogenous mechanism of regulation, limiting the number of
vegetative cells within a population (Van Den Hoek et al., 1995).
8.2.3GASES
Algae assimilate inorganic carbon during photosynthesis in two steps: (1)

during light reaction, solar energy or other sources are converted to chemical
energy with oxygen as by-product; and (2) during dark reaction, the chemi-
cal energy is used to assimilate carbon dioxide and converted into sugars
(Fig. 8.2) (Larsdotter et al., 2006). This can be simplified as follows:
2H2O + 2NADP+ + 3ADP + 3Pi + light 2NADPH +

2H++ 3ATP + O2 (8.1)
3CO2 + 9ATP + 6NADPH + 6H+ C3H6O3phosphate +

9ADP + 8Pi + 6NADP + 3H2O (8.2)
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FIGURE 8.2 A generalized structure of the Calvin cycle (Igamberdiev and Kleczkowski,
2011).
The concept of fixating the carbon into biomass through photosynthe-

sis is a sustainable way of sequestering CO2. Important considerations are
the growth rates and photosynthetic efficiency, resource requirements such
as land, nutrients, and water, resistance to environmental stress, as well as
the possibility of acquiring useful end products from cultivation. Compared
to conventional forest, agricultural and aquatic plants, algal growth rate is
approximately 50 times higher with better CO2 fixation and more efcient
carbon capture, utilizing small growth areas and lower energy consumption
and costs. However, the use of algae is only considered feasible if they are
used as biofuel feedstock rather than merely as a carbon sequester. Turning
CO2 emissions into a fuel via photosynthesis ensures recycling of carbon and
reducing the demand for virgin resources (Packer et al., 2009). With simple
metabolism and reproduction system, algal species could grow under harsh
and varied conditions, with no necessity for land or freshwater making CO2
biofixation economically viable (Packer et al., 2009).
In addition to pure CO2, ue gases from industrial plants can also be
used as feeds. For coal-fired thermal power plants, algal conversion of pho-
tosynthetic function and solar energy has been explored in two ways: (1)
to use CO2 gas separated from the flue gas; and (2) to use the flue gas di-
rectly. Direct use of ue gas in the cultivation system does not adversely
affect algal growth and the production has been shown to be reliable, pos-
sible (Suali and Sarbatly, 2010), and advantageous due to energy saving, but
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may encounter problems such as high temperature, CO2 concentration up to

15%, and the presence of SOx and NOx. Since flue gases from industries such
as steel-making plants and thermal power stations contain about 500 times
higher concentration of CO2 [1020% (v/v)] than that in the air, there may
be inhibition of algal growth with requirement for large amount of nutrients
such as nitrogen and phosphorus but low CO2 conversion due to short gas
retention time. Screening and selection of suitable algal strains having toler-
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ance to high CO2 concentration have been extensively carried out (Mata et
al., 2010). The focus is to identify suitable algal strains that can grow under
high CO2 concentration while producing lipid. The desired strains should
have the following characteristics: (1) high growth rate and biomass pro-
ductivity, (2) high tolerance to trace amount of acidic components from NOx
and SOx, and (3) the ability to sustain growth even under extreme culture
conditions (e.g. high temperature of water due to direct introduction of flue
gases) (Chisti, 2007). Moderate SOx and NOx contents (a few tens of ppm)
can be tolerated by algae (Brown, 1996; Lee et al., 2000; Lee et al., 2002),
but higher concentrations may have moderate (Negoro et al., 1993) to strong
inhibitory effects, depending on culture conditions and species (Yanagi et
al., 1995). The causes of toxicity are not clear as these molecules can act
directly on the cell physiology, or indirectly by changing the properties of
culture medium. The deleterious effects of SO2 (Matsumoto et al., 1997) and
NO (Lee et al., 2002; Jin et al., 2005) can be significantly altered, if the pH
of the media is regulated within the physiologically acceptable ranges.
Microalgal species suitable for CO2 xation include Chlorella, Spirulina
platensis, Emiliania huxley, Phaeodactylum, and Nannochloropsis sp.
(Negoro et al., 1993). The challenge of limited accessibility of land for large
scale CO2-capturing from industrial or power plants by microalgae can be
overcome by sophisticated area-efficient techniques to recycle CO2 (Suali
and Sarbatly, 2010). A pilot scale system has been successfully developed
to look into CO2 recycling where Scenedesmus obliquus is shown to tolerate
high concentration of CO2 up to 12% (v/v) with optimal removal efficiency
of 67% (Li et al., 2011). CO2 tolerance of Chlorella vulgaris is enhanced by
gradual increase of CO2 concentration while S. obliquus, Chlorella kessleri,
and Spirulina sp. have exhibited good tolerance (up to 18% CO2) indicat-
ing great potentials for CO2 sequestration from CO2-rich streams (Ho et al.,
2010). CO2 consumption rate of 549.9mgL1d1 for maximum S. obliquus
biomass productivity of 292.5mg/L and lipid productivity of 78.73mgL1d1
(38.9% lipid content per dry weight of biomass), has been reported in two-
stage system with 10% CO2 (Ho et al., 2010). A study on Dunaliella tertio-
lecta, Chlorella vulgaris, Thalassiosira weissflogii, and Isochrysis galbana,
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representing four different phyla, grown with CO2-enriched air or with a

mixture of gases mimicking the composition of a typical cement flue gas
(CFG) has suggested no effect of CFG. Dusts added at realistic concentra-
tions also do not show any impact on growth. In the second stage, the culture
exposed to the CFG receives an increasing concentration of dust characteris-
tic of cement industry at concentration two ranges of magnitude higher and
microalgal growth is inhibited (Amlie et al., 2013). Dust in flue gases may
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contain toxic compounds such as soot (Matsumoto et al., 1997) or trace met-
als (Borkenstein et al., 2011) and at inhibitory level can kill the cells.
8.2.4 SOLID WASTES
Nutrient-rich wastes from animal farms can pose great environmental chal-
lenges with regards to regional eutrophication and global warming. Excess
runoff and discharge of nutrients such as 18% of N from farms and 25%
of P from animal wastes have partly caused water quality deficiency in
the Chesapeake Bay (the largest estuary in the United States) (Chesapeake
Bay Foundation, 2004). Animal waste is a rising source of GHG emissions,
including methane and nitrous oxide. The U.S. Environmental Protection
Agency (EPA; http://www.epa.gov/methane/) estimates that GHG emissions
from animal wastes have inceased by almost 60% between 1990 and 2009
(EPA, 2010). There is therefore a considerable interest in leveraging possible
synergies between algae-derived energy production and the animal waste
management initiative for environmental sustainability and economic con-
sideration. Co-digestion of Spirulina platensis with waste activated sludge
(WAS) could improve volatile solid (VS) reduction, but Chlorella sp. has a
slight negative effect on dewaterability of the digestate as compared to WAS
alone (Yuan et al., 2012). The major aspect in co-utilizing the solid waste
with microalgae is to achieve optimized C/N ratio for biogas production.
Olive mill solid waste (OMSW) is a pollutant coming from olive oil extrac-
tion by the two-phase centrifugation system and contains high organic mat-
ter content and unbalanced C/N ratio (31:1), resulting in reduced methane
yields in the anaerobic digester. Dunaliella salina used as co-substrate with
the OMSW in anaerobic digestion has enhanced substrate biodegradability
when D. salina is increased from 25 to 50%. Maximum methane production
of 330mLg1 VS is achieved at co-digestion mixture of 75% OMSW-25%
D. salina, keeping C/N ratio at 26.7/1 (Fernndez-Rodrguez et al., 2014). In
another study, co-digestion of algae biomass residue and fats, oil, and grease
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(FOG), each at 50% of the loading, allowing organic loading rates (OLR) up
to 3g1 VSL1 d1 has resulted in a specific methane rate of 0.54Lg1 VS d1
and a volumetric reactor productivity of 1.62Lg1 VS d1. Lipid-rich FOG is
the key factor to achieve high methane yields, accounting for 6883% of the
total methane produced (Park and Li, 2012).
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8.3BIOENERGY
The worlds primary energy need is projected to grow by 55% between 2005
and 2030, where fossil fuels will remain as the major source (International
Energy Agency, 2009). Coal reserves are predicted to last over 200 years
(Khan et al., 2009b) and will most likely account for half of the worlds base-
line electricity generation by 2015, before oil production reaching its peak
by 2020 (Almeida et al., 2009). The main concerns are related to economic,
ecological, and environmental impact of carbon fossil fuel and whether or
not the conventional fuels should be left underground unmined and replaced
with greener options. Alternative renewable energy such as biodiesel (Naik
et al., 2010) and the bio-refinery set-up utilizing nutrient-rich wastes and
capturing and utilizing CO2 from flue gases can partly address the issue on
CO2 mitigation (Stewart et al., 2005). For biodiesel production, the required
amount of biomass can be huge and the production cost should fall below
$400/tonne of biomass to be economically feasible. This is still far from the
price currently reported in a full-scale plant, where the cost for even a me-
dium-scale plant still 173 times more expensive (Chisti, 2010; Acin et al.,
2012). The concept of using algal biomass as a potential source of biofuel is
therefore promising and now being taken seriously because of the fluctuat-
ing petroleum prices and the concerns about global warming and climate
change (Gavrilescu and Chisti, 2005). It has become pertinent to improve
the economics of bioenergy generation from algae by understanding and
improving the algal biology through genetic and metabolic engineering, the
use of hybrid bioreactors for a more controlled environment (Chisti, 2007;
Abdullah et al., 2015b) and efficient downstream processes. A conceptual
model for integrated microalgal biomass and biofuel production is shown
in Figure 8.3. Renewable biofuels that can be developed based on algae
include biodiesel (from algal oil), biomethane (by anaerobic digestion of the
algal biomass), photobiologically produced biohydrogen, and bio-ethanol
(by fermentation of the algal carbohydrates) (Gavrilescu and Chisti, 2005;
Spolaore et al., 2006; Park et al., 2012).
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FIGURE 8.3 A conceptual model for integrated microalgal biomass and biofuel production
(Demirbas, 2011).
8.3.1BIODIESEL
Biodiesel is a promising alternative to petro-diesel for vehicles and internal

combustion engines (Sgroi et al., 2005). Lipids, presently extracted for bio-
diesel production, are mainly derived from oily seeds such as soybean, palm,
castor bean, peanut, sunflower, corn, rapeseed, and cotton. Cultivation of
these oil crops does not actually replace the natural energy resources as there
may be a need for large available areas, suitable soil, high-quality water,
seasonality and in some cases causing demineralization, salinization, desert-
ification, soil erosion, reduction of water sources, and extensive use of pesti-
cides (Gerpen, 2005; Knothe, 2005; Cheng et al., 2014). In a tropical climate
region, the mean annual productivity of microalgal biomass is 1.53kgm3d1,
with a mean lipid content of 30%. The concentration per hectare of the total
area is around 123m3 for 90% of the year, since the remaining 10% may be
for perpetuation and cleansing of the reactors, for the yield of biodiesel from
microalgae around 98.4m3ha1. For the production of 5.4 billion m3 of bio-
diesel, an area of approximately 5.4Mha must therefore be cultivated, which
represents only 3% of the area currently used for cultivation of plants for
biodiesel production. This is a possible scenario even with algal lipid content
of 15% dry weight (Chisti, 2008). To meet the required demand and for 5%
biodiesel (B5) addition to mineral diesel oil, the production of vegetable oils
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may be boosted by 50100%, which is a difficult goal to achieve as it rep-

resents a proportional increase in arable land with oil crops, and the current
agricultural productivity makes it harder to increase. As the concentration
of fatty acids and productivity of algae are much higher than that of plants,
the effort to increase oil production with energy crop cultivation would not
be so great (Maa and Hanna, 1999). Biodiesel output per required land area
is estimated at: corn 145kg oil ha1, soybeans 375kg oil ha1, palm oil
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5000kg oil ha1, and algae 80000kg oil ha1 (Skjanes et al., 2007). The
amount of land needed for the corresponding production using microalgae
would be around 100200 times less (Costa et al., 2011).
Algal cultivation can be an exceptional source of lipids and fatty acids
(Colla et al., 2004; Johnson and Wen, 2009; Cheng et al., 2014). Among mi-
croalgae species, the levels of 2050% lipids are quite common. Chlorella
has been reported with 50% lipids and Botryococcus achieves 80% lipids
(Powell and Hill, 2009). The variations can be attributed to different grow-
ing conditions especially the CO2 levels and methods of extraction of lipids
and fatty acids. In areas where algae are grown for biodiesel production
alongside fossil-fuel power stations, CO2 from flue gases can be utilized and
the lipid content has been reportedly increased (Brown and Zeiler, 1993;
Sawayama et al., 1995). The alkane chain distribution of microalgae is very
similar to that of mineral diesel (Miao et al., 2004). Analyses of the satu-
rated fraction of biofuel from Chlorella protothecoides demonstrate that the
alkane chain reaches 1030 carbons, while the alkane chain of the saturated
fraction of biofuel from Microcystis aeruginosa records 1028 carbons.
Microalgal biofuel also has lower O content and a higher H/C ratio than
biofuel from plants, sunflower and cotton although the content in the former
may be lower. The H/C and O/C mean molar ratios of microalgal biofuel are
1.72 and 0.26, while the plant biofuel is 1.38 and 0.37, respectively (Miao et
al., 2004). High O content is not attractive for the production of transporta-
tion fuels. Biofuels from C. protothecoides and Microcystis aeruginosa have
high calorific values of 30 and 29MJkg1, respectively, due to the high C
and H content and low O content, conferring higher stability, lower viscos-
ity, and lower density than biofuel from plants. This high H content of algal
biofuel is due to the chlorophyll and proteins (Miao et al., 2004).
8.3.2BIOMETHANE
Microbial activities annually generate some 590880 million tonnes of

methane released into the atmosphere worldwide with about 90% coming
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from the biogenic sources. Methane is over 20 times more effective in

trapping heat in the atmosphere than CO2 over a 100-year period (EPA,
2010). Methane can be used as fuel gas and converted to produce electric-
ity (Vergara-Fernandez et al., 2008) and CO2 can be removed should pure
methane is to be used (Hankamer et al., 2007). Anaerobic digestion of plant-
based lignocelluloses and organic waste materials such as cow dung, pig
slurry, effluent from slaughter houses, palm oil mill effluent (POME), and
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landfill produce biomethane and biohydrogen (Ahmad et al., 2003; Basri et
al., 2009). There are four sequential stages in anaerobic digestion process:
hydrolysis, acidogenesis, acetogenesis, and methanogenesis. During hydro-
lysis, complex organic biopolymers (e.g. carbohydrates, lipids, and proteins)
are hydrolyzed and broken down into soluble sugars. Fermentation carried
out by bacteria converts sugars into alcohols, acetic acid, volatile fatty acids
and gases containing H2 and CO2. The acids are primarily metabolized by
methanogenesis into CH4 (6070%) and CO2 (3040%). Anaerobic produc-
tion based on algae similarly produces a mixture of methane (5575%) and
CO2 (2545%). The main advantages of algae-based over the conventionally
used plant biomass are that algae are grown in a liquid medium where the
space available for cultivation is three-dimensional and the absence of lignin
and lower cellulose content which lead to good process stability and high
conversion efficiencies (Harun et al., 2010a). Furthermore, anaerobic diges-
tion is suitable for high moisture content (8090%) organic wastes which are
the characteristic of wet algal biomass, and the remaining biomass can be
reprocessed into fertilizers for sustainable agricultural practices, thus econo-
mizing the production costs. The conversion of algal biomass into biogas
even recovers energy through the extraction of lipids for biodiesel produc-
tion (Prasertsan, 1996; Li et al., 2008), or high-value biocompounds (Khan
et al., 2005; Abdullah et al., 2015b).
8.3.3BIOHYDROGEN
Hydrogen has wide applications in fuel cells, liquefaction of coal, and

upgrading of heavy oils. The technological viability is dependent on the
development of cost-effective, large-scale, sustainable production systems
capable of replacing classical steam reforming of natural gas, petroleum
rening, and coal gasication (Rupprecht et al., 2006). Efficient containers
and absorbers/adsorbers for liquid hydrogen are needed to minimize leak-
age and risk of explosion. Hydrogen can be produced by steam reformation
of bio-oils, dark and photo fermentation of organic materials, and photolysis
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of water catalyzed by special algal species (Kapdan and Kargi, 2006; Ran et
al., 2006; Wang et al., 2009a). Biohydrogen production by photosynthetic
microorganisms requires a simple solar reactor such as a transparent closed
box, with low energy requirements as opposed to thermochemical or elec-
trochemical technique via solar battery-based water splitting which need
high energy requirements. Photosynthetic production of H2 from water via
biological process converts sunlight into useful chemical energy and the
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underlying phenomenon is discovered long time ago (Gaffron and Rubin,
1942), but the biotechnology progress is slow. Cyanobacteria are able to
diverge the electrons emerging from the two primary reactions of oxygenic
photosynthesis directly into the production of H2 based on the use of solar
energy and water (Tamagnini et al., 2007). Immobilized cells of Clostridium
acetobutylicum are used to ferment various microalgae (Arthrospira pla-
tensis, Nannochloropsis sp., Dunaliella tertiolecta, Galdieria partita,
Chlorella vulgaris, Cosmarium sp., Nostoc sp.) where the highest produc-
tivity of 0.35mmol H2 L1 h1 has been reported with Nannochloropsis sp.
(Efremenko et al., 2012). The use of 2.5g Nannochloropsis with short ther-
mal treatment and without acid addition elevates productivity to 1.07mmol
H2 L1 h1 (Nobre et al., 2013). Biohydrogen production from C. vulgaris and
D. tertiolecta yields of 10.8mL H2 g1 VS and 12.6mL H2g1 VS, respec-
tively, using untreated anaerobic digested-sludge as inoculum (Lakaniemi
et al., 2011). High H2 production of 81mLg1 alga is obtained after 65h
fermentation with C. vulgaris (acid hydrolyzed) by C. butyricum (Liu et
al., 2012).
In recent years, efforts have been directed toward decreasing the costs re-
lated to microalgae culture systems for the production of biofuels. Dark fer-
mentation for biohydrogen generates CO2 emissions and soluble metabolites
(e.g. volatile fatty acids) with high COD as the by-products, which necessi-
tate further treatments. Mixotrophic culture of an isolated Chlorella vulgaris
ESP6 could be utilized to simultaneously consume CO2 and CODs from
dark fermentation, converting them to valuable biomass. Light intensity is
adjusted to 150mmolm2s1 and food to microorganism (F/M) ratio of 4.5
to improve the efficiency of assimilating the soluble metabolites. The mixo-
trophic microalgae culture reduces the CO2 content of dark fermentation ef-
fluent from 34 to 5% with nearly 100% consumption of soluble metabolites
(mainly butyrate and acetate) in 9 days. The obtained microalgal biomass
is hydrolyzed with 1.5% HCl and subsequently used as the substrate for H2
production with Clostridium butyricum CGS5, giving a cumulative H2 pro-
duction of 1276mlL1, a H2 production rate of 240mlL1 h1, and a H2 yield
of 0.94mol mol1 sugar (Liu et al., 2013). Simple and inexpensive strategy
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to bio-prospect and cultivate mixed indigenous chlorophytes with high car-

bohydrate content for biomethane and biohydrogen production have been
developed. Mixed microalgae from four different water-bodies in Queretaro,
Mexico are grown in bold basal mineral medium and secondary effluent
from a wastewater treatment plant using inexpensive photo-bioreactors.
Large variations in microalgal genera diversity are observed based on dif-
ferent culture media and nitrogen sources. In secondary effluent, Golenkinia
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sp. and Scenedesmus sp. proliferate, while the carbohydrate content varies
between 12 and 57%, with the highest volumetric productivity achieved at
61mgL1d1 and 4.6gm2d1, respectively. The results indicate that mixed
microalgae are a good feedstock for biomethane and biohydrogen produc-
tion (Glenda et al., 2014).
8.3.4BIO-ETHANOL
Ethanol has been globally considered as an alternative to petrol fuel as it

reduces the levels of CO2 and CO emission, lead, sulfur, and particulates
(Willke and Vorlop, 2004). The largest bioenergy program that has tak-
en place is in Brazil for sugar cane ethanol (Prolcool), which begins in
1976. Since the 1980s, ethanol has been an established alternative to fos-
sil fuels in Brazil and produced mainly from sugar and starch (sugar cane,
corn). The use of ethanol as a gasoline substitute avoids the emission of
9.56106tonnes of carbon per annum (about 15% of Brazils total emis-
sions). The government encourages ethanol production from sugar cane and
the adaptation of motors to the Otto cycle to run on pure ethanol (hydrated
alcohol with 96% ethanol and 4% water) or gasohol (78% gasoline and 22%
anhydrous ethanol). Alcohol addition increases the gasolines octane and
removes the highly toxic tetraethyl lead additive. Ethanol has a calorific
value of 22MJL1, while gasoline is 33MJL1, but the higher octane rat-
ing of ethanol and the compatibility to the engines and injection systems
mean that the technical equivalence of ethanol per liter of gasoline is about
1.15 (Rupprecht, 2009). There are, however, issues related to environmental
sustainability of bioenergy after the growth of ethanol in the world market
especially with regards to the competition for agricultural land for food. The
value of agricultural commodities reaches an unprecedented high in 2006,
mainly those of grains. In 2008, the United States produces 600 million litres
of alcohol from cereals for beverages where the conversion utilizes more
energy than the sugar conversion into alcohol. This can even result in nega-
tive yields, which precludes the use of cereals from an energy point of view.
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Thus, identifying alternative sources of bio-ethanol feedstock is of high pri-

ority (Xuan et al., 2009).
The common methods for bio-ethanol production are fermentation (bio-
chemical process) or gasification (thermo-chemical process) (Singh and
Gu, 2010). Traditionally, ethanol is produced by fermentation of biomass
such as from energy crops and organic wastes (Xuan et al., 2009). The bio-
mass preparation can be carried out with mechanical press or enzymatic cell
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wall break down to make carbohydrates more accessible, as well as break-
ing down the large molecules. When cells are disrupted, Saccharomyces
cerevisiae is added for fermentation, where sugar is converted into ethanol
and purified by distillation (Amin, 2009). The energetic yield of converting
sugar into ethanol is estimated at 40%. Alcoholic fermentation is one of the
end products for pyruvate at the end of the glycolytic pathway. It consists
of anaerobic conversion to ethanol and CO2 in two steps. In the first step,
pyruvate is decarboxylated by pyruvate decarboxylase, releasing CO2 and
forming acetaldehyde, which is then reduced to ethanol by alcohol dehy-
drogenase (Lehninger et al., 2004; Park et al., 2012). CO2 can be recycled
during fermentation for residual biomass in anaerobic digestion for biometh-
ane production, such that in essence all the organic matter is accounted for
(Harun et al., 2010b; Harun et al., 2010c; Singh and Gu, 2010).
Algal feedstock can be advantagous as they are less resistant to conversion
into simple sugars than plant biomass. Algal species such as Chamydomonas
sp., Chlorella sp., Oscillatoria sp., Cyanothece sp., S. platensis, Chlorella
vulgaris (Ueno et al., 1998; Branyikova et al., 2011), and Chlamydomonas
reinhardtii UTEX 90 (Choi et al., 2010) build-up their energy reserves in
starch, which is an efficient carbohydrate feedstock. After oil extraction
from the biomass, fermentation ensues utilizing glucoamylase, amylase,
and yeast, bacteria or fungi to convert sugars to ethanol and CO2 with used
water recycled (Dismukes et al., 2008). Enzymatic hydrolysis of C. vulgar-
is FSP-E biomass (containing 51% carbohydrate per dry weight) gives a
glucose yield of 90.4% (or 0.461g g1 biomass). The separate hydrolysis
and fermentation (SHF) and simultaneous saccharification and fermentation
(SSF) processes convert the algal hydrolysate into ethanol with 79.9% and
92.3% theoretical yield, respectively. Hydrolysis with 1% sulfuric acid is
efficient in saccharifying C. vulgaris FSP-E biomass, achieving a glucose
yield of nearly 93.6% with starting microalgae biomass concentration of
50gL1. Using acidic hydrolysate of C. vulgaris FSP-E as feedstock, the
SHF produces 11.7gL1 ethanol at 87.6% theoretical yield. Chlorococum
sp. achieves bio-ethanol production of around 38% dry weight (Harun et al.,
2010c), while Chlorella vulgaris can achieve conversion efficiency above
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65% (Ueno et al., 1998). These show the feasibility of using carbohydrate-
producing microalgae as feedstock for bio-ethanol fermentation (Ho et al.,
2013), attributable to their high carbon composition and direct availability
for fermentation or after pre-treatment.
8.4 LARGE-SCALE APPLICATION
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8.4.1 FACTORS FOR OPTIMAL PRODUCTIVITY
Successful cultivation of microalgae needs favorable environmental condi-

tions, which may differ from species to species. The main parameters influ-
encing biomass productivity include nutrients, light exposure, intensity and
wavelength, temperature, CO2 concentration, pH, salinities, and mixing.
8.4.1.1NUTRIENTS
In large scale algal cultivation, providing optimum nutrient balance is essten-

tial for optimum growth rate and maximum lipid productivities. Inorganic
elements, macronutrients, vitamins, and trace elements build-up algal cells.
The composition variations and different environmental conditions should
affect the performance of anaerobic digestion based on their digestion pos-
sibility. The mineral composition must meet the nutrient necessities of the
anaerobic microflora. Major components in algal composition are carbon,
nitrogen, phosphorus, and metals such as iron, cobalt, and zinc (Grobbelaar
and Richmond, 2004). The required macronutrients are nitrogen and phos-
phorus (16N:1P ratio) and silicon (Richmond, 2004). The composition of
proteins (652%), lipids (723%), and carbohydrates (523%) is strongly
dependant on species (Brown et al., 1997). The normal C/N ratio for fresh-
water microalgae is 10.2 (Christenson and Sims, 2011). For some species,
the high proportion in proteins is characterized by a lower C/N ratio as com-
pared to terrestrial plants. The combination of different substrates is a strat-
egy to increase the performance of a digester by ensuring optimal influent
composition and enhanced biogas productivity. When C/N is lower than 20,
there is an inequality between carbon and nitrogen necessary for anaero-
bic microflora, leading to nitrogen release and may become inhibiting from
the accumulation of volatile fatty acids (VFAs) (Leadbeater, 2006; Yen and
Brune, 2007).
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At 120150gL1 KNO3, 1215gL1 Na2HPO4, and 45gL-1 FeCl3 in

conway media formulation in seawater, N. oculata achieves the highest cell
density of 72106 cells mL1 with maximum biomass of 0.8gL1 and 35%
lipid, but T. suecica only attains 46.5106 cells mL1 although almost com-
parable 0.7gL1 biomass and 27% lipids. The maximum specific growth
rates are generally 0.160.18d1 with doubling time of 3.784.62 days in
250mL shake flask and 5300L tanks. Both I. galbana and P. lutheri achieve
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17106 cells mL1 with 0.7gL1 dry weight and 2437% lipid. The lower
cell density suggests that cell division may be lower, but the cellular com-
position making up the dry weight can be higher. Under nutrient deficiency
conditions at 1065 g L-1 KNO3, 37.5 g L-1 Na2HPO4, and 2.5gL1 FeCl3, the
cell growth (g L1) of N. oculata (0.64), T. suecica (0.49), I. galbana (0.54),
and P. lutheri (0.38) is much reduced, but the lipid accumulation remains in
the range 23.637.3% (Shah, 2014).
8.4.1.2 LIGHT INTENSITY
Microalgae carry out photosynthesis and cellular division in the presence of

light that accessibility and light intensity may affect the success or failure
of the cultures. The light-harvesting antennae of algal cells are efficient and
can absorb all the lights striking them, even if not used for photosynthe-
sis (Richmond, 2004). The net growth may become zero at low intensities,
but photosynthesis increases with increasing intensities until a point with
maximum growth rate (saturation point). Most algae get light saturated at
about 20% of solar light intensities. Increasing light intensity beyond this
point may not affect the growth rate and instead can lead to photo-oxida-
tion, damaging the light receptors, and thereby reducing the photosynthetic
rate and productivity (photo-inhibition) (Molina et al., 1999). A study on
cyanobacterium Aphanothece nageli in a bubble column closed photobio-
reactors (PBRs) shows that a linear reduction is found in the CO2 fixation
rate and biomass production with reduction in light regime, while the 12:12
(night:day) cycle results in average biomass production and carbon fixation,
suggesting the pre-adaptation of microalgae to light regime charges (Jacobe-
Lopes et al., 2009; Kesaano et al., 2014). Light intensity as high as 400mol
photons m2s1 has resulted in the highest biomass yield of Scenedesmus sp.
(3.88gL1) with equally high lipid (41%), neutral lipid content (32.9%), ole-
ic acid (4352%), palmitic acid (2427%), and linoleic acid of 711% (Liu
et al., 2012). Although this may be good in terms of productivity, utilizing
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high light intensity or prolonged photoperiod may defeat the purpose of de-
veloping green-alternatives with reduced additional energy operating cost.
8.4.1.3TEMPERATURE
Generally, increase in temperature leads to exponential algal growth until an
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optimum level is reached, after which cell growth starts declining. Ambient
temperature fluctuations can result in diurnal temperature differences as
much as 20C which can affect productivity (Molina et al., 1999). This
affects photosynthesis and creates such condition under which photo-inhibi-
tion may occur during low level of light intensity and sub-optimal tempera-
tures. Generally, constant temperatures above the optimal range may cause
cell death, but temperatures below the favourable range, excluding freezing
conditions, may not. Low biomass during dark periods can therefore be en-
hanced by increasing the temperatures. Maximum formation of bio-ethanol
from Chlorella sp. cultivated at 30C is 448molg1 dry weight, but is much
reduced to 196molg1 dry weight at 20C. Ethanol production decreases
when temperature is increased to 35C and is completely inhibited at 45C.
Enzyme activity at 35C is lower than at 25C, indicating that enzymes
may be denatured at too high a temperature but activated within acceptable
temperature range (Ueno et al., 1998). Anaerobic digestion can operate in
both mesophilic (35C) and thermophilic (55C) conditions but the differ-
ence is in the reaction rate constant where mesophilic digestion of Ulva sp.
results in 180mLg1 VS of methane, but with slower breakdown of organic
compounds (Otsuka and Yoshino, 2004; Chynoweth, 2005).
8.4.1.4 GAS EXCHANGE
Roughly, 4550% of algal biomass is made up of carbon. If oxygen concen-

trations increase above the saturation level, photo-oxidative damage may
occur to the chlorophyll reaction centers which inhibit photosynthesis and
decrease productivity (Molina et al., 1999; Pulz and Gross, 2004). With low
percentage of CO2 in the air (0.033%), algal growth rate can be limited if
additional carbon is not supplied. Generally, CO2 is blended with air in aer-
ated cultures or injected into the cultures through gas exchange systems in
PBRs or sumps in open raceways. To reduce the losses of CO2 in open sys-
tem, bubbling can be affected through air stones, with perforated pipes and
plastic dome exchangers, injection into deep sumps, trapping the CO2 under
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floating gas exchangers, and maintaining high alkalinities in the culture wa-
ter (Richmond, 2004).
8.4.1.5 SALINITY AND pH
Although microalgae can tolerate wide range of salinities, changes due to
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evaporation and rainfall are the major factors affecting the growth of marine
microalgae in open system (Richmond, 2004). During hot conditions, salin-
ity will increase due to evaporation and decrease during rainfall. Microalgae
are also sensitive to pH changes that control of pH may be essential to main-
tain high growth rate. Fresh and marine algal species grow at pH 7.610.6.
Some freshwater species grow well up to 10.6, while some are badly affect-
ed by higher pH. The optimum initial pH 8 and salinity of 35ppt in conway
media and seawater achieve the cell dry weight of 0.82, 0.72, and 0.58gL1
and lipid content of 35.7, 33.5, and 37.3%, respectively, for N. oculata, T.
suecica, and P. lutheri. At 25ppt NaCl, irrespective of pH tested (pH 69),
the biomass remains below 0.5gL1 and the lipid content below 25%. This
may suggest the role of osmotic stress in regulating influx or efflux of nutri-
ents and ions for improved cell growth and lipid accumulation (Shah et al.,
2014b).
8.4.1.6MIXING
In large scale cultivation, almost all available light may be absorbed only by
a thin top layer of algal cells. This can be avoided by having proper mixing to
keep the cells in motion, keeping consistent exposure to light and nutrients.
While light reduction inside the reactor is not influenced by mixing, there
is a complex interaction between culture mixing and the light attenuation as
each single algal cell passes through dark and light zones of the reactor in a
more or less statistical manner (Barbosa et al., 2003). Mixing is necessary
to prevent algae sedimentation, avoiding cell attachment to the reactor wall
for equal cell exposure to the light and nutrients, improving gas exchange
between the culture medium and the air, and reducing the boundary layer
around the cells, facilitating increased uptake, and exudation of metabolic
products (Lou and Al-Dahhan, 2004; Richmond, 2004; Kommareddy and
Anderson, 2005; Carvalho et al., 2006).
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8.4.2 REACTOR ENGINEERING
8.4.2.1 OPEN AND CLOSED SYSTEM
For CO2 sequestration, issues that need to be addressed include how effi-
ciently the microalgae could use CO2 in order to avoid excess release into
the atmosphere, the design of effective reactor system, and irrigation, plant-
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ing, fertilization, and harvesting. Since the 1950s, open ponds consisting of
natural waters (lakes, lagoons, and ponds) and artificial ponds or containers
have been used for algae cultivation (Borowitzka, 1999). The major advan-
tage of open pond is it does not compete for land with accessible agricultural
crops, but can be implemented in areas with marginal crop production po-
tential (Chisti, 2007). It also has lower energy input requirement (Rodolfi et
al., 2008), and easy regular maintenance and cleaning (Ugwu et al., 2008)
with greater potential to return large net energy production (Rodolfi et al.,
2008). Raceway ponds, typically closed loop, with mixing and oval shaped
recirculation channels, between 0.2 and 0.5m deep to stabilize algae growth
and productivity, are the most frequently used artificial system (Fig. 8.4).
Raceways ponds are usually built in concrete, but compacted earth-lined
ponds with white plastic have been reported (Jimnez et al., 2003). In a con-
tinuous production cycle, algal broth and nutrients are introduced at the front
of the paddlewheel and circulated through the loop to the harvest extraction
point. The paddlewheel is in continuous operation to provide mixing and
FIGURE 8.4 Plan view of a raceway pond. Algae broth is introduced after the
paddlewheel, and completes a cycle while being mechanically aerated with CO2. It
is harvested before the paddlewheel to start the cycle again (Chisti, 2007).
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avoid sedimentation. The CO2 requirement is usually from the surface air,
but submerged aerators can be installed to improve distribution (Razzak et
al., 2013). For open and outdoor culture, the ability to control temperatures,
however, is limited due to atmospheric temperature, solar irradiance, and
humidity. To sequester industrial CO2 outputs, it has to take into account
that during night time and cloudy days, algal reproduction rates slowdown,
thus taking up less CO2. Installation of gas storage facilities to cope with the
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influx of CO2 at night may be necessary.
Pollution and contamination risks may preclude open pond for the prepa-
ration of high-value products in the cosmetics and pharmaceutical industries
(Ugwu et al., 2008). Various species in open system with different utiliza-
tion efficiencies may also directly release excess CO2 into the atmosphere,
while closed systems allow for easy control of cultivation and CO2 release
(Brennan, 2010). A closed-loop system with innovative uses of resources
can lead to both more sustainable and cheaper production. PBR technol-
ogy can overcome some of the major problems associated with open-pond
systems. PBRs such as tubular, flat-plate, and column reactors allow culture
of single-species for prolonged periods with lesser risk of contamination
(Chisti, 2007). Comparison of different closed PBR systems and biomass
productivities is shown in Table 8.1. Flat-plate PBRs especially have re-
ceived much attention for mass cultures due to the large surface area exposed
TABLE 8.1 Comparison of Different Closed Photobioreactor Systems.

Photobioreactor Light Capacity Algal Strain Biomass (g/L) References
Type Source (L) a
mg/(L.d)b
Tubular Solar 500 Scenedesmus 284b Hulatt and
radiation obliquus Thomas (2011a)
Artificial 1.4 Dunaliella 830b Hulatt and
tertiolecta Thomas (2011b)
Artificial 0.26 Anabaena 750b Yoon et al.
variabilis (2011)
Artificial 2 Chlorella sp. 111.8b Rasoul-Amini et
al. (2011)
Airlift Artificial 3 Haematococ- 4.09a Kaewpintong et
cus pluvialis al. (2007)
Artificial 170 Chaetoceros 0.80a Krichnavaruk et
al. (2007)
Artificial 3.7 Chlorella 750b
vulgaris
Artificial 3.2 Scenedesmus 2.27a Sarah and Jones
sp. Susan (2014)
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Photobioreactor Light Capacity Algal Strain Biomass (g/L) References

Type Source (L) a
mg/(L.d)b
Bubble column Artificial 200 Chlorella 31.55b Wang et al.
ellipsoidea (2014)
Artificial 1.8 Cyanobium sp. 0.071a Henrard et al.
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(2011)
Artificial 3.5 Spirulina 4.13a De Morais and
Costa (2007)
Artificial 50 Fistulifera sp. 0.50a Reiko et al.
(2014)
Artificial 1.8 Sc. obliquus 2.12a De Morais et al.
(2007)
Artificial 1.5 Spirulina 1.83a Ankita et al.
platensis (2014)
Flat plate Articial 3.4 Dunaliella 1.5a Barbosa et al.
(2005)
Solar 60 Chlorella 41.3b Feng et al.
radiation zofingiensis (2011)
Articial 15 Chlorella 1.3a Huang et al.
pyrenoidosa (2014)
Articial 440 Chlorella 124b Qiang et al.
pyrenoidosa (2014)
to illumination (Samson et al., 1985; Ugwu et al., 2008), high densities of

photoautotrophic cells (>80gL1) (Hu et al., 1998), low accumulation of
dissolved oxygen, and high photosynthetic efficiency as compared to tubular
PBRs (Richmond et al., 2000). The reactors are made of transparent materi-
als for maximum solar energy capture, and a thin layer of dense culture flows
across the flat plate, allowing radiation absorbance in the first few millimetre
thickness (Hu et al., 1998; Richmond et al., 2008). Closed flat panels mixed
by bubbling air can achieve overall groundaerial productivity as high as
0.27gL1d1 using 500 L flat-plate glass PBR with 440 L culture volume
(Richmond and Cheng, 2001).
Tubular PBRs consist of an array of straight transparent tubes that are
usually made of plastic or glass. This tubular array or the solar collector
captures the sunlight for photosynthesis (Fig. 8.5). The solar collector tubes
are generally less than 0.1m in diameter to enable light to penetrate into
a significant volume of the suspended cells. Algal broth is circulated from
a reservoir (such as the degassing column) to the solar collector and back
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to the reservoir (Chisti, 2007, 2008). Tubular PBR is typically operated as

a continuous culture during daylight and cannot be scaled-up indefinitely.
There is also a design limitation on the length of the tubes, which affects the
potential O2 accumulation, CO2 depletion, and the pH difference (Eriksen
et al., 2008). Large closed tubular PBRs include the 700m3 plant in Klotze,
Germany (Pulz et al., 2001) and the 25m3 plant at Mera Pharmaceuticals,
Hawaii (Olaizola, 2000). The performance of column PBRs compares fa-
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vourably with tubular PBRs (Sa et al., 2002) as they offer efficient mixing,
high volumetric mass transfer rates, low cost, compacted and controllable
growth conditions (Eriksen et al., 2008). The vertical column is aerated from
the bottom, and illuminated through transparent walls (Eriksen et al., 2008),
or internally (Suh et al., 2003).
FIGURE 8.5 A tubular photobioreactor with fence-like solar collectors (Chisti, 2007, 2008).
8.4.2.2 LIGHT PENETRATION
Artificial production should attempt to replicate and improve the optimum

natural growth conditions where phototrophic algae take up sunlight, absorb
CO2 from the air, and nutrients from aquatic habitat. Algae need light within
the photosynthetically active radiation (PAR) to obtain energy by photosyn-
thesis (Fernandes et al., 2010). The wavelength of the PAR ranges from 400
to 700nm, which is equal to the visible light (Kommareddy and Anderson,
2003). The attenuation of light intensity in the PBR is dependent on its
wavelength, cell concentration, reactor geometry, the light penetration dis-
tance and the path length, and the algal light absorption (Grima et al., 1994;
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Fernandes et al., 2010; Bitog et al., 2011). In a dense culture, the gradient
of light varies along the radius of the PBR (Grima et al., 1994; Bitog et al.,
2011). The use of natural conditions for commercial algae production should
take advantage of using sunlight (Janssen et al., 2003). For outdoor produc-
tion systems, light is generally the limiting factor (Pulz and Scheinbenbogan,
1998) as this will depend on sunlight availibility which varies with diurnal
cycles and seasons; thereby limiting the viability of commercial production
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to areas with high solar radiation. To address the limitation, artificial means
of employing fluorescent lamps are almost exclusively used for cultivation
at pilot scale stages (Janssen et al., 2003). It becomes essential to know the
absorption spectra of main algal accessory pigments present in different
quantities in different algal groups. Diatoms generally have photosynthetic
pigments that contain chlorophylls a and c, and fucoxanthin whereas green
algae contain chlorophylls a and b, and zeaxanthin (Brennan et al., 2010;
Bitog et al., 2011). Artificial lighting allows for constant production, but
at considerably higher energy input. Frequently the electricity supply for
artificial lighting is derived from fossil fuels thus negating the primary aim
of developing a price-competitive fuel and increasing the systems carbon
footprint. Because of this, although tubular PBRs are deemed more suitable
for outdoor mass cultures since they have larger surface area to sunlight,
large-scale production plants are ideally based on the combination of mul-
tiple reactor units to compensate for any limitation of individual unit such as
the possible alternate use of sunlight and artificial lights.
8.4.2.3 GAS INJECTION
Aeration of CO2-rich gas through PBR provides CO2 to the algae, aids in
deoxygenating the suspension, and provides mixing to increase the cycle
frequency. For very dense cultures, CO2 originating from the air can be lim-
iting for algae that pure CO2 supplemented to the air supply is a necessity.
CO2 addition buffers the water against pH changes as a result of the CO2/
HCO3 balance (Chen and Durbin, 1994). From economic point of view, a
high aeration rate will lead to higher running costs and therefore not always
recommended for large-scale application (Zhang et al., 2002; Bitog et al.,
2011). Air enriched with 5 or 10% (v/v) CO2 at 0.025vvm (volume of air/
medium volume/time) may be cost effective for mass culture (Zhang et al.,
2002). In a flat panel PBR, optimum aeration rate of 0.05v/v has been pro-
posed as sufficient to improve the mixing and mass transfer (Sierra et al.,
2008).
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8.4.2.4 HYDRODYNAMIC CONDITIONS
Hydrodynamic conditions play major role in ensuring light intensity de-

livery, sufficient CO2 transfer, maintaining uniform pH (Kommareddy
and Anderson, 2005), and enhancing biomass productivity (Lou and Al-
Dahhan, 2004). Poor hydrodynamic condition leads to cell aggregation and
three phase system (solidliquidgas) that is prone to reduce mass transfer
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(Panda et al., 1989). The productivity of S. dimorphus in a plate PBR is
reportedly three times lower than the determined highest productivity of
0.54gL1d1, attributable to the lower air volume in the plate PBR, and
consequently poor mixing regime. However, too high a mixing rate may
lead to shear-induced injury on cells and affects cell viability (Panda et al.,
1989; Thomas and Gibson, 1990; Gudin and Chaumont, 1991; Carvalho et
al., 2006). In a bubble column and airlift reactor, hydrodynamic condition is
characterized by axial dispersion coefficient, mixing and circulation time,
and Bodenstein number (Miron et al., 2004). The longer duration for air
to reside in the medium with smaller bubbles in the bubble column reac-
tors should result in higher mass transfer. A vertical glass tube with 5cm
diameter and 2.3m height (4.5L) established as a bubble column reactor
with good light penetration achieves Monoraphidium productivity as high
as 23gm3d1 (Miyamoto et al., 1988). Bubble column has shorter mixing
time than airlift reactor, but an airlift reactor is favoured because of its
efficiency and biomass productivity (Fan et al., 2007; Oncel and Sukan,
2008; Ranjbar et al., 2008). Airlift reactor has more defined fluid flow and
relatively higher gasliquid mass transfer rates, while a bubble column is
likely to cause uneven cell density along the length of the reactor, which
may induce cell starvation and death (Fan et al., 2007). The light penetra-
tion path is also wider for bubble column than airlift PBR because of the
longer path to the center of the column, and the cloud effect caused by cha-
otic rising bubbles in the column (Oncel and Sukan, 2008). The presence
of a draft tube in airlift reactor results in a more effective mixing because
of the internal loop for the culture to circulate through the draft, and down
through the annulus between the housing column and outside the draft tube
(Oncel and Sukan, 2008). Bubbles rising inside the draft tube provide less
turbid zone in the annulus region, enabling better exposure to light. The
growth and the productivity in a translucent vertical airlift PBR is reported
at 109264gm3d1 for Nannochloropsis and between 32.595.3gm3d1 for
the Chlorella strain (James and Al-Khars, 1990).
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8.4.2.5 HYDRAULIC RETENTION TIME (HRT) AND OLR
Retention time is the time required to degrade the organic matter completely
depending on the process temperature and batch composition, and it is relat-
ed to the microbial growth rate. The average retention time for waste treated
in a mesophilic plant is 1530 days and a bit shorter for thermophilic plant
(Ekama et al., 2008). There are two types: the solid retention time (SRT) for
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the average time the bacteria (solids) are in the anaerobic digester, and the
HRT. These should be sufficient for the active populations, especially the
methanogens, and not limiting the hydrolysis which is usually the limiting-
step of the overall conversion of complex substrates to methane. HRT and
OLR are the main parameters in anaerobic digestion processes. OLR is the
amount of VSs to be fed into the digester each day in a continuous process.
As the OLR increases, the biogas yield increases to some extent. Above the
optimal OLR, the VS degradation and biogas yield decrease due to overload-
ing (Babaee et al., 2011). For slow biodegradability, HRT is an important
deciding factor (Yen and Brune, 2007). When operated at high OLR and
HRT, the methane yield is stable and maximal, but at lower OLR or mini-
mum HRT, the methane yield is reduced. Optimal OLR and HRT depend on
the type or composition of the algal substrate. When the cells are directly in-
oculated into the anaerobic process, accessibility of the intracellular content
to the anaerobic microflora is limited by the resistance of the algal cell wall
to hydrolysis. Thus, characteristics of the species make the difference for a
given OLR or HRT (Yen and Brune, 2007; Sialve et al., 2009). For efficient
conversion of organic matter, OLR and HRT must be chosen depending on
the type or composition of the algal substrate.
8.4.2.6 OPERATING CONDITIONS
Table 8.2 summarizes the conditions and the corresponding methane conver-
sion yield. Two main approaches can be developed: (1) a multi-specific bio-
mass harvested from waste water treatment pond (Leadbeater, 2006); and (2)
a mono-specific biomass grown in the laboratory (Munozn and Guieysse,
2006). The methane yield varies from 0.09 to 0.45Lg VS1 depending on
the species and culture conditions, where the CH4 proportion is 6975%
regardless of species and operating conditions. The most significant factor
impacting CH4 proportion is pH, which controls the speciation of the carbon-
ate system and the release of CO2. If the pH is high from NH3 release during
digestion, the gas content will shift more to CH4. The oxidation state of the
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TABLE 8.2 Thermal Pretreatment for Microalgae Biogas Production.

Microalgae Reactor Methane Pretreat- Solubi- Methane References
Species Conditions Yield (L ment lization Yield
CH4 g Conditions Increase Increase
VS1) (%)
Thermal CSTR, 28 0.270b 100 oC; 8 h n.d. 33 Chen and
pretreatment days Oswald
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Microalgal HRT (1998)
biomassa
Scenedesmus BMP 0.076c 70, 90 oC; 7, 11-fold 12, 220 Gonzlez-
biomass 3h Fernndez et
al. (2012)
Microalgal CSTR, 20 0.180 75, 95 oC; n.d. 67, 72 Passos
biomassa days 10 h and Ferrer
HRT (2014)
Chlorella sp. BMP 0.336 120 oC; 29% 20 Cho et al.

and Scenedes- 30 min (2013)
mus sp.
Nannochlo- CSTR 0.130 100120 oC; n.d. 108 Schwede et
ropsis salina 2h al. (2013)
Acutodesmus BMP 35 33 0.97 0.26 Alzate et al.
obliquus and (2012)
Oocystis sp.
Scenedesmus BMP 0.180 170 oC; 8 10-fold 81 Keymar et
biomass bar; 30 min al. (2013)
Chlorella BMP 0.156 160 oC; 20 4.5-foldd 65 Mendez et
Scenesmus min al. (2014)
a
Microalgal biomass grown in wastewater treatment open ponds; bData expressed in L
biogas/g VS; cData expressed in L CH4/g COD; dData from solubilization of carbohydrates.
biomass which drives the proportion of methane released also influences

the biogas quality. Microalgae have somehow received less consideration as
compared to macroalgae as substrates for anaerobic digestion, although they
contain sulphurated amino acids that digestion releases lower amount of hy-
drogen sulphide than the organic substrates. There is, however, the potential
presence of ammonia in the biogas due to the high protein content (~50
60%) that hydrolysis may lead to high ammonium concentration which can
be toxic to methanogens (Munozn and Guieysse, 2006). The increase in tem-
perature from 15 to 52C improves methane production by Spirulina max-
ima, but the energetic balance becomes negative if the energy supplied for
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heating is considered. The productivity, mutually with the VS reduction, is

enhanced with increment upto 35C. For multi-specific algae, a temperature
increase from 35 to 50C improves the rate of algal biodegradability from
5 to 10%. Maximal methane productivity is achieved at 40C suggesting
mesophilic temperatures as optimal for anaerobic digestion of microalgae
(Chen, 1987; Sialve et al., 2009).
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8.4.2.7 REACTOR SCALE AND CONFIGURATION
The effects of photoperiod, salinity and pH on growth, and lipid content

of Pavlova lutheri microalgae for biodiesel production in small-scale and
large-scale open-pond tanks have been reported (Shah et al., 2014b). The
cultures grow well under 24h illumination in 250mL flask with the high-
est cell density of 13.314.1106 cells mL1 and dry weight of 0.45gL1,
achieving max of 0.12d1 and lipid content of 35% as compared to 0.1d1
and 15% lipid in the dark. The salinity is optimum for the cell growth at
3035ppt, but the lipid content of 3436% is higher at 3540ppt. Algal
growth and lipid accumulation is optimum at pH 89. Cultivation in 5L and
30L tanks achieve max of 0.130.14d1 as compared to 0.12d1 in small-
scale and 300L cultures. Comparing between 5L PBR and 300L tank under
19.324h illumination and light intensity of 162198mol photons m2s1,
the highest cell density and biomass are shown by N. oculata at 82.6106
and 63.7106 cells mL1 density with 0.96 and 0.72gL1 biomass, respec-
tively, followed by T. suecica (59106, 42.7106 cells mL1 density with
0.73, 0.58gL1 biomass, respectively), and I. galbana and P. lutheri (19.6
21.2106, 15.115.9106 cells mL1 density with 0.520.66gL1 biomass,
respectively). The lipid contents, however, are higher in 5L PBR at 40.1 and
41.8% as compared to 30.7 and 32.1% in 300L open tank for N. oculata and
P. lutheri, respectively. Fatty acid profile for N. oculata suggests that hep-
tadecanoic acid C17:0 (13.7%) and oleic acid C18:1 (7.4%) are enhanced in
PBR, but palmitic acid C16:0 (22.1%) and palmitoleic acid C16:1 (9.9%)
are reduced. Although the total saturated fatty acids (SFA) (57.0%) and
monounsaturated fatty acids (MUFA) (17.7%) are comparable to previous
study on optimum pH and salinity, PUFA (22.3%) is enhanced. For P. lutheri
in PBR, palmitic acid C16:0 (34.4%) is higher than in 300L tank, while
both eicosapentaenoic acid (EPA) C20:5 (8.4%) and docosahexaenoic acid
(DHA) C22:6 (6.9%) are slightly increased with the total SFA (47.9%) and
MUFA (30.9%) comparable, but PUFA (18.9%) is elevated (Shah, 2014).
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8.4.3 INTEGRATED PROCESS ENGINEERING
8.4.3.1 HARVESTING AND EXTRACTION
The reason algal fuels have yet to replace the fossil fuels is due to the cost of
production and the major bottleneck for algal-based bulk commodities lies
in efficient cell harvesting method. The feasibility for commercialization
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is hampered by high energy input requirement especially in the upstream
processes. Life cycle assessment (LCA) on microalgae biodiesel produc-
tion shows negative energy balance due to high energy input to harvest and
dry the biomass (Sander et al., 2010). The economics can be improved with
efficient cultivation technique and simple, low energy, and cost-effective
downstream processing. Harvesting from either open pond or PBR also need
to separate out the media and algae in the quickest way. Different physical-,
chemical-, and biological-based methods can be applied depending on the
type of algae, the requirements of the cell sizes and biomass concentrations
(Salim et al., 2012). Among conventional methods are centrifugation, filtra-
tion, gravity sedimentation, flotation, and flocculation. Continuous centrifu-
gation is currently the preferred process as it is rapid and efficient (Rawat
et al., 2011), but requires high energy and a primary concentration step.
Filtration making use of packed bed filters can be used with or without ad-
ditional pressure and works best at low algal cell concentrations (Greenwell
et al., 2010). Membrane processes have long been applied in different stages
of algal cultivation and processing. These include cross-flow microfiltration,
ultra-filtration, dialysis, forward osmosis, membrane contactors, and mem-
brane spargers. Cross-flow, micro, and ultra-filtrations may have the same
efficiency as centrifugation, but possibly at a much lower cost (Brennan and
Owende, 2010; Greenwell et al., 2010; Mata et al., 2010). It can be imple-
mented both as a standalone and as a coupled system [in membrane biomass
retention PBRs (BR-MPBRs) or membrane carbonation PBRs] (C-MPBRs)
(Bilad et al., 2014a). For smaller suspended algae, tangential flow filtra-
tion is more sufficient than dead-end filtration, but the major drawbacks are
membrane fouling, huge costs of replacement (Uduman et al., 2010), and
high power requirements (Danquah et al., 2009). Filtration by microstrain-
ers is another commonly used solidliquid separation technique but the
problems encountered include incomplete solids removal and membrane
fouling by bacterial biofilms. Although the first problem may be solved by
using flocculation, regular cleaning or membrane replacement generate siz-
able costs (Rawat et al., 2011). The main benefit is in having no chemical
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additions, but the high power requirements do not make centrifugation and
filtration attractive for large-scale applications (Uduman et al., 2010).
Sedimentation is a low cost harvesting option that can typically give con-
centrations of 1.5% solids (Uduman et al., 2010). Because of the fluctuating
density of algal cells, reliability is also low (Shen et al., 2009). At settling
rates of 0.12.6cmh1, sedimentation is relatively slow, and much of the
biomass may deteriorate during the settling time (Greenwell et al., 2010).
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Gravity sedimentation is simple and highly energy-efficient (Rawat et al.,
2011), but only work for a relatively large size and that grow to high densi-
ties such as Arthrospira sp., or when the pH is increased and/or chemical
flocculants are added to the water (Knuckey et al., 2006; Chen et al., 2011).
The latter, however, incur additional cost. To induce flocculation, organic
and inorganic flocculants can be applied (Vandamme et al., 2013). Another
option is to induce auto-flocculation by interrupting or limiting CO2 supply
(Demirbas, 2010). Induced flocculation may only be successful for fresh-
water species (Vandamme et al., 2013), while flocculation of marine micro-
algae has been suggested as not-feasible (Vandamme et al., 2013). A study
has actually shown that cationic polymeric flocculants are viable options
to pre-concentrate marine cultivated microalgae before further dewatering
(Lam et al., 2014). Different cationic polymeric organic flocculants have
been tested on Phaeodactylum tricornutum and Neochloris oleoabundans
grown under marine conditions. The effects of 10ppm of the commercially
available Zetag 7557 and Synthofloc 5080H flocculants on P. tricornutum,
followed by 2h sedimentation, show a recovery of 98 and 94%, respec-
tively. The same flocculants and dosage for harvesting N. oleoabundans only
achieve 52 and 36% recovery, respectively. Use of bioflocculant in combina-
tion with sedimentation can reduce energy demand based on centrifugation
from 13.8 to 1.34MJkg1 W (Salim et al., 2012). Chitosan is an example of
promising flocculant due to its high efficacy, low dose requirements, and
short settling time (Naim et al., 2013). The recovery efficiency of C. vulgaris
at 120mgL1 chitosan achieves the highest harvesting efficiency (HE) of
92% within 3min, with the highest HE (99%) at pH 6.
Direct air flotation (DAF) is often used as an efficient clarification step,
notably when treating water containing hydrophobic matter and algae
(Demirbas, 2010; Sturm and Lamer, 2011). It involves injecting air at the
bottom of a water column to form an upward stream of bubbles. Tiny air
bubbles may attach to the algal surface and carry them to the surface, form-
ing a concentrated layer of foam which can be separated by skimming. The
main cost is related to the power required for air injection but chemical floc-
culation is often necessary prior to DAF which further increases the total
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harvesting costs (Christenson and Sims, 2011). Flotation under vacuum us-
ing a vacuum gas lift can be carried out at different airflow rates, bubble
sizes, salinities, and harvest volumes. HE and concentration factor (CF) in-
crease by around 50% when the airflow rate of the vacuum gas lift is reduced
from 20 to 10Lmin1. Reduced bubble size enhances HE and CF by 10
times when specific microbubble diffusers are used or when the water salin-
ity is increased from 0 to 40%. The reduction in harvest volume from 100 to
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1L increases the CF from 10 to 130. An optimized vacuum gas lift allows
partial microalgae harvesting using less than 0.2kWhkg1, thus reducing
the energy costs by 10100 times as compared to complete harvesting pro-
cesses, albeit at the expense of a less concentrated biomass (Bertrand et al.,
2013). Ozoflotation is used to recover microalgae grown in treated waste-
water where 79.6% as TSS of biomass can be harvested with ozone dose of
0.23mg mg-1 of dried biomass. The amount of lipid extracted and fatty acids
methyl esters (FAME) recovered doubles with ozone doses of 0.120.23mg
mg-1 of dried biomass as compared to when using centrifugation. The oxi-
dative stability of biodiesel can also be enhanced by the effect of ozone on
the degree of FAME saturation (Velasquez-Orta et al., 2014). Separation us-
ing ozoflotation is advantageous over dissolved air flotation and gas bubble
flotation because it does not require flocculant or lower pH (Ya-Ling et al.,
2010; Nguyen et al., 2013).
Magnetic separation can be applied for quick, simple, efficient, and reli-
able capture of cells and biomolecules from liquid solution by the functional
magnetic particles driven by an external magnetic field (Yang et al., 2009).
It can save time and energy associated with harvesting (Ling et al., 2011). A
simple and rapid harvesting method by in situ magnetic separation has been
developed where Fe3O2 nanoparticles are added to culture both leading to
the cells of Botryococcus braunii and Chlorella ellipsoidea adsorbed and
then separated by an external magnetic field. Maximal recovery efficiency
of more than 98% is achieved at 120rpm stirring within 1min. Maximal
adsorption capacity on Fe3O4 nanoparticles are 55.9mg dry biomass mg1
particles for B. braunii and 5.83mg dry biomass mg1 particles for C. el-
lipsoidea. Appropriate pH and high nanoparticle dose are favorable for good
cell recovery. The functional nanocomposites provide a base for efficient
microalgae harvesting with advantages such as rapid execution, low energy
consumption, and improves water-use in the algal harvesting process. Fe3O4
nanoparticles functionally coated with polyethylenimine (PEI) contain high
concentration of ANH2 groups, for efficient harvesting of microalgae. The
functional magnetic nanocomposites are 12nm in diameter with 69.77emu/g
of saturation magnetization. For harvesting Chlorella ellipsoidea cells, the
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nanocomposite dosage of 20mgL1 achieves 97% HE within 2min, and

increasing the temperature could increase the HE. The adsorption capacity
of the Fe3O4PEI nanocomposites for the microalgal cells reaches 93.46g
DCW g1 nanocomposites. The adsorption mechanism between naked Fe3O4
nanoparticles and the microalgal cells is through electrostatic attraction and
nanoscale interactions (Yi-Ru et al., 2014). For simultaneous algal biomass
harvesting and cell disruption, cationic surfactant-based harvesting and cell
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disruption (CSHD) method may be effective. With CSHD, the HE is more
than 91% in less than 5min and 97% in 90min. Moreover, CSHD exhibits
powerful ability to disrupt the cells with lipid recovery from the cells in-
creases by 133% allowing the extraction of up to 100% of the total lipids
from wet microalgal biomass with 80% water content (Wen-Can and Jong-
Duk, 2013).
8.4.3.2 IMMOBILIZATION AND RECYCLING
Immobilizing microorganisms in a variety of matrices has been applied

to enhance nutrient and heavy metal removal and to treat hazardous con-
taminants (Lebeau and Robert, 2006; Muoz and Guieysse, 2006; Moreno-
Garrido, 2008). Similar to biofiltration, there is a physical barrier between
microorganisms and the surrounding environment. The most common way
is through gel entrapment and encapsulation in polymers, in which natural
polysaccharides such as agars, carrageenans, and alginates are used such
as for microbial inoculants in agriculture (Bashan, 1998) due to their low
toxicity and high transparency (Moreira et al., 2006; Ignacio et al., 2008;
Moreno-Garrido, 2008; Cao et al., 2010). The microorganisms can be im-
mobilized alive within the polymer because its pores are smaller than the
microorganisms, while the fluid flows through and sustain cell metabolism
and eventual growth (Cohen, 2001). Comparable heavy metal uptake to the
free, non-immobilized biomass (Alejandro et al., 2010) has been reported for
biomass immobilized in Ca alginate (Sag et al., 1995) and polyacrylamide
(Nakajima et al., 1982; Wong et al., 1993). Most immobilization techniques
can be easily modified and applied to algae, adding a design factor for the
requirement of light. The polymer is mixed with microalgae cells and conse-
quently stabilized with divalent ions to form immobilized microalgal beads
through a nozzle. Freshwater C. vulgaris has been immobilized in alginate
beads and found suitable for simplifying the overall separation process (Lan
and Lee, 2012a). Since the immobilized beads are relatively large in size as
compared to the free cells, a simple filtration method (e.g. sieving) would be
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sufficient to separate the beads from water without significant amount of en-
ergy input. Handling of microalgae biomass will become easier and feasible
to be implemented in commercial scale. Applications are diverse including
for organic pollutants and heavy metal removal and biosensor for toxicity
measurement (Moreira et al., 2006; Ertgrul et al., 2008; Ruiz-Marin et al.,
2008; Vijayaraghavan et al., 2011).
Co-immobilization of microalgae and nutrients such as plant-growth-
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promoting bacteria is a solution for the low growth rate of immobilized
microalgae (Gonzalez and Bashan, 2000). An entrapment matrix in which
algal cells are embedded and grown can be employed at the beginning of
the cultivation. For entrapment matrix using filamentous fungi, pelletization
with Aspergillus niger under photoautotrophic and heterotrophic growth
conditions is used to immobilize and grow the freshwater C. vulgaris (Zhang
and Hu, 2012) where 63 and 24% of C. vulgaris are harvested, respectively.
Pelletization of oleaginous filamentous fungi with microalgae may contrib-
ute to the enhancement of the total oil yield and the fatty acid quality. For
cell recycle, a new and effective microalgae cultivation and pre-harvesting
have been developed using a membrane PBR (MPBR) in which the bioreac-
tor is coupled to membrane filtration. The membrane completely retains C.
vulgaris and the biomass can be partly recycled into the bioreactor to main-
tain a high biomass concentration, enhancing the flexibility and robustness
of the system. MPBR can operate at both higher dilution and growth rates,
resulting in 9-fold increase at 0.42gL1 biomass. Pre-harvesting is achieved
by applying variable CFs in the filtration stage. The permeate is recycled to
the reactor as feed medium without affecting the algal growth, offering a
substantial 77% reduction in the water footprint (Bilad et al., 2014b). Owing
to the higher biomass productivities, the harvesting costs can be consider-
ably reduced.
8.4.3.3 WASTE TREATMENT AND BIOENERGY CO-GENERATION
The integrated microalgaebacterial system can improve the feasibility

of microalgae biomass production for its further valorization (Gonzlez-
Fernndez et al., 2011). Two 5L PBRs used to treat wastewater from potato
processing industry (RPP) and from a treated liquid fraction of pig ma-
nure (RTE) inoculated with Chlorella sorokiniana and aerobic bacteria at
242.7C and 6000 lux for 12h per day of light supply achieve the highest
biomass of 26.3mgL1d1 with RTE wastewater but with lipid content reach-
ing 30.2% in RPP and only 4.3% in RTE. Methane yield is highly influenced
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by the lipid content and the substrate/inoculum ratio where maximum meth-
ane yield of 518mLg1 COD is achieved at 30% lipid and the substrate/
inoculum ratio of 0.5 (Hernndez et al., 2013). In a study where auto/mixo-
trophic growth is evaluated by using domestic wastewater (WW) amended
with glycerol, C. vulgaris and Botryococcus terribilis show biomass pro-
ductivities of 118 and 282mgL1d1, producing 18 and 35mgL1d1 of lipids,
respectively, at the highest glycerol supplements (50mM). If scaled-up to
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200m3d1 of WW for 240 days per year, the estimated biomass and lipid
yields turn out to be 5.6 tonnes y1 and 0.9 tonne y1, for C. vulgaris, or 13.5
tonnes y1 and 1.6 tonnes y1 for B. terribilis, respectively. The mixotrophic
production of lipids can generate high-quality biodiesel based on the estima-
tion fatty acids profiles and the whole process can be combined with the
production of methane and bio-ethanol in a bio-refinery set-up (Cabanelas
et al., 2013).
Co-digestion leads to the dilution of toxic compounds by maintaining
reaction under toxic threshold level. Some co-substrates can stimulate enzy-
matic synthesis for improved anaerobic digestion yield. Methane production
rate of 1.61L L1d1 has been reported under mesophilic condition when
algal sludge is mixed with 60% waste paper with the OLR of 5 gVS L1d1
(Yen and Brune, 2007). Digestion of algal cell walls with waste paper has
a positive effect on the anaerobic digestion, an indication of the increased
cellulase activity stimulated by specific nature of the waste paper. Increasing
the C/N ratio from 4.2 to 6.2 using co-digestion of S. maxima with sewage
sludge, enhance methane yield to 60%. Co-digestion of algae with effluent
from canning facility and protein-extracted algae achieve optimal methane
production of 62% at C/N ratio between 25 and 35. The optimal C/N ra-
tio is between 20 and 35, close to the prescribed range known to have a
positive effect on the methane yield. Lower ratios lead to potential inhi-
bition due to the presence of excessive ammonia released whereas higher
ratios may lead to nitrogen limitations (Golueke et al., 1957). Algal biomass
can be fermented directly with co-fermentation of residual algae biomass
after lipid extraction. Produced methane is comparable to the pig manure
substrate co-cultivation with Chlorella sp. producing 0.45m methane kg1
dry weight and the reported yield methane is typically around 0.3Lg1 VS,
which is about half of the theoretical maximum based upon biochemical
composition of biomass (Kelly and Dwarjanyn, 2008). The low yield can
be attributed to the recalcitrance of a few algal species to biodegradation
and inhibition of microbiological conversion by ammonia released from the
biomass. Mesophilic reactors have shown concentrations below ammonia
toxicity with HRT of 2030 days and OLR of 12kg VS m3d1 (Schwede et
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al., 2013; Passos and Ferrer, 2014). This is more critical under thermophilic
conditions with higher possibility of NH3 release. The tough algal cell wall
may prevent high methane production, since organic matter retained in the
cytoplasm is not easily accessible to anaerobic bacteria. Many organic sub-
strates such as activated sludge and lignocellulosic biomass similarly con-
sist of a complex structure, which reduces hydrolysis rate in the anaerobic
digestion process (Carrre et al., 2010; De La Rubia et al., 2013; Monlau et
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al., 2013). Suitable pre-treatment must be developed to solubilize particulate
biomass and improve anaerobic digestion rate and extent. Thermochemical
and mechanical pre-treatments can solubilize biomass by breaking down
cell walls which are resistant to biodegradation. These result in significant
increase in methane production rates (ca. 30%) for sewage ponds treated
with microalgal biomass (Chen and Oswald, 1998).
The cost of closed system is substantially higher than the open-pond sys-
tem (Carvalho et al., 2006). The unit cost of producing Dunaliella salina,
one of the commonly cultivated algae strains in an open-pond system is
about $2.55kg1 of dry biomass, which is considerably too high to justify the
production for biofuels. LCA and life cycle costing (LCC) have examined
the integrated algae bioenergy production and nutrient management on small
dairy farms. The most important challenge is in the procurement of low cost
and low energy-intensive nutrients, most notably nitrogen and phosphorus.
Nutrient procurement can represent up to 50% of energy consumption during
algae cultivation when fertilizers are used (Clarens et al., 2010; Stephenson
et al., 2010). Four cases are being considered: a reference land application
scenario (REF), anaerobic digestion with land-application of liquid digestate
(AD), and anaerobic digestion with recycling of liquid digestate to either an
open-pond algae cultivation system (OPS) or an algae turf scrubber (ATS).
LCA indicates that all the three improved scenarios (AD, OPS, and ATS)
are environmentally good as compared to REF, exhibiting increases in net
energy output up to 854GJ y1, reductions in net eutrophication potential
up to 2700kg PO4-eq y1 and reduction in global warming potential up to
196Mg CO2-eq y1. LCC reveals that the integrated algae systems are much
more financially attractive than either AD or REF, where the net present
values (NPV) are estimated at $853,250 (OPS), $790,280 (ATS), $211,126
(AD) and $62,279 (REF). However, these results are highly dependent on
the sale price for nutrient credits. Comparison of LCA and LCC results indi-
cates that robust nutrient credit markets or other policy tools are required to
align financial and environmental management, preferably with the ability
for energy production systems and foster widespread adoption of sustainable
nutrient management systems (Zhang et al., 2013).
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8.5 CASE STUDY PALM OIL MILL WASTES
8.5.1 OIL PALM WASTES AND UTILIZATION
It is estimated that more than 50 million tonnes of biomass are generated

from the palm oil industry in Malaysia alone, mostly from the oil extraction
process. This is about 4kg of dry biomass from every kg of palm oil pro-
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duction, which comes in the form of the mesocarp fiber, empty fruit bunch
(EFB) palm oil mill sludge (POMS), palm kernel cake (PKC), decanter
cake, and POME. Most have not been extensively commercially re-used by
the industry (Habib, 1979; Wu, 2009). Other solid residues generated during
harvesting and replanting are shells, fronds, and trunks from the plantation
area. In 2010, out of 88.74Mt of fresh fruit bunches (FFB) processed, ap-
proximately 87Mt of biomass being produced, excluding the fronds and
trunks (Wu et al., 2007). With high moisture content of 6070%, EFBs are
difcult to use as fuel for power boilers but the shells, mesocarp and fibers
can be used as boiler fuel to produce steam and electricity for the mills and
for power generation. Partial EFB and decanter cake are utilized as fertiliz-
ers and soil cover materials in plantation areas. In Thailand, about 60 crude
palm oil mills produce approximately 1.24 million tonnes of crude palm oil
from 6.4 million tonnes of FFBs (Paepatung et al., 2006), resulting in the
million tonnes per year of shell (0.13), decanter cake (0.27), fibers (0.894),
and EFB (1.53). Shells are sold as solid fuel to other industry such as cement
factories (Paepatung et al., 2006). For old mills, the EFBs applied in the
plantation as fertilizer by mulching or disposed off in the landfill or burned
in the incinerator to produce potash (Chavalparit et al., 2006; Ludin et al.,
2009). PKC is rich in carbohydrate (48%) and protein (19%) and is used as
cattle feed or processed into chicken feed. As PKC is nitrogen decient, ad-
ditional nitrogen addition such as using poultry manure, with goat manure as
supplement to convert it into compost (Ismail, 2004).
8.5.2 POME TREATMENT
POME is a viscous brown liquid with fine suspended solids, with pH rang-
ing between 4 and 5 (Poh and Chong, 2009). It is composed of high or-
ganic content mainly oil and fatty acids, carbohydrates (29.55%), proteins
(12.75%), nitrogenous compounds, lipids, and a considerable amount of cel-
lulose and non-toxic minerals which can be a good source for microbial fer-
mentation (Wu et al., 2007). The percolation of POME into the waterways
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and eco-systems remain a major concern (Foo and Hameed, 2010). The
high COD and BOD can cause considerable environmental problems and
destruction of aquatic biota if discharged without proper treatment (Cheng
et al., 2010; Singh and Dhar, 2011). The characteristics and the parameter
limits for POME discharge into water courses in Malaysia are summarized
in Table 8.3. Figure 8.6 shows different treatment systems used for POME
treatment (Yi et al., 2012) which include: (a) anaerobic/facultative ponds
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(Wong, 1980; Rahim and Raj, 1982); (b) tank digestion and mechanical aer-
ation; (c) tank digestion and facultative ponds; (d) decanter and facultative
ponds; (e) physicochemical and biological treatment (Andreasen, 1982);
(f) evaporation (Ma, 1993) and clarification pond coupled with filtration and
aeration (UNEP, 1994).
TABLE 8.3 Characteristics and Parameter Limits for POME Discharge into Water Courses
in Malaysia.
Parametera Meanb Rangec Parameter Limits Major Quantity
for Watercourse Constituentse (g/g dry
Discharged sample)f
pH 3.5 3.45.2 5.09.0 Moisture 6.75
Crude protein 9.07
Temperature 80 8090 45 Crude lipid 13.21
BOD 3days 30C 24117
b
10,250 100 Ash 32.12
43,750
COD 65272 15,000 Carbohydrate 20.55
1,00,000
Total solids 11,500 11,500 Nitrogen-free 19.47
79,000 extract
Suspended Solids 68367 5,000 400 Total carotene 20.07
54,000
Volatile Solids 32743 9,000
72,000
Oil and Grease 3546 13018,000 50
Ammoniacal 480 150c
Nitrogen
Total Nitrogen 385 1801400 200c
Units in mg/L except pH and Temperature (C); The sample for BOD analysis is incubated
at 30C for 3 days; Value of filtered sample; a,bSource: Ma (1999) and Ahmad et al. (2014a,
2014b, 2014c); c,dSource: EQA (2001); e,fSource: Habib et al. (1979).
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FIGURE 8.6 POME treatment systems (Yi et al., 2012).
At present, 85% of POME treatment is based on anaerobic and faculta-

tive pond system, followed by open tank digester attached with extended
aeration in a pond (Vijayaraghavan et al., 2007; Subramaniam et al., 2008;
Fang et al., 2011). Although require relatively little energy to operate, con-
ventional POME treatment has several disadvantages such as extensive land
area, long retention time, low treatment effectiveness where only a fraction
of nitrogen and phosphorus in the wastewater removed, more sludge produc-
tion, and release of GHG in form of CO2 and CH4 (rpez et al., 2009; Wu et
al., 2010; Lam and Lee, 2011). The under-sized treatment systems in most
mills are also unable to cope with increasing POME volume (Cheng et al.,
2010; Singh and Dhar, 2011). New technologies that have been explored in-
clude membrane technology, up-flow anaerobic filtration, up-flow anaerobic
sludge blanket (UASB), and up-flow anaerobic sludge fixed film (UASFF)
bioreactor. The main challenge is to balance environmental protection with
economic viability and sustainable development (Ahmad et al., 2003), and
meeting the tough environmental regulations.
8.5.2.1 PONDING (LAGOON) SYSTEM
Ponding system consists of the sequence of anaerobic, facultative, and aero-

bic ponds with low investment cost (Tong and Bakar, 2004; Yacob et al.,
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2006). There is less energy to manage due to limited mechanical mixing, op-
erational control and monitoring (Yacob et al., 2006), but requires large land
area to accommodate the ponds. Facultative and aerobic ponds are necessary
to decrease the organic content in the wastewater before being discharged
into rivers (Yacob et al., 2006; Poh and Chong, 2009). For anaerobic ponds,
the optimum depth ranges from 5 to 7m with 3045 days HRT; for faculta-
tive ponds, 11.5m depth with 1520 days HRT (Tong and Bakar, 2004;
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Yacob et al., 2006) and a shallower depth of 0.51m for aerobic ponds with
24 days HRT (Yacob et al., 2006). The raw POME feeding to the anaerobic
digestion and aerobic/facultative treatment system normally have BOD and
COD concentrations in a narrow range of 20,00025,000mgL1 and 45,000
50,000mgL1, respectively. Overtime, there will be scums developed on the
POME surface and the building-up of solid at the bottom. The sludge and
scums will clump together, thus reducing the treatment efficiency. These
need continuous de-sludging by using submersible pumps or excavators to
maintain efficiency.
8.5.2.2 AEROBIC TREATMENT
Aerobic treatment systems may differ in the type of oxygen (aeration sys-
tem) and the designed loading rates in the form of facultative ponds (matu-
ration ponds), oxidation ponds, aerated lagoons, and polishing ponds. The
common method is the aerobic pond, and a few palm oil mills use the more
advanced activated sludge system (Environmental Management Guideline
for the Palm Oil Industry, 1997). Facultative ponds, oxidation ponds, and
polishing ponds establish oxygen supply by photosynthetic activities of
algae and plants by absorbing CO2 from the atmosphere and releasing O2
for bacteria or vice versa. Aerated lagoons are artificially aerated, where
high temperature of the pond enhances the biochemical reactions, result-
ing in increased substrate removal even at low oxygen solubility in water
(Environmental Management Guideline for the Palm Oil Industry, 1997).
Large quantities of GHGs including methane and carbon dioxide are pro-
duced from open ponds and tanks raising environmental concern. The total
emission can be substantially reduced if CH4 is completely recovered in
sealed anaerobic reactors or simply burnt to convert into CO2 (Yacob et al.,
2006).
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8.5.2.3 ANAEROBIC TREATMENT
Anaerobic treatment systems have more advantages over aerobic treatment

or other methods as they are almost energy free with low capital cost. There
is a high organic load, excluding the acidification step and low formation
of surplus sludge. No secondary treatment is required if the treated effluent
is used for irrigation. However, if the treated effluent is discharged into the
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river, secondary treatments are essential in the form of aerobic treatment.
During anaerobic process, dissolved organic substrates are mostly converted
into biogas (a mixture of around 60% CH4 and 30% CO2). Only very little
substrate is converted into biomass. The biomass should be separated as
biological excess sludge or volatile suspended solids (VSS). Under usual
operating conditions, less than 0.3kg VSS kg1 BOD is removed (Shilton et
al., 2008).
8.5.2.4 OPEN DIGESTING TANK
Open digesting tank is used when limited land area is available for pond-
ing system (Poh and Chong, 2009). An open digester tank is made up of
mild steel with variety of volumetric capacities ranging from 600 to 3600m3
(Yacob et al., 2006). Shorter HRT is required ranging from 20 to 25 days as
compared to 3045 days for ponding system. However, the treated POME
from the digester will still have to go through the facultative pond, followed
by aerobic ponds for more degradation of organic material. Similar to pond-
ing, no mechanical mixing is installed and the biomethane is released directly
to the atmosphere (Tong and Bakar, 2004; Poh and Chong, 2009). Methane
emission rate of 518.9kgd1 has been registered in open digesting tank as
compared to 1043kgd1 in anaerobic pond, with 54.4% methane (Yacob et
al., 2006). Open-pond and open digesting tank treatment systems therefore
are not certified for Carbon Emission Reduction (CER) trading. One of the
advantages of using open digester tank is in the removal of solids build-up at
the bottom continuously, thus maintaining the desired treatment efficiency
(Tong and Bakar, 2004). The dewatered sludge can be used as fertilizer. The
major drawback is corrosion of the steel structures due to long contact with
hydrogen sulfide. Incidents such as bursting and collapsed digesters have
been reported (Yacob et al., 2006).
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8.5.2.5 CLOSED DIGESTING TANK
Clean development mechanism (CDM) promotes closed anaerobic digest-

ing tanks where biogas is captured and straightly used for flaring, boiler fuel
or power generation (Tong and Bakar, 2004). A closed digesting tank has
comparable design with an open digesting tank, except that a fixed or float-
ing cover is included and set with other facilities like gas collector, safety
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valves, and monitoring devices (Sulaiman et al., 2009). Methane production
rate from a large scale closed digesting tank has been reported at 5019kgd1
with 62.5% methane which is much higher than from the open-pond and
open digesting tank (Tong and Bakar, 2004). The system contains three per-
manent stirred-tank reactors, in which two reactors are installed with float-
ing roof and another reactor with a fixed cover. The total capacity of the
three reactors is 7500m3 with HRT of 18 days. The system is equipped
with dual-function mixing mechanism: the pump-aided circulation and the
gas lifting mixing. The system has been operating continuously for over 20
years. The high efficiency of the design allows the highest recovery of bio-
methane potential from the optimally controlled POME digestion. The total
biomethane captured and utilized as boiler fuel has been estimated to be
1407 tonnes y1, which is equivalent to 29,547 tonnes y1 of CO2-equivalent
reduction (Hassan et al., 2004).
8.5.2.6 CONTINUOUS STIRRED-TANK REACTOR
Continuous stirred-tank reactor (CSTR) is the same as a closed-tank digester

but with a mixer. The mechanical agitators mix the biomass thus improv-
ing the biogas production. It has been in use since early 1980s for POME
treatment by Keck Seng Oil Palm Mill (Malaysia) Berhad in Masai, Johor
(Tong and Bakar, 2004). CSTR has also been applied for coke wastewater
treatment in aerobic conditions (Vazquez et al., 2006) and for dilute dairy
wastewater treatment (Chen and Shyu, 1996). The CSTR in Keck Sengs
mill has approximately 83% COD removal efficiency with 63% methane,
while the CSTR treating dairy wastewater has 60% COD removal efficiency
with 2377% methane. High COD removal from POME between 94 and
98% has been achieved using CSTR at lab scale (Ugoji, 1997). The lower
COD removal at the plant scale can be assumed due to the large volume of
feed resulting in inefficient mixing. The existing CSTR has been improved
by incorporating a biolm support system (BSS) within a low-density nylon
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mesh, rolled into cylinders and inserted into the CSTR. The BSS functions as
a support media for biomass growth and the efciency of CSTR is improved
without the need for biomass recycling (Ramasamy and Abbasi, 2000).
8.5.2.7 UASFF REACTOR
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The drawbacks of anaerobic treatment are long HRT and long start-up peri-
od. The problem with long HRT can be rectified by using high-rate anaerobic
bioreactors. The long start-up period can be shortened by using granulated
seed sludge (Wu, 2010; Lam and Lee, 2011) or utilizing seed sludge from
the same process or maintaining suitable pH and temperature in the high-rate
anaerobic bioreactor for growth of bacteria consortia (Lorestani, 2006; Wu,
2010). Table 8.4 shows methane emission rate from anaerobic digestion of
POME. UASB reactor and anaerobic lter have been integrated to form a
hybrid bioreactor called up-flow UASFF. While eliminating the respective
drawbacks, the hybrid reactor has the advantages of both reactors. UASFF is
better in terms of biomass retention, reactor stability at the shock loadings,
and operation at high OLRs, whilst overcoming the problems of clogging
TABLE 8.4 Methane Emission Rate from Anaerobic Digestion of POME.

Reactor Type Conditions HRT CH4 Emission COD References
(days) Rate (L g1 Removal Ef-
COD removed) ficiency (%)
Single stage stirred 6.2 0.325 96 Borja and
digester Banks (1994)
Modified anaerobic 6 0.42 95 Faisal and
baffled bioreactor Unno (2001)
Up-flow anaerobic Mesophilic 3 0.346 97 Najafpour et
sludge-fixed film 38 C al. (2006)
(UASFF)
Single stage Mesophilic 17 0.15a 97 Yacob et al.
3742 C (2006)
Continuous stirred Mesophilic 7 0.30a 71 Choorit and
tank reactor (CSTR) 37 C Wisarnwan
(2007)
Continuous stirred Thermo- 5 0.27a 70 Choorit and
tank reactor (CSTR) philic Wisarnwan
55 C (2007)
Self-estimation.
a
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and biomass washout in anaerobic lter and UASB (Borja and Banks, 1994;
Ayati and Ganjidoust, 2006). Investigations using UASFF include the treat-
ment of wood fiber wastewater, sugar wastewater, dairy wastewater, wash
waters from virgin olive oil purication, and POME treatment (Najafpour et
al., 2006; Wu, 2010). Anaerobic treatment with UASFF reactor can reduce
up to 95% COD at an average OLR of 15g COD L1d1. A 96% COD re-
moval has been obtained at an OLR of 10.6g COD L1d1 at COD concen-
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tration of 42,500mgL1 and HRT of four days (Lorestani, 2006; Siew, 2006;
Wu, 2006). COD removal efficiency has been over 70% except for wood
ber wastewater as wood ber is harder to degrade although the methane
production is satisfactory at 0.346Lg1 COD. The internal packing and high
ratio of efuent recycle are the main factors controlling the constancy of
the UASFF reactor in POME treatment. Internal packing effectively retains
biomass in the column while effluent recycle produces internal dilution to
reduce effects of high OLR (Borja and Banks, 1994; Najafpour et al., 2006).
8.5.2.8 MEMBRANE TREATMENT
Membrane technologies in combination with coagulation/flocculation as

pre-treatment achieve 78% water recovery from POME (Ahmad et al., 2003;
Ahmad et al., 2011). The analyses of the reclaimed water show that the water
qualities fulfill the drinking water standard set by the U.S. EPA. The study
shows that membrane fouling is reversible and primarily due to cake forma-
tion. The treated effluent has a high-quality and crystal clear water that can
be recycled or used as the boiler feed water or as the source of drinking water
(Ahmad et al., 2003). A pilot plant has been developed for POME treatment
where the first stage involves coagulation, sedimentation, and adsorption
and second stage involves combination of ultra-filtration and reverse os-
mosis. Results show a reduction in turbidity, COD, and BOD upto 100, 99,
and 99.4%, respectively, with a final pH of 7. Membrane ultra-filtration is
also used as the tertiary treatment method. Combination of filtrationultra-
filtration treatment is the best overall treatment efficiency, with a reduction
of 93.4% for total nitrogen, suspended solids turbidity, and colour content.
For combination of centrifugationultra-filtration, the average removal ef-
ficiency is only 86.4%, while coagulationultra-filtration treatment only
achieves an average of 67% removal (Wong et al., 2002).
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8.5.2.9 FIBROUS-BED COLUMN
Adsorption is attractive because of its simplicity, convenience, and effi-

ciency. Biosorbents from renewable sources are economical and biode-
gradable, and have potential for industrial applications (Li et al., 2011;
McManamon et al., 2012). Methods for heavy metal removal such as
chemical precipitation, chemical oxidation/reduction, reverse osmosis,
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electrodialysis, and ultra-filtration from aqueous solutions have low ef-
ficiency, high cost, and generate toxic wastes. The performance of raw
Ceiba pentandra (L.) gaertn. [raw kapok fibers (RKF)] for oil sorption
and POME treatment have been compared with structurally modified ka-
pok [NaOH-treated (SKF), surface-modified kapok fiber (SMKF)], raw
bentonite clay, and HCl-treated bentonite. For filtration under gravity at
0.08gcm3, SKF shows high POME sorption of 82gg1, but lower diesel
sorption of 23gg1. With HCl-treated bentonite, POME sorption at 69gg1
is only slightly higher than diesel sorption of 60gg1. Both RKF and raw
bentonite achieve higher removal efficiency of BOD, COD, YOC, and TN
at 7498% and 7294%, respectively, than with SKF at 6680%, and HCl-
treated bentonite at 6480% (Abdullah et al., 2015a). Fibrous bed filtra-
tion column studies have been carried out to evaluate the performance of
RKF in removing heavy metals (Fe, Mn and Zn) from POME and residual
oil from Crude Palm Oilwater emulsion at varying flow rate (510 mL
min-1) and packing density (0.040.08gcm3). The best result is obtained
at low flow rate (5mLmin1) and high packing density (0.08gcm3) of
RKF achieving the removal percentage for Fe, Mn, and Zn of 98.8, 99.4,
and 98.6%, respectively. For all packing densities and flow rates, the COD
reductions exceed 99% whilst turbidity reduction was 90.795.6%. RKF
is therefore a promising adsorbent for POME remediation and the removal
of metallic ions from POME with excellent capability to reduce COD and
turbidity of effluent contaminated with residual oil (Afzaal, 2014; Afzaal
et al., 2014). Eco-friendly extraction of cellulose from EFB has been re-
ported (Nazir et al., 2013), and the extracted cellulose has been successful-
ly surface-modified with acetic acid and EDTA to be used as biosobent for
heavy metal removal from water. The highest Pb (II) removal is achieved
with EDTA-surface modification at 236.7mgg1 (Nazir, 2013). Higher
sorption capacity is also achieved for the removal of Mn (II), Ni (II), and
Cu (II) ions with EDTA-surface modification (39.354.2mgg1) than ace-
tic acid (36.445.3mgg1) (Daneshfozoun et al., 2014).
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8.5.3 ALGAL BIOMASS CO-UTILIZATION
8.5.3.1 AEROBIC AND ANAEROBIC TREATMENT
Sustainable energy management in palm oil mill has entered a new dynamic
era with the opportunity of culturing microalgae using POME (Khan et al.,
2005c; Khan et al., 2009b; Procedia, 2009). Algal treatment replacing con-
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ventional tertiary POME treatment can offer an oxygenated effluent and an
ecologically safe, less expensive, and more efficient mean to remove nu-
trients and metals. Microalgae as a tertiary treatment of nitrogen and phos-
phorus not removed during anaerobic digestion can reduce eutrophication at
point sources better than can be achieved by conventional treatment (rpez
et al., 2009; De-Bashan et al., 2010). During digestion, bacteria consume the
oxygen released by microalgae to decompose organic matter, giving out car-
bon dioxide, ammonia, and phosphates, which are assimilated by microalgae
and methane released as energy. Sludge from wastewater treatment plant
can be co-cultured with algae to enhance remediation but unlike activated
sludge for secondary effluents treatment, algae can eliminate nitrogen and
phosphorus without organic carbon requirement (Aslan and Kapdan, 2006).
Microalgae culture or sludge can be used as a diet supplement for live feed
culture (Li et al., 2008a; Li et al., 2008b) or digested for bio-ethanol conver-
sion or harvested for biodiesel or biocompounds.
The wastewater treatment utilizing the algalbacterial system is capable
of removing about 80% COD (Weiland, 2010). Utilizing N. oculata and
Chlorella sp., the highest removal of COD (9598%) and BOD (9098%)
TOC (8086%) and TN (80%) are achieved after seven days of anaero-
bic treatment as compared to the treatment without microalgae (Table 8.5)
(Ahmad et al., 2014b; Ahmad et al., 2014c). POME treated with anaerobic
co-cultivation of T. suecica achieves high removal efficiency of COD, BOD,
TOC, and TN after three and seven days HRT at 8795%, 8495%, 6790%,
7380%, respectively (Ahmad et al., 2014a). The lower removal efficiency
of COD (53%), BOD (73%), TOC (49%), and TN (48%) are achieved on
day 3 of aerobic treatment without microalgae. Filtered POME composition
in sea water at different levels (1, 5, 10, 15, and 20%) used as an alternative
medium for algal cultivation has attained enhanced cell growth and lipid
accumulation (Shah et al., 2014a; Shah et al., 2016). At 10% POME for N.
oculata and T. suecica with high max (0.21 and 0.20d1) and lipid content
(39 and 27%), respectively, were achieved after 16 days of flask cultivation.
The POME/seawater media also achieve high removal of COD (93.695%),
BOD (9697%), TOC (7175%), TN (78.890.8%), and oil and grease
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(9294.9%). The major fatty acids composition of lipid recovered from N.

oculata and T. suecica cultivated in 10% POME composition with sea water
are pentadecanoic acid (C15:0), palmitic acid (C16:0), stearic acid (C18:0)
for SFA; and palmitolic acid (C16:1) and oleic acid (C18:1) for MUFA.
The total SFA (59.24, 68.74%); MUFA (15.14, 12.26%); and PUFA (9.07,
8.88%) are obtained for N. oculata and T. suecica, respectively. N. oculata
contains high palmitic acid (C16:0) at 28.22% and palmitolic (C16:1) at
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9.37% while T. suecica contains high palmitic acid (C16:0) at 36.48% and
pentadecanoic acid (C15:0) at 9.21%. In PUFA profile, the highest percent-
age of linolenic acid (C18:3) is found in N. oculata (4.54%) and T. suecica
(5.11%). Algal cultivation in 10% filtered POME with sea water therefore is
suitable for improved cell growth as well as MUFA and PUFA production.
The percentage of fatty acids content of microalgae can be tuned based on
the growth phases from which the cultures are harvested. With high satu-
rated and MUFA s, N. oculata and T. suecica are potential candidates for the
production of biodiesel (Shah et al., 2016).
TABLE 8.5 Aerobic and Anaerobic Treatment of POME with and without Microalgae.
Removal Efficiency (%)
Aerobic Treatment Anaerobic Treatment
Days 3 Days 7 Days 3 Days 7
Parameters
Without algae
Without algae
Without algae
Without algae
Chlorella sp.
Chlorella sp.
Chlorella sp.
Chlorella sp.
N. oculata
N. oculata
N. oculata
N. oculata
T. suecica
T. suecica
T. suecica
T. suecica
pH 7.8 7.9 7.9 7.8 7.7 7.8 7.6 7.7 6 6.3 7.2 7.2 5.7 5.6 6.8 7.1
BOD 73 82 81 83 77 84 88 86 78 90 86 67 87 98 95 90
COD 53 65 73 69 62 90 87 96 73 83 86 87 87 97 98 95
TOC 49 56 67 59 58 65 75 78 62 63 68 67 70 80 86 80
TN 48 60 67 59 61 64 75 64 69 73 59 73 70 80 78 80
Anaerobic cultivation of C. vulgaris and Scenedesmus dimorphus with

POME for eight days HRT removes 50.5 and 86% COD; 61.6 and 86.5%
BOD; and 61 and 99.5% TN, respectively (Techobanoglous, 2002). The pre-
viously reported removal efficiencies of TOC (76.6%) and TN (84%) are
achieved in the treatment of industrial wastewater by C. vulgaris (Godos and
Bianco, 2009; Hee-Jeong and Seung-Mok, 2012; Zhou, 2012). Increasing
C. vulgaris content from 1 to 10gL1 increases the removal rate of BOD
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to 8089%, COD to 7882%, TN to 8185%, TP to 3236%, NH3N to

9997%, and PO4P to 4549% (Hee-Jeong and Seung-Mok, 2012). Shorter
HRTs of two days have been reported for 89% BOD and 88% COD reduction
using C. vulgaris grown in seed and animals feed production wastewater at
30C (Residua, 2007). The algae-based STP achieves total BOD removal of
82% (Thani et al., 1999) and 76% COD removal from piggery wastewater in
high-rate algal ponds (Ranga Rao et al., 2007). A study with C. protothecoi-
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des similarly achieves the removal efficiency of 78.3% COD, when algae is
grown in concentrated soybean wastewater (Zhou, 2012). However, lower
COD removal (5979%) has been reported by combining the high-rate algal
pond, using filamentous green algae and an artificial wetland (Beccari et al.,
1996).
8.5.3.2 BIOMETHANE PRODUCTION
The most extensive study using anaerobic digestion technology has been
on the oil palm wastes, which can be utilized to meet the energy require-
ment of the palm oil mills (Lorestani, 2006). Through the implementation
of CDM, the mills could earn carbon credits as revenue by utilizing the
methane gas (Poh and Chong, 2009). The performance of an anaerobic di-
gester for biogas production can be improved by optimizing factors such as
the quality of POME, inocula composition, co-substrate addition, pH, tem-
perature, OLR, reactor type and design, retention time, and pre-treatment
process. With Chlorella sp. after three days HRT, the highest biomethane
yield (5276mLL1 POME d1) and the highest specific biogas production
rate (0.129m3kg1 COD day1) are achieved at 2mLmL1 POME and EFB
of 0.12gmL1 POME. With N. oculata, the biomethane is slightly lower
(4812mLL1 POME day1), but the specific biogas production rate is com-
parable (0.126m3kg1 COD d1) (Ahmad et al., 2014a; Ahmad et al., 2014b;
Ahmad et al., 2014c). With reduced amount of EFB (0.06gmL1 POME), but
high mono-algal N. oculata and Chlorella sp. cultured separately (2mLmL1
POME), comparable biomethane yield (44434524mLL1 POME d1) and
the specific biogas production rate (0.1200.122m3kg1 COD d1) are ob-
tained. At lower amount of Chlorella sp. (1mLmL1 POME) but high EFB
(0.12gmL1), lower biomethane yield (3816mLL1 POME d1) and specific
biogas production rate (0.105m3kg1 COD d1) although comparable to N.
oculata, and both at all times register higher production than T. suecica. With
anaerobic co-cultivation of T. suecica, EFB, and POME, the biomethane
production is 3965mLL1POME d1 and the specific biogas production is
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0.116m3kg1 COD d1. Without microalgae, the specific biogas production is

high (0.127m3kg1 COD d1) but with lower biomethane yield (3642mLL1
POME day1) (Ahmad et al., 2014a). These, however, are still much higher
than the reported methane production of 5731170mLL1d1 from co-di-
gestion of Scenedesmus sp. and Chlorella sp. separately, with 50% waste
paper (Yen and Brune, 2007). Without microalgae, the highest biomethane
is 3650.3mLL1 POME d1 but with equivalent specific biogas production
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rate of 0.121m3kg1 COD d1 at high EFB (0.12gmL1) POME. Without
both algae and sludge inocula, no biomethane is produced and CO2 (190mL
CO2 L1 POME d1) is much lower with some hydrogen detected (78mL H2
L1 POME d1) although the specific biogas production rate (0.130m3kg1
COD d1) remains the same. High microalgae and EFB co-digestion with
POME, at the correct ratio of POME and sludge inocula lead to 1.11.4-fold
higher biomethane production than without microalgae co-digestion. The
specific biogas production rate, however, remains consistent between 0.094
and 0.129m3kg1COD day1 (Ahmad et al., 2014a; Ahmad et al., 2014b;
Ahmad et al., 2014c).
Microalgae display major characteristic of methanogens by generating
biogas containing methane as an integral part of their energy metabolism, re-
moving over 80% BOD by anaerobic treatment (Satyawali and Balakrishnan,
2008). The lipid content of 23.730.4% under slightly above mesophilic
conditions at 48oC has resulted in higher biogas production (0.128m3kg1
COD d1) after three days HRT (Ahmad et al., 2014c). The higher the lipid
content of the cell, the higher will be the potential for biomethane produc-
tion, as these can serve as nutrients for bacteria, and for microalgae to work
in tandem with bacteria to breakdown the EFB and POME. Algal biomass
containing between 2 and 22% lipid produces methane yield at 0.470.80m3
CH4 kg1 VS in anaerobic digestion (Cirne et al., 2007; Li et al., 2011). The
productivity can be increased by mixing the proteinaceous algal biomass
with carbon rich waste such as primary sewage sludge (Chua and Oh, 2010)
and EFB and POME to increase the C/N ratio of digester feeding to balance
the high nitrogen concentration of algal sludge for methane production (Yen
and Burne, 2007). The C/N ratio of 29:1 is close to the range known to have
a positive effect on the biomethane yield which is at the range of 20:130:1.
The optimized C/N ratio of 20:125:1 has been reported for the co-digestion
of algae and waste paper (Yen and Burne, 2007). The recommended op-
timal ratio for anaerobic bacterial growth is 25:1 (Yen and Burne, 2007;
Tabatabaei et al., 2011; Saleh et al., 2012; Thong et al., 2012). Co-digestion
of EFB with POME enhance microbial biodegradability and has resulted
in 2532% higher methane production at mixing ratios of 0.4:1, 0.8:1, and
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2.3:1 on the VS basis than with EFB alone (Thong et al., 2012). An active
microbial activity supported by the readily biodegradable organic compo-
nents in POME enhances hydrolytic capacity of the digestion contributing to
higher release of hemicellulose from the lignocellulosic component of EFB
(Kaparaju and Felby, 2010).
The addition of microalgae and EFB also enhances the buffering capac-
ity of the digester. Co-digestion is beneficial because potential toxic NH4 is
9781771883627
diluted which allows improved OLR and enhanced biogas yield (Satyawali
and Balakrishnan, 2008). With excess VFAs, the acidogens grow rapidly
and produce more volatile acids to reduce the pH. Methanogenesis may not
occur as it requires pH around 6.57.5 and the methanogens may not be able
to keep up with the changes and degrade acids as fast as they are generated.
These may lead to low methane production (Poh and Chong, 2009). Several
studies have also looked at microalgal co-digestion with sludge under me-
sophilic and thermophilic conditions where methane production at 0.42Lg1
VS is much higher under mesophilic than thermophilic conditions (0.17
0.32 Lg1 VS) (Yadvika et al., 2004). The variations in solids retention times
(SRTs) between 11 and 30 days do not affect gas production (Sialve et al.,
2009). The major drawback is the energy to maintain thermophilic condi-
tion. Economical integrated processes that combine algae cultivation and
wastewater treatment system for methane production should therefore be the
most suitable approach.
8.6 CONCLUSION AND FUTURE OUTLOOK
Integrated algal wastewater treatment provides GHG abatement with a com-

bination of low energy wastewater treatment, renewable fuel production,
high-value biocompounds, and fertilizer recovery. The production of bio-
diesel, biomethane, biohydrogen, and bio-ethanol as co-products or high-
value biocompounds has immense potential of decreasing the total cost of
algal biofuels production thereby improving economic feasibility. Mixed
bacterialalgal co-immobilized systems in water treatment plants is ideal for
methane or hydrogen production. For these, selection of algal strain/consor-
tium, climatic conditions, existing infrastructure, logistic considerations as
well as overall availability of waste resources in sufficient quantity are im-
portant considerations. A transparent hybrid photobioreactor utilizing both
incidents light directly from the sun, as well as collected and distributed
light can achieve higher biomass production with heterotrophic/mixotrophic
growth systems and waste water media. Hybrid bioreactor that utilizes the
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design can make up deficiencies for algal growth in systems such as green-
house reactors, raceway cultivators, and ponds. Novel consideration is the
fiber optic photon units strategically placed in the growth section within the
bioreactor for possible growth throughout the entire daylight time period,
and not just when the sun is directly overhead. Since a wastewater treatment
system is viable for efficient tertiary-level treatment, a refined full-scale al-
gal production, harvesting and biofuel conversion technologies should be
9781771883627
the major consideration for future green energy generation.
KEYWORDS
Aerobic treatment
Algae
Anaerobic treatment
Biodiesel
Bioenergy
Bio-ethanol
Biohydrogen
Biomass utilization
Biomethane
Immobilization
Industrial wastes
Palm oil mill effluent
Product purification
Reactor engineering
Waste remediation
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9781771883627
AUTHOR COPY
CHAPTER 9
BIOAMBIANT PRESERVATION OF
RAW HIDES USING PLANT-BASED
9781771883627
MATERIALS A GREEN TECHNOLOGY
TO REDUCE TANNERY WASTE WATER
POLLUTION
PRAFULLA NAMDEO SHEDE1, ASHISH VASANTRAO POLKADE2,
PRADNYA PRALHAD KANEKAR3, PRASHANT KAMALAKAR
DHAKEPHALKAR4, and SEEMA SHREEPAD SARNAIK5
1
Department of Microbiology, MES Abasaheb Garware College,
Karve Road, Pune 411004, India
2
Microbial Culture Collection, National Centre for Cell Science, First
floor, Central Tower, Sai Trinity Building Garware Circle, Sutarwadi,
Pashan, Pune 411021, India
3
Department of Biotechnology, Modern College of Arts Science and
Commerce, Shivajinagar, Pune 411005, India
4
Bioenergy, MACS Agharkar Research Institute, G.G. Agarkar Road,
Pune 411004, India
5
Microbial Sciences Division, MACS Agharkar Research Institute,
G.G. Agarkar Road, Pune 411004, India
CONTENTS
9.1 Introduction......................................................................................226
9.2 Pollution Problems Faced by Leather Industry................................231
9.3 Alternative Methods to Salt Curing Current Scenario..................232
9.4 Contemporary Biotechnology for Leather.......................................238
9.5 Components Responsible for Putrefaction of Raw Buffalo Hide....239
9.6 Plant-Based Materials as Ideal Bioambiant Preservatives...............241
Keywords..................................................................................................248
References.................................................................................................249
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9.1INTRODUCTION
In looking for a covering material for himself, his hut and food, primitive
man turned either to leaves from plants or to the skins of animals he killed.
The latter were usually chosen for clothing, as they were bigger, stronger,
and warmer. As per British Standard Definitions, hide is the raw skin of a
mature or fully grown animal of the larger kinds, such as cattle and horses;
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also camels, rhinoceroses and whales and skin is the raw skin of a mature,
fully grown animal of the smaller kinds, like goats, sheep, pig, and others.
The flayed skins or hides of dead animals are used as raw materials for
leather making. Leather is nothing but the skins or hides whose protein has
been stabilized through the process of tanning.
9.1.1 MAIN SOURCES OF RAW MATERIAL FOR LEATHER

MANUFACTURING
The outer flayed covering, obtained from literally any animal and containing
considerable amount of specific proteins can possibly serve as raw material
for leather manufacturing. The skins could be acquired from animals like
sheep, goat, pig, reptile mostly snakes and lizards, crocodile, birds and fish-
es, or the immature animals of the larger species, such as calves and colts.
The hides could be gained from buffalo, bull, cow, horse, camel, elephant,
and whale. The biological subfamily Bovine, comprising ten different genera
of medium to large-sized animals including buffalo, is the most prominent
contributing toward the hides produced worldwide. According to the Food
and Agriculture Organization Statistics, world bovine (cattle and buffalo)
population is constantly increasing. In 2013, the cumulative heads of cattle
and buffalos available as livestock alone were 1.69 billion. Its worldwide
distribution is detailed in Figure 9.1.
FIGURE 9.1 Worldwide distribution of bovine population (Source: Food and Agriculture
Organization of The United Nations Statistics Division).
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Raw Hide Bioambiant Preservation - A Green Technology 227
9.1.2 STRUCTURE AND COMPOSITION OF BOVINE HIDE
The bovine hides processed worldwide are mainly acquired from the African
and Asian water buffalos scientifically known as Bovidae bubalis. The bo-
vine hide can be mainly divided in to two principle layers from leather mak-
ing point of view as epidermis and corium. Epidermis or the outer layer
also called the striated epithelium and the corium, inner layer also called as
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dermis, cutis vera or true skin. These two layers are quite different in their
structure and functions. The epidermis, which is a very thinner layer cover-
ing the underneath corium is removed during the leather processing. Corium,
the main layer of hide constitutes about 98% of its thickness and comprises
mainly of the fibrous protein bundles. About 9095% of the corium layer of
most of the hides is converted into the leather. The main components of the
skins/hides are shown in Figure 9.2.
FIGURE 9.2 Cross-section of bovine hide (Sharphouse, 1983).
A fresh hide consists of water, protein, fatty materials, and some mineral
salts. Of these the most important for leather making is the protein. This
protein may consist of many types of structural (fibrous) and non-structural
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(globular) forms. The important one is collagen, the fibrous protein that on
tanning gives leather. Corium consists of a network of collagen fibers very
intimately woven and joined together. The approximate composition of a
freshly flayed hide is given in Figure 9.3. Overall, the freshly flayed hide
could be a feast for bacteria if it is not processed instantly in the beam house
and/or tannery to prepare leather from it.
FIGURE 9.3 Approximate composition of bovine hide.

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9.1.3 CHIEF PROCESSES USED IN LEATHER MANUFACTURING
Leather manufacturing in particular is an age-old skill acquired by our an-

cestors. This whole process is roughly divided into three parts as before
tannage, tannage, and after tannage processes. Although modern leather pro-
cessing is very much dependent on automated machineries, the process re-
mains the same. These chief processes are described in Table 9.1. There are
many variations on this simple line of processing. Their choice determines
the character of the leather produced. Their in-depth study forms a firm base
for Leather Technology.
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TABLE 9.1 Chief Processes Used in Leather Manufacture* (Shede, 2007).

Chief Process Sub Processes Function
Before tannage Flaying Removes the skin from animal
Curing Preserves skins during transport or storage
Washing Restores cured skins to natural raw condition
Liming Loosens hair, fat and flesh
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Unhairing Removes hair
Fleshing Cut away unwanted fat and flesh
Deliming Neutralizes alkali from liming process
Bating Make the skin softer and cleaner
Pickling Brings the skin to right acidity for tannage
Tannage Tanning Hides are tanned by whichever method appropriate such
as using vegetable tans or chrome or alum or oil
After tannage Washing Removes surplus tan
Setting Out Removes wrinkles and flatten the leather
Oiling Makes the grain flexible and of good colour
Stuffing Waterproofing
Drying Removes surplus water
Rolling Makes leather firm and flat
Splitting Achieves uniform thickness
Washing Makes free from surplus tan
Neutralizing Adjusts acidity
Dyeing Obtains required color
Fatliquoring Achieves softness
Setting Out Removes creases and surplus moisture
Staking Softens the leather
Seasoning Improves appearance
Glazing Pressurized polishing
Plating Gives smooth, flat surface
Embossing Produces fancy design
*Not all these processes may be given to a particular type of hide or skin; the order of after
tannage processes varies for different leathers.
9.1.4 SOCIOECONOMIC INFLUENCE OF LEATHER INDUSTRY
The word industry in its own has a meaning. It not only conveys vision, cre-
ativity, business, trade, capital, raw materials, hard work, and employment
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all the same also wastes, pollution, and waste management irrespective of
the prefix added to it and the leather industry is no exception for this. The
figures stated for raw material of leather and finished leather products im-
port-exports with respect to Indian and Global perspectives are striking to
both economists and sociologists (Fig. 9.4). The revenue generated in the
import export of leather raw material and finished products is considered to
be influencing developing countrys economy.
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FIGURE 9.4 Worldwide distribution of bovine hide production in 2013 (Source: Food and
Agriculture Organization of The United Nations Statistics Division).
The total number of small scale and large-scale tanneries worldwide is

immense. The report commissioned by ETSU on behalf of Department of
Trade and Industry, Government of India on the topic Evaluation of en-
ergy from waste investment opportunities in India and prepared by FEC
Consultants Ltd., and Delphi International Ltd., in the year 1997 estimated
1935 tanneries in India. It also states that in India, tanneries have developed
mostly in a concentrated fashion around specific centers for historical and
social reasons. The state-wise distribution of tanneries in India in the year
2006 is given in Table 9.2.
The Indian leather sector receives direct investment from foreign coun-
tries and is a source for 10% global leather requirement with a production
value over US $4 billion (annual export value over US $2 billion). As per
the Council for Leather Export (CLE), India, the tanneries in India have
more than 2.5 million employees, 30% of which are women. The industrial
setups and/or tanneries distributed throughout the globe on one hand offer
employment while in many cases create environmental problems by means
of waste generated.
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TABLE 9.2 State-wise Distributions of Large-Scale Tanneries in India.

State Area Number of Tanneries
Tamil Nadu Chennai, Ranipet, Ambur, Pernampet, 520
Vaniyambadi, Dindigul, Trichy
West Bengal Tangra, Topsia, Tiljala 400
Uttar Pradesh Kanpur, Unnaon, Agra 200
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Maharashtra Mumbai, Dharavi, Aurangabad, Pune 200
Karnataka Banglore, Dharwar, Belgaum, Humnabad, 180
Bijapur
Rajasthan Beawar, Bhinwal 150
Punjab Jalandhar, Nakodar 50
Gujarat Idar, Bhavnagar, Ahmdabad, Rajkot, 50
Wadhwan
Andhra Pradesh Hyderabad, Vijaynagaram, Karimnagar, 30
Warangal
Haryana Ambala, Jind 20
Bihar Muzaffarpur, Mekemaghat 15
Jammu and Kashmir 10
9.2 POLLUTION PROBLEMS FACED BY LEATHER INDUSTRY
Leather industry makes use of hides and skins, which are believed to be
byproducts of meat industry. Considering the global production of these
hides and skins, leather industry becomes the worlds largest industry based
on byproducts. As stated earlier, leather processing in general involves a
series of unit operations that can be classified into three groups: (i) beam
house operations or pre-tanning which clean the hides or skins, (ii) tanning
which permanently stabilizes the skin or hide matrix, and (iii) post-tanning
and finishing operations where aesthetic value is added to leather. At each
stage, various chemicals are used and a variety of materials are expelled,
which ultimately lead to the environmental stresses (Suresh et al., 2001;
Thanikaivelan et al., 2003; Sivakumar et al., 2005; Song et al., 2011). This
could be the reason for placing leather industry in the list of most polluting
industries worldwide.
In terms of biological oxygen demand (BOD), chemical oxygen demand
(COD), and total dissolved solids (TDS), almost 70% of the pollution origi-
nates from pre-tanning operations. Almost all sulfide emissions originate
from dehairing operations. In addition, chrome-tanning operations result in
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large emissions of chromium and sulfate ions. With the total annual global
processing capacity of 9109 kg hides and skins, it is estimated that 30
401010 L of liquid effluent is generated (Thanikaivelan et al., 2004). Salt
used for preserving the hides and skins generates huge amounts of pollution
in terms of TDS and chlorides (Boopathy and Sekaran, 2014). According
to Kanagaraj et al. (2005a), the effluent generated in the soaking operation
alone accounts for 40% of chlorides and 55% of TDS in the entire leather
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processing operations.
The discharge limit for TDS is quite stringent in India and the restric-
tions from the global leather buyers and countries are also severe. The socio-
economic status of leather industry and the pressure from various pollution
monitoring agencies has forced the leather sector to look for various cleaner
pre-tanning and tanning methodologies. These methodologies are based on
the partial or complete replacement of tanning agents, dehairing chemicals,
and the curing agents, especially the alternative preservative methods that
will eliminate the use of salt as a preservative.
9.3 ALTERNATIVE METHODS TO SALT CURING CURRENT

SCENARIO
In olden days, every man would tan or dress each skin/hide as he acquired
it and before it putrefied or the expert tanner would do it for him. As the de-
mand for leather and the special qualities in it became greater, the processes
became more complicated and required special skills, tools, processing plant
and machinery. This led to the growth of tanneries collecting skins/hides
over large areas, and producing and selling their special products all over the
country and eventually the world.
This raised a special problem that if transport of the raw skins/hides took
days, weeks or months before they were tanned, putrefaction would start
and the skins/hides would be useless when they arrive at the tannery. The
need arose for simple methods of stopping this putrefaction, methods which
would not so alter the skins from the raw state that the tanner could not tan
or color them, as he required. These are referred to as curing or preserving
methods and are well designed considering the type of material to be pre-
served and the factors leading to its putrefaction. These curing methods are
almost same for all types of skins/hides with certain exceptions. The most
favored preservative throughout the world was common salt, chemically
termed as sodium chloride or NaCl. The traditional usage of this common salt
in the process of preservation has got certain limitations in present situation
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as discussed earlier. Salt is one of the most notorious components of any

effluent to treat and it is said that the only cost effective way to remove it
from an effluent is to avoid putting it in the first place. Secondly, the rate of
salt penetration in the hides also decides the curing efficiency of the salting
process (Cooper et al., 1972). Moreover, halophiles are one more factor that
demands an alternative to salt. The halophilic bacteria capable of growing at
high salt concentrations were evident on salt cured hides and skins (Stuart
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and Frey, 1938; Woods et al., 1971; Kallenberger, 1984) and this is generally
called as red heat amongst the tanners. Considering this, various attempts
were made by different workers (Kanagaraj and Chandra Babu, 2002) to use
compounds other than salt as preservatives of the raw hides and skins or to
engage different physical methods to achieve preservation of raw material.
9.3.1 PHYSICAL METHODS OF CURING
These are the methods wherein the skins and hides were preserved by physi-
cal treatments. One more conventionally used method of preservation other
than salting is drying of skins and hides. Drying, in particular is such method
where the putrefaction is stopped by removal of water so that only 1014%
moisture is retained in hide, which eventually ceases the bacterial activity.
Roughly the drying could be achieved by two ways as sun drying the hides
are hung in the sun and shed drying the hides are dried in open sided, cov-
ered shed, designed to keep off the direct heat of sun however allows good
ventilation. According to Sharphouse (1983), drying has got certain limita-
tions if the drying is too slow which is possible in cold and wet climate. The
putrefaction may occur before the moisture content is low enough to stop the
bacterial action and if the drying is too fast and the temperature is too high,
part of the wet skins will start to gelatinize to glue like material making the
skins hard and brittle. Another disadvantage of drying is dried hides require
careful handling and packing, as they are hard and mere bending may cause
them to crack. Apart from this, the dried hides and skins are susceptible to
insect attack. Hence drying could not be the method of choice for the com-
mitted tanner.
Storing the raw skins and hides at low temperatures termed as chilling
or freezing is also considered as the alternative to salt curing. Haines (1981)
has studied the effect of chilling and freezing on preservation of cattle hides.
He found that the hides can be safely held at 0C for three weeks without any
additional treatment, whereas at 10C the hides could be preserved for three
months. Chilling and freezing, although a common practice in developed
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countries for preservation of meat and byproducts of meat. It is highly im-

possible to use this method in any developing country, not even India for its
demand of high cost and energy inputs in the total process of preservation.
Considering the share of underdeveloped and developing countries in the
production of raw hides and skins which is more than half of the total global
production capacity, one should not solely depend on such an expensive
method. Even if the developed countries favor such an energy intensive and
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pricey method to cope up with the stringent pollution control norms of their
own countries or province, these methods do have some lacunae in those
countries as well. The failure of machinery or the interruption in power sup-
ply to the machinery maintaining such low temperature could lead to the
putrefaction of chilled and frozen hides and skins. Secondly, it becomes very
difficult to transport huge amounts of raw hides and skins in chilled or frozen
conditions. Hence chilling and freezing could not be the methods of choice
for the tanner from the undeveloped and/or developing countries with cer-
tain exceptions from the developed countries.
9.3.2 CHEMICAL METHODS OF CURING
These are the methods where both proprietary products and standard chemi-
cals were used in place of salt as biocides or biostatic compounds to pre-
serve the raw skins and hides. Reports for use of such chemicals in place
of common salt dates back to 80s however in actual sense, the intellect to
minimize the pollution caused by salt is unintentionally owned by Kritzinger
and Vanzyl (1954). They have reported the preservation of hides with used
salt. Their main concern was to make available the salt to curers when there
was less salt supply due to excessive rains or drought. Hence they proposed
reuse of salt as preservative thereby decreasing the concentration of salts in
soak liquors and eventually in the tannery effluent. Although, they failed to
mention their achievement in their research article, no doubt it was the first
indirect attempt to minimize the pollution caused by salt in the tannery efflu-
ents. Further, various attempts were made directly to minimize the pollution
problems caused by the salt as a raw skin and hide preservative.
The approach used by most of the researchers in this case was, use of
the chemicals with known antibacterial activities. In most of the cases, these
chemicals possessed bactericidal activities with few exceptions where the
chemicals used were bacteriostatic in nature. Several of them were structur-
ally similar to one another and were from the same class or family of chemi-
cals however poses different properties. Mostly the inorganic and organic
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salts of different metals such as zinc, sodium, potassium, nickel, and mer-
cury were engaged in these studies. These biocides were effective in terms of
preservation of raw skins and hides. They could preserve the skins and hides
for the period of two days to six months, depending on the chemical used.
Table 9.3 portrays the attempts made by various researchers for replacement
of common salt with other chemicals as short-term preservatives of raw
hides and skins. The above-mentioned chemicals are yet again problematic
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with the environment and health perspective. Majority of these substances
comes under the hazardous chemical categories as toxic or very toxic, harm-
ful or irritant, corrosive and dangerous for the environment (Table 9.4).
TABLE 9.3 Evaluation of Chemical Preservatives for Skin and Hide Preservation.
Chemicals Raw Period References
Material (days)
Sodium bisulfate DextrasetUN* Hides 814 Hopkins et al. (1973)
Benzalkonium chloride Skins 214 Sivaparvathi and Nandy (1974)
Sodium trichlorophenate
Sodium pentachlorophenate
Sodium silicofluoride
Sodium chlorite
Zinc dimethyldithiocarbamate
Zinc chloride
Zinc borate
Nickel chloride
Mercuric chloride
p-Chloro-m-cresol
Acetic acid Hides 630 Hopkins and Bailey (1975)
Sodium sulfite
Sodium bisulfite
Sodium chlorite Hides 68 Haffner and Haines (1975)
Gloquat C* (a quaternary
compound)
Glokill 77* (linear and cyclic
hydroxylamine)
Vantocil IB* (polymeric biguianide)
Vantoc CL* (lauryl dimethyl benzyl
ammonium chloride)
Organic biocide# Skins/ 90 Venkatachalam et al. (1982)
Inorganic biocide# Hides
Sodium carbonate Hides 8 Rao and Henrickson (1983)
Phenol, Borax Skins/ 180 Selvarangan and Shanmugas-
Hides undaram (1984)
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Chemicals Raw Period References
Material (days)
p-Chloro-m-cresol Skins 1214 Sivaparvathi et al. (1993)
Zinc dimethyl dithiocarbamate
B-hydroxyquinoline
Cetrimide
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Benzalkonium chloride
Boric acid
Sodium pentachlorophenate
Potassium chloride Hides 180 Bailey (1995)
Silica gel, p-Chloro-m-cresol Skins 14 Kanagaraj et al. (2001)
Boric acid, Sodium metabisulphate Skins 1430 Kanagaraj et al. (2005a),
Kanagaraj et al. (2005b)
Cetyl trimethyl ammonium bromide Skins 12 Ganesh Babu et al. (2009)
*Brand name of the product; Chemical nature not revealed.
#
TABLE 9.4 Safety Hazards Involved in the Use of the Alternative Chemical Preservatives.
Toxic/Very Toxic Harmful/Irritant Corrosive Dangerous for the
Environment
Sodium chlorite, Zinc dimethyldithiocarba- Sodium bisulfate, Zinc chloride,
nickel chloride, mate, sodium sulfite, benzalkonium nickel chloride,
mercuric chloride, sodium bisulfite, hydroxyl- chloride, mercuric chloride,
p-chloro-m-cresol, amine, biguianide, zinc chloride, hydroxylamine,
phenol sodium carbonate, borax, acetic acid, cetrimide
cetrimide, hydroxylamine
8-hydroxyquinoline
Secondly the expenses involved in the use of such chemical preservative

is very high as the chemicals to be used are required in bulk that too in the
maximum pure form to achieve utmost possible efficacy in preservation. In
nearly all of the above cases, increased cost and decreased preservative effi-
ciency was observed as compared to common salt. Another disadvantage of
these alternative chemicals is the difficulty in the handling of the chemicals
by the curers. The ease experienced in the handling of common salt is not
possible for these chemicals, as they may require some special skills and
care by handlers or the curers, being hazardous in nature. Most importantly
the main goal of salt less preservation could not be achieved, as these chemi-
cal substances themselves are the salts of certain metals. Their use as an
alternative preservative in the tannery definitely reduced the TDS problem
from soak liquors although could not eliminate it completely.
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9.3.3 BIOLOGICAL METHODS OF CURING
These are the methods wherein compounds obtained from biological me-
dium is used as preservatives for raw skins and hide and are termed as bio-
preservatives. One such compound is bacteriocin or antibiotic, a small
peptide with antibacterial activity. Several bacteria are reported to produce
various bacteriocins. Hanlin et al. (1995) has used bacteriocins, nisin, and
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pediocin AcH obtained from lactic acid bacteria as antibacterial for the pres-
ervation of cattle hides. While concluding, they have mentioned that if a
shift in the bacterial population occurs on rawhide, the bacteriocins will
need to be supplemented with other components. In these studies, they have
used sodium dodecyl sulfate (SDS) at 0.25% w/v of the mixture. Addition
of this detergent in the preservative mixture makes it unfavorable from the
viewpoint of environment conscious tanners. An approach to inhibit the col-
lagenolytic test bacterium Vibrio alginolyticus that could degrade the fibrous
matrix from hide was used by Berwick et al. (1990). In their studies they
made use of -lactam, aminoglycosides, and tetracycline type antibiotics.
Tetracycline type antibiotics were most effective against the test organisms
and they also reduced the hide biodeterioration rate significantly to store the
raw hide till ten days.
Antibiotics as preservative agents offer certain advantages over the meth-
ods discussed previously. These advantages include their biodegradability,
efficacy at low concentrations and their relative ease of production by bacte-
rial fermentation. On the contrary, if the equipments and facilities required
for fermentation are not owned by the tanners, then the use of antibiotics as
preservatives will increase the cost of leather manufacturing. The possibility,
that the small-scale tanners could not necessarily afford the setup and run-
ning cost of fermentation facilities, has to be considered. In such cases the
tanners will have to depend entirely on the commercial antiseptic formula-
tions from the pharmaceutical company. The bulk purchase of antibiotics
may reduce the cost of hide preservation however the nature of antibiotics in
many cases will force the tanners to make necessary arrangements for their
storage at low temperatures that again involves capital investment. Most im-
portantly the tannery effluent containing the unused and washed antibiotics
if introduced in natural habitats and/or water bodies may create health prob-
lems. The microorganisms, susceptible to these antibiotics will be constantly
exposed to their high dosage, making them resistant to such antibiotics. All
these factors make it a non-user-friendly method from the point of view of
attentive tanners.
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In 1981, the very first attempt was made to preserve the raw skins and
hides using plant-based material. Venkatachalam et al. (1981) have used the
herb Decalepis hamiltonii for this purpose. The boiled root extract when ap-
plied on flesh side could preserve the hides and skins for 35 days, whereas
the soaked hides and skins in the decoction were found to remain in good
condition for at least two weeks. Further they found that to make it more
effective, this aryl alcohol has to be mixed with inorganic salts and bases.
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This mixture could preserve the hides and skins for a month. The preserva-
tion period of two weeks is not sufficient to count as a short-term storage
period with respect to tanners logic of leather processing, whereas addition
of inorganic salts and bases to the extract deflects the main aim of salt less
preservative. Thus, by making it practically unsuitable as biopreservative,
the research on these lines was brought to a standstill. Although the search
of biopreservative for raw skins and hides to entirely replace the salt was
kicked off in last two decades, for one reason or the other, it has been sur-
rendered to the baselines where it started way back in the early 90s.
9.4 CONTEMPORARY BIOTECHNOLOGY FOR LEATHER
The biological methods engaging the biopreservatives were thought to have

advantage over salt, alternative chemicals, and the physical methods. They
do not just reduce the TDS, salinity, and chlorides from soak liquors how-
ever they completely eliminate such problems from the soak liquors. The
efforts taken by the researchers to discover and develop the biopreservatives
of raw skins and hides are of great significance. Though their hard work
could not give any realistic method of biopreservation, it was good enough
to suggest the right approach to invent the effective biopreservative.
The approach talked about could be termed as biotechnological ap-
proach. In the tanning industry, biotechnology is not a new concept. For
several years, enzymes have been extensively used at all stages in the leather
making, with the exception of curing and actual tanning process. At pres-
ent, the biotechnological methods are being used with great accuracy in
soaking, dehairing, bating and degreasing procedures. With this situation,
salt less biopreservation of raw skins and hides is not less than a challenge.
Thanikaivelan et al. (2004) in their review on recent trends in leather pro-
cessing, has discussed a few biotechnological products and processes along
with probability of their success. Bioproducts for ambient preservation of
raw skins and hides is amongst them.
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In the task of inventing any preservation method, one needs to know the
nature of material to be preserved, adverse changes occurring in the material
when stored without any preservative and the components responsible for
such changes. The information available on raw buffalo hide as a material to
be preserved has to be gathered through various ways, such as the changes
occurring on the raw buffalo hide during ambient storage. The components
responsible for these changes could be studied by considering the protein-
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aceous nature of raw buffalo hides. In this quest the naturally occurring anti-
microbial compounds could be used. Olasupo et al. (2003) has reported few
such compounds active against the Escherichia coli and Salmonella enterica
serovar Typhimurium. Curing agent, which is a plant-based substance and
advantageous in terms of biodegradability and eco friendly nature over alter-
nate chemicals other than sodium chloride and expensive biocides/antibiot-
ics, could be identified from the available pool of knowledge. Its efficacy as
a biopreservative for raw buffalo hide could be checked before concluding it
as a worthy biopreservative.
9.5 COMPONENTS RESPONSIBLE FOR PUTREFACTION OF RAW

BUFFALO HIDE
Occurrence of putrefaction of the raw skins and hides, if not cured by physical
or chemical method, is a traditional understanding. One of the components
responsible for the putrefaction is a bacterium, was the vital information
made available after the inception of bacteriology. Leather making is one
such trade where the solution to problem was available far before the prob-
lem was identified. Application of common salt was the solution to avoid the
problem of growth of putrefying bacteria on the raw hides and skins.
As mentioned earlier, buffalo hide is considered to be a by-product of
meat industry. The process of flaying involves handling of hides and contact
of hide with flaying knifes. The flayed hides are washed to remove dirt and
blood. Even in the process of flaying, plenty of water is being continuously
poured on the half flayed carcasses of buffaloes. All these factors could be
the potent sources of bacterial contamination. Apart from this, the state of
animal to be slaughtered, its post- and pre-mortem history such as feeding
habits and diseases harbored also decides the load of microorganisms on the
flayed hides.
However the work done in this context and emphasizing the above-
mentioned factors are not related to the raw buffalo hides. For instance,
Venkatesan et al. (1973) studied microbial flora associated with goat skins
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and found facultative anaerobes on fresh goat skin. Botteldoorn et al. (2003)
showed high degree of carcass cross-contamination with Salmonella sp.
from the slaughterhouse environment in case of slaughtered pigs. How the
environment of slaughterhouse influence microbial contamination could
be understood by the studies done by Adeleye and Adebiyi (2003) on mi-
crobiological assessment of a Nigerian abattoir. They observed that almost
throughout the year the runoff from slaughtering slabs consisted 108cfu/mL
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bacteria, which may lead to contamination of hides and animal carcasses.
Acinetobacter sp. was reported from the fresh beef samples by Eribo and Jay
(1985). One more factor stated above is pre-mortem history of the animal
to be slaughtered may produce contamination of hides which was proved
by Byrne et al. (2000) where they washed the cattle to be slaughtered with
power hose for 3 min to remove all visible faecal matter on the live animal.
They observed that this kind of pre-slaughter washing reduced incidences of
Escherichia coli O157:H7 transfers from hide to the carcass during slaughter.
Apart from these bacterial components, the enzymes produced by these
bacteria and the autolytic enzymes were considered to be important factors
that lead to putrefaction of skins and hides. The protein rich raw buffalo
hides are prone to attack by various proteolytic enzymes. Various bacteria
are reported to produce proteases that could damage the raw buffalo hides.
Few of them are known to posses the ability to produce specific group of
enzymes that selectively degrade a particular element from the raw hide.
With this perspective the enzymes collagenase, elastase, keratinase, and
others could be of grave importance in putrefaction of raw buffalo hide.
The collagenase produced by Clostridium histolyticum is one such enzyme
that degrades the triple helical region of native collagen under physiological
conditions. Yoshida and Noda (1965), Kono (1969), Markel et al. (1975),
Dreisbach and Markel (1978) are some researchers who have worked on
bacterial collagenase. However, Vanwart and Steinbrink (1981) and Barrett
et al. (1989) have discussed continuous spectrophotometric and fluorimetric
assay for bacterial collagenase. Koshy et al. (1999) has suggested an alterna-
tive assay method measuring collagenolytic activity using Hacetylated col-
lagen. Huang and Abramson (1975) and Yoshioka et al. (1987) are the ones
amongst many who have demonstrated presence of mammalian collagenase.
Keratinase is one such enzyme which degrades keratin and the organisms
producing keratin have been reported by Onifade et al. (1998), Wang and
Shih (1999), Gradisor et al. (2000), Yamamura et al. (2002), Gessesse et al.
(2003), Singh (2003) and Macedo et al. (2005). Few of these researchers
have discussed the use of keratinase produced by these organisms for the
process of dehairing however hardly any of them has considered their role
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in putrefaction of raw buffalo hide. Elastase is another enzyme, which could

degrade the elastin from raw buffalo hide. He et al. (2004) have described
the bacteria producing this enzyme. Along with these enzymes, there are
many more which can act on one or more than one element of raw buffalo
hide leading to unwanted changes. These are esterase (Deasy et al., 1968),
pepsin (Steven, 1966), lipases, phosphatase, oxidase, dehydrogenase, and
glycolytic enzymes. Polkade (2007) has extensively studied the microflora
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of raw buffalo hides by both culture dependent and culture independent ap-
proach. Polkade et al. (2013) developed a suitable nutrient medium contain-
ing skin extract to study microflora of buffalo hide. The studies identified 65
distinct morphotypes of bacteria. Many of these organisms were found to be
proteolytic, amylolytic, and lipolytic becoming cause of hide putrefaction.
The changes occurring in the hide due to these components will lead to
release of certain degradation products. Protein being the major constitu-
ent, main degradation products of raw buffalo hides or skins could be the
amino acids. They have been detected from the preserved skins and hides
to evaluate the effective preservation (Sivaparvathi et al., 1993). Another
degradation product from the raw buffalo hide could be free fatty acids re-
leased as a result of fat hydrolysis. Shede et al. (2008) have studied changes
occurring in number and types of bacteria in raw buffalo hide on standing.
Their activity contributes to the putrefaction of the hide. Shede et al. (2009)
showed that keeping qualities of hides are always dependent on the total
microbial flora associated with the freshly flayed and/or stored hides and the
biochemical changes brought about by these microorganisms during short-
term storage at ambient temperature.
9.6 PLANT-BASED MATERIALS AS IDEAL BIOAMBIANT

PRESERVATIVES
It is estimated that there are 250,000500,000 species of plants on earth

(Borris, 1996), a very small share of which is used by humans and animals as
their food. Customarily many of them are also used as a source of medicine.
Finding healing powers in plants is an ancient idea. One such possession
shown by plentiful plant species is antimicrobial activity.
Plants have an almost limitless ability to synthesize secondary metabo-
lites. In many cases, these substances serve as plant defense mechanisms
against predation by microorganisms, insects, and herbivores. Functional
antimicrobial phytochemicals can be divided into several categories as phe-
nolics (Proestos et al., 2005) and polyphenols, quinones, flavones, flavonoids
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and flavonols, tannins, coumarins, alkaloids, lactins, and polypeptides. Other

than these, there are few complex mixtures containing chemicals that prove
to be antimicrobial in nature. Latex of papaya is one such complex mixture
containing enzyme, alkaloid, and terpenoids. Plant derived oils also possess
antimicrobial activities (Varel and Miller, 2001). Different parts of the plant,
viz. root, leaves, fruits, stem, bark, flowers, seeds, and fruits may contain
such kind of activities.
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Several researchers have tested numerous plant species for their antimi-
crobial activities. In most cases, they used organic solvents for extraction of
different plant parts with certain exceptions wherein aqueous extracts have
been used. Aloe vera (Pawar et al., 2005), Thespesia populnea (Shastry et
al., 2005), Eclipta alba (Kothari and Shrivastava, 2005), Euphorbia fusi-
formis (Natarajan et al., 2005), Zingiber officinale, Bosenbergia pandurata,
Curcouma longa (Thongson et al., 2004), Puerariae radix (Kim and Fung,
2004), Cymbopogon citratus, Ocimum basilicum, Ocimum gratissimum,
Thymus vulgaris (Nguefack et al., 2004), Larrea divaricata (Quiroga et al.,
2004), Eucalyptus maculata (Takahashi et al., 2004), Aquilegia vulgaris
(Bylka et al., 2004), Erythrina poeppigiana (Sato et al., 2003) and Withania
somnifera (Poonkothai et al., 2005) are few plants screened for their antibac-
terial and antifungal activities. The test organisms for their studies were from
the genera Staphylococcus, Streptococcus, Pseudomonas, Proteus, Listeria,
Salmonella, Bacillus, Klebsiella, Escherichia, Micrococcus, Enterococcus,
Bordetella, Aspergillus, Trichoderma, Curvularia, Pichia, Saccharomyces,
and Candida. Most of them were pathogenic in nature. The plant parts used
in these studies were different for each screened plant. In many of the above
cases it was observed that the single plant material shows antimicrobial ac-
tivity against a wide array of organisms. This could strengthen the idea of us-
ing plant substances as antimicrobial agents to preserve the raw buffalo hide.
Typically the trend pursued to explore plants with antibacterial activi-
ties is based on the traditional knowledge involving ethno-botanical infor-
mation. Indigenous medicinal plants and their antibacterial activities were
of great interest to scientists from Iran (Bonjar et al., 2003, 2004a, 2004b,
2004c, 2004d), Ivory Coast (Okepekon et al., 2004), East Africa (Fabry
et al., 1998), South Africa (Elgorashi et al., 2003), Asia (Almas, 2001),
Taiwan (Wang and Huang, 2005) and Yemen (Ali, 2001). They found al-
most each plant tested was having the antibacterial and/or antifungal activity
for screened microbial cultures. In the Indian continent, there are plenty of
herbs, shrubs, and trees with strong antibacterial activities. However keep-
ing in mind the proposed global use of selected plant materials in leather
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industries Plumbago sp., Lawsonia sp., and Azadirachta sp. should be the
plants of preference.
9.6.1 AZADIRACHTA SP.
The genus Azadirachta consists of two species of Indomalayan origin. The
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species Azadirachta indica A. Juss. (syn. Melia azadirachta Linn.) is com-
mon in India. Vernacularly it is known as Neem tree (English Margosa
tree). It is a large, evergreen tree of about 1218 m high. Leaves of these
plants are imparipinnate, alternate, 2038 cm long with 819 leaflets. The
plant is distributed throughout India (Almeida and Almeida, 2005), in de-
ciduous forests and also widely cultivated. It is a hardy tree, grows well
in saline soils and drought conditions and is propagated from seeds. It can
grow in all types of soil. Within one year, the seedling grows up to a height
of 120 cm. Rapid multiplication through leaf culture has been found suc-
cessful. The parts used of this plant are bark, leaf, flower, seed, and oil.
About 100 compounds, mostly triterpenoids of protolimonoids, limonoids,
tetranortriterpenoid--hydroxy butenolides, pentanortriterpenoids, a hexan-
ortriterpenoid apart from a few nontriterpenoid constituents have been re-
ported from various parts (Sharma et al., 2000a). The plant shows different
pharmacological activities as anticancer, antiviral, spasmogenic, antibacte-
rial, antineoplastic, antifungal, antihelminthic, mosquito larvicidal, hypo-
glycemic, pesticidal, insecticidal, nematicide, vermicide, antitubercular,
antimicrobial, diuretic, antiprotozoal, and antimalarial. The plant substances
are nontoxic in nature. Neem is a hardy tree, grows well in saline soils and
drought conditions, propagated from seeds, root suckers and stem cuttings.
It can be grown in all types of soil. Rapid multiplication through leaf culture
has been found successful.
Different researchers have reported the antibacterial and antifungal ac-
tivities of neem oil. Das et al. (1999), Baswa et al. (2001), Coventry and
Allan (2001), and Fandohan et al. (2004) are few of them. SaiRam et al.
(2000) have demonstrated antiviral activity of neem against Poliovirus along
with antibacterial and antifungal activities. Parida et al. (2002) showed in-
hibitory effect of neem leaves on dengue virus. Biswas et al. (2002) and
Subapriya and Nagini (2005) have commented on medicinal properties
and different activities of neem leaves and antiseptic nature is one of them.
Antimicrobial effect of neem on certain foliar pathogens was studied by
Bipte and Musaddiq (2005). The above-mentioned studies were carried out
mainly on the pathogenic cultures and Staphylococcus aureus, Escherichia
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coli, Pseudomonas aeruginosa, Xanthomonas sp., and Candida albicans

were few of them (Okemo et al., 2001).
The phytochemical investigations of Azadirachta sp. have revealed pres-
ence of various chemical constituents. Tetracyclic triterpenoids from the
leaves of Azadirachta indica have been reported by Siddiqui et al. (2004);
azadirachtin-A, nimbin and salannin by Babu et al. (2006). The leaf extract
of neem has been reported to be nontoxic, non-mutagenic and possesses
9781771883627
immuno-stimulant, hepato-protective, anti-oxidant, anti-genotoxic, and an-
ti-cancer activities (Gangar et al., 2006). The leaves of neem mainly yield
quercetin (flavonoid) and nimbosterol (b-sitosterol) as well as number of
liminoids (nimbin and its derivatives). Fresh matured leaves yield an odor-
ous viscous essential oil, which exhibits antifungal activity. The principal
constituents of neem leaf include protein (7.1%), carbohydrates (22.9%),
minerals, calcium, phosphorus, vitamin C, carotene, and others. However
they also contain glutamic acid, tyrosine, aspartic acid, alanine, proline, glu-
tamine, and cystine like amino acids, and several fatty acids (dodecanoic,
tetradecanoic, elcosanic, and others). Shede (2007) has demonstrated use of
leaf extract of Azadirachta for preservation of buffalo hide.
9.6.2 PLUMBAGO SP.
The genus Plumbago consists of ten species distributed in tropical and

warm region. Out of these, three are reported in India as Plumbago zeyl-
anica Linn., P. indica and P. auriculata. Vernacularly it is known as Chitrak
(English Ceylon leadwort, White flowered leadwort). These are perennial,
sub-scandent shrubs with 60120 cm height. P. zeylanica has white flow-
ers and P. indica (syn. P. rosea) has red flowers. Cultivated as ornamental
plant throughout India, wild in Peninsular India and West Bengal. The parts
used of this plant are root and root bark. Plant contains number of naph-
thoquinone derivatives, viz. plumbagin, 3-chloroplumbagin, 3,3-biplumba-
gin, elliptinone, chitranone, zeylinone, isozeylinone, droserone, plumbagic
acid, plumbazeylanone, naphthalenone, and isoshinanolone (Sharma et al.,
2000b). The plant shows different pharmacological activities as antipyretic,
antibacterial, antifungal, anticancer, anticoagulant, antitumor, and hepato-
protective. The plant substances are nontoxic in nature. The plant is propa-
gated by seeds or by cuttings of side shoots. Well-drained sunny situation
and mild climate are preferable. It can also be propagated through tissue
culture technique.
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Quite a lot of scientists have described chitrak as an antibacterial plant.

Beg and Ahmad (2000) has tested Plumbago zeylanica against multidrug
resistant bacteria Staphylococcus aureus, Salmonella paratyphi, Escherichia
coli and Shigella dysenteriae of clinical origin. Chatterjee and Pakrashi
(1995), Paiva et al. (2003) and Singh et al. (2004) also gave such infor-
mation about the antimicrobial activity of chitrak. The plant Plumbago sp.
has been studied extensively for its chemical composition and their spe-
9781771883627
cific activities. Presence of binaphthoquinone in Plumbago zeylanica was
explained by Okamoto et al. (2001). Lupenone, lupeol acetate, plumbagin
and trilinolein are few constituents isolated from Plumbago zeylanica by
Nguyen et al. (2004). Lin et al. (2003) described the cytotoxic effect of con-
stituents like naphthoquinones and plumbagic acid glucosides from chitrak.
DNA cleavage by plumbagin was illustrated by Fujii et al. (1992). Further
Knecht et al. (2000) has reported inhibition of enzyme dihydroorotate dehy-
drogenase (EC 1.3.99.11) by plumbagin. Production of plumbagin from cell
cultures of Plumbago rosea L. was also reported by Komaraiah et al. (2001,
2002, 2003) and Panichayupakaranant and Tewtrakul (2002). Shede (2007)
has extensively studied effect of root powder extracts of Plumbago sp. on
microflora as well as their hydrolytic enzymes suggesting its potential in
bioambient preservation of hides.
9.6.3 LAWSONIA SP.
The genus Lawsonia is monotypic, that means it possess only one spe-
cies, Lawsonia inermis Linn. (syn. Lawsonia alba Lamk.). Vernacularly
it is known as Mehndi (English Henna). The plant is a glabrous, much
branched shrub or small tree. Geographically distributed in North Africa,
tropical and old world (Mabberley, 2000). Cultivated as ornamental plant
and naturalized all over India. The parts used of this plant are root, leaf,
flower, and seeds. The plant contains lawsone, esculetin, fraxetin, isoplum-
bagin, scopoletin, betulin, betulinic acid, hennadiol, lupeol and its related
compounds, lacoumarin, laxanthone I, II and III, flavone glycosides and two
pentacytic triterpenes (Sharma et al., 2000c). The plant shows different phar-
macological activities as antibacterial, antifungal, anti-inflammatory, antitu-
berular, and antitumor. The plant substances and the compounds present in
it are nontoxic in nature (Marzin and Kirkland, 2004). Mehndi grows on any
type of soil. It tolerates little alkalinity in the soil. Propagation is done using
seeds and cuttings.
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The plant is reported to inhibit certain bacteria (Musa et al., 2011a),

viz. Micrococcus luteus, Serratia marcescens, Klebsiella pneumonia and
Bordetella bronchiseptica (Bonjar, 2004d), Escherichia coli (Bonjar,
2004b), Staphylococcus aureus (Bonjar, 2004a), Xanthomonas sp. (Satish et
al., 1999), and few water pathogens (Kumar and Gopal, 1999). Malekzadeh
(1968) and Habbal et al. (2005) has also shown the antibacterial activity of
Lawsonia sp. Muhammad and Muhammad (2005) have used Lawsonia iner-
9781771883627
mis in the management of burn wound infections.
The literature stated various constituents from Lawsonia sp. and the ac-
tivities of these constituents have been also studied in great details (Musa
et al., 2011b, 2011c, 2012). Takeda (1988) reported phenolic glucosides
and Bakkali et al. (1997) showed presence of lawsone in Lawsonia inermis
cell cultures. Leung and Foster (1996) have mentioned presence of lawsone
(2-hydroxy-1,4-naphthoquinone) 1,4-naphthoquinone, gallic acid and tannin
with antimicrobial activities from mehndi plant. The genotoxic effect of this
lawsone from mehndi was assessed by Kirkland and Marzin (2003). Also
there are reports of enzyme inhibition by lawsone. Trypsin (Yogisha et al.,
2002) and dihydroorotate dehydrogenase (Knecht et al., 2000) were found to
be inhibited by lawsone. Extensive studies have been done by Shede (2007)
on antimicrobial activity of Lawsonia sp. leaf extract. The microflora of raw
buffalo hide and their hydrolytic enzymes were inhibited by Lawsonia. The
bioambiant preservation of raw buffalo hide using Lawsonia sp. leaf extract
was demonstrated.
Leather industry, holding a concrete place in Indian and Global economy is

under severe threat because of the waste generated and the upcoming strin-
gent laws and regulations of waste disposal posed by concerned governing
bodies. To survive in such conditions, leather sector was provoked to move
away from traditional processing techniques. Use of bioprocessing methods
instead of chemical processes was one such move, which was exercised in
the process of soaking, dehairing, bating, and degreasing and reduction in
the amount of hazardous waste was achieved. Nevertheless salting which
contributes to almost half the TDS from composite tannery waste continues
to be a traditional processing routine. Attempts made to minimize these pol-
lution problems by replacing salt with other chemicals are appreciable al-
though found inadequate to cope up with two major fronts as cost of leather
production and quality of leather produced.
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Although the plant materials are studied in great details for their chemi-
cal constituents, recently new additions to list of constituents has been re-
ported (Siddiqui and Kardar, 2001; Nguyen et al., 2004). This supports our
assumption that few or many more new constituents with varied biological
activities could be discovered from these plant materials.
Previous studies (Shede, 2007) specify that the organic extracts of chi-
trak root and mehndi leave possess antibacterial activities. The solubility
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patterns of identified antibacterial principle from these plants also support
this data. Plumbagin and lawsone, the major antibacterial constituents from
chitrak and mehndi, respectively are known to be soluble more in non-po-
lar solvents than that of polar solvents. Considering this, the antibacterial
activity shown by aqueous (polar solvent) extract in the recent studies is
of immense importance. Investigation of solubility pattern of antibacterial
principle(s) from aqueous extracts in organic solvents revealed that their sol-
ubility is more in ethyl acetate than chloroform. This conclusion is based on
the percent yield of residues obtained from solvent fractions after extraction.
Nevertheless, the extent of antibacterial activity shown by them is nearly
comparable. Investigation of antibacterial activities, in plant substances after
exhaustive Soxhlet extraction revealed that the antibacterial activity from
chitrak root powder and mehndi leaf powder is reduced after ethanol extrac-
tion whereas, chloroform extraction could not affect the activity. This is evi-
dence in support of the information that the active antibacterial principle(s)
from chitrak root and mehndi leaf powders used in recent studies are more
soluble in polar solvent than that of non-polar solvent. These pieces of infor-
mation strengthen the possibility of antibacterial principle(s) being different
than known naphthoquinone derivatives.
The conservative way to preserve the raw buffalo hides is use of common
salts leading to high TDS ratio in the tannery waste. The approach to replace
this common salt with other chemicals was not appreciated at tannery levels.
The literature states the defects and changes occurring in the buffalo hide
when preserved with common salt and the alternative chemicals. There are
no reports on the changes in hide stored at ambient temperature without any
preservatives. Hence it is required to study these kinds of changes, which
could be of help in evaluating the efficiency of any alternative preservative.
The work carried out on the alternative chemical, biological, and physical
methods of preservation states that many of these methods yield inferior
quality leather. Hence it becomes of immense importance that the alternative
preservation method devised yields good quality leather.
Biopreservation, the process employing bio-based preservatives for pres-
ervation of raw buffalo hides has not been explored to its best. The insight in
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biopreservation gives an enormous prospect to plant substances with the bio-

cidal activities. Such plant substance was reported to preserve the hide but
with aid of chemicals. Hence more promising plant substances are needed to
be screened. Plenty of plant substances are known to have the bactericidal
as well as the bacteriostatic activities. The basis for plant selection should be
their abundance, ease of propagation, inexpensive cultivation methods, and
broad-spectrum antibacterial activities. Considering this, Azadirachta sp.,
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Plumbago sp., and Lawsonia sp. possibly could be the finest source of long
waited biopreservatives.
In the light of socio-economic and environmental aspects of Leather
industry, biotechnological intervention appears to play an important role.
Preservation of animal skins and hides before they reach tannery for leath-
er making is an important step. Environmental friendly biotechnological
method of preservation of skins and hides becomes a desirable choice. The
literature available on preservative activity of different plant species opens
up a field of invention. Considering this benefit and the socio-economic rel-
evance, Government of India launched a research project in Biotechnology
for Leather under Council for Scientific and Industrial Research New
Millennium Indian Leadership Initiative program during 20022011.
Discovering new plant species and developing plant material based products
for preservation of skins and hides could be the future areas of research in
leather industry.
KEYWORDS
Ambient storage
Antimicrobial activity
Azadirachta sp.
Bioambient preservation
Hide degradation
Hide microflora
Hides and skins
Lawsone
Lawsonia sp.
Leather deterioration
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Leather industry
Leather making
Microbial degradation
Microbial succession
Napthoquinone
9781771883627
Natural products
Phenolics
Pickling of hides
Plant-based materials
Plumbagin
Plumbago sp.
Salting of hides
Slaughter house
Tannery
Tannery waste
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CHAPTER 10
PHYTOREMEDIATION OF ORGANIC
CHEMOPOLLUTANTS
9781771883627
PRADNYA PRALHAD KANEKAR1, SEEMA SHREEPAD SARNAIK2,
PARAG AVINASH VAISHAMPAYAN3, and PRAFULLA NAMDEO
SHEDE4
1
Department of Biotechnology, Modern College of Arts, Science and
Commerce, Shivajinagar, Pune 411005, India
2
Microbial Sciences Division, MACS Agharkar Research Institute,
G.G. Agarkar Road, Pune 411004, India
3
Biotechnology and Planetary Protection Group, Jet Propulsion
Laboratory, California Institute of Technology, Pasadena, CA 91109,
USA
4
Department of Microbiology, MES Abasaheb Garware College,
Karve Road, Pune 411004, India
CONTENTS
10.1 Introduction....................................................................................258
10.2 Phytoremediation...........................................................................259
10.3 Constructed Wetlands.....................................................................263
10.4 Phytoindicators and Test Systems..................................................263
10.5 Applications of Phytoremediation.................................................264
10.6 Applications of Constructed Wetlands for Different
Organic Pollutants..........................................................................278
10.7 Conclusion and Future Perspectives..............................................279
Keywords..................................................................................................280
References.................................................................................................281
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10.1INTRODUCTION
Over the past 50 years, rapid growth of population, mining and industrializa-
tion significantly contributed to extensive soil, air, and water contamination.
Many industrial wastes contain toxic chemicals, both organic compounds,
such as petrochemicals, polyaromatic hydrocarbons (PAHs), dyes, pesticides,
explosives and others, and inorganic compounds like heavy metals, nitrates,
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sulfates and chlorides, and others which are harmful to various forms of life.
Therefore it becomes necessary to remediate these contaminated sites. Some
of the remediation methods include chemical methods, such as use of differ-
ent chemicals for removal of these contaminants, physico-chemical methods
include techniques like reverse-osmosis, membrane filtration, and others.
However, these treatment methods are costly and difficult to adapt in case of
soil contaminated sites. Therefore biological methods of remediation gener-
ally called as bioremediation technologies become suitable in such cases.
Some of organic pollutants are toxic and difficult to degrade compounds.
Because of their insolubility, they accumulate in sediments and thus become
a serious problem.
Bioremediation is a biotechnology that uses microorganisms to de-
grade and detoxify pollutants causing environmental contamination. This
technology is applied for treatment of wastes. The bioremediation can be
carried out at the site of contamination (in situ) or the wastes are trans-
ported at some suitable site for treatment (ex situ). Exploration of plants
for remediation is an emerging cost-effective approach. The strategies
involving plants are commonly called phytotechnologies which include
phytoremediation. Phytotechnologies are defined as the use of plants to
remediate, treat, stabilize, or control contaminated substrates, and phy-
toremediation is one of these, specifically dedicated to the removal or
destruction of the contaminant. Phytotechnologies and phytoremediation
exploit the natural plant physiological processes. Environmental pollution
by organic nitro- and chlorinated compounds, hydrocarbons, pesticides,
and others, is caused mainly by human activities (Megharaj et al., 2011).
Microorganisms are employed to develop bioremediation technology for
degradation of organic pollutants. This technology suffers from some
limitations in applications to contaminated sites. Other technologies like
composting, phytoremediation, and related others are suggested. Finding
sources of pollutants is important to know their status in sediments. Perelo
(2010) has suggested adapting new efficient strategies like phytoremedia-
tion, bioaugmentation, and others.
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Phytoremediation of Organic Pollutants 259
10.2PHYTOREMEDIATION
Phytoremediation describes the treatment of environmental problems

(through the use of plants that mitigate the environmental problem) with-
out the need to excavate the contaminant material and dispose of it else-
where. For treatment of sites contaminated with pesticides, heavy metals,
explosives, hydrocarbons, and other related chemopolluants, plant systems
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that can degrade or stabilize them are used to develop phytoremediation
technology. Though phytoremediation is a promising alternative to physi-
cal and chemical remediation method it is still a nascent technology. More
fundamental research is also urgently required to better understand, control,
and fully exploit the metabolic diversity of plants. Certain plant species are
endowed with the property to accumulate or degrade the pollutants. Their
physiological processes like absorption and translocation can be employed.
An integrated process involving microbial activity, plant physiology, envi-
ronmental engineering, and few others may be fruitful. Phytoremediation is
a cost effective, environmental friendly green technology (Schwitzguebel et
al., 2002; Schwitzguebel, 2004).
Adler (1996) suggest cleanup of polluted water and soil using plants.
McCutcheon and Jrgensen (2008) described phytoremediation as the use of
green plants to treat and control wastes in water, soil, and air which is an im-
portant part of the new field of ecological engineering. Various factors like
characteristics of pollutants and contaminated soil or water, nutrient avail-
ability, hydrology, toxicity of the pollutants to the plants, bioavailability, and
others, govern the application of phytoremediation process. Long time and
area treatment is restricted to rhizosphere and shallow water bodies. At low
concentrations of pollutants, plant ecosystems like wetlands, grasslands can
be effective.
10.2.1 ADVANTAGES AND LIMITATIONS OF

PHYTOREMEDIATION
10.2.1.1ADVANTAGES
Phytoremediation technology seems to be more advantageous technology as

compared to other ones. The phytoremediation technology uses the natural
resources like plants, therefore it becomes a cost effective technology as
it dose not require high amount of capital for raising the technology. The
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plants can be easily developed and monitored for their tolerance to paricular
contaminant. The training can be given to layman and no highly qualified
personnel are required. When this phytoremediation technology is used for
remediation of sites contaminated with heavy metals, the plants can be used
for recovery of economically important or highly precious metals. These
valuable recovered metals can be used anywhere. This technique is also
known as phytomining. Since this technology uses the natural resources
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like plants and naturally occurring organisms in their vicinity, this tech-
nology preserves the environment in a more natural state. Many a times
this technology can be applied directly at the site of contamination either
soil or water and no need of ex situ application or excavation of soil. One
more advantage of phytoremediation technology is environmental friendli-
ness. Traditional methods disrupt soil structure and reduce soil productivity,
whereas phytoremediation can clean up the soil without causing any kind
of harm to soil quality.Thus it is a clean, efficient, inexpensive, and envi-
ronmentally non-disruptive technology as against other chemical or other
technologies.
10.2.1.2LIMITATIONS
Although it is a clean and green technology, this technology also has some
limitations. Phytoremediation is limited to the surface area and depth occu-
pied by the roots. Since growth of plants is slow and comparatively low bio-
mass is obtained from them, this technology requires long time for treatment
and thus requires a long-term commitment. Leaching of pollutants leading
to groundwater pollution can not be fully controlled since the plant systems
are used in tretament. Thus the efficiency of phytoremediation of ground-
water is affected. Tolerance of the plants is different in case of different
contaminants, therefore the survival of the plants is affected by the toxicity
of the contaminated land and the general condition of the soil or water site.
Since there is bio-accumulation of contaminants, especially metals, into the
plants which then pass into the food chain, such plants cannot be used in
food chain and require the safe disposal of such affected plant material. This
remediation process is dependent on a plants ability to grow and thrive in
an environment that is not ideal for normal plant growth. Phytoremediation
may be applied wherever the soil or static water environment has become
polluted or is suffering from ongoing chronic pollution.
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10.2.2 PROCESSES INVOLVED IN PHYTOREMEDIATION
During phytoremediation, different plant physiological processes are ex-

ploited to remove or destroy the contaminants from the contaminated sites
(Cunningham et al., 1995, 1996; Singh and Jain, 2003). A range of processes
mediated by plants or algae is useful in treating environmental problems.
Phytoremediation can occur through the following routes.
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10.2.2.1PHYTOEXTRACTION
Certain plants have ability to take up pollutants from soil, water bodies, and
others. The contaminants are absorbed through root system and transported
to leaves or stem. This plant physiological process is termed as phytoextrac-
tion. Some plants can accumulate pollutants in large quantity till the time
they are harvested. Compared to the volume of contaminated site, plant mat-
ter that extracts the pollutants is small and therefore easy for disposal. Thus
the phytoextraction process is being globally employed. After the process,
the cleaned soil can support other vegetation. Mining with plants or phy-
tomining is also being experimented. Willow plants were used by Greger
and Landberg (1999) for phytoextraction purpose.
10.2.2.2PHYTOSTABILIZATION
In this process, the mobility of contaminants in the environment is reduced

from the contaminated site. Chemical modification due to plant metabolic
activity of the pollutant may also result in immobilization, like phytostabili-
zation of the pollutant. Phytostabilization focuses on long-term stabilization
and containment of the pollutant. The pollutants are adsorbed or accumulated
in root zone of the plant system, where they are precipitated and stabilized.
The pollutants are restricted to roots only and therefore are not available to
animals or human beings. In mining regions, the mine tailings are covered
by vegetation for stabilization of the pollutants (Mendez and Maier, 2008).
10.2.2.3PHYTOTRANSFORMATION
This process involves chemical modification of the pollutant as a direct re-

sult of plant metabolism, often resulting in their inactivation, or degradation,
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like phytodegradation. In phytotransformation process, toxic compounds

like explosives, pesticides are transformed to nontoxic compounds by some
plants, like Cannas. However, there is no complete degradation of the pol-
lutants in phytotransformation. Microorganisms present in rhizosphere may
degrade the organic compounds. Thus, plants primarily reduce toxicity of
the pollutants. Subramanian et al. (2006) have studied phytotransformation
of TNT.
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10.2.2.4PHYTOSTIMULATION
In this process, the microorganisms associated with plant roots enhance the
soil microbial activity for the degradation of contaminants. This process is
essentially a rhizodegradation of pollutants. Some aquatic plants support
growth of microorganisms and stimulate them to degrade the chemical com-
pounds. Rupassara et al. (2002) have studied degradation of atrazine via
phytostimulation by the plant hornwort.
10.2.2.5PHYTOVOLATILIZATION
In this plant physiological process, the pollutants are first transformed to

volatile compounds which are released into the air.
10.2.2.6RHIZOFILTRATION
This process involves filtering water through a mass of roots to remove

toxic substances or excess nutrients. The pollutants remain absorbed in or
adsorbed by the roots. Plants capable of absorbing pollutants are cultivated
and used to minimize concentration of pollutants. However, it is likely that
the pollutants are incorporated in plant biomass and returned to environment
with death or harvesting of plants.
10.2.3 PLANTS USED IN PHYTOREMEDIATION
A variety of plants are used for the purpose of phytoremediation. These in-
clude both aquatic plants and terrestrial plants. Most commonly used aquatic
plants include species of Hydrilla, Pistia, Wolffia, Echornia, such as water
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hyacinth, Ceratophyllum (hornwort), Salvinea (watermoss), Vallisneria

(eelgrass, tape grass), Najas, and Potamogeton (pondweed). The terrestrial
plants used in phytoremediation technique include species of Ambrosia,
Apocynum, Barley, Brassica, Cannabis, Carex, Festuca, Helianthus,
Lupinus, Melilotus, Moringa, Phalaris, Phragmites, Poplar, Polypogon,
Salix, Thlaspi, Tradescantia, Tree bog, Vicia and Vetiver, and others.
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10.3 CONSTRUCTED WETLANDS
Wetlands are transitional areas between land and water. The boundaries
between wetlands and uplands or deep water are therefore not always dis-
tinct. The wetlands include a wide variety of ecosystems like marshes, wet
meadows, floodplains, swamps, and others. The pollutants like metals are
filtered in natural wetlands. This mechanism of biofiltration is simulated in
constructed wetland (CW). Wetlands are very productive ecosystems with
efficient photosynthesis and transpiration. Phytoremediation in wetlands has
been described by Williams (2002).
Different processes like plantmicrobe interactions, response of mi-
crobes to soil environment and interaction of all these elements in the natural
ecosystem like wetlands are yet to be fully understood. Wetlands also serve
the purpose of offering shelter to wildlife, land reclamation, restoring habi-
tats disturbed by storm water runoff, mining wastes, and related others. The
mechanisms of plantmicrobe interaction in wetlands have been studied by
Stottmeister (2003).
10.4 PHYTOINDICATORS AND TEST SYSTEMS
Plant species vary in their response to various pollutants occurring in their

environment. Effects of various chemopollutants on plant systems have been
studied (Dixit and Merle, 1985; Sarbhoy et al., 1991). Before selecting plant
species for the purpose of developing phytoremediation technology, it is es-
sential to understand the behavior of plant systems in presence of pollutants.
Some plant species, such as Allium sepa L. has been evaluated as a test sys-
tem for industrial pollution (Dixit and Merle, 1985). Examples of plant spe-
cies studied as indicators of pollution or test systems for evaluating toxicity
of pollutants are summarized in Table 10.1.
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TABLE 10.1 Plant Species Used as Indicators of Pollution and Test Systems to Evaluate
Toxicity of Pollutants.
Organic Pollutant Plant Species References
Atrazine (herbicide) Leguminous plants (Vigna unguiculata) Vaishampayan and
Kanekar (2007)
Mancozeb (fungicide) Gliricidia sepium (Jacg.) Kanekar et al. (1998)
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Methyl violet dye Wolffia arrhiza (L.) Wimmer, Spirodella Kanekar et al. (1993a,
polyrrhiza (L.) Schleiden, Acacia nilotica 1993b)
(L.) Del, Casuarina aquisetifolia Forst
Thallium Lemna minor Kwan and Smith (1988)
Pesticides Pisum sp. Sarbhoy et al. (1991)
Industrial effluents Allium cepa (L.) Dixit and Merle (1985)
10.5 APPLICATIONS OF PHYTOREMEDIATION
One of the major applications of phytoremediation is to treat polluted soil

or water bodies. This process has been demonstrated for treatment of metal
mining wastes that contaminate soil and water. Phytoremediation has been
shown to be studied globally for pollutants like pesticides, nitroexplosives,
metals, crude oil, and related others. Some of the plants, such as mustard,
hemp, and alpine pennycress have been found to be successful in accu-
mulation of pollutants occurring in high concentration at contaminated
sites. Phytoremediation has been demonstrated as an efficient process in
restoration metal mining sites, dumping places of polychlorinated biphe-
nyls (PCBs). This technology is becoming a desirable fruitful option in
last twenty years for remediation of sites contaminated with heavy, toxic
metals.
10.5.1 PHYTOREMEDIATION OF ORGANIC POLLUTANTS
Environmental pollution with xenobiotics is a global problem and the de-

velopment of phytoremediation technologies for the plant-based cleanup
of contaminated soils is therefore of significant interest. Since it is a new
technique, meager data are available on actual use of plants for remedia-
tion of wastewaters containing pollutants like pesticides, dyes, nitroaromat-
ics, and other hazardous compounds or remediation of soils contaminated
with these toxic and chemicals. However, researchers over the globe studied
some plant species for phytoremediation of organic pollutants. The work is
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summarized in Table 10.2. This research forms basis to develop phytoreme-

diation technologies.
TABLE 10.2 Plant Species Studied for Phytoremediation of Organic Chemopollutants.

Organic Plant Species Studied for References
Chemopollutant Phytoremediation
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Atrazine Corn and sorghum Shimabukuro (1968)
Atrazine Sorghum Lamoureux et al. (1973)
Atrazine Panicum dichotomiflorum De Prado et al. (1995)
Atrazine Poplar Burken and Schnoor (1997)
Nitroexplosive TNT Switch grass, brome grass Peterson et al. (1998)
Nitroexplosive RDX Myriophyllum aquaticum, Bhadra et al. (2001)
Catharanthus roseus
Atrazine Corn Cherifi et al. (2001)
Nitroexplosive TNT Abutilon avicennae (Indian mallow) Chang et al. (2004)
Herbicide atrazine Vetiveria zizanioides L. Vaishampayan (2004)
Antibiotic tetracycline Pistia sp. Gujarathi (2005)
Tannery waste water Pharagmites australis Calheiros et al. (2007)
Phenol Brassica napus (hairy roots) Coniglio et al. (2008)
RDX Constructed wetland Low et al. (2008)
Nitroexplosive TNT Vetiveria sp. Das et al. (2010)
Textile dye malachite Blumea malcolmii Hook. f. (cell Kagalkar et al. (2011)
green culture)
Naphthalene Eichhornia crassipes Nesterenko-Malkovskaya et
al. (2012)
Textile effluent Leucaena leucocephala Jayanthi et al. (2014)
10.5.1.1 NITRO COMPOUNDS INCLUDING NITROEXPLOSIVES

AND WASTE WATERS CONTAINING NITRATES
Nitroexplosives are difficult to degrade and exert toxicity. They cause large
scale contamination of land and groundwater. Under this situation, phytore-
mediation becomes a method of choice. Phytoremediation of groundwater/
sites contaminated with explosives was described by Best et al. (1997, 2006).
Peterson et al. (1998) studied the germination of switch grass and brome
grass, in soil contaminated with Trinitrotoluene (TNT). Results of their stud-
ies indicated that switch grass was more tolerant of TNT than smooth brome
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grass, but the establishment of both species may be limited to soil containing
less than 50 mg/kg of extractable TNT.
Both aquatic and terrestrial plants have been studied for phytoremedia-
tion of nitroexplosives like TNT, HMX, RDX, and others. Plants namely
pondweed, arrowroot, contrail, poplar have been employed for remediating
TNT contamination in a CW (Rodgers and Bunce, 2001). The plant sys-
tem could degrade 0.019 mg/L TNT per day. TNT removal rates increased
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with increased plant density and removal kinetics increased with increasing
temperature up to 34C (Medina et al., 2000). Algae nitella (stonewort) and
Myriophyllum aquaticum (parrot feather) exhibited 30 mg/L rate of TNT
removal in a hydroponic experiment. Several agricultural and indigenous
terrestrial plants were examined for their capacity to accumulate and de-
grade High Melting Explosive (HMX). Traces of mononitroso-HMX were
detected in contaminated soil extracts and leaf extracts. Mechanism for
HMX translocation and accumulation in foliar tissue was concluded to be
aqueous transpirational flux and evaporation (Groom et al., 2002). These
reports brighten the potential of applying phytoremediation for explosives.
Phytoremediation is more rugged than microbial bioreactors with respect to
physical conditions and changes in contaminant loading.
Vanek et al. (2007) applied phytoremediation techniques for selected ex-
plosives. Work on phytoremoval of TNT was carried out by Medina et al.
(2000) and Hannink et al. (2001). Groom et al. (2002) studied the accumula-
tion of HMX by indigenous and agricultural plants grown in HMX contami-
nated water. Kanekar et al. (2003) have mentioned use of some aquatic and
terrestrial plant species for phytoremediation of TNT, HMX, and others.
In study by Bhadra et al. (2001), M. aquaticum removed RDX from the
aqueous medium. RDX levels decreased by approximately 75% in the pres-
ence of live plants compared to about 10% in the presence of dead plant mat-
ter. RDX disappearance in the presence of dead plant matter typically rep-
resents that fraction was sorbed into biomass. This showed that biological
processes contributed to the depletion of RDX on exposure to M. aquaticum.
However, HMX was not metabolized in natural systems by M. aquaticum.
Rylott et al. (2009) developed an environment friendly, low-cost phy-
toremediation technique by applying recent knowledge of the biochemistry
underlying endogenous plant detoxification systems and the use of genetic
engineering to combine bacterial explosives-detoxifying genes with the phy-
toremediatory benefits of plants, such as poplar and perennial grasses. In
view of slow and incomplete phytoremediation of organic pollutants, such
as explosives, Aken (2009) suggested an innovative approach of develop-
ing transgenic plants to accomplish the phytoremediation at accelerated rate.
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According to his work, bacterial genes encoding enzymes involved in de-

rivatization of explosives has to be introduced in higher plants.
Chang et al. (2004) carried out experiments on phytoremediation of soil
contaminated with TNT by growing Indian mallow (Abutilon avicennae) in
a soil column reactor with 2 kg of TNT contaminated soil (120 mg TNT/
kg) in the top and 18 kg of uncontaminated soil in the bottom. The results
showed that planting Indian mallow in TNT contaminated soil enhanced
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TNT reduction both by stimulating microbial activity that enhances micro-
bial TNT transformation, and by direct uptake and phytotransformation of
TNT. Das et al. (2010) in their earlier experiments have shown the high
affinity of Vetiver grass for TNT and the catalytic effectiveness of urea in
enhancing plant uptake of TNT in hydroponic media. Further the authors
have demonstrated complete removal of TNT in urea-treated soil was ac-
complished by Vetiver at the low initial soil-TNT concentration (40 mg/kg),
in soil-pot-experiment masking the effect of urea. When the TNT concentra-
tion was doubled (80 mg/kg), there was significant increase in removal of
TNT by Vetiver. Thus the authors have concluded that Vetiver grass in the
presence of urea effectively removes TNT from soil.
Podlipn et al. (2015) thought that the 2,4-dinitrotoluene (2,4-DNT),
which is used mostly as explosive, belongs to the hazardous xenobiotics and
soils and waters contaminated with 2,4-DNT may be cleaned by phytoreme-
diation using suitable plant species. The authors studied the ability of crop
plants namely hemp, flax, sunflower, and mustard to germinate and grow on
soils contaminated with 2,4-DNT. The authors found the lethal concentra-
tion of 2,4-DNT for growth of these plant species around 1 mg/g. In hydro-
ponicic experiments, the authors have observed that, the above-mentioned
plant species were able to tolerate concentration of 2,4-DNT as high as 200
mg/L, which is close to maximal solubility of 2,4-DNT in water. The authors
have recommended the possible use of these crop plants for phytoremedia-
tion of nitroaromatic compounds. The uptake and removal of nitrobenzene
by Mirabilis jalapa L. has been reported by Zhou et al. (2012). The plants
could grow in presence of 10 mg nitrobenzene per kg soil without any ad-
verse effect. Thus the plant has potential in phytoremediation of soil con-
taminated with nitrobenzene.
Nitramines are thought to be metabolized by plants. Transformation
of RDX by plant tissue cultures is suggested by Aken et al. (2004a) via a
three step process involving a light-independent reduction of RDX to MNX
(hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine) and DNX (-dinitroso-) de-
rivatives by intact plant cells; a plant/light mediated cleavage of hetero-
cyclic ring of RMX, MNX, or DNX into CH2O and CH3OH and further
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mineralization of the chloride metabolites by intact plant cells. In nature,

plantmicrobe symbiotic association occurs which contributes to transfor-
mation of xenobiotics. Symbiotic bacterium Methylobacterium sp. isolat-
ed from Poplar plant tissues was found to mineralize HMX (Aken et al.,
2004b). Thus plantmicrobe symbiotic phytoremediation of nitroexplosives
can be possible.
Ability of agricultural and terrestrial plant species to degrade HMX was
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explored by Groom et al. (2002). The growth of wheat and ryegrass was
found to be rapid in presence of HMX. However these plants being edible
plants, their use in phytoremediation may not be advisable. Terrestrial plant
species namely Poplar and Glyricidia were explored for phytoremediation
of HMX wastewater (Dautpure, 2007). These plants removed nitrate from
microbially treated HMX wastewater to considerable extent. These studies
indicated that an integrative process involving microbial degradation fol-
lowed by phytoremediation can be exploited for treatment of nitroexplosive
wastewaters.
Nitrate removal by plants was studied by a few research workers.
Phytoremediation of nitrates was described by Bose and Srivastava (2001).
Dautpure (2007) has reported the use of phytoremediation technique for re-
moval of nitrates from high nitrate containing wastewater generated during
production of HMX. In the studies, the effect of high nitrate containing waste-
water on germination of some legume seeds was seen. The terrestrial plant
species studied were Silver Oak (Grevillea robusta) and Poplar (Populus
salicaceae) and the aquatic plant species included Hydrilla (Hydrilla verti-
cillata Casp.), Water lettuce (Pistia stratiotes Linn.) and Duckweed (Lemna
minor Linn.). The authors found that the silver oak and Hydrilla plants could
tolerate high concentration of nitrate present in the wastewater containing
HMX. In some other studies, Dautpure (2007) found the aquatic plants like
Ceratophyllum, Vallisneria, Salvinia and Hydrilla could be the suitable can-
didates for developing phytoremediation technology for treatment of high ni-
trate containing wastewaters generated during production of HMX. Kanekar
et al. (2014) have reviewed work on bioremediation of nitroexplosive waste.
10.5.1.2PESTICIDES
Pesticides are widely used for protection of crops and contribute signifi-
cantly to environmental pollution. Atrazine is one of the herbicides ap-
plied in the agricultural fields to get rid of the weeds. Atrazine is a toxic
herbicide and therefore remediation of sites contaminated with atrazine is
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necessary. Vaishampayan (2004) studied extensively plantmicrobe interac-

tive degradation of Atrazine. Arthrobacter sp. isolated from rhizosphere of
a grass plant Vetiveria zizanioides was found to be efficient in degradation
of atrazine (Vaishampayan et al., 2007). The grass plant Vetiver (Vetiveria
zizanioides) with its robust root system was found to be resistant to atra-
zine. Pot culture studies showed tolerance of Vetiver plant up to 10,000 ppm
(Vaishampayan, 2004). VetiverArthrobacter sp. culture interactive reme-
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diation of soil spiked with atrazine was effective and faster as compared to
that by the plant or the bacterial culture alone.
Merini et al. (2009) in their in vitro studies on tolerance to atrazine by dif-
ferent plant species and found Lolium multiflorum as a novel tolerant species
which was able to germinate and grow in the presence of 1 mg/kg of the herbi-
cide. The authors also studied the mechanisms involved in atrazine tolerance
such as mutation in psbA gene, enzymatic detoxification via P450 or chemical
hydrolysis through benzoxazinones, and others and demonstrated that atrazine
tolerance is conferred by enhanced enzymatic detoxification via P450. Due to
its atrazine degradation capacity in soil and its agronomical properties, L. mul-
tiflorum is a candidate for designing phytoremediation strategies for atrazine
contaminated agricultural soils, especially those involving run-off.
Amaya-Chvez et al. (2006) studied the removal efficiency of methyl
parathion (MeP) by a common cattail Typha latifolia L. from water and arti-
ficial sediments. The authors observed high efficiency of cattails for removal
of MeP from water and sediments relative to controls. Cattails may thus
prove to be a good candidate for developing a phytoremediation system for
MeP-contaminated water and artificial sediments. The plant species Solanum
nigrum L. was found to have potential in remediation of a persistent, leachy
fungicide metalaxyl (Teixeira et al., 2011). The plant was found to complete
its life cycle in the presence of metalaxyl stress. It accumulates the fungicide
in the plant part above ground and thus the plant tissue with high concentra-
tion of the fungicide can be disposed off. The authors also suggest that an-
tioxidant response in form of proline accumulation, increase in activities of
guaiacol peroxidase and glutathione-s-transferase could be the mechanism
of tolerance of the plant to the fungicide. Thus the plant Solanum nigrum is a
potential candidate for phytoremediation of a fungicide metalaxyl.
lvarez et al. (2012) studied removal of the organochlorine pesticide,
lindane from minimal medium (MM) by two Streptomyces native strains,
while growing on maize root exudates (REs) as a primary carbon and energy
source. Their studies showed that phytostimulation of organochlorine pes-
ticide degrading actinobacteria by maize REs may be a successful strategy
for the remediation of lindane-contaminated environments. Mimmo et al.
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(2015) developed a plant-based biotest (RHIZOtest) to study the phytoex-

traction capacity of Italian ryegrass (Lolium multiflorum L.) to remove a
trichloro azine herbicide, terbuthylazine (TBA) from aqueous solution at
three different concentrations of TBA, that is 0.5, 1.0, and 2.0 mg/L. The
authors have observed that the ryegrass can remove TBA and concluded that
RHIZO test could be adequate in testing removal of herbicides from aque-
ous/soil solutions.
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Accelerated mineralization of organic compounds was shown in rhizo-
spheric soil (Piutti et al., 2002). There was no attempt to establish plantmi-
crobe interaction for remediation of organic pollutants especially atrazine.
Vetivermicrobial interactions for remediation of atrazine-contaminated soil
were extensively studied by Vaishampayan (2004). Following the success-
ful screening and establishing potential of Vetiver in atrazine removal from
soil, a plant microbial interaction effect was studied in remediation of soil
contaminated with atrazine.
During the course of the bench scale studies, an atrazine degrading cul-
ture Arthrobacter sp. strain MCM B-436 was isolated from rhizosphere of
Vetiver plant. To establish a plantmicrobial interaction model, Vetiver and
the microbial culture isolated from its rhizosphere was selected. Soil used in
this experiment was dried and passed through 2 mm sieve and 500 g black
cotton soil was filled in 1 L capacity glass bottles. Vetiver plants of average 3
months age were procured from a nursery nearby Pune. Then, 100 g biomass
of Vetiver plants were planted in 500 g black cotton soil filled in 1 L capacity
glass bottles (Fig. 10.1).
FIGURE 10.1 Vetiver-microbial interactions for removal of atrazine from soil: (1) soil +
25 ppm atrazine + vetiver, (2) soil + vetiver (without atrazine), (3) soil + 25 ppm atrazine +
vetiver + culture, (4) sterile soil + 25 ppm atrazine, (5) sterile soil + 25 ppm atrazine + culture,
(6) soil + 25 ppm atrazine + culture (7) soil + 25 ppm atrazine (Vaishampayan, 2004).
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Plants were allowed to grow for 15 days to acclimatize the experimental

conditions. All the bottles were kept in the glasshouse conditions. All the
plants were watered regularly throughout the experiment. The following ex-
perimental sets were run with five replicates for each treatment: (1) Soil+25
ppm atrazine + Vetiver; (2) Soil + Vetiver (without atrazine); (3) Soil + 25
ppm atrazine + Vetiver + microbial culture; (4) Sterile soil + 25 ppm atra-
zine; (5) Sterile soil + 25 ppm atrazine + culture; (6) Soil + 25 ppm atrazine
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+ culture; (7) Soil + 25 ppm atrazine. Vetiver plants grown in soil without
atrazine exposure served as control to compare with Vetiver plants in atra-
zine spiked soil for normal growth. Atrazine spiked in sterile and non-sterile
soil served as control to determine atrazine losses due to physico-chemical
and microbial factors, respectively. For sterile soil controls, soil was steril-
ized by autoclaving at 121C for 20 min for three times, at an interval of
24 h. Sterility of the soil was determined by plating soil slurry on Standard
plate count agar. Plates were incubated at 37C for 24 h and after incubation
period presence of bacterial colonies was observed. No bacterial growth on
the plates suggested proper sterilization of the soil.
After acclimatization of plants for 15 days, soil was exposed to atrazine.
Soil was spiked with commercial grade atrazine dissolved in water to attain
25 ppm concentration in the soil by spraying. Immediately after atrazine
treatment, 5 g of black cotton soil based bioinoculum of Arthrobacter sp.
strain MCM B-436 was added to 500 g soil to attain culture density to 108
CFU/g. After addition of bioinoculum and spraying of atrazine, soil was wa-
tered to ensure uniform distribution of atrazine and the culture. All the glass
bottles were kept under glass house condition with average 30 2C tem-
perature and natural light conditions. Soil samples were collected from all
the experimental sets for 4 days with interval of 24 h. From each soil sample
(50 g) atrazine was extracted with dichloromethane extraction method and
residual atrazine concentration was determined by HPLC.
After 4 days of incubation, the soil planted with Vetiver along with bac-
terial culture showed complete removal of atrazine. Vetiver plants alone
showed 31% atrazine removal from the soil. Bacterial culture was able to
remove 94% and 90.8% atrazine from unsterile and sterile soil, respectively.
Plant microbial interaction showed rapid atrazine removal when com-
pared with culture and plants alone. When the results of the 3rd day, were
compared, culture and plants alone showed 65% and 24% atrazine removal
respectively, while plant microbial interaction showed 80 % atrazine remov-
al. Sterile soil spiked with atrazine showed 6% atrazine removal after 5 days
of exposure, which could be attributed to adsorption and other physical fac-
tors. Normal flora of soil contributed to 14% atrazine removal (Fig. 10.2).
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FIGURE 10.2 Vetiver-microbial interactions for removal of atrazine from contaminated
soil (St. = sterile) (Vaishampayan, 2004).
Field level experiment was carried out in Sugarcane farm nearby Pune.
The field site selected for the experiment had no history of atrazine applica-
tion. Design of field scale treatment protocols was on the basis of results of
the initial laboratory scale experiments. The dimensions of the experimental
plots were 1m 1m 0.5m (Fig. 10.3). Soil was excavated from the plots
and a plastic (polythene) lining was given to cover the entire plot area. The
distance between two adjacent plots was 1 m. The objective of the plastic
lining was to (1) avoid contamination of the soil adjacent to the experimen-
tal plots with atrazine, (2) avoid percolation/seepage of atrazine down the
soil column with water, and (3) define soil volume used in the experiment.
The entire volume of soil removed was replaced back after the plastic lin-
ing was laid. The total soil volume per experimental plot was found to be
approximately 500 kg. Ten experimental plots were prepared for Vetiver-
microbial interaction experiment. Vetiver plants approximately 7 months old
were procured from a nursery nearby Pune. Vetiver plants having biomass of
approximately 500 g were planted in each plot. Plants were allowed to grow
for 15 days for acclimatization to field conditions.
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FIGURE 10.3 Diagrammatic representation of field experimental plots (Vaishampayan, 2004).
A field-scale study consisting of ten (0.5 m3) treatment plots was de-
signed to test five treatment protocols in duplicate. A random design was
used to assign plots for different treatments to avoid bias in any environ-
mental factor. Experimental sets were as follows: (1) Soil not spiked with
atrazine and planted with Vetiver plant (Vegetation control); (2) Soil spiked
with atrazine and planted with Vetiver plants; (3) Soil spiked with atrazine,
planted with Vetiver and atrazine degrading culture; (4) Soil spiked with
atrazine and atrazine degrading culture; and (5) Soil spiked with atrazine (no
Vetiver and no atrazine degrading culture). Soil planted with Vetiver plants
without atrazine served as vegetation control. Soil was spiked with aque-
ous solution of commercial grade atrazine (25 ppm) by spraying. Bacterial
suspension was sprayed to attain Arthrobacter sp. strain MCM B-436 load
1 108 CFU/g soil. The bacterial culture was added in the soil without any
nutrient amendments. Soil spiked with atrazine without culture and Vetiver
plantation served as control to determine the atrazine removal by native mi-
croflora, chemical degradation and soil bound un-extractable atrazine. All
the plots were irrigated regularly throughout the experiment. The experiment
was carried out under natural conditions (temperature and light). Soil spiked
with atrazine without culture treatment and Vetiver plantation showed 6%
atrazine removal from the soil within 8 days. This loss of atrazine may be at-
tributed to the role of indigenous microflora, chemical degradation, and soil
bound non-extractable atrazine.
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Experimental plots with Vetiver plants showed 45.07% loss of atrazine.

Soil spiked with Arthrobacter sp. strain MCM B-436 culture showed 90.24%
removal on the 8th day (Fig. 10.4). Vetiverculture (Arthrobacter sp. strain
MCM B-436) interaction contributed to 96% removal of atrazine from the
soil. Though the difference in atrazine removal was marginal in plantmi-
crobe interaction and culture treatment, the rate of atrazine degradation in
the plant microbial treatment was significant. As on the 4th day, atrazine
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removal was 27.53% in the plots treated with culture alone and 53.39% in
plots with plant-culture together.
FIGURE 10.4 Vetiver-microbial interactions for the removal of atrazine from field soil
(Vaishampayan, 2004).
10.5.1.3 OTHER ORGANIC COMPOUNDS
Dyes and dye industry wastewaters contribute significantly to environmen-

tal pollution. Both aquatic and terrestrial plants respond to different dyes.
Kanekar et al. (1993a, 1993b) tested aquatic plants and terrestrial plants to
evaluate toxicity of methyl violet wastewater. Aquatic plants Wolffia arrhiza
(L.) Wimmer and Spirodela polyrhiza (L.) Schleiden indicated toxicity of
untreated methyl violet wastewater. Terrestrial plant species Acacia nilot-
ica (L.) Del and Casuarina equisetifolia Forts could be grown on micro-
bially treated methyl violet waste-water. Decolorization of textile industry
waste containing different dyes was studied by Kagalkar et al. (2011) using
cell cultures of Blumea malcolmii Hook. 93.4% of decolorization was ob-
served in case of malachite green within 24 h. Thus the plant cell culture
shows potential in phytoremediation of dyes. Plant biomass of Leucaena
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leucocephala was found to be useful in treating textile effluent as reported

by Jayanthi et al. (2014).
Cunningham et al. (1996) carried out studies on phytoremediation of
soils contaminated with organic pollutants. Phenolic compounds present in
the drainage from several industries are harmful pollutants and represent a
potential danger to human health. The hairy roots of Brassica napus pos-
sess enzymes such as peroxidases which oxidize phenols. The hairy roots
9781771883627
were found to be tolerant to high concentration of phenol and hence could
be used for remediation of water containing phenol (Coniglio et al., 2008).
Jha et al. (2013) attempted degradation of phenol, a common pollutant found
in wastewater of many industries using hairy roots, induced in ornamental
variety of Helianthus annuus (sunflower). The authors also found complete
removal of phenol by these hairy roots and proposed a possible pathway of
rhizodegradation of phenol based on the data collected by identification of
metabolites by GC-MS. Shi et al. (2012) investigated the toxicity, uptake,
accumulation, and removal of 2,4-dichlorophenol (2,4-DCP) in four Salix
matsudana clones and feasibility of phytoremediation using S. matsudana
clones in hydroponic system. The authors observed removal of 2,4-DCP
from aquatic environment rapidly and efficiently by these clones without
any adverse effect on the plants.
Maqbool et al. (2012) reported the effect of bioaugmentation by free and
immobilized bacterial culture on the rhizodegradation of petroleum-pollut-
ed soil using Sesbania cannabina plant. The authors have observed higher
and rapid rhizodegradation with indegenous bacteria than bioaugmented
bacterial cultures and concluded that natural plantmicrobe interaction in
the rhizosphere of S. cannabina was more efficient in degrading TPH to the
petroleum hydrocarbons. Al-Baldawi et al. (2013) developed a phytotoxic-
ity test of bulrush (Scirpus grossus) to assess its ability to phytoremediate
diesel contamination in simulated wastewater at different concentrations
(0,8700, 17,400, and 26,100 mg/L). The authors observed that S. grossus
was able to reduce total petroleum hydrocarbon (TPH) by 70.0 and 80.2%
at the concentrations of 8700mg/L and 17,400mg/L, respectively. The
concentration of diesel > 26,100 mg/L was found to be lethal for S. gros-
sus after 14 days. The plant Scirpus grossus was found to survive in water
contaminated with diesel as evidenced by increase in biomass. Al-Baldawi
et al. (2014) investigated the optimum conditions for TPH removal from
diesel-contaminated water using phytoremediation treatment with Scirpus
grossus through a Box-Behnken Design. The optimum conditions were
found to be a diesel concentration of 0.25% (Vdiesel/Vwater), a retention time
of 63 days and no aeration with an estimated maximum TPH removal from
AUTHOR COPY
water and sand of 76.3 and 56.5%, respectively. The authors recommended
the use of S.grossus, a Malaysian native plant for remediation of waste-
water containing hydrocarbons. Polycyclic aromatic hydrocarbons (PAHs)
represent one of the major groups of organic contaminants in the aquat-
ic environment. Duckweed (Lemna minor L.) is a common aquatic plant
widely used in phytotoxicity tests for xenobiotic substances. Zezulka et al.
(2013) studied physiological, biochemical and other changes occurring in
9781771883627
duckweed plants exposed to fluoranthene for 410 days. The authors ob-
served that FLT did not influence the growth and content of photosynthetic
pigments in duckweed, however, histological changes occurred only at the
level of root cells and tissues. They further observed that FLT treatment had
no adverse effect on the chlorophyll fluorescence parameters and activity of
some enzymes like ascorbate peroxidase, catalase, superoxide dismutase,
and related others, was found to be enhanced in presence of FLT. Kirk et al.
(2005) studied rhizospheric microbial community to enhance degradation
of organic contaminants. They investigated the effect of ryegrass in com-
bination with alfalfa in soil contaminated with 31,000 ppm of petroleum
hydrocarbons.The denaturing gradient gel electrophoresis (DGGE) analy-
sis of PCR-amplified partial 16S rDNA sequences showed increase in the
number of rhizosphere bacteria especially petroleum degrading bacteria in
the hydrocarbon-contaminated soil with ryegrass and ryegrass/alfalfa mix-
ture. The authors had concluded that plants altered the microbial population
which were plant-specific and could contribute to degradation of petroleum
hydrocarbons in contaminated soil.
Nesterenko-Malkovskaya et al. (2012) investigated the potential of an
aquatic plant, the water hyacinth (Eichhornia crassipes) devoid rhizospher-
ic bacteria, to reduce naphthalene (a polyaromatic hydrocarbon) present in
wastewater and wetlands. The plants enhanced the removal of pollutants
through their consumption as nutrients and also through microbial activity of
their rhizospheric bacteria. Naphthalene removal by water hyacinth coupled
with natural rhizospheric bacteria was 100% after 9days; however without
rhizospheric bacteria, the removal was only up to 45% during 7days. In
the removal of naphthalene from water, the contribution of aquatic plant
Eichhornia crassipes is much more than its rhizospheric bacteria.
Among the persistent organic pollutants, dioxins get bioaccumulated in
food chain causing a considerable risk to human health. Removal of dioxins
using plants was suggested. Uptake of 2,3,7,8-tetra-chlorinated dibenzo--
dioxin (TCDD) by the plant Arabidopsis thaliana was found to be more
in roots than shoots. The phytotoxicity of the compound was observed in
the plant in form of biological damages (Hanano et al., 2014). The authors
AUTHOR COPY
thought that better understanding of the plants ability to detoxify dioxins

would help to improve their use as a safe bioremediators.
Macek et al. (2000) reviewed various phytoremediation technologies,
paying special attention to removal of organic xenobiotics like polychlo-
rinated biphenyls (PCBs) and the application of in vitro systems for basic
research in the role of plants for the remediation of sites polluted by PCBs
and enhancement in their effectiveness.The authors have mentioned vari-
9781771883627
ous aspects of metabolism of xenobiotic compounds in plant cells, the role
of enzymes involved, and the cooperation of rhizospheric microorganisms
in accelerating remediation of these toxic organic recalcitrant compounds.
They have also discussed the application of this phytoremediation approach
as well as the possibility of introduction of foreign genes into plant genome
that can enhance the rate of the bioremediation of these toxic organic com-
pounds. Meagher (2000) reported the removal of organic pollutants such as
PCBs from soils by transgenic plants containing genes for bacterial enzymes.
Wang et al. (2014) studied the effect of polybrominated diphenyl ether
congener (BDE-47) on the growth and antioxidative responses of the seed-
lings of Kandelia obovata and Avicennia marina in hydroponic system and
observed that Avicennia was more tolerant to BDE-47 than Kandelia, as
its antioxidative enzymes could better counter-balance the oxidative stress
caused by the pollutant.
Lao et al. (2003) investigated the metabolic fate of [UL-14C]-3,4-
dichloroaniline (DCA) in Arabidopsis root cultures and soybean plants
over a 48 h period following treatment via the root media. DCA was rapidly
taken up by both species and metabolized, predominantly to N-malonyl-
DCA in soybean and N-glucosyl-DCA in Arabidopsis. The difference in the
routes of DCA detoxification in the two plants could be explained partly
by the relative activities of the respective conjugating enzymes, DCA-N-
malonyltransferase activity in soybean plants. In Arabidopsis sp., DCA-N-
glucosyl transferase activity was dominant. The studies reveal differential
regulation of DCA-N-glucosyltransferase enzyme in the two plant species.
Gujarathi (2005) demonstrated the use of Pistia sp. as bioindicators for
effective and complete treatment of wastewater generated during produc-
tion of antibiotics like tetracycline. Dordio et al. (2009) conducted studies
to assess ability of Typha sp. to withstand and remove clofibric acid (CA),
a metabolite of blood lipid regulator drugs from water. Exposure to higher
CA concentrations did not affect Typhas photosynthetic pigments but there
was overall increase in enzyme activity (ascorbate and guaiacol peroxidases,
catalase, and superoxide dismutase). The authors have suggested ability of
Typha for phytoremediation of CA contaminated waters.
AUTHOR COPY
10.6 APPLICATIONS OF CONSTRUCTED WETLANDS FOR

DIFFERENT ORGANIC POLLUTANTS
Vymazal (2009) reviewed the use of horizontal flow constructed wetlands

(HFCW) for wastewater treatment for more than 30 years. The author has
also mentioned that municipal HFCW focus not only on common pollutants
but also on special parameters such as pharmaceutical compounds, chemi-
9781771883627
cals causing endocrine disruption or linear alkylbenzene sulfonates (LAS).
At present, HFCW are used to treat many other types of industrial wastewa-
ters arising from oil industry, pulp and paper mill, textile industry, tannery,
distillery and winery industries, and others. In particular, the use of HFCW is
becoming very common for treatment of food-processing wastewaters, and
various runoff waters from agriculture, airports, highway, greenhouses, plant
nurseries, and others, as well as to treat leachate arising from landfill. In ad-
dition to the use of HFCW as a single unit, they are also employed in com-
bination with other types of CWs. The enzymes peroxidases of Phragmites
australis (POD) were found to degrade Acid Orange 7 (AO7), an azo dye in
a vertical flow constructed wetland (VFCW) indicating potential of the plant
in tretament of the dye waste (Davies et al., 2005).
Calheiros et al. (2007) studied the survival of different plant species, such
as Typha latifolia, Canna indica, Stenotaphrum secundatum, Phragmites
australis, and Iris pseudacorus in subsurface HFCW receiving tannery
wastewater. Only Phragmites australis and Typha latifolia could be estab-
lished successfully resulting in high removal of organic content from the
wastewater. Davies et al. (2009) studied phytoremediation of a textile azo
dye (AO7) in a pilot scale CW using most widely used plant in CWs, such as
Phragmites australis, and found the removal of the dye to be feasible even
at concentrations of AO7 as high as 748 42 mg/L. The authors have also
carried out studies on the gene-expression for the Phragmites australis while
integrated in the AO7 wastewater treatment.
Low et al. (2008) investigated the ability of down-flow CWs to remediate
hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) contaminated water at vary-
ing loading rates over a period of 2 years. Significant RDX removal occurred
(8996%) for all loading rates ranging from 160 to 1600 mg/m2/day at a hy-
draulic retention time of approximately 2 days. RDX degradation occurred
in both NO3 and SO42 dominated electron acceptor zones. These results
support the use of CWs for the remediation of low-level RDX-contaminated
water. Darryl et al. (2008) had also applied down-flow CW mesocosms for
treatment of RDX.
AUTHOR COPY
Verlicchi and Zambello (2014) reviewed the occurrence of 137 pharma-

ceutical compounds in the effluent from various types of CW treating ur-
ban wastewater. Investigations of 136 treatment plants including free water
systems, horizontal and vertical subsurface flow beds, were reported by the
authors. The uptake of selected pharmaceuticals occurring in sediments and
gravel by common macrophytes was also reviewed suggesting novel ap-
proach for removal of persistent organic compounds.
9781771883627
Phytoremediation is an emerging technology which uses plant systems for

degradation and detoxification of chemopollutants. Terrestrial plant species
like Poplar, Vetiver, Arabidopsis are found to be effective in removal of
pollutants. Constructed wetlands are natural ecosystems that are commonly
used for filtration of pollutants from agricultural runoffs. Among the organic
pollutants, nitroexplosives, like TNT, RDX, HMX, and pesticides namely
atrazine are extensively studied for their removal from contaminated sites
using phytoremediation technologies.
Breeding programs and genetic engineering are powerful methods for
enhancing natural phytoremediation capabilities, or for introducing new
competence into aquatic and terrestrial plants. Genes for such improved
phytoremediation capacity may originate from a microorganism or may be
transferred from one plant to another variety better adapted to the environ-
mental conditions at the cleaning site. In past genes specifically encoding a
nitroreductase from a particular bacterium were inserted into tobacco and
showed faster removal of TNT and enhanced resistance to the toxic effects
of TNT. Researchers have also discovered a mechanism in plants that allows
them to grow even when the pollution concentration in the soil is lethal for
untreated plants. Some naturally occurring biodegradable compounds such
as exogenous polyamines allow plants to tolerate concentrations of pollut-
ants 500 times higher than untreated plants and to absorb more pollutants.
Plants are known to accumulate and translocate particular types of con-
taminants; plants can also be effectively used as biosensors to detect subsur-
face contamination, thereby allowing investigators to quickly demarcate the
trail of contaminants. Phytoscreening may lead to more optimized site inves-
tigations and reduce contaminated site cleanup costs. Phytoremediation can
become an ecological solution to remediate sites contaminated with organic
chemicals.
AUTHOR COPY
KEYWORDS
Atrazine
Bioaugmentation
Dichloroaniline
9781771883627
Dioxins
Hydrilla
Hydrocarbons
Methyl
Parathion
Naphthalene
Nitroexplosives
Pesticides
Petroleum hydrocarbons
Phenolic compounds
Phytoaccumulation
Phytodegradation
Phytoextraction
Phytoremediation
Phytoscreening
Phytostabilization
Phytostimulation
Phytotoxicity
Phytotransformation
Phytovolatilization
Plant microbe interaction
Poplar
Rhizodegradation
Rhizofiltration
Vetiver
AUTHOR COPY
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CHAPTER 11
BIOCONVERSION OF PALM
OIL AND SUGAR INDUSTRY
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WASTES INTO VALUE-ADDED
POLYHYDROXYALKANOATE
NOOR FAZIELAWANIE MOHD RASHID1, AIN FARHANA MOHD
YATIM1, AL-ASHRAF AMIRUL2, and KESAVEN BHUBALAN3
1
School of Marine and Environmental Sciences, Universiti Malaysia
Terengganu, Kuala Terengganu 21030, Terengganu, Malaysia
2
School of Biological Sciences, Universiti Sains Malaysia, Pulau
Pinang 11800, Malaysia
3
Malaysian Institute of Pharmaceuticals and Nutraceuticals, MOSTI,
Bayan Lepas 11700, Pulau Pinang, Malaysia
CONTENTS
11.1 Introduction....................................................................................288
11.2 POME............................................................................................288
11.3 Molasses.........................................................................................291
11.4 Polyhydroxyalkanote.....................................................................293
11.5 Production of PHA from POME....................................................295
11.6 Production of PHA from Molasses................................................298
11.7 Conclusion and Future Outlook.....................................................300
Keywords..................................................................................................301
References.................................................................................................301
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11.1INTRODUCTION
Among the key features of tomorrows plastic are eco-friendly bioplastics that
can be degraded in shorter time and sustainability to ecosystem. Microbial
polyhydroxyalkanoate (PHA) has attracted research and commercial inter-
est because of their biodegradability, thermoplastic properties, and synthesis
from renewable resources. The high PHA production cost is a major fac-
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tor that hinders commercialization process. The production cost commonly
involves fermentation set-up, carbon feedstocks of the bacteria, electricity
as well as chemical usage (Lee, 2008). However, carbon feedstock is con-
sidered to make-up the major fraction of production cost. Therefore, efforts
are taken to identify cheap and renewable carbon substrates. Agricultural
industry wastes and by-products have been investigated in detail as poten-
tial carbon sources. The aim of reducing the total production cost of PHA
is to increase the commercialization potential and demand in global market
for this biodegradable polymer. In recent years, the use of organic wastes,
such as palm oil mill effluent (POME) (Hassan, 1996), municipal wastewa-
ter (Coats et al., 2011), biodiesel wastewater (Dobroth et al., 2011), and food
processing waste effluent (Venkateswar et al., 2012) provide double benefits
because the wastes were converted into environmentally friendly biodegrad-
able polymer. Several types of microbial-derived PHA have shown great
potential to be commercialized in industrial scale, medical application, and
utilized as substitutes to petrochemical-based plastics.
11.2POME
By year 2014, the palm oil industries in Indonesia and Malaysia have grown
rapidly and both nations have become the leading producers in the world,
replacing Nigeria as a main producer in the last three decades. Figure 11.1
shows the palm oil producers in the world. From a humble source of the
edible oil, palm oil has given us a very meaningful knowledge in the appli-
cation from every part of its plant (Foo and Hameed, 2010). Oil palm cur-
rently accounts for more than a half of the total cultivated land in Malaysia,
and its oil production is one of the highest among the producing countries.
The contribution of the oil palm industry to Malaysias economic develop-
ment has indeed been impressive. Based on the statistics obtained from the
Malaysian Palm Oil Council, Malaysia currently accounts 39% of world
palm oil production, and 44% of world exports (MPOC, 2014). The palm oil
industry generates large quantities of by-products composed of triglycerides
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Bioconversion of Palm Oil and Sugar Industry Wastes 289
and fatty acids, suitable for microbial utilization. Other than that, Malaysia
also accounts for 12 and 27% of the worlds production and exports of oils
and fats. Being one of the biggest producers and exporters of palm oil and
palm oil products, Malaysia has an important role to play in fulfilling the
growing global need for oils and fats sustainably (MPOC, 2014). The high
abundance of palm oil has shown the potential primary and secondary of
resources for PHA biosynthesis.
FIGURE 11.1 World palm oil producers (MPOC, 2014).

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POME is considered as one of the most polluting agro-industrial effluent
due to its high values of biochemical oxygen demand (BOD) and chemical
oxygen demand (COD) (Ahmad et al., 2011). Based on Figure 11.2, the re-
newable resources from palm oil extraction generate about 50 million tonnes
of POME, 9 million tonnes of fiber, 16 million tonnes of empty fruit bunch-
es, and 4 million tonnes of shells. POME is brownish in color with a dis-
charge temperature of between 80 and 90C, acidic, viscous, and colloidal
suspension that contain 9596% of water, 0.60.7% of oil, and 24% sus-
pension solids (Foo and Hameed, 2010). The wastewater of POME cannot
be discharged directly into the drainage due its high levels of BOD and COD
that is harmful to environment. The wastewater has to be removed in order
to prevent interfaces in water treatment units and stability of aquatic biodi-
versity and also to avoid problems in biological treatment stages (Ahmad et
al., 2005). POME is a strong wastewater from palm oil mills and has been
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identified as a potential source to generate renewable bioenergy through an-

aerobic digestion. The raw treated POME has an extremely high content of
degradable organic matter, which is due in part to the present of unrecovered
palm oil (Ahmad et al., 2003). Application of the renewable palm oil bio-
mass for PHA production will help to reduce the overall production cost and
address the waste management issue of these materials. Table 11.1 summa-
rizes the information on palm oil and oil palm industry in Malaysia.
FIGURE 11.2 Palm oil derivatives and biomass generated from palm oil extraction
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processes (Hassan et al., 2013).
TABLE 11.1 Information on Palm Oil and Oil Palm Industry in Malaysia.
Derivative Description Value References
Oil palm Total planted area (million hectares) 4.92 MPOB (2014)
Plantation density (palms/hectare) 135 USDA (2012)
Economical life span (years) 30 USDA (2012)
Fresh fruit Weight (kg) 1525 MPOB (2014)
bunch (FBB)
Number of fruits/FFB 10003000
Average number FFB/palm 10
Palm oil Production volume (million tonnes/annum) 4.1 MPOB (2014)
Average yield (million tonnes/hectare/annum) 4.0
Average market price (US$/tonne)
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Derivative Description Value References

Edible use fraction (%) 65
Amount of by-product/waste generate by palm
oil mills (million tonnes/annum)
Empty fruit bunch 16
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Mesocarp fiber 9
Trunk 9
Shell 4
Palm oil mill effluent 50 Hassan et al.
(2013)
Cost of production (US$/tonne of palm Sime Darby
products) (2009)
Estate cost 256.36
Mill cost 62.81
11.3MOLASSES
Molasses is a sugar-rich co-product stream produced during the extraction

of sugar from sugar cane and sugar beets (Du et al., 2012). The global sugar
production is exceeds 165 million tonnes per year. In 2012, 80% of global
sugar production is produced from sugar cane, while remaining 20% is pro-
duced from sugar beet. Currently, Brazil is the worlds largest producer of
sugarcane, accounting for one-thirdof world production. Asian production,
which includesIndia,China, andThailand, accounts foranother one-third
of theworld production. India is the worlds second largest producer of sug-
arcane and China is the fourth largest.A vast global market for sugarcane
derivatives keeps the industry booming. Sugar is prevalent in the modern
diet and increasingly a source of biofuels and bioplastics.
Sugar cane industry generates some by-products namely molasses, ba-
gasse, and press mud (Fig. 11.3). Molasses is generated during the centri-
fuging of sugar crystals, whereby the yield per ton of cane is approximately
44.5%. Bagasse is residual woody fiber of the cane, which used for several
purposes, especially used as fuel for the boilers in the generation of process
steam. Solid mud contained high amount of potassium, sodium, phospho-
rous, and organic matters produced during sugar cane processing.
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FIGURE 11.3 Simplified process flow of refined sugar production (Koller et al., 2010).
Molasses from both sugarcane and sugar beets is a major component

of animal feed. Molasses composed of very high sugar content of mainly
sucrose, glucose, and fructose, 41, 6.7, and 3.2% w/w, respectively, and
other growth promoters, such as vitamins and biotin (Koller et al., 2010).
Molasses and saccharose obtained will be used for the production of PHA-
rich biomass and production of catalytically active biomass. Meanwhile, ba-
gasse will be hydrolyzing into convertible sugars, namely glucose, xylose,
and arabinose, which directly used for PHA production. Zhang et al. (1994)
noted that molasses normally sells approximately 3055% of the cost of
glucose.
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11.4POLYHYDROXYALKANOTE
Polyhydroxyalkanote (PHA) is among the most widely investigated group

of biodegradable polymer due to its natural, renewable, and biocompatible
nature (Verlinden et al., 2011). It is considered to be a potential substitute
for some commercially available petro-chemical derived plastics. PHA is
produced intracellularly by various types of bacteria including Cupriavidus
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sp., Aeromonas sp., Acinobacter sp., Azobacter sp., Bacillus sp., Klebsiella
sp., Pseudomonas sp., and recombinant Escherichia coli (Steinbchel et al.,
1992; Koller et al., 2010). Among the different strains tested, Cupriavidus
necator (previously known as Alcaligenes eutrophus, Ralstonia eutropha, or
Wautersia eutropha) has been studied most extensively due to its ability to
accumulate large amount of PHA from simple carbon sources, for example,
glucose, fructose, and acetic acid (Khanna and Srivastava, 2005).
PHAs are reported as biodegradable (100% biodegradable), insoluble
in water, biocompatible, non-toxic, piezoelectronic, thermoplastics, and/or
elestomeric (Shivakumar, 2012). Their characteristic feature makes them
as possible alternatives to replace petroleum-based polymers (Andreeen
and Steinbchel, 2010). PHA is degraded aerobically by microorganism
to CO2 and H2O upon disposal, thus reduce the issue of greenhouse gas
emissions. PHA can be produced from renewable carbon source, thus al-
low the sustainable production process (Albuquerque et al., 2007). PHA
assembles various types of monomers with approximately 150 structures.
It can be classified according to their branching monomers. Short-chain
length PHAs (scl-PHAs) consist of 35 carbon atoms, while medium-
chain-length PHAs (mcl-PHAs) and long-chain-length PHAs (lcl-PHAs)
composed of 614 and more than 14 carbon atoms, respectively (Anderson
and Dawes, 1990).
Poly(3-hydroxybutyrate) [(P3HB)], a scl-PHA is the most common type
of PHA produced by bacteria in the natural environment. The presence of
P(3HB) was first identified in Bacillus megaterium by Lemoigne at the
Pasteur Institute in Paris in the mid-1920s (Lemoigne, 1926). P(3HB) is
the intracellular granule, which synthesized by bacteria under unbalanced
growth conditions, such as high carbon concentration and limited concentra-
tion of N, P, S, or some trace elements, such as Mg, Fe, and Ca (Shivakumar,
2012). Under normal condition, N sources will be used by the bacteria to
synthesize protein and is an essential element for growth. Limitation of N
sources will inhibit the TCA cycles enzyme, such as isocitrate dehydroge-
nase, which eventually slows down the TCA cycle. As a result, acetyl-CoA
is channeled for the synthesis of P(3HB) (Shivakumar, 2012). In bacteria,
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P(3HB) can be accumulated up to 80% of dry cell weight (DCW) as mem-

brane enclosed inclusion (Khanna and Srivastava, 2005). P(3HB) has a wide
range of application in packaging, medical, insecticides, herbicides, cos-
metic world, and disposable personal hygiene due to its physical properties,
which remarkably similar to those conventional plastics (Ojumu et al., 2004;
Kulpreecha et al., 2009; Chee et al., 2010). Advancement of PHA applica-
tion in medical industry for bone replacement, wound dressings, and surgi-
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cal pins were progressing studies.
P(3HB) are commercially produced by various types of cheap substrate,
namely methanol (Suzuki et al., 1986), ethanol (Alderet et al., 1993), starch
and whey (Kim, 2000; Ghaly, 2003), and beet molasses (Page, 1992a,b).
Nevertheless, Page and Knosp (1989) proved that unrefined carbon source,
such as cane molasses and beet molasses also promote biopolymer produc-
tion comparable to, or even better than refined sugars. Shivakumar (2012)
noted that polyhydroxybutyrate (PHB) production using fermentation re-
sulted in a very high production cost, thus making their use unattractive. The
cost of carbon source, fermentation strategy, and recovery process/down-
stream processing contributes to the high manufacturing cost for biopolymer
production. Halami (2008) noted that carbon source for P (3HB) production
can reach approximately 50% of the total production cost. Research cur-
rently focuses on the use of waste agriculture residue, namely starch, whey,
molasses, CSL, bagasse, and soy meal as well as POME to downsize the
production cost. Currently, the application of biopolymer for industrial pro-
duction is restricted due to its extremely high-cost. Hence, the viable solu-
tion strategy to overcome this problem is by utilizing the waste materials
generated from agro-based industry. Choi and Lee (1999) and Kim (2000)
noted that the use of industrial waste and byproducts as sole carbon source
for PHA production can reduce approximately 4050% of total production
cost. The utilization of waste materials provides a viable strategy for the pro-
duction of biopolymer and overcoming the disposal problem as well. Other
parameters which contribute significantly to the total production cost are
bacterial strains, fermentation strategy, and recovery process as well.
Various types of wastes have been tested for the production of PHA by
numbers of bacteria, namely waste lipids (waste cooking oil), glycerol (by-
products of biodiesel production), molasses (by products of sugar cane indus-
try), whey (by-products in the manufactures of cheese), wastewater (munici-
pal wastewater), and lignocellulosic raw materials. Table 11.2 summarizes
the PHA fermentation by different cheap substrate as a carbon source.
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TABLE 11.2 PHA Production Using Sustainable Raw Materials.

Carbon Sources Strains PHA Content References
(%)
Lipids
Residual oil C. necator H16 41.3 Fchtenbusch et al. (2000)
P. oleovorans 38.9
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Tallow P. resinovorans 15 Cromwick et al. (1996)
Olive oils Aeromonas caviae 612 Doi et al. (1995)
Palm kernel oil, crude C. necator 5 Loo et al. (2005)
palm oil, palm acid oil
Crude palm oil Erwinia sp. USMI-20 46 Majid et al. (1999)
Vegetable oil Pseudomonas sp. 23.5 Song et al. (2008)
strain DR2
Whey and hydrolyzed whey
Whey Recombinant 70 Park et al. (2002)
Escherichia coli
CGSC 4401
Hydrolysates whey C. necator 37 Marangoni et al. (2002)
Molasses
Sugar cane molasses Bacillus megaterium 61.62 Kulpreecha et al. (2009)
BA-019
Beet molasses Azobacter vinelandii 76 Chen and Page (1997)
UWD
Soy molasses Pseudomonas 517 Solaiman et al. (2006)
corrugata 388
Lignocellulosic wastes
Xylose P. cepacia ATCC 60 Ramsay et al. (1995)
17759
Hemicellulosic Burkholderia cepacia 161 Keenan et al. (2006)
hydrolysates ATCC 17759
11.5 PRODUCTION OF PHA FROM POME
POME is organic waste water, which has high carbon content and low ni-
trogen content. With these characteristics, the POME becomes a suitable
carbon replacement for PHA biosynthesis (Hassan et al., 2013). This enables
the bioconversion of environment polluting waste into value added material.
The abundant supply of agro-industrial waste has all so far resulted in the
successful accumulation of PHA, and there is a growing knowledge base
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in the use of these feedstocks (Chanprateep, 2010). Anaerobic digestion of

POME produced a series of volatile fatty acid (VFA), namely formic acid,
acetic acid, propionic acid, and butyric acids, which became a potential plat-
form chemical for biofuel and material production (Cheng et al., 2010). It is
likely to improve POME conversion efficiency and energy utilization further
if bacterial PHA production can be integrated into the existing mill VFA-
producing units (Mumtaz et al., 2010). Several studies proved that polymers
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from POME-derived VFAs are comparable to those obtained from commer-
cially available organic acids. POME has been used as a carbon sources by
numbers of bacteria, such as Rhodobacter sphaeroides (Hassan et al., 1996,
1997a, 1997b), Comamonas sp. (Mumtaz et al., 2010; Zakaria, 2010), and
C. necator (Din, 2012). Those studies from POME cultivation have been
reported for two-stage cultivation or pure culture systems in a simple and
direct way. Both organic removal and PHA-producing microbial organisms
are cultivated in the same system, which is called a hybrid fed-batch system
(Din, 2012).
Mainly, PHA productions focus on the scaling-up of production and im-
provement of material properties. Based on Junpadit et al. (2014), POME
produced high DCW and PHA content compared to others carbon sources. It
might due to the high fatty acid content (VFA and LCFA). Fatty acid was de-
graded by -oxidation, which is the main metabolic route for the PHA syn-
thesis. In most cases, the organic carbon sources are converted into VFA in
aerobic activated sludge in the first step, and then converted into the PHAs by
mixing cell cultures in the second step (Du, 2012). PHA-producing bacteria
from Junpadit and Boonsawang (2010) showed that POME gave the highest
PHA monomer composition containing the 3-hydroxybutyrate (3HB), 3-hy-
droxyvalerate (3HV), 3-hydroxyhexanoate (3HHx), and 3-hydroxydecano-
ate (3HD) of 534, 552, 488, and 41 mg/L, respectively. Although the fi-
nal PHA concentrations are still low at the current investigated conditions,
PHAs could accumulate to approximately 50% of the cell dry weight in
some cases (Liu, 2011).
The production of PHA from Comamonas sp. EB172 has shown a posi-
tive result to POME as a carbon sources. The combination of POME treat-
ment and PHA production can provide a zero discharge system for palm
oil mills (Hassan, 2002). The fed-batch cultivation of Comamonas sp. was
cultivated in 2L fermentation, the development of cell biomass and PHA
accumulation recorded were 9.8g/L and 59 (wt %) of PHA after 60 h of
incubation (Mumtaz, 2009; Zakaria, 2010). The incorporation of P(3HV)
monomer unit (21mol%) in the poly(3-hydroxybutyrate-co-3-hydroxyval-
erate) [P(3HB-co-3HV)] copolymers was obtained in this study due to the
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presence of propionic acid in the mixture of organic acids from anaerobi-

cally treated POME. Zakaria (2010) proved that high PHA accumulation
was achieved from mixed microbial cultures by feeding them a high concen-
tration of VFAs from anaerobically fermented POME. The maximum PHA
content was 40% of the DCW and more than 80% of COD was removed.
Based on the result, it has shown that Comamonas sp. with POME is ongo-
ing to meet the market potentials and reduction of PHA production cost.
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POME can be used as a carbon sources for PHA production, but the pH
in the anaerobic treatment of POME has to be maintain at seven, so that no
formic acid and biogas but only acetic and propionic acids will be produced
(Hassan, 1996). As reported, the highest yield of PHA with 50% and PHA
content with over 65% was obtained at pH 7. The control of agitation, pH,
and time in the cultivation of R. sphaeroides by using POME as a carbon
sources was required in order to achieve maximum yield and PHA content.
C. necator are known to be able to accumulate a large amount of PHA when
nitrogen and phosphorus limited also able to use residual oil from POME as
the source of carbon to produce PHA (Hassan, 2013). By using a fed-batch
production of PHA, C. necator produced 1876% of PHA contents but the
overall PHA productivity obtained was less than PHA obtained by other
researchers (Hassan, 1997a, 1997b). This might due to the unstable cell con-
centration when concentrated acetic acid separated from POME was incor-
porated into the standard medium. Hence, further research and development
to explore advanced technology to produce PHAs with consistent quality
from wastewater treatment are required to ensure that target applications
are achieved. The proposed strategy should not only reduce the overall PHA
production costs, but also help to solve the waste management problems
in the palm oil industry. Table 11.3 summarizes the PHA production using
POME as carbon source.
TABLE 11.3 Biosynthesis of PHA Using POME as Carbon Source.

Microorganism PHA Yield PHA Content (% References
(g/L) cell dry weight)
Comamonas sp. EB172 0.31 85.8 Mumtaz et al. (2010);
Zakaria et al. (2010)
Rhodobacter sphaeroides 0.5 67.0 Hassan et al. (1996),
Hassan et al. (1997a, 1997b)
C. necator 0.32 45.0 Hassan et al. (1997a, 1997b)
Anaerobic bacteria Junpadit et al. (2014)
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11.6 PRODUCTION OF PHA FROM MOLASSES
11.6.1 SUGAR CANE MOLASSES
The use of sugar cane molasses, which is by-products of sugar cane indus-
tries, may help to reduce the cost production of biopolymer (Mona et al.,
2001). It contained high amount of sugar (over 50% of dry weight) and
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widely used for production of PHA using various cultivation techniques
(Albuquerque, 2007). Wu et al. (2001) have isolated Bacillus sp. JMa5 from
molasses contaminated soil and further investigates the use of molasses as
a sole carbon source for PHB production. Bacillus sp. JMa5 grew up to
30g/L of CDW after 8h of incubation in fed-batch fermentation with the
initial molasses concentration of 210g/L. However, after 30h of incuba-
tions, cell reached approximately 70g/L. Wu et al. (2001) concluded that
aeration plays important roles in synthesizing P(3HB). High P(3HB) content
was detected when culturing in non-baffled flask compared to baffled flask,
which were 42 and 22%, respectively. This can be concluded that aeration
allows the cell growth and PHB production was parallel.
Chaijamrus and Udpuay (2008) further investigated the use of sugar
cane molasses and corn steep liquor (CSL) for P(3HB) production using B.
megaterium ATCC 6748. Sugar cane molasses and CSL were used, as they
provide sufficient amount of carbon and nitrogen source for bacteria growth.
For the first parameter, sugar cane molasses at a level of 16% w/w and 0.5%
of NH4Cl were used, meanwhile for the second parameter, the experiment
was conducted using 06% of CSL instead of NH4Cl and 4% of molasses.
The highest production of DCW obtained was 7.2g/L using 4% of molasses
and 6% of CSL. For P(3HB) production, the highest concentration was ob-
tained after 48h of incubation (43% w/w, dry matter) using 4% of molasses
and 4% of CSL as C and N source, respectively. However, DCW produced
is only 5g/L, thus can be concluded that bacterial growth increased with the
increasing of CSL, whereas P(3HB) accumulation decreased.
Kulpreecha (2009) reported the production of PHB by B. megaterium
BA-019 using shake flask and fed-batch culture. The bacterium was isolated
from soil in Thailand. In batch culture, approximately 8.78g/L of DCW and
42.1% of P(3HB) content were achieved, respectively, after 12h of incuba-
tion. Meanwhile, the DCW was increased up to 72.6g/L when culturing
using PH-stat-batch culture at 24h of cultivation. Using PH-stat-batch cul-
ture, the P(3HB) content also increased to 61.62% with productivity of 0.45
(g/L/h).
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Mona et al. (2001) have reported the use of different concentration of

sugarcane molasses and CSL as sole carbon and nitrogen source for produc-
tion of P(3HB) by B. megaterium strain. Different sugarcane molasses levels
ranging from 1 to 5% were used. DCW obtained was ranging from 0.8 to
1.6g/L after 48h of incubation. Meanwhile, PHA and P(3HB) contents were
ranging from 20 to 50%.
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11.6.2 SUGAR BEET MOLASSES
Sugar beet molasses contains 3050% (w/w) of sucrose. After hydrolysis, the
sucrose will decompose to glucose and fructose. Liu (1998) has investigated
the production of P(3HB) using beet molasses by recombinant Escherichia
coli (HMS174/Ptz18u-PHB). Beet molasses was successfully replaced
glucose in synthesizing PHB. In a batch culture, approximately 2060g/L
of beet molasses was used. The maximum DCW and P(3HB) production
achieved were ranging from 6.5 to 16.7g/L and 68 to 85% w/w, respective-
ly. Meanwhile, using 5 L fed-batch culture, maximum DCW obtained was
39.5g/L with 80%w/w of P(3HB) content after 19h of incubation.
Chen and Page (1997) have reported P(3HB) production by Azobacter
vinelandii UWD using two-stage fermentation process. Aeration was used to
promote cell growth and suppressed the P(3HB) for the first stage, and lower
aeration of raw sugar medium containing fish peptone was used to promote P
(3HB) production in second stage. P(3HB) yield of 36g/L and productivity
of> 1g/L/h were obtained using 5% of beet molasses. P(3HB) content was
reported at 76wt%, after40h of incubation. Table 11.4 summarizes the PHA
production from sugar cane and sugar beet molasses by different bacteria us-
ing different cultivation strategies.
TABLE 11.4 PHA Production by Various Microorganisms Using Sugar Cane and Sugar
Beet Molasses by Different Cultivation Techniques.
Strain Carbon Cell Cultivation PHA PHA Cultivation References
Source Density Time (h) Type Content Strategy
(g/L) (%)
Bacillus Cane 30 8 P 2535 Batch Wu et al.
sp. JMa5 molasses (3HB) culture (2001)
B. megate- Cane 5 45 P 43 Batch Chaijamrus
rium ATCC molasses (3HB) culture and Udpuay
6748 (2008)
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Strain Carbon Cell Cultivation PHA PHA Cultivation References

Source Density Time (h) Type Content Strategy
(g/L) (%)
B. mega- Cane 8.78 12 P 61.62 Batch Kulpreecha
terium molasses (3HB) culture et al. (2009)
BA-019
9781771883627
B. Cane 40 48 P 46.3 Batch Mona et al.
megaterium molasses (3HB) culture (2001)
Recom- Beet 6.516.7 80 P 6885 Batch Liu et al.
binant E. molasses (3HB) culture (1998)
coli
Recom- Beet 39.5 19 P 80 PH-DO-stat Liu et al.
binant E. molasses (3HB) fed-batch (1998)
coli culture
Azobacter Beet 36 40 P 76 Two-stage Chen and
vinelandii molasses (3HB) cultivation Page (1997)
UWD
11.7 CONCLUSION AND FUTURE OUTLOOK
Bioconversion of agricultural industry wastes into value added products

is indeed a brilliant way to solve waste accumulation in the environment.
PHA, which holds much potential as an alternative to chemically synthe-
sized plastics is a value added material that can be produced using agri-
cultural industry wastes. Considering economic, environmental, and social
issues, the ultimate goal is to obtain an economically viable PHA production
system based on clean and safe processes. The final commercial PHA based
products can be environmentally compatible, leading to a truly sustainable
manufacturing process. This chapter has highlighted the use of POME and
molasses from sugar industry as carbon feedstock for PHA production. It
is evident that these waste materials can be converted into biodegradable
plastic, a value added product. Some strains have shown their ability to
convert these wastes into PHA; however, researchers are still trying to iso-
late better PHA producing strains from the environment. This effort is also
complemented by designing suitable fermentation processes for high yield
PHA production. Major producers of palm oil, cane, and beet sugars, such
as Malaysia, Indonesia, Thailand, Brazil, and India may exploit the readily
available waste products as carbon feedstock for PHA production. Research
can be geared toward fully exploiting this renewable carbon feedstock to
AUTHOR COPY
its maximum and developing sustainable PHA production systems. Much

potential and great economic revenue can be seen in conversion of organic
substrate from agricultural wastes to PHA. With technological and scientific
developments in production of bio-based as well as biodegradable products,
such as PHA, a positive attitude toward sustainable green technology and
increased usage of green products in future is evident.
9781771883627
KEYWORDS
Agro-industrial wastes
Biopolymer
Microbial fermentation
Oil palm
Palm oil mill effluent
Poly(3-hydroxybutyrate)
Polyhydroxyalkanoate
Renewable raw material
Sugar beet molasses
Sugar cane molasses
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CHAPTER 12
INFLUENCE OF ENVIRONMENTAL
FACTORS ON THE PREVALENCE OF
9781771883627
POSTHARVEST DETERIORATION OF
RAPHIA AND SHEA FRUITS IN NIGERIA
OKUNGBOWA FRANCISCA IZIEGBE1 and
ESIEGBUYA OFEORITSE DANIEL2
1
Department of Plant Biology and Biotechnology, University of Benin,
Benin City, Nigeria
2
Plant Pathology Division, Nigerian Institute for Oil Palm Research
(NIFOR), Benin City, Nigeria
CONTENTS
12.1 Introduction....................................................................................308
12.2 Raphia Palm...................................................................................309
12.3 Causes of Postharvest Diseases of R. hookeri Fruits ....................309
12.4 Traditional Methods for Management of Postharvest
Disease of R. hookeri Fruit............................................................313
12.5 Emerging Technologies for Postharvest
Disease Control of R. hookeri Fruit...............................................313
12.6 Shea Tree........................................................................................317
12.7 Influence of Environmental Conditions on Quality
of Processed Shea Butter...............................................................318
12.8 Traditional Strategies for Control of Postharvest
Disease of Shea Nuts and Kernels.................................................321
12.9 Future Prospects for Management of Postharvest
Diseases of Seeds...........................................................................322
Keywords..................................................................................................323
References.................................................................................................323
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12.1INTRODUCTION
Coates and Johnson (1997) stated that losses due to postharvest disease
may occur at any time during postharvest handling from harvest to con-
sumption. When estimating postharvest disease losses, it is important to
consider reductions in fruit quantity and quality, as some diseases may not
render produce unmarketable yet reduce product value. Apart from direct
9781771883627
economic considerations, diseased produce poses a potential health risk
by mycotoxigenic fungi belonging to some genera such as Penicillium,
Alternaria and Fusarium, which are known to produce mycotoxins un-
der certain conditions. Losses due to postharvest disease are affected by a
great number of factors including: commodity type, cultivar susceptibility
to the causal agents of postharvest disease, postharvest storage environ-
ment, maturity and ripeness stage, disease control methods, and handling
methods.
Virtually all postharvest diseases are caused by fungi and bacteria. In
some root crops and brassicas, viral infections present before harvest can
sometimes develop more rapidly after harvest. In general, however, viruses
are not an important cause of postharvest disease. The so-called quiescent
or latent infections are those where the pathogen initiates infection of the
host at some point in time (usually before harvest) but then enters a period of
inactivity or dormancy until the physiological status of the host tissue chang-
es in such a way that infection can proceed (Coates and Johnson, 1997). The
dramatic physiological changes which occur during fruit ripening are often
the trigger for reactivation of quiescent infections. Examples of postharvest
diseases arising from quiescent infections include anthracnose of various
tropical fruits caused by Colletotrichum spp. and grey mould of strawberry
caused by Botrytis cinerea.
The other major groups of postharvest diseases are those which arise
from infections initiated during and after harvest. Often these infections oc-
cur through surface wounds created by mechanical or insect injury. Wounds
need not be large for infection to take place and in many cases may be mi-
croscopic in size. Common postharvest diseases resulting from wound infec-
tions include blue and green mould (caused by Penicillium spp.) and transit
rot (caused by Rhizopus stolonifer). Bacteria such as Drutnia carotovora
(soft rot) are also common wound invaders. Many pathogens, such as the
banana crown rot fungi, also gain entry through the injury created by sever-
ing the crop from the plant.
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Environmental Factors on Postharvest Deterioration of Fruits 309
12.2 RAPHIA PALM
In Nigeria, Raphia palms grow wild in the lowland forest region and swamps
of the South as well as river courses of the Savannah region of the North
(Ndon, 2003). Raphia palms are peculiar for their hepaxanthic flowering and
so a trunk usually flowers and fruits only once and dies after 335 years of
vegetative growth (Otedoh, 1985). Economic products of Raphia palm include
9781771883627
building materials such as bamboos, Raphia fiber, thatch, pissava and palm
wine. Most of the species are tapped for wine by tapping the young terminal
inflorescence (Otedoh, 1982). The wine is now successfully bottled for com-
mercial purposes at the Nigerian Institute for Oil Palm Research (NIFOR),
Benin City and other places in Nigeria such as Federal Institute of Industrial
Research, Oshodi (FIIRO), Lagos. The palm wine is also used in distilling
local dry gin. The trunk of the Raphia palm has been strongly recommended
for paper making as well as for producing soft tissue paper because of its good
quality (Odeyemi, 1984). According to Ndon (2003), the R. hookeri palm is
the most popular among the twenty species of Raphia palm that have been
identified. Its advantage over the other species includes its ability to mature
between 3 and 6 years and also being able to yield 1151145 L of palm wine
within its life time when compared to the other species of the palm.
Other economic importance of R. hookeri fruits as reported by Ndon
(2003) includes its oil which can be used for cooking and making of confec-
tionery, the mature and ripe fruit which serves as food for coastal people of
Akwa Ibom State, Nigeria. Ndon (2003) also reported that the fruit contains
plant growth hormones which can be applied in tissue culture and saponin
that can be used to stupefy fish. Literature shows that some saponins are tox-
ic to cold-blooded organisms and insects at particular concentrations. Most
saponins, which readily dissolve in water, are poisonous to fish.
The healthy ripe mesocarp of R. hookeri fruits have also been reported
to posses some phytochemical agents such as phenols, flavonoid, alkaloids
saponin oxalate, quinones, and other nutritional components such as mois-
ture, minerals, fat, protein, carbohydrates, and other mineral components
which are of beneficial and nutritional purposes to man (Murray et al., 2000;
Esiegbuya, 2012, 2013).
12.3 CAUSES OF POSTHARVEST DISEASES OF R. HOOKERI FRUITS
There are two major types of postharvest diseases of R. hookeri fruits in-
cludes the black seed rot (Fig. 12.1) and dry seed rot (Fig. 12.2) diseases.
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These postharvest diseases are of importance because they have the abil-
ity to affects the scale, mesocarp, and endocarp of the Raphia fruits, thus
destroying the embryo and thereby making the seed unsuitable for planting
(Esiegbuya et al., 2013a, 2013b, 2013c, 2013d). These diseases are of eco-
nomic importance because the seed is the only means of propagation of the
palm. The causes of these postharvest diseases on the R. hookeri fruit are as
a result of the following:
FIGURE 12.1 Black seed rot (arrow) of R. hookeri fruits. 9781771883627
FIGURE 12.2 Dry rot seedrot disease affecting the scale (a) and mesocarp (b) of R. hookeri
fruits.
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12.3.1 SURFACE WOUND
Surface wound of R. hookeri fruits are mainly caused during harvesting

when the fruit bunches are allowed to fall to the ground thereby creating
wound on the body of the fruit. This allows the entrance of postharvest
pathogens. Chalara paradoxa causing the black rot postharvest disease
of R. hookeri palm was isolated from the spaces between the scales of the
9781771883627
fruits, from the mesocarp and occasionally from the testa and embryo.
According to Oruade and Ekundayo (1992) the pathogen can only pen-
etrate the fruit through wound. The authors also stipulated that the inability
of the C. paradoxa to penetrate the hard part of the scale and unwounded
fruits reveals it to be a weak pathogen. The quick transformation of the C.
paradoxa from the microconidia to macroconidia with thick walls within
48 h allows the pathogen to survive under adverse conditions. Chalara
paradoxa and other fungi associated with the postharvest disease of the
R. hookeri fruits include Aspergillus niger, Fusarium sp., Botryodiplodia
theobromae, Penicilium, and Trichoderma. These are common moulds
found in the air and soil and easily contaminate fruits and affect the me-
socarp and endocarp of the Raphia fruits, thus destroying the embryo and
thereby making the seed unsuitable for planting (Esiegbuya et al., 2013a,
2013b, 2013c, 2013d).
Xylaria feejensis the causal agent of the dry seed rot on the other hand
posses the ability to penetrate intact fruit but its pathogenicity is also
enhanced when the surface of the fruit is compromised (Esiegbuya et
al., 2013a, 2013b, 2013c, 2013d). Xylaria feejeensis is an ascomycete
of Class Sordariomycetes, Family Xylariaceae and Order Xylariales. The
ecophysiological features of the Xylariaceae indicate a xerophilous life-
style of their ancestors (Rogers, 2000) which partly explains the ability
of X. feejeensis to cause postharvest dry seed rot on the fruit because it
has the ability to thrive well on the all parts of the fruit (scale, meso-
carp, testa, and embryo) and also forming a mycelia weft around the fruit
thus preventing the invasion by other pathogens (Esiegbuya et al., 2013a,
2013b, 2013c, 2013d). As pointed out by Whalley (1996) the Xylariaceae
have long been considered to be wood-destroying saprobes, aside from
a few facultative tree parasites (Ostry and Anderson, 2009). Saprophytic
Xylariaceae are considered to be white-rot fungi, owing to their ability to
degrade lignin, but they even can degrade cellulose very effectively (Wei
et al., 1992).
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12.3.2 VIRULENCE ABILITY OF THE PATHOGEN
The virulence of these pathogens has been shown to be enhanced by environ-

mental factors such as relative humidity and temperature under storage condi-
tions. Locally, harvested bunches of R. hookeri fruits are usually left exposed
on farmland or along corridors of farmhouse. This unwholesome practice
allows conditions for creation of mechanical injuries to the fruits and also
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proliferation of the storage pathogens. Esiegbuya et al. (2014) reveals that
environmental factors such as relative humidity and temperature have been
found to significantly favor the prevalence and incidence of the black rot and
dry rot diseases of Raphia palm caused by C. paradoxa and X. feejensis in
storage. After two week of storage of 500 samples of R. hookeri fruits under
room temperature, the percentage occurrence of black rot, dry rot, and the un-
infected fruit was 48.54, 17.48, and 33.98%, respectively. However, after one
and three months of storage the disease incidence of the black rot, dry rot, and
healthy increased from 65.37 to 72.81%, 23.95 to 27.18%, and 10.68 to 0%,
respectively, for the two storage periods (Figs. 12.112.3). The high intensity
of these diseases in storage showed that environmental factors (relative hu-
midity and temperature) played a significant role on the disease intensity and
severity. The study also showed that the intensity of the black rot disease was
more when compared to the dry rot disease. Fungi associated with postharvest
diseases are important in that they can cause, among others, the following: (i)
loss of viability of seeds; (ii) tainting, leading to decrease in market value of
seeds and fruits; (iii) Deterioration of seed in storage; (iv) disease which result
in rots of fruits and seeds; and, (v) introduction of new pathogen through fruit
and seeds into areas where such pathogen are unknown.
FIGURE 12.3 Healthy R. hookeri fruits showing exposed mesocarp (arrow)

(Esiegbuya, 2013e).
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12.3.3 NUTRITIONAL STATUS OF THE FRUIT
As earlier stated, the ripe mesocarp of R. hookeri fruits serve as delicacies

in the Southern part of the Nigeria. Comparative study of the proximate and
mineral composition of healthy and black and dry rot infected R. hookeri
fruits caused by the agents of these postharvest diseases showed a signifi-
cant decrease in all the food components analyzed for, except for ash which
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showed a slight increase. The decrease in proximate and mineral composi-
tion of the fruit, indicate utilization of the food components by the posthar-
vest pathogens and also ensure its survival in the pathogenicity of the fruit
(Esiegbuya et al., 2013a, 2013b, 2013c, 2013d). Other authors have also re-
ported on the changes in the mineral contents of fruits as a result of infection
caused by microbes (Pathak, 1997). The Ascomycetes are also reported to be
able to degrade mineral elements on substrates in which they grow (Lawal,
2011; Lawal and Fagbohun, 2012).
12.4 TRADITIONAL METHODS FOR MANAGEMENT OF

POSTHARVEST DISEASE OF R. HOOKERI FRUIT
Two methods are commonly used which include fungicide application and
storage of fruit endocarp in water. Fungicides such as benlate, dithane M45
and captan have been proposed by Oruade-Dimaro (1990) for the control of
the mycelia growth of C. paradoxa causing the black seed rot disease of R.
hookeri fruits. However, due to the risks of fungicides application to man
and the environment, the traditional method of storing the seed of the fruits
on a basin of water was adopted. This is done by totally removing the scale
and the mesocarp from the fruit and the seed is then stored on plastic rubber
containing water till the next planting season. The water is changed at inter-
vals. This method is not environmentally friendly because the water serves
as a breeding ground for mosquito larva.
12.5 EMERGING TECHNOLOGIES FOR POSTHARVEST DISEASE

CONTROL OF R. HOOKERI FRUIT
12.5.1 BIOLOGICAL CONTROL
Increasing consumer concerns over the presence of chemical residues in

food have prompted the search for non-chemical disease control measures.
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Fungicides used before and after harvests are of particular concern because
they are applied close to the time of consumption. However due to the high
risk of fungicides to humans, animals, environment, and the increasing rate
at which pathogens develop resistance against fungicides, new approaches
to control the black rot postharvest diseases are currently under investiga-
tion. Okogbenin et al. (2014) have proposed the use of biocontrol agent such
as Aframomum sceptrum for the control of the postharvest black rot disease
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of R. hookeri fruit caused by C. paradoxa.
Aframomum species have been reported to be fungitoxic against fungi
such as A. niger, P. digitatum, Helminthosporium solani, and Mucor piri-
formis, against E. coli, Klebsiella spp., and Salmonella spp. (Doherty et al.,
2010; Chiejina and Ukeh, 2012). The antimicrobial properties of Aframomum
species reported by some authors are attributed to the phytochemical con-
stituents such as flavonoids, phenolics, tannins, saponin, terpernoids, cardiac
glycosides, and alkaloids present in the seeds. The ability of the different ex-
tracting solvent of A. sceptrum to inhibit the mycelia growth of C. paradoxa
was attributed to the presence of phytochemical agents such as phenols,
reducing sugar, steroids, oxalate, and alkaloids present in the different ex-
tracts. These extracts have been reported by Matasyoh et al. (2007) to have
the ability to diffuse through the cell membranous structures of fungal cells
and cause damage to the cell thereby altering or lowering the physiological
activities of the cell.
The presence of phenolic compounds in these extracts indicates that
the seed extract of the plants can serve as antimicrobial agents. Phenols
and phenolic compounds have been extensively used in disinfection and
remain the standard with which other fungicides are compared (Doherty et
al., 2010). According to Doherty et al. (2010) alkaloids rank as the most ef-
ficient therapeutically significant plant substance. Pure isolated plant alka-
loids and their synthetic derivatives are used as basic medicinal agents for
their analgesic, antispasmodic, and bactericidal effects (Stary, 1998). They
exhibit marked physiological activity when administered to animals. While
the potential for biological control of postharvest diseases clearly exists,
future success relies on the ability to achieve consistent results in the field
and after harvest. It will be necessary to enhance the efficacy of biological
control agents against postharvest disease and commercialize the technol-
ogy involved.
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12.5.2 MANIPULATION OF ENVIRONMENTAL STORAGE

CONDITIONS
Studies have shown that the causal agents of the black rot of R. hookeri can
be controlled by manipulation of environmental storage conditions such
as temperature, lighting, relative humidity, and pH. The temperature re-
sponse of the C. paradoxa was found to be similar to what is found in most
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fungi whose temperature optimum lies between 25 and 35 C (Cochrane,
1958). The optimum temperature for the germination of C. paradoxa was
found to be between 20 and 30 C (Esiegbuya et al., 2013a, 2013b, 2013c,
2013d). Dede and Okungbowa (2007) have also reported on the growth of
C. paradoxa at different temperatures on various growth media, surviving
under a temperature range of 1528 2 C. San-Juan (1997) and Bachiller
(1998) also observed a temperature range of 2530 C as the optimum
for C. paradoxa (date palm and coconut isolates, respectively). According
to Esiegbuya et al. (2014) continuous light regime for 24 h was found
to slow down the mycelia growth of C. paradoxa isolated from R. hook-
eri under ambient temperature. Bell et al. (2005) reported that illumina-
tion will sometimes increase or more commonly reduce the rate at which
fungi spread across an agar surface. Such effects are sometimes due to
the photochemical destruction of components of the medium but in other
instances a direct effect on metabolism seems likely. Other isolates of C.
paradoxa from date palm and coconut palm was also found to grow well at
all the three conditions of light but sporulation was much less or nil, under
continuous darkness (San-Juan, 1997; Bachiller, 1998). The survival of C.
paradoxa under a wide range of relative humidity varying from 55100%,
with the relative humidity of 100% producing the highest mycelial growth
and 55% slowing down the growth of the pathogen reveals that storing the
R. hookeri fruits under environmental conditions with low relative humid-
ity, high temperature, and complete lighting system will reduce the post-
harvest fungal deterioration of the R. hookeri fruits (Oruade-Dimaro and
Ekundayo, 1992; Esiegbuya et al., 2013a, 2013b, 2013c, 2013d). There is
however need to investigate the influence of these factors on the viability
of the seed (Figs. 12.412.6).
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9781771883627
FIGURE 12.4 Incidence of black rot and dry rot on R. hookeri fruits two weeks after
harvest at room temperature condition.
FIGURE 12.5 Incidence of black rot and dry rot on R. hookeri fruits one month after
harvest at room temperature condition.
FIGURE 12.6 Incidence of black rot and dry rot on R. hookeri fruits three months after
harvest at room temperature condition (Esiegbuya, 2012).
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12.6 SHEA TREE
Shea butter tree (Vitellaria paradoxa) is indigenous to Sub-Saharan Africa

and belongs to the family Sapotaceae. It grows in the wild and has a huge
economic and ecological potential. Shea butter is naturally rich in Vitamins
A, E, and F (Okullo et al., 2010). Shea butter is widely utilized for domestic
purposes such as cooking, skin moisturizer, and commercially as an ingredi-
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ent in cosmetic, pharmaceutical, and edible products (Alander, 2004). The
fruit when very ripe can be eaten raw. Traditionally, Shea butter are used
as cream for dressing hair, protecting skin from extreme weather and sun,
relieving rheumatic and joint pains, healing wounds/swelling/bruising, and
massaging pregnant women and children. It is also used in treatments of
eczema, rashes, burns, ulcers, and dermatitis. Lovett (2004) concluded that
Shea butter is a highvalue export to Europe and the United States, where it
is considered a luxury. Maranz and Wiesman (2003) stated that at least 500
million production trees are accessible in West Africa, which equates to a
total of 2.5 million tons of dry kernel per annum (based on 5 kg dry kernel
per tree).
It is also asserted that over two million people in 13 African countries
process the commodity for cash and consumption. Shea butter is mostly
processed manually in small villages in Nigeria. Shea butter processing is
done by village women, and the method which they use is one passed down
through generations. Moreover, there is no estimate of the overall balance
between cost of input and the economic output of Shea butter, as the process-
ing is not only arduous, labor-intensive, and time consuming, it also requires
large amounts of water and firewood.
Nigeria is the largest producer of Shea nuts in Africa. According to
USAID (2010) Nigerian production of Shea nuts in 2002 was 57% of the
total Shea nuts production among some West African countries (Ghana, Cote
d Voire, Togo, Benin, Mali, and Burkina Faso). In that same year, Nigerian
production was six times more than Ghana production. But the export rate
of the Nigerian Shea butter in 2005 was 0% while that of Ghana was 15,000
million tons (USAID, 2004). According to the USAID report, one of the
challenges facing the Shea industry in Nigeria is lack of certification of the
product and weak organization of the processors. This has lead to the low
penetration level of the Nigeria Shea butter in the international markets. The
inefficiency of the processing techniques lowers the quality of Shea butter
available in the market. Shea butter processing in West Africa involves mini-
mum mechanical input, heavy drudgery and high input of firewood, which
has a direct effect on the quality of Shea butter (Carette et al., 2009). The low
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quality of Shea butter is thus a concern, as it falls below international stan-

dard. Consequently, demand is decreasing and the potentials of Shea butter
in alleviating rural poverty especially the women and children involved in
the processing is dwindling, necessitating an assessment of the processing
techniques. In Nigeria, lack of quality control system during the processing
of the kernel and Shea butter at the rural end of the chain is one the major
challenges of the Shea industry. The quality control of the Shea industry is
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affected mainly by poor postharvest practices. This is as a result of favorable
environmental conditions. Recent research supported by USAID and com-
missioned by TechnoServe-Ghana, has shown that the first three steps in the
post-harvest processing (accumulation of fresh Shea nuts, heating the fresh
nuts and drying the kernel) are the critical determinants of kernel quality, for
example Free Fatty Acids (FFA), Peroxide Value (PV), and fungal levels.
Subsequent steps during extraction, can only maintain quality, which if
low, will almost certainly necessitate the need for refining before use in the
Western marketplace (USAID, 2004).
12.7 INFLUENCE OF ENVIRONMENTAL CONDITIONS ON

QUALITY OF PROCESSED SHEA BUTTER
The quality of fats and oils is dictated by several physical and chemical pa-
rameters that are dependent on the source of oil, geographic, climatic, and
agronomic variables of growth in the case of plant oils as well as processing
and storage conditions. The quality of processed Shea kernels and butter are
also affected by some postharvest conditions which are as a result of envi-
ronmental influence.
12.7.1 TIMELY PICKING OF SHEA FRUITS
The first factor affecting the quality of the Nigeria Shea industry is the delay
in picking of the Shea fruits. This results in the fruits being colonized by
aflatoxin producing fungi (Atehnkeng et al., 2013). The colonization of Shea
fruits by fungi is as a result of the favorable environmental conditions such
as temperature and moisture. Since Shea fruits are harvested during rainy
season, the temperature and moisture conditions are favorable for fungi to
thrive. In order to overcome this challenge, timely picking of Shea fruits
from the bush is recommended. However this requires contingent arrange-
ment because of the tedious and clumsy nature of the work in addition to
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being faced with some challenges such as snakebite, poor visibility, covering
long distances, harassment by monkeys, and other reptiles (Nahm, 2011). As
a result of the difficulties associated with Shea fruit collection, the proces-
sors insist on processing every kernel picked irrespective of its quality status
that is a major determinant of Shea butter quality.
Moisture and temperature influence the initiation, development of infec-
tious diseases and succulence of host to diseases in many interrelated ways.
9781771883627
It may exist as rain or irrigation water on the plant surface or around the
roots, as relative humidity in the air, and as dew. Moisture is indispens-
able for the germination of fungal spores and penetration of the host by the
germ tube. It is also indispensable for the activation of bacterial, fungal,
and nematode pathogens before they can infect the plant. Moisture, in such
forms as splashing rain and running water, also plays an important role in the
distribution and spread of many of these pathogens on the same plant and on
their spread from one plant to another.
12.7.2 SHEA FRUIT GERMINATION
The Shea fruit germinates quickly when it falls to the ground. This is because
the fruit has no dormancy period; it germinates within few days of dropping
from the tree (Jker, 2000). Environmental conditions such as moisture and
temperature are thought to play a role in the quick germination of the fruit.
Temperature affects cellular metabolic and growth rates. Seeds of different
species and seed from same plant germinate over a wide range of tempera-
tures. Seeds have temperature range within which they will germinate and
they will not germinate at temperature above or below this range. Many
seeds germinate at temperatures slightly above room temperature, others
germinate only in the response to alternation in temperature between warm
and cool temperature.
Germination process in seed sometimes may be a nuisance due to the
depletion of the target reserves or a strategy to enhance some nutritive con-
stituents of the seed. According to Munshi et al. (2007) the quantity of phos-
pholipids, glycolipids, and sterols in cotyledons and embryonic axes in fast
germinating seeds increased progressively between the 1 and 6 days after
sowing (DAS) compared with the slow growing seeds. The fatty acid com-
position in cotyledons of fast growing seeds showed increased levels of pal-
mitic and oleic acids 6 and 8 DAS, while a decline in palmitic and stearic ac-
ids as well as accumulation of oleic and linoleic acids were observed in slow
growing seeds. On the other hand, Urbano et al. (2004) studied the effects
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of germination of Pisum sativum, for 2, 4 or 6 days, with and without light,

on the proteolytic activity, the contents of soluble protein and non-protein
nitrogen, and the amount of available starch of P. sativum L. as well as their
nutritive utilization by growing rats, and concluded that germination of peas
for 2 days would be sufficient to significantly improve the palatability and
nutritive utilization of protein and carbohydrates from P. sativum.
According to Obubizor et al. (2013) depletion of butter in the germi-
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nated kernel, was due to the butter being mobilized and consumed during the
germination process. This led to depletion in the value of the lipid content
of Shea kernel, and elevated the FFA by seven-folds, the PV by 81, while
iodine value decreased when compared to the ungerminated. Low FFA im-
proves shelf life of butter. Such butter therefore attracts a premium price.
The elevated free fatty value could be attributed to the germination process
even though it is a known fact that hydrolysis could proceed via microbial,
enzymatic and autocatalytic pathways. According to the author, for maxi-
mum butter yield, all the conditions necessary for Shea kernel germination
must be eliminated or reduced to the barest minimum and prompt processing
of the collected fruit must be considered as an important requirement.
12.7.3 SHEA NUT DRYING/STORAGE
The Shea nuts are usually sun dried for 12 weeks and dehusked to obtain
the Shea kernel which is further sun dried for another 12 weeks. Although
the Shea kernels can sometimes be baked to concentrate the oil in the ker-
nel and lengthen the storage period, this has been discouraged because it
is a limiting factor to quality of Shea butter. Methods of solar drying on
polythene sheeting have been developed in some African countries, but they
have limited durability (FAO and CFC, 2005). According to USAID (2004),
the Shea kernels can be stored for several years without spoilage by main-
taining its moisture content between 6 and 7%. This is so because the drying
process inactivates enzymes responsible for the build-up of fatty acids in the
seed kernel (USAID, 2004).
According to Esiegbuya et al. (2014) during the drying stage and storing
of Shea nuts and kernels, they undergo various forms of postharvest dete-
rioration, namely: nut cracks/holes, nut discoloration, kernel discoloration,
and kernel deterioration. The possible source of the above problems in the
Shea nuts and kernels is ascribed mainly to poor environmental storage con-
ditions. The poor storage conditions lead to mechanical injury, fungal bio-
deterioration and discoloration. It also creates opportunity for insect larvae
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which have the ability to bore holes through the nuts and kernels, thus ex-
posing the latter to microbial contamination.
It has been reported by Agboola (1992) and Mlambo et al. (1992) that
species of insects that are associated with various stored seeds in Nigeria
and other parts of Africa cause appreciable damage to stored rubber seeds by
boring holes in the kernels, after gaining entrance through the micropyle, in
the hard testa. Healthy Shea nuts and kernels are coffee brown in color, their
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discoloration to black/dark color is as a result of the activities of postharvest
fungi. According to JICA (2007) Shea nuts will turn black if the nuts are not
dry well or if they are wetted by rain or when direct sunlight is not available.
Poorly dried or black nuts fetch lower prices in the market than well-dried
kernels.
According to Igeleke and Ekpebor (1986) fungi such as A. flavus and
niger growing on Palm kernels are known to impact various colors to the
seeds and also increase the free fatty acid content. Aspergillus niger, A. per-
sii, and A. flavus are common moulds associated with postharvest discol-
oration and deterioration of stored Shea kernels in Nigeria (Esiegbuya et
al., 2014). Members of this group are common and widespread. They have
been reported to produce mycotoxins including malformin and naphthopy-
rones and some strains are known to produce ochratoxin. Members of this
species aggregate have been implicated in human and animal infections in-
cluding superficial and local infections (cutaneous infections, otomycosis,
and tracheobronchitis), infections associated with damaged tissue (aspergil-
loma, osteomyelitis), pulmonary infections, and clinical allergies (allergic
bronchopulmonary aspergillosis, rhinitis, and Farmerss lung). However, the
majority of infections relate to immunocompromised individuals while the
Aspergillus flavus strain has been reported to produce both aflatoxins B and
G (Klich, 2002).
12.8 TRADITIONAL STRATEGIES FOR CONTROL OF

POSTHARVEST DISEASE OF SHEA NUTS AND KERNELS
12.8.1 USE OF STORAGE BAGS AND SACKS
Shea kernels are stored in sacks, woven baskets, and plastic buckets that are
stored either in house, granary, or kitchen floors. Sometimes the kernels are
hung in houses or kitchens instead of floors. In West Africa, jute bags from
cocoa industry are widely used. Over the past decades, polythene bags or
sacks have come into wide use for storage of Shea kernels. However, this
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has been reported to stimulate fungal growth important for quality because
they do not allow air circulation (FAO and CFC, 2005). Moreover, because
of the recalcitrant properties of Shea nuts, its storage is very difficult (Karin,
2004). The tightly woven plastic mesh also does not allow free circulation of
air, and condensation of kernel moisture over a diurnal temperature gradient
stimulates development of fungal spores leading to rapid contamination of
the stored Shea nuts. The situation is made all the worse by the fact that the
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bags are often stored directly on the earthen floor of a house (USAID, 2004).
Storage of Shea kernels in sack bags, also contributes to the high incidence
of the postharvest fungal deterioration of the kernels such as kernel discol-
oration and kernel deterioration. The deteriorations were as a result of the
favorable moisture content of the kernels which was above 10% (Esiegbuya
et al., 2014).
12.8.2 PROPOSING BETTER ENVIRONMENTAL STORAGE

CONDITIONS FOR SHEA NUTS AND KERNELS
The moisture content most favorable for the storage of Shea nut is 812%
humidity/0.30.6 water activity. The adsorption is measured in conditions
of ambient temperature (25 C) and does not reach a certain value (superior
to 0.75). It is advisable to store Shea kernels with a moisture content of
10 2%. This corresponds to an activity of water of 0.30.5 (Kapseu and
Ngongang, 2002).
12.9 FUTURE PROSPECTS FOR MANAGEMENT OF POSTHARVEST

DISEASES OF SEEDS
Due to the high risk of fungicides to humans, animals, environmental and

the increasing rate at which pathogens develop resistance against fungi-
cides, and also the difficulty in commercializing the technology of biologi-
cal control agents, there is the need to develop an environmental growth
storage models for pathogens causing postharvest diseases of fruits of high
economic value. The environmental factors influencing the growth for a par-
ticular spoilage pathogens can be used to develop a storage screen house or
box for against the spoilage pathogens. The internal environmental condi-
tions such as humidity, temperature and different lighting conditions of the
storage screen house/box can be designed to hinder the growth of spoilage
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pathogens and also maintaining the viability, physiological, and nutritional

status of the fruits.
KEYWORDS
9781771883627
Biodeterioration
Chalara
Discoloration
Germination
Mycotoxins
Postharvest quality
Raphia palm
Shea butter
Shea tree
Xylaria
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(CTA) Nertherlands, Mather, S. B., Jorgensen, J., Eds., Seed Pathology Technical Centre
for Agricultural and Rural Cooperation (CTA): Netherlands, 1992, pp 109116.
Munshi. S. K.; Sandhu, S.; Sharma, S. Lipid composition in fast and slow germinating sun-
flower (Helianthus annuus L.) seeds. Gen App Plant Physiol 2007, 33(34), 235246.
Murray, R. K.; Granner, D. K.; Mayes, P. A.; Rodwell, V. W. Harpers Biochemistry, 25th
Edn., McGraw-Hill, USA, 2000.
Nahm, H. S. Quality characteristics of West African shea butter (Vitellaria paradoxa) and
approaches to extend shelf. MS thesis, The State University of New Jersey: USA, 2011.
Ndon, B. A. The raphia palm, economic palm series. Concept Publication: Lagos, Nigeria,
2003, pp 1156.
Obubizor, J. U.; Abigor, R. D.; Omoriyekemwen, V.; Okogbenin, E. A.; Okunwaye, T. Effect
of processing germinated shea kernels on the quality parameters of shea (Vitellaria para-
doxa) butter. J Cereals and Oilseeds 2013, 4(2), 2631.
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A. In vitro control of Chalara paradoxa isolated from Raphia hookeri (Mann and Wendel)
fruits using diethyl ether, acetone and methanol extracts of the seeds of Aframomum scep-
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S. A. Physico-chemical characteristics of shea butter (Vitellaria paradoxa C. F. Gaertn.)
oil from the Shea districts of Uganda. Afr J Food Agric Nut Develop 2010, 10, 20702084.
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causing fruit rot of raphia palm in Nigeria. Trop Sci 1992, 33, 2736
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CHAPTER 13
SOIL REMEDIATION AND

ECOLOGICAL RESTORATION FROM
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HEAVY METAL POLLUTION AND
RADIOACTIVE WASTE MATERIALS
USING FUNGAL GENETIC AND
GENOMIC RESOURCES
JEYABALAN SANGEETHA1, DEVARAJAN THANGADURAI2,
MUNISWAMY DAVID3, JADHAV SHRINIVAS3,
ABHISHEK CHANNAYYA MUNDARAGI2,
PAIDI MURALI KRISHNA3, ETIGEMANE RAMAPPA HARISH3,
PRATHIMA PURUSHOTHAM2, and SWAPNA KISHOR DESHPANDE2
1
Department of Environmental Science, Central University of Kerala,
Kasaragod, Kerala 671316, India
2
Department of Botany, Karnatak University, Dharwad 580003,
Karnataka, India
3
Department of Zoology, Karnatak University, Dharwad 580003,
Karnataka, India
CONTENTS
13.1 Introduction......................................................................................328
13.2 Biomagnification of Heavy Metals and Loss of Soil Biodiversity.....329
13.3 Radioactive Waste: Potential Soil, Air and Water Pollutant............334
13.4 Fungal Genetic and Genomic Resources.........................................337
13.5 Potential Use of Fungi for Pollution Abatement..............................343
13.6 Mycoaccumulation of Xenobiotics..................................................344
13.7 Mycoremediation, Mycosorption, and Mycofilteration...................347
Keywords..................................................................................................351
References.................................................................................................352
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13.1INTRODUCTION
Soil executes pivotal role in our environment by providing an active atmo-

sphere for existence of life on earth. A wide array of diverse organisms that
are living on earth is haphazardly dependent on soil for growth, develop-
ment, and survival. Soil is the niche habitat for numerous types of micro-
and macroorganisms which assists the processes of biogeochemical cycling,
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degradation of organic/inorganic wastes and other natural activities that fur-
ther helps in maintenance of ecological balance. Despite the fact that soil
acquires major significance on earth, it is being polluted by number of haz-
ardous activities by human beings. Urbanization and industrialization are
the main agencies of environmental pollution. The exudates from industries
includes several non-degradable chemicals, oils, toxic materials, synthetic
dyes, heavy metals, acidic contents, and radioactive wastes from nuclear
energy plants are the main sources that brings a threatening outcomes to-
ward life on earth. In addition to this, the new advancements in the field of
agriculture, pharmaceutical, and other sectors are also causing damage to
the soil habitats. It was assumed that, if this dangerous practice continues for
few more years, there will be a severe destruction may occur in the environ-
ment that eventually leads to an irreversible destructive impact on environ-
ment and the living creatures on earth.
Metals are said to be the major contaminants of nature. The indiscrimi-
nate use of these metallic elements by human beings made excessive release
of certain harmful metals like cadmium, nickel, copper, zinc, lead, and other
metals into the environment (Dixit et al., 2015). These heavy metals con-
taminate soil, aquatic environment, as well as food. By its long-term per-
sistence in nature they may cause biomagnification. There is a possibility of
occurrence of many harmful diseases in human, animals, as well as plants.
In the past decades, many types of uncertain effects were seen by the metal
accumulation.
Another major environmental pollution is radioactive wastes from nu-
clear atomic energy plants. The presence of heavy metals and radioactive
wastes in the environment has to be removed in order to obtain healthy na-
ture. In order to have contamination free environment restoration of heavy
metal and radioactive is required. To preserve the soil from these kinds of
pollutants, new and innovative physical, chemical, and biological methods
are being developed which not only aims at removal of pollution, but also
aims at long-term management of contaminated sites (Francis and Dodge,
1998). There are various methods which have been designed to reduce
the amount of heavy metals and radioactive wastes from the soil. Acidic
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Fungal Bioresources for Soil Remediation and Ecological Restoration 329
mixtures of many organic acids were used to treat contaminated soil and to
extract the pollutants from it.
In order to overcome this unpredictable impact on living biota, number
of experiments is performed. In this direction, some of the microorganisms
that are beneficial having application in degrading the heavy metal contami-
nants have been used and this led the invention of new approach called bio-
remediation. Some beneficial microorganisms are capable in remediating the
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impurities of heavy metals and nuclear wastes from the environment. The
bacterial species such as Pseudomonas putida are used in degradation of oil
spills and some of the fungal species are also supports in bioremediation pro-
cess. The spillage of oil on surface of earth can be decomposed by spreading
a layer of mycelium on to the polluted soil. Mycelium in fungi facilitates
the mycofiltration of chemicals thereby flowing of chemical pollutants into
other habitats could be prevented. The saprophytic nature of these fungal
species favors the elimination of organic wastes by decaying them. Use of
microorganisms in general, fungi in particular, for pollution control is one of
the beneficial approaches, because it is low-cost, low-maintenance biologi-
cal solution to remediate toxin and heavy metals from the environment.
Biotechnological applications are helpful to obtain microbial strains
with high efficiency toward pollution control. The genetic engineering ap-
proaches and strain improvement techniques are useful to develop a micro-
bial system with good ability to degrade environmental pollutants. For the
sake of bioremediation there requires several genetic systems to be exploited
(Sayler et al., 1988; Menn et al., 2000). The technique of protein engineering
also gives prominent results in bioremediation. Overall, this chapter pro-
vides a complete vision to understand the importance of fungal strains and
their role in soil remediation, techniques involved in mycoremediation of
environmental pollutants such as heavy metals and radioactive wastes, dif-
ferent sources of fungi to be used for soil remediation and applications of
fungal strains for ecological restoration.
13.2 BIOMAGNIFICATION OF HEAVY METALS AND LOSS OF

SOIL BIODIVERSITY
Pollution may be defined as an introduction of unwanted and unacceptable

compounds in to the natural system which in turn may prevent natural pro-
cesses in the environment and may have undesirable health effects. In that
sense, heavy metal pollution is defined as pollution due to the metal whose
elemental density is greater than 57 g/cm3 and more than 23 atomic weight
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and number and poisonous even at low concentration (Duffus, 2002). Rapid
urbanization, industrialization, mining and implementation of synthetic met-
al containing pesticides in a broad spectrum for pest management systems in
developing countries, root up arsenic, cadmium, and lead metal ions in soil
(Sherene, 2010). Heavy metals enter into the environment by natural phe-
nomena like volcanoes, weathering, paedogenesis from rock surfaces and
anthropogenic activities for human benefits (Jankaite et al., 2008; Mbah and
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Anikwe, 2010). In terms of soil pollution, anthropogenic causes are intense
in terms of heavy metal pollution due to introduction of several contaminants
like fertilizer, pesticides, and other xenobiotics. Ecotoxicologists have found
in the past that natural resources such as air, soil, and water were polluted by
heavy metals in and around of industrial and mining areas through discharg-
ing its wastes into water columns like water reservoirs, rivers, lakes, and
canals (Natesan and Ranga Rama Seshan, 2011). The subsequent squeezing
of heavy metals along with industrial wastes leads the soil and water pollu-
tion (Verma and Dwivedi, 2013). Particle matter in air may sometimes get
deposited to soil by conveyance of particles from air to soil (Malizia et al.,
2012). Geo-accumulation of heavy metal progressively increased in urban
soil and road dust in cities (Wei and Yang, 2010).
However, the application of metal composed pesticides passively in-
creased for wild pests control throughout worldwide. Due to lack of aware-
ness on pesticide compassion and its application limitations, the excessive
applied pesticides fetch the toxic metals to non target organism through di-
rect inhalation or water streams (Wuana and Okieimen, 2011). For example,
cadmium containing pesticide used in a high range through the world, viz.
Excoecaria agallocha mangrove species are in first line bio-indicator for
heavy metal water pollution; the metal ions zinc, copper, and lead in the
root, stem, and leaf of Excoecaria agallocha in north east coast of Bay of
Bengal in and around Indian Sundarbans mangrove ecosystem (Chakraborty
et al., 2014). Cadmium accumulation rising day by day anthropogenic activ-
ities such as tobacco smoking, mining, smelting, and refining of nonferrous
metals, fossil fuel combustion, incineration of municipal waste (especially
cadmium-containing batteries and plastics), manufacture of phosphate fer-
tilizers, and recycling of cadmium-plated steel scrap and electric and elec-
tronic waste process magnify Cd in both aquatic and terrestrial ecosystems
(WHO, 2010).
The modern urbanization and industrialization progressively increased
automobile usage expel toxic metals particles into air in the form of dust,
strengthen air pollution and increasing intake of arsenic, cadmium, copper,
chromium and lead particles during inhalation. People who live in industrial
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waste and smoke discharged location are subsequently exposed to airborne

lead from combustion of solid waste, coal, and oils, emissions from iron
and steel production and lead smelters, and tobacco smoke (Wuana and
Okieimen, 2011). Thermal power plants and metal smelters contribute ma-
jority of arsenic and mercury in to environment due to burning of arsenic
rich coal (Sahu et al., 2012). The continuous intake of toxic air damage
airways by surface mucus layer precipitation and stimulate inflammations
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(Fahy and Dickey, 2010).
Nevertheless, mining activities inflate the socioeconomic life of a coun-
try, but the resulted waste effluents are discharged in open area. Mining ore
tailings are major source of toxic metals (Guan et al., 2014). The soil erosion
is a natural phenomenon during higher rainfalls. The overburden ore tailing
leachate runoff into water streams containing metal ions causes water with
metal pollution (Reza and Singh, 2010) and accumulation in river sediments
(Natesan and Ranga Rama Seshan, 2011; Nachiyunde et al., 2013). Gold
mine ore dumps are the major source of toxic metals such as cadmium, chro-
mium, lead, zinc, copper, arsenic, selenium, and mercury which can con-
taminate the environment through soil and water pollution (Cobbina et al.,
2013).
Among developing countries, industrialization due to mining activities
and exploitation of soil and water resources stands controversial because
of socioeconomic status of the local people (Verma et al., 2012). In some
extent mining operations and their waste disposal methods are considered
the main sources for the environmental degradation. After mining, the left-
over debris like rocky wastes is mainly dumped in waste dumping yards.
Overburden deposits generated from mining activities constitute a potential
risk to the environment fetching through leachate of potentially toxic ele-
ments hosted by a variety of minerals present in the mine-waste materials
(Armienta et al., 2003). During metallurgy, ore tailings contain metal ions
which are drastically increased while dumped (Jung, 2008). Mining activi-
ties change the geochemical nature of crop land, which induces the soil acid-
ification, impairing the soil vegetation and degeneration of aquatic natural
life (Christiana, 2012).
Historical mining background countries suffering from heavy metal con-
tamination. For example in India, Gold Mine, Hutti and Greenstone Belt,
Mangalur in Karnataka discharged dumps in open area causes heavy im-
pact on agricultural lands and leads to the magnification of heavy metals in
around mining areas since long many centuries (Chakraborti et al., 2013).
Mine ore tailing show high nutritional quality and clay, hence which is ad-
vised in compost, school play ground preparations. While in some extent
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used in concrete composition mixture, enhance the flexible strength of con-

cert (Skanda Kumar et al., 2014). Arsenic and cadmium content in school
children toes and nails were reported in playing school ground prepared
by mine ore tailings (http://www.healthyschools.org). The Environmental
Protection Agency classifiedchromated copper arsenic (CCA) wood chips
as hazardous wastes. This possessed an incredible 8131654 ppm arsenic
(www.health.usnews.com).
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Industrial wastes contain good nutritional values and also high percent-
age of metal ions. Due to high nutritional quality farmers used these soils
and liquid wastes as compost preparation and irrigation for cultivation of
common vegetable (bulbs). The consequent application of these industrial
wastes leads biomagnifications of metal ions in green vegetable Coriandrum
sativum, Spinach (Spinacia oleracea) and Amaranthus sp. (Chiroma et al.,
2014). Plants were grown in metal polluted sites escort the bioaccumulation
of metal ion content in root, leaves and stem and also elevation of low mo-
lecular antioxidants such as ascorbate, glutathione, and flavonoids (Geneva
et al., 2014).
Metal magnification in soil reduces the bacterial growth rate and diver-
sity. Survival and colony size of several burrowing insects like ants is altered
due to disturbances in ecological aspects of soil (Grze, 2010). Toxic metal
ions induce stress on cop plants by elevation of free radicals. Lead contami-
nation in capsicum cultivation, despite a reduction in the plant growth and
chlorophyll content and elevation of malondialdehyde (MDA), super oxide
dismutase (SOD) and proline contents corresponding to the concentration
of the metal ion (Britto et al., 2011). The aquatic metal pollution may alter
the natural physiology of aquatic lives (Baby et al., 2010; Zaki et al., 2013).
Metal toxicity to fresh water is considered to regulate chemical communi-
cation between freshwater habitants and this alteration in chemical com-
munication could further devastate relationship between two species in an
ecosystem (Boyd, 2010).
Heavy metal soil pollution reducing soil enzyme activity is a major cause
of depletion of soil flora and fauna (Jose et al., 2011). The toxic metal ion
transferred into food web in the form of biomagnification and through drink-
ing water shows serious problems in vertebrates growth and sexual matura-
tions. It has been also reported that heavy metal toxicity could lead to endo-
crine disruption and DNA expression by molecular signaling. For example
Zn fingers of the estrogen receptor, Zn can be replaced by several heavy met-
al molecules such as copper, cobalt, Ni, and Cd activities in mice (Georgescu
et al., 2011). Cadmium chronic exposure induces free radical concentration,
rennin angiotensin system abnormalities (RAS), microalbuminuria and Na+
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K+ ATPase depression in human being (Gulati et al., 2010). Heavy metal pol-
lution could cause epigenetic modification in DNA mechanisms (Rzymski
et al., 2015). According to the study of Benbrahim-Tallaa et al. (2007), Cd
induced prostate epithelial malignant transformation through DNA hyper-
methylation at the global and gene specific levels. People who are living and
predominantly exposing to cadmium, lead, and mercury metal contaminated
location suffering from infertility (Joffe, 2003).
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Aquatic toxic metal contamination may alter the fish physiology through
elevation of free radicals, vital organelle cellular damage, and some extent,
over exposure leads to death (Annabi et al., 2013). Metallothionein protein
accumulation is a hall marker of metal exposure in fishes (Zaki et al., 2013)
due to higher sensitivity of fishes and hence fishes are considered as bioindi-
cator of effect of heavy metals (Authman, 2015).
Phytoinhalation is ubiquitous detoxification strategy in many plants at
heavy metal stress conditions, elevation of glutathione (GHS) and phyto-
chelatin synthase (PCS) enzymes activities are major indication of metal
contamination which induces physiological stress. Metals like Al, Cu, Zn,
and Fe abundance in soil influence on hyper activation of ascorbate oxidase
(ASO), guaiacol peroxidase (GPX), ascorbate peroxidase (APX) and super-
oxide dismutase (SOD) activities and alter the plasma membrane phospho-
lipids composition in Zygophyllum sp. (Morsy et al., 2012). Considerable
reduction in chlorophyll, sugar, and protein contents were observed at road
sites receiving higher toxic load is a significant bioindication of heavy metal
pollution (Rai and Panda, 2015). The decline yielding capacity of common
commercial vegetable cultivation in metal contaminated sites passively in-
creases the accumulation of heavy metals. For example, plants (Matricaria
recutita) grown in Cd, Pb, and Zn contaminated sites, the elevation account
of APX in the above ground parts, glutathione peroxidase and guaiacol per-
oxidase in the leaves and dehydroascorbate reductase and glutathione-S-
transferase in the flowers are for the toxic tolerance (Popova et al., 2012;
Nadgorska-Socha et al., 2013).
The rapidly growing and uncontrolled population creates scarcity of
essential natural resources which has become a major problem and shows
significant impact on natural ecological degradation (Jahan, 2008; Anand,
2013). There are huge findings on heavy metal pollution and its toxicity
throughout the world (Authman, 2015). Especially in developing countries
lack of awareness on metal and its toxic potential in physiological life cycle,
people are consequently exposing and applying excessively to the croplands.
The anthropogenic activities are becoming essential for saturation of liveli-
hood. In overall the above activities, our mother planet (Earth) is going to
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be contaminating with various pollutants. Hence, this is the time to raise the
alarm on pollution and toxicity and early implementation of eco-friendly
things and bioremediation, bioleaching and biodegradation on pollution af-
fected locations.
13.3 RADIOACTIVE WASTE: POTENTIAL SOIL, AIR AND WATER
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POLLUTANT
Waste that emits harmful level of nuclear radiation is radioactive waste. None
of the place on the Earth is free from natural radioactive background and
every inhabitant on this earth is constantly exposed to both natural and arti-
ficially occurring ionizing and non-ionizing radiation. Cosmic rays from the
celestial body, radioactive minerals widely distributed in rocks, sediments
and soils, radionuclides normally incorporated into our bodys tissues, and
radon and its products are the sources of natural radiation (Vogiannis and
Nikolopoulos, 2015). Also exposed to ionizing radiation from man-made
sources, mostly through medical procedures like X-ray diagnostics, civilian
war, nuclear industries and nuclear explosion tests especially when carried
out in the atmosphere, leakage of radiations from nuclear reactors, and other
nuclear facilities (Rao, 2001).
Radioactivity was discovered over several years beginning with the dis-
covery of X-rays in 1895 by Wilhelm Conrad Roentgenand continuing with
such people as Henri Becquerel and the Curie family (http://www.nobel-
prize.org). After the Second World War, radioactivity was artificially and
intentionally been introduced into nature through modern warfare and nucle-
ar war head testing contaminating air, soil, and water bodies. Further, civil
nuclear program for clean energy and biomedical research has its fare share
in contamination (Rao, 2001; Shruti, 2010).
The nuclear residual waste from natural sources, from past mining, and
its related operations, produced more than 1000 EBq/year of radioactive
waste in the atmosphere and it is estimated to be around several million
tons at many places and its radioactivity more or less equal to 0.001 EBq.
Thousands of such sites are scattered all over the world. Exploiting nuclear
energy in civil nuclear program has contributed major nuclear wastes in past
50 years, estimating 1000 EBq and is still on the growth by around 100
WBq/year (Gonzalez, 2000). Approximately 30 tons of high-level radio-
active waste is generated by a mega nuclear energy plant with a capacity
of 1000 MW electricity. A total of 440 tons of intermediate-level and 460
tons of low-level radioactive waste. While the coal fired power plant release
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nearly 400 tons of heavy metals and six million tons of greenhouse gases,
500,000 tons of mixture of sulfur and nitrogen oxides and about 320,000
tons of ashes to generate 1000 MW electric power (Rashad and Hammad,
2000). The ashes generated by thermal plants have the potential to pose hu-
manity into the risk higher than that of collective dose of radiation due to
nuclear waste from nuclear plants. Yet common perception among people is
against those nuclear electric plants in several countries (Rao, 2001).
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13.3.1 POTENTIAL CAUSES OF RADIOACTIVE WASTES
Radioactive soil pollution is commonly defined as excessive presence of

radiation in soil system due to obvious anthropogenic or certain natural
causes. Such solid waste dumping has become abundant landfill because of
the indiscriminate discarding of solid waste on the earth. Nuclear industries,
nuclear explosion tests, and nuclear bomb attack especially when carried
out in the atmosphere are responsible for increasing the background level of
radiation throughout the world and major cause of radiation pollution in soil.
During atmospheric nuclear explosion tests, a number of long-lived radionu-
clides are released into the atmosphere (Simon et al., 2006). This radioactive
dust (also known as radioactive fallout) gets suspended in air at a height of
67 km above the earths surface and is dispersed over long distances by
winds from the test site and some of the radioactive isotopes given off during
nuclear test which affects the human body. The best example of fallout is the
nuclear bomb attack on Hiroshima and Nagasaki, Japan in 1945 by United
States of America during World War II. The radiation from the blast was so
intense that it would leave shadows of the materials in way and as many
as 2,25,000 people have lost their live in later five years of the blast due to
radiation (John and Pastore, 1987; Simon et al., 2006).
Naturally occurring radioisotopes such as radon-222, potassium (K-40)
and carbon (C-40), uranium, thorium, and radium are found in soil in small
quantity is another source of radiation pollution in soil. Radiation due to
Potassium-40 is the sole source of pollution for all potassium containing
systems in the soil. Crops grown on such soil contain radioactive elements
like carbon-14 and potassium-40 (http://mragheb.com). The unusable and
unwanted waste products from nuclear industries, atmospheric fallout of the
radioactive waste and radioisotopes from the natural sources may contain
radionuclides, often settle down by rain and get mixed with soil and water.
Through bioaccumulation and biomagnification, it passes on from organism
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to organism finally reaching to human, where it can cause serious health

hazards (Eisenbud and Gesell, 1997).
13.3.2 RADIATION POLLUTION AND ITS TOXIC EFFECTS
Whether or not, artificial and natural, the potential risk of radiation to hu-
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man beings is noteworthy. The effect of these radiations was first reported
in early Twentieth Century when person working in uranium mines suffered
major health problems including cancer (Rao, 2001). The effects vary from
organism to organism, level of radioactivity of nuclear isotopes and how
much and how fast a radiation dose is received. On contradiction, it was
fascinating to hear, some workers have reported low doses of radiations are
in fact beneficial to organisms in terms of increased longevity, better disease
resistance, and greater life span. But higher doses of radiation effect studies
came only after Hiroshima attack in 1945 and people received major doses
of radiation (Krishnan, 2014). About 12% of all the cancers that have devel-
oped among those survivors are estimated to be related to radiation effect
(Jane and Orient, 2014). While, a chronic dose is a relatively small amount
of radiation received over a long period of time may induce somatic and ge-
netic effect which include the development of eye cataract, cancers, and ge-
netic or heritable effects appear in the future generation. An acute radiation
dose (a large dose delivered during a short period of time) may result in ef-
fects which are observable within a period of hours to weeks (Strom, 2004).
Radioactive particles in its most threatening form, forms ions which it
reacts with biological molecules and has potential to damage genetic mate-
rial of all organisms. Free radicals are then formed in the process which will
start scavenging on biological materials like proteins, carbohydrates, and
fats. A longer exposure to radioactive radiations can damage the DNA cells
that results in cancer, genetic defects for the generations to come and even
death (http://www.cna.ca).
13.3.3 BIOREMEDIATION OF RADIOACTIVE WASTES
Radioactive pollution in soil is defined as any site that has been exposed to
naturally occurring or artificial radionuclides which may ultimately cause
health and environmental hazards. It is imperative that radioactive waste is
proven to be a severe threat toward environmental and human health (Kumar,
2004). Hence, there is an urgent need to develop efficacy management with
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respect to radioactive pollution management. According to Evrard (2012)

there are three aspects in radioactivity management policies; site manage-
ment according to the future perspective or anticipated uses; to record the
past pollution hazards and remediation; and provide up to date information
to the public with respect to concerning associated hazards.
Eight years after the publication of Controle Magazine devoted to the
management of sites contaminated by radioactive substances, DGSNR
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(General Directorate for Nuclear Safety and Radiation Protection) came in
force and ASN was tasked with the management of sites contaminated by
radioactive materials and it became noticeable that an initial inventory of
national and international practices was needed, in order to identify the ma-
jor obstacles and the changes that were required. Hence, ASN organized its
first national symposium on Radioactive contamination: how to deal with
polluted sites? jointly with Ministry of Ecology on May 4, 2004 and also
drafted the First National Radioactive Material and Waste Management Plan
during 2006 for appropriate management of radioactive pollution. Again in
2007, ASN created National Commission for Assistance in Radioactive Field
(CNAR) and recently launched operation radium diagnosis guide concern-
ing the management of sites potentially polluted by radioactive substances
(Evrard, 2012).
Management of polluted sites has gone from surveying the sites to more
global aspects in past 20 years. This global approach allows faster and more
sustainable management of the sites, by involving all the stakeholders as
early as possible in the polluted site management process. Various measures
were however initiated, to allow effective management of radioactive wastes
which involves segregation, characterization, handling, treatment, condi-
tioning, and monitoring prior to final storage or disposal even more trans-
parent and efficient management of these forms of pollution (Wattal, 2013).
However, mycoremediation is considered recently as an eco-friendly way of
treating hazardous radioactive pollutants and is gaining attention nowadays
and also it offers an efficient and cost effective way to treat contaminated
ground water and soil.
13.4 FUNGAL GENETIC AND GENOMIC RESOURCES
Many fungi survive in extreme environmental conditions. These are rec-

ognized as extremotolerant fungi. Regrettably, the mechanisms involved in
their ability to withstand harsh conditions against the abiotic and biotic stress
have been poorly understood. Nevertheless, these fungi have evolved with
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exceptional resistance power to support their existence. Hence these may

perhaps act as useful resources for developing novel compounds and en-
zymes for bioremediation. It is very important to analyze the genes respon-
sible for the sorption and specificity to heavy metals. Stress-tolerant fungi
such as extremophiles have evolved with a resistant power to withstand harsh
environments and could be novel genetic resources for the decontamination
of heavy metal polluted environment. The extremophiles can be further clas-
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sified in to thermophiles (4050 C), psychrophiles (1620 C), acidophiles
(pH 1.04.0), alkalophiles (pH>9.0), xerophiles or osmophiles (water activ-
ity (aw) of 0.850 such as Wallemia sebi) and halophiles (tolerant to high
salt concentrations such as Wallemia ichthyophaga) (Padamsee et al., 2012;
Zajc et al., 2013). These extremotolerants indeed could be useful genetic
resources for the detoxification or biosorption of heavy metals.
Certain fungi inhabiting soil are exposed to harmful radiations such as
the ionizing and non ionizing radiations and these fungi are termed as pig-
mented fungi which have dark pigmentation due to the protective nature of
fungi against radiations. According to Dadachova and Casadevall (2008),
these fungi utilize the radiations as source of nutrition for their growth.
Moreover, pigmented fungi such as Cladosporium cladosporioides and
Paecilomyces lilacinus have been found at the site of Chernobyl Nuclear
Power Plant (Zhdanova et al., 2004). These extremophiles can be hopeful of
potential genetic resources. With powerful and novel functional screening
strategies extremotolerant fungi could be a promising new opening for the
identification of stress tolerant genes (Li et al., 1997; Trincone, 2011).
In the last two decades, with the advent of genomics and proteomics, an
unprecedented growth has been achieved in understanding and characteriz-
ing fungal biodiversity and fungal biotechnology (Giaever and Nislow, 2014;
Bianco and Perrotta, 2015). The power of fungal genetics and genomics has
been transformed drastically due to the availability of mutations mapped
and defined by available advanced genetic tools (Magee et al., 2003; Nagy
et al., 2003). Saccharomyces cerevisiae is a best experimental model for eu-
karyotic studies. Genomic studies help us to better understand the complex
biotrophic interactions and also in sustenance of natural environment. This
was accomplished with the advent of two pioneering sequencing technolo-
gies. MaxamGilbert method and the Sanger method are the two sequenc-
ing tools developed in the 1970s. Sanger sequencing be the most followed
standard method for genome sequencing in the past and future few decades
too. Now, next generation sequencing technologies is being employed in
DNA sequencing such as the Pyrosequencing (454), Illumina sequencing
(Solexa), ABI SOLiD sequencing and the single molecule sequence (Helicos
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HeliScope) (Zhang et al., 2011). Goffeau et al. (1996) decoded genome se-
quence of the yeast S. cerevisiae in their classical publication entitled Life
with 6000 genes, which was the first work to be reported on fungal ge-
nomics. Furthermore, the first ectomycorrhizal genome to be sequenced was
Laccaria bicolor in 2008 (Martin and Selosse 2008). The genome of fungus
Tuber melanosporum is the largest fungal genome (125 Mbp) decoded till
date (Martin et al., 2010). Genome annotations of model fungi (S. cerevisiae,
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Aspergillus nidulans, Neurospora crassa, and Schizosaccharomyces pombe)
lead to gene discovery and identication of novel gene activities (Seiler and
Plamann, 2003; Galagan et al., 2005).
Though genomics era has begun in last decade, in upcoming years, it
will pave the way in creating and understanding complex fungal pathways
and bioprocesses for producing biofuels and biochemicals (Nevoigt, 2008;
Dellomonaco et al., 2010). Comparative genomics is one such approach
which deals with the study of two or multiple genomes, comparing them ei-
ther through nucleic acid sequence or protein sequence to map gene positions
(Koonin and Galperin, 2003). Until now more than 100 fungal genomes have
been sequenced and many more are in progress with the target to achieve
1000 fungal genomes in near future; particularly, those fungi that are very
important in industrial processes, bioenergy, and medical sciences. Major
fungal genomic research institutes such as the Broad Institute, the National
Human Genome Research Institute (NHGRI) and the U.S. Department of
Energys (DOE) Joint Genome Institute (JGI) are concurrently involved in
the decoding of genomes of many important fungi. JGIs fungal genome
project (FGP) is the major program which is involved in the extensive re-
search on fungal genomics. This project has collaborated with major re-
search institutes across the globe. In contrast, one of the key initiatives of
FGP, the Genome Encyclopedia of Fungi is working to track down the ge-
nomes of fungi that are considered to be important in three thrust areas, plant
feedstock health, biorefinery and fungal diversity. The sequenced genomes
are deposited and maintained in the database MycoCosm (http://www.jgi.
doe.gov/fungi) which is a fungal genomics resource portal that promotes
users in submission, annotation and analysis of vast fungal genome data
(Grigoriev et al., 2014). The following fungi have already been sequenced
by NHGRI in the last decade: Aspergillus nidulans, Batrachochytrium
dendrobatidis, Candida albicans, Candida tropicalis, Candida guillier-
mondii, Candida lusitaniae, Chaetomium globosum, Coprinus cinereus,
Coccidioides immitis, Cryptococcus neoformans Serotype A, Cryptococcus
neoformans Serotype B, Geomyces destructans, Histoplasma capsulatum,
Lodderomyces elongisporus, Myceliophthora thermophila, Pneumocystis
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carinii, Podospora anserine, Rhizopus oryzae, Schizosaccharomyces japon-

icus, Schizosaccharomyces octosporus, Saccharomyces cerevisiae RM11
1A, Ustilago maydis, and Unicinocarpus reesii. High-throughput genomic
assays, has enabled and resulted in understanding structure, functions, com-
plex mechanisms, and variations among different genomes, genes of interest
and their expression in model organisms and gene regulation in develop-
mental processes.
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Fungi are the most promising tools for biosorption as they have distinctive
properties to sequester the toxic heavy metals from environment (Hossain et
al., 2005; Ji et al., 2005; Romero et al., 2005; Baxter and Cummings, 2006;
Monrroy et al., 2007). Yeast and filamentous fungi are able to bind heavy
metals because of the special cell wall structure that they posses. Chitin is
a major component in framework of cell wall composed of polysaccharides
such as glycans, chitin, chitosan, mannans and phosphormannans. Though
there are several established methods or chemical processes to reduce the
heavy metal pollution and toxicity the fungal biosorption is quite accept-
ed and popular as it is efficient, eco-friendly and cost effective (Rao and
Bhargavi, 2013; Saraf and Vaidya, 2015). The mechanisms involved in
heavy metal adsorption are methylation, reduction and dealkylation (Gadd,
1993). Recent studies indicate that the removal and recovery of toxic heavy
metals has been successful by the innovations in the bioresource and biopro-
cessing technologies, many fungal strains have been used to recover these
toxic heavy metals in lab scale bioreactors within short period of time. The
approach is astonishing as the fungi used is the waste biomass from any food
and pharmaceutical industries, the dead biomass absorb the heavy metals
and recovery is also thought to be simple and effective. Many fungal strains
and biomass has been tried out to assess their potentiality to sequester the
heavy metals but still there are certain limitations such as the experiments
have been carried out in the lab scale wherein still the in situ application is
lacking and also more research has to be carried out in mathematical model-
ing, kinetics and suitability toward the environmental factors. These limi-
tations could be overcome by adapting modern technologies and genomic
techniques.
The enzyme Phytochelatin synthase (PCS) is a 95 kDa tetramer belongs
to dipeptidyl transferases that was first characterized by Grill et al. (1989).
PCS ((-Glu-Cys)n-Gly) catalyzes the synthesis of phytochelatin from
GSE. Phytochelatin (earlier known as cadystins) are structurally similar
to GSE. These are heavy metal binding peptides that protect cells against
heavy metals toxicity. PCS genes from fungi have been characterized in
species belonging to Ascomycota and these genes are sparsely distributed
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in most of fungi. Heavy metal cations such as Cd, Cu, Zn, Pb, and anions
like nickel, mercury, and arsenate are the causes for activating PCS in cells
and tissues. The molecular weights of various PCS enzymes deduced from
DNA sequences range from 4070 kDa and the genes have been charac-
terized in the following fungi and other organisms: Schizosaccharomyces
pombe (SpPCS), Dictyostelium discoideum (DdPCS1), Arabidopsis thali-
ana (AtPCS1), Triticum aestivum (TaPCS1) and Caenorhabditis elegans
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(CePCS1) (Cobbett, 2000a). Together, these observations indicate that PCS
is primarily regulated by induction of heavy metal cations such as zinc and
cadmium. The best activator tested was Cd, followed by Ag, Bi, Pb, Zn,
Cu, Hg, and Au cations. PCS expressed in E. coli or in S. cerevisiae was
activated to varying range of Cd, Cu, Ag, Hg, Zn, and Pb ions. Shen et al.
(2015) reported PCS in Sporobolomyces sp. strain IAM 13481 belonging to
Pucciniomycotina subphylum of the Basidiomycota.
Metallothioneins (MTs) are low molecular weight cysteine-rich polypep-
tides which bind heavy metals. MTs are widely distributed among living
organisms and involved in heavy metal tolerance of many eukaryotes (Kagi
and Nordberg, 1979; Butt and Ecker, 1987; Bellion et al., 2007). Margoshes
and Vallee (1957) first reported purified MTs from horse kidney, who was
working with bioregulation of cadmium and zinc. Pulido et al. (1966) isolat-
ed MTs from human liver. Since then, MTs have been isolated from different
organisms including many fungi and several groups have reported copper-
inducible protein from fungi. Depending upon their distribution in cell they
have been characterized into three subfamilies such as cytosolic, micro-
somal, and mitochondrial GST or kappa-class GST subfamilies (Sheehan
et al., 2001; Hayes et al., 2005; Frova, 2006; Shen et al., 2015). Based on
amino acid/nucleotide sequences, immunological, kinetic, structural proper-
ties and other aspects, they have been further classified in to nine classes of
cytosolic GSTs in fungi: GTT1, GTT2, Ure2p, MAK16, EFB1, GSTFuA
(Mathieu et al., 2013; Thuillier et al., 2013), phi (Morel et al., 2013), omega,
and glutathionyl hydroquinone reductase (GHR) (McGoldrick et al., 2005).
Lindegren and co-workers for the first time demonstrated that a single gene
was responsible for copper sensitivity (cupl') and copper resistance (CUP1"),
in which MTs play key role in Copper (Cu) absorption, storage and homeo-
stasis. Saccharomyces cerevisiae contain a single Cu-MT gene present in
a CUP1 locus (Fogel et al., 1982) that encodes a Cys-rich protein which
results Cu sorption in the fungi. This ability to bind might be due to Cu ions
chelation, which is determined. Hence, high CUP1 expression levels result
in increased Cu-binding capacity (Butt et al., 1984). More recently, MT-
encoding gene from the AM fungi such as Pisolithus tinctorius, Gigaspora
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rosea and Gigaspora margarita have also been reported (Stommel et al.,
2001; Voiblet et al., 2001; Lanfranco et al., 2002). Bellion et al. (2007) re-
ported Pimt1 gene coding for a metallothionein from the ectomycorrhizal
fungus Paxillus involutus.
Glutathione S-transferases (GSTs) earlier known to be ligandins are the
metabolic isozymes that are involved in detoxification of xenobiotics and
heavy metals by catalyzing their conjugation to GSE (sulfur-containing
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tripeptide thiol). Earlier reports suggested that the formation of the GSH
Cd conjugate seems to regulate cadmium absorption in S. cerevisiae cells
(Gomes et al., 2002). In Saccharomyces cerevisiae Yeast Cadmium Factor
1 (YCF1) and in S. pombe Heavy-Metal Tolerance 1 (Hmt1) genes en-
code vacuolar ATP-binding cassette membrane proteins that transport bis-
(glutathionato)-cadmium and PCCd conjugates in to vacuole, play sig-
nificant role in cadmium detoxification (Ortiz et al., 1995; Li et al., 1997;
Cobbett, 2000b). Yeast such as S. cerevisiae protects itself from non-essen-
tial elements such as cadmium would help to understand the mechanisms of
metal ion detoxication. Adamis et al. (2004) investigated that two genes,
GTT1 and GTT2 of S. cerevisiae conferred tolerance to cadmium absorption
than the control strain, and proposed that the formation of the cadmium-
glutathione complex is dependent on that transferase. The technique reverse
Northern blot to assess molecular response of GSTs in arbuscular mycorrhi-
zal fungi Glomus intraradices has been well-demonstrated (Waschke et al.,
2006). Fraser et al. (2002) reported gene from Aspergillus nidulans that is
similar to URE2 gene of Saccharomyces cerevisiae encodes a GSTs that ac-
counted in heavy metal and xenobiotic resistance. In a similar study twenty-
four GST genes from the transcriptome of a metal-tolerant Exophiala pisci-
phila a dematiaceous fungus also known as dark septate endophyte (DSE)
closely related to the heavy metal tolerance has been documented (Shen et
al., 2015). Vallino et al. (2005) investigated the genetic basis of heavy met-
al tolerant ericoid mycorrhizal species Oidiodendron maius, isolated from
roots of Vaccinium myrtillus growing in soil heavily contaminated with high
zinc concentrations. Martino et al. (2007) studied genetic transformation of
metal tolerant Oidiodendron maius and proposed that genetic transformation
could be useful in understanding responsible genes and potential application
in bioremediation strategies.
The potential use of fungi for the detoxication or biosorption of
heavy metals in polluted environments is being increasingly exploited.
Understanding the phytochelatins (PTs), metallothioneins (MTs), and gluta-
thione (GSE) genes and their biosynthetic pathway on metal tolerance and
sorption will soon lead to indications as to their advantageous in this attempt.
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The mechanisms by which cells defend themselves against the heavy metals
involve complex biosynthetic pathways. The structural and functional simi-
larities of genes in microorganisms suggest that understanding molecular
mechanisms and how fungi detoxify or biosorp heavy metals would aid to
develop potential resource for bioremediation of heavy metal contaminated
sites (Hossain et al., 2012).
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13.5 POTENTIAL USE OF FUNGI FOR POLLUTION ABATEMENT
Fungi are unique group of microorganisms and occur ubiquitously in nature.

From the ancient days to till today, fungi occupy a unique place in human
life. They have a wide range of applications from cookery to industry. Fungi
being used for multipurpose, they are considered as true and natural ecosys-
tem engineers (Jones et al., 1994). Fungal cells have totipotent nature and
hence they can reproduce for unlimited period. The concept of degrading un-
wanted, harmful pollutants by fungi was thought by observing decomposing
nature of fungi by spreading and decaying materials such as wood, papers,
textiles, leather, and other various wastes. Decomposition of polythene by
the fungal species such as white rot fungi, enzymes from Aspergillus flavus
and Mucor rouxii NRRL 1835 and Penicillium simplicissimum were used
(Singh, 2006). In addition fungi are proved to be the well decomposers of
effluents from dye industries, paper and pulp industries. In order to achieve
complete degradation of pollutants by the use of fungi, it is necessary to un-
derstand the decomposing nature of fungi, fungal byproducts, effects after
fungal decomposition and availability of fungi being used.
The process of absorption of heavy metals by fungi can be termed as
mycosorption. This process has gained importance in environmental protec-
tion and metal recovery. Application of fungal strains to attain biosorption of
polluted materials becomes one of the recent trends in area of environmental
pollution control. Process of mycosorption is a pseudo-ion-exchange meth-
od, in which the metal ion exchanged for a counterion in biomass (Singh,
2006). The filamentous and aquatic fungi possess more affinity and capacity
to absorb heavy metal contaminants from the environment. This approach of
heavy metal uptake was described for the first time by Michelot and further
demonstrated by cultivating mushrooms to achieve bioaccumulations. Some
of the marine fungi namely, Corollospora lacera and Monodictys pelagi-
ca are identified to accumulate lead and cadmium extracellular in mycelia
(Taboski et al., 2005). The process of biosorption and metal recovery can
be increased by providing a stirrer magnetic field externally (Gorobets et
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al., 2004). Ion exchange, adsorption, chelating, crystallization, precipitation,

and entrapment are few mechanisms involved in biosorption of heavy met-
als. A detailed account on the aspect of biosorption of heavy metals using
fungi is discussed in this chapter.
In addition to heavy metals, the radioactive wastes from nuclear energy
plants became a major threat to the environment. The flow of radioactive
pollutants into the environment is a dangerous criterion which affects the
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human health and other living organisms on earth. Not only nuclear atom-
ic energy plants, but environment also polluted by the use of nuclear and
atomic weapons during wars. U, Th, Pu, 90Sr, 135Cs, and Tc are the major
radioactive contaminants of environment. Some fungal strains have ability
to colonize and remove these radioactive contaminants from the soil. Most
interestingly, mushrooms are useful in detaching the radioactive wastes from
the environment.
13.6 MYCOACCUMULATION OF XENOBIOTICS
Traditionally, chemical recovery processes are followed to extract heavy

metals from environments, but the process is considered expensive and inef-
fective (Anahid et al., 2010). However, most common biological methods of
remediating recalcitrant pollutants from environment are by the use of soil
microbes including symbiotic fungi and/or arbuscular mycorrhiza alone or
in association with plants. These fungal species have a greater potential in
increasing plant uptake of heavy metals. Gonzaga et al. (2006) have reported
mycorrhiza as a potential candidate in bioremediation strategies for heavy
metals as they act by elevating the plants ability to accumulate phospho-
rous and other static elements. It is a well-established fact that mycorrhiza
are the integral and functional part of plant root systems. These fungi ei-
ther act by attenuating plant heavy metal toxicity or by increasing the metal
uptake. Weissenhorn and Leyval (1995) observed and reported toxic levels
and higher accumulation of heavy metals in plants as a result of arbuscular
mycorrhizal colonization. On the contrary, Heggo et al. (1990) reported less
or no metal accumulation in plants as a result of mycorrhizal colonization.
Anahid et al. (2010) studied different species of fungi in bioleaching of
ores and spent wash containing toxic metal pollutants (Mo, V, Mn, W, and
Zn). The study revealed that Penicillium simplicissimum was highly resis-
tant capable of withstanding up to 8000 ppm concentration as compared with
different isolated species. Abdel-Aty et al. (2013) investigated biosorption
behavior of Pb and Cd to blue green A. sphaerica. They concluded stating
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mechanism of biosorption was chemisorptions as from the experimental

data obtained from Freundlish and Langmuir isotherms and amino-, car-
boxyl-, hydroxyl-, and carbonyl groups were responsible for the biosorption
as from the data obtained by FTIR analysis. A. sphaerica could demonstrate
maximum biosorption of 111.1 mg/g and 121.9 mg/g for Pb and Cd at opti-
mum operating conditions, respectively.
There exist two ways of metal uptake by living and/or dead cells. The
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first way is by surface binding of metal ions to cell surface or to extra cellular
materials. The second way involves active transport of metal ion across the
cell membrane which is metabolism dependent and termed as active uptake
or bioaccumulation (Volesky, 1990). Metal uptake by surface adsorption is
common to both live and dead cells, whereas the active uptake is a mea-
sure exclusive to live cells since it requires metabolic processes. This may
include production of certain metal-binding proteins within the cell. Thus,
metal uptake may happen in different ways, based on whether or not the cell
is alive or dead.
Further, certain physical factors like cell age, composition of growth me-
dia, pH of solution and temperature determine bioaccumulation of heavy
metals. Cell age is a factor for fungi by which bioaccumulation of metals
can occur. It has been observed during lag phase, there is an increase in bio-
accumulation and accumulation comes to halt as culture reaches stationary
phase. Volesky and May (1995) observed that bioaccumulation of uranium
by bakers yeast was 2.6 times higher at 12 h of incubation than with 24 h
incubation. Bioaccumulation of heavy metals may also differ with respect to
culture conditions and growth media composition. Avery and Tobin (1992)
investigated Sr2+ uptake by culture of S. cerevisiae. They concluded that up-
take of Sr2+ in presence of glucose (2% w/v) was more as compared without
the glucose amendment in the media. Further, they reported that growth me-
dia controls the composition and structure of cell wall, which in turn affects
the bioaccumulation.
The pH is another most important factor determining metal uptake by
fungi. Various fungal strains are pH sensitive. Cd biosorption on the other
hand, is pH dependent too. Barros et al. (2003) observed four different or-
ganisms, A. oryzae, A. niger, F. solani, and Candida utilis which showed
maximum biosorption rate at acidic range. This pH dependent biosorption
can be explained on the basis of proton-competitive adsorption reaction
(Huang et al., 1991).
It has been known for decades that fungi can interact with heavy metal
present in environment by secreting their extracellular enzymes. This even-
tually causes a physiological response through uptake of heavy metals by
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fungi. One of the species involved is white rot fungi, which can uptake and
accumulates heavy metals in their mycelia. Favero et al. (1991) have report-
ed that Pleurotus ostreatus was able to accumulate 20 mg g1 dry weight Cd
from liquid medium containing 150 ppm Cd with at least 20% of accumulat-
ed Cd deposited intracellularly. Sanglimsuwan et al. (1993) have reported a
seven day growth study with Daedalea quercina in potato dextrose medium
having 5 mM Cu and Zn. The fungus accumulated 10 g g1 dry weight Cu
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and 5 g g1 dry weight Zn.
Preferentially, heavy metal uptake is species specific. Gabriel et al. (1994)
studied four different species of white rot fungi, D. quercina, Ganoderma
applanatum, Stereum hirsutum, and Schizophyllum commune and their toler-
ance levels to Al, Cd, Pb, and Ca at 1 mM each. During an eight day study,
they reported that most Pb was found in S. hirsutum (90.6 M g1), most Cd,
Al, and Ca in G. applanatum (272, 600, and 602 M g1, respectively). From
equimolar solution (1 mM), Pb was preferentially accumulated by all fungi
except G. applanatum that accumulated more Al. On the contrary, although
research data provide sufficient evidence regarding heavy metal accumula-
tion by fungi, the same is not obvious in natural systems. Purkayastha and
Mitra (1992) observed that Cu was taken up much preferably by Volvariella
volvacea and Pb was taken up very low in liquid cultures. Whereas, when
grown on wheat substrate, the fungus demonstrated high uptake of Pb and
low uptake of Cd and Cu.
Through the course of hyperaccumulation, heavy metals interact with
fungi affecting their physiological, enzymatic, and reproductive processes.
This may cause changes in community structures in ecosystem. It is there-
fore obvious that different species of fungi differ in the degree of their heavy
metal tolerance. Sanglimsuwan et al. (1993) studied minimum inhibitory
concentrations (MIC) for 21 strains of 16 species. They reported MIC were
lowest in case of Hg (0.050.2 mM), Cd (0.55 mM) and Co (15 mM)
whereas higher in case of Ni (0.77 mM), Zn (51 5mM) and Cu (320
mM). Baldrian and Gabriel (1997) also studied 15 wood rotting species, in
which S. hirsutum was the most Cd tolerant and slow growing I. obliquus
was the most sensitive. This variation in heavy metal resistance can occur
among strains in a single species as well. Major differences in Cu tolerance
have been found among isolates of brown rot fungi. 40 mM Cu is the toler-
ance level for about 35 strains of Antrodia vaillantii, whereas, other could
only resist 3 mM Cu (Collett, 1992).
In general, many factors determine bioaccumulation of heavy metals by
fungal biomass with pH being a more sensitive variable. Cultivating fun-
gal biomass is relatively easy and it can be used again and again. More
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precisely, metal scavenging by fungal biomass is considered better than any

other means of conventional remediation. This mycoaccumulation processes
could be a better replacement for existing metal-remediation processes or
could be considered as supplementary for existing treatment technologies.
13.7 MYCOREMEDIATION, MYCOSORPTION, AND
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MYCOFILTERATION
Fungi are diverse group of organisms due to its unique nature in its morphol-
ogy, physiology and genetic features; fungi having greater ability of decom-
posing several enzymes, heavy metals and toxic substances, which can be
utilized as a nutritional source by them. Intense industrial activity and ur-
banization led to disposal of heavy metals which causes deleterious effect on
animals and humankind. Fungi are capable of degrading hazardous chemical
substances like chlorinated hydrocarbons, heavy metals such as chromium,
cadmium, lead, gasoline, zinc, arsenic, and benzene through several applica-
tions ranging from biological, chemical, and engineering techniques from
the contaminated sites.
Mycoremediation is a process of using fungi for the conversion of high-
ly contaminated soils to less contaminated state. Due to the production of
diverse enzymes like lipases, pectin and cellulose, the organic matters are
easily degraded by fungi (Gupta and Shrivastava, 2014). A number of mush-
room, Pleurotus platypus, Agaricus bisporus, Calocybe indica, Calvatia
excipuliformis, Hygrophorus virgineus, Boletus edulis, Lepiota rhacodes,
Lepis tanuda, Pleurotus sajor-caju, Pleurotus ostreatus, Psalliota campes-
tris and Russula delica shows significant role in remediation of heavy met-
als (Nilanjana, 2005; Elekes and Busuioc, 2013). Fungal treatment utilizes
low cost than using any other physical and chemical methods (Jalc, 2002).
Mycosorption is a technique used to detoxify the environmental contami-
nants by fungi, and the organisms responsible for detoxification are con-
sidered as mycosorbents. A wide variety of living and dead biomass like
bacteria, algae, fungi, and plants are known to tolerate and accumulate heavy
metals (Singh, 2006). Gorobets et al. (2004) showed that mycosorption in-
volves various external factors (type of metal, ionic form in solution and the
functional site) and tends to be exothermic. Other factors like pH, tempera-
ture, biomass concentration, type of biomass preparation and initial metal
ion concentration, are also important in evaluating mycosorption.
Mycoremediation involves the physico-chemical and biological meth-
od of remediating toxic substances; a physico-chemical process includes
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precipitation, coagulation, a reduction process, ion exchange, membrane

processes such as ultrafiltration, electrodialysis; its implementation is costly
and effectiveness is limited. Biological method is a novel approach in the
mycoremediation due to its lack of adverse effect on environment and the
low cost implementation (Prasad et al., 2012). Some fungal species like
Basidiomycetes, Ascomycetes are capable of degrading lignocellulosic ma-
terials which are present on wood, paper and other effluents (Pozdnyakova,
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2012). White rot fungi degrade lignin, Phanerochate chrysosporium is
one which fragments lignocellulosic macromolecules into simpler forms
(Novotny et al., 2004). Pleurotus ostreatus is an edible mushroom, fruiting
body of the organisms helps in degrading the organic polymers (Rhodes,
2014). Several soil residing fungi which shows significance in remediation
of heavy metals as listed in Table 13.1.
TABLE 13.1 List of Fungi Exploited for Mycoremediation of Heavy Metals through
Mycosorption and Mycofiltration.
Heavy Toxic Effects Fungi Used in References
Metals Mycoremediation
Antimony Respiratory disorder, cardiovas- Scopularioopsis Jenkins et al.
cular disorder, diarrhea, vomiting, brevicaulis, Phae- (1988), Paul et al.
ulcer, dermatitis, disturbance in olus schweinitzii (2001)
menstruation, breakage in human
leukocytes, cot death or sudden
infant death syndrome (SIDS)
Arsenic Bronchitis, dermatitis, bone Penicillium Mamisahebei et al.
marrow depression, hemolysis, chrysogenum (2007)
hepatomegaly
Cadmium Kidney damage, bronchitis, Aspergillus Barros et al. (2003)
gastrointestinal disorder, cancer, cristatus
hypertension, itai-itai disease,
weight loss
Chromium Carcinogenic, mutagenic, terato- Penicillium cane- Chhikara et al.
genicity, epigastria pain nausea, scens, Pleurotus (2010), Donghee et
vomiting, severe diarrhea, produc- ostreatus al. (2005)
ing lung tumor
Cobalt Cardiomyopathy, pnemonoconio- Saccharomyces Mapolelo and Torto
sis, goiter cerevisiae (2004)
Copper Neurotoxicity, acute toxicity, diz- Pleurotus Jha et al. (2012)
ziness, diarrhea ostreatus
Iron Nausea, vomiting, anemia, ab- Neurospora crassa Rashmi et al.
dominal pain, nephropathy (2001)
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Heavy Toxic Effects Fungi Used in References
Metals Mycoremediation
Lead Anemia, brain damage, anorexia, Rhizopus nigri- Osman and
malaise, loss of appetite, liver, cans, Trichoderma Bandyopadhyay
kidney, gastrointestinal damage, longibrachiatum (1999)
mental retardation in children
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Manganese Parkinson-like syndrome, respira- Penicillium Townsley et al.
tory disorder, neuropsychiatric spinulosum (1986a), Ross and
Townsley (1986)
Mercury Damage to nervous system, Aspergillus Murugesan et al.
protoplasm poisoning, corrosive flavus, Aspergillus (2006)
to skin, eyes, muscle, dermatitis, fumigatus
kidney damage
Nickel Chronic bronchitis, reducing lung Aspergillus niger Ahmad et al. (2006)
function, lung cancer
Selenium Caustic burns, pneumonitis, hypo- Alternaria Thompson-Eagle et
tension, brittle hair and nails, red alternata al. (1991)
skin, paresthesia, hemiplegia
Silver Hemorrhage, bonemarrow sup- Cladosporium Pethkar et al.
pression, pulmonary edema, cladosporiodes (2001)
hepatorenal necrosis, argyria,
blue-grey discolouration of skin,
nails, mucosae
Strontium Lung cancer, skin inflammation Saccharomyces Avery and Tobin
cerevisiae (1992)
Thallium Vomiting, diarrhea, painful Mariannae sp., Sun et al. (2015)
neuropathy, coma, instability, Trichoderma
alopecia viride, Trichoder-
ma asperellum
Uranium Radiation-induced erythema, Penicillium Galun et al. (1983)
impairment of kidney digitatum
Zinc Short term metal-fume fever, Aspergillus niger Kumar et al. (2011)
gastrointestinal distress
It is a similar process of remediating heavy metals by fungal mycelia to

filter toxic substances and microorganisms from water in soil (Sagar, 2015).
According to Taylor and Stamets (2014), mushrooms are ubiquitous in na-
ture, where the mycelium network produces enzyme acids that breakdown
hydrogen and carbon chains. Mycofiltration shows significant role in filtering
pathogens (viruses, bacteria, and protozoa), chemical toxins, and silt/heavy
metals from the contaminated soil and water. Stropharia rugoso-annulata
AUTHOR COPY
is an ideal model for mycofiltration, which reduces fecal coliforms by 99%

contaminant in 2448 h using mycological filters.
Nutrient from soil is supplied to plant roots through mycorrhiza, which
are symbionts (Smith and Read, 1997). Soil arbuscular mycorrhiza is a sym-
biotic association between higher plants and soil fungi, mycelium forms
an bridge to plant and soils, which also translocate various elements to the
plants. An arbuscular mycorrhizal fungus exerts potentiality on the soil met-
9781771883627
al contamination (Finlay, 2008). Heggo et al. (1990) demonstrated that ar-
buscular mycorrhiza reduces heavy metal concentrations in soil and the con-
centrations of Zn, Cd, and Mn in plant leaves; ecto-mycorrhizal and ericoid
mycorrhizal fungi can increase the tolerance of their host plants to heavy
metals when the metals are present at toxic levels. Several investigations are
evident that mychorrhizal fungi can colonize plant root extensively even in
heavy contaminated soil (Sambandan et al., 1992).
The heavy metals and radioactive wastes are long term persisting pollut-
ants in nature; they cannot be degraded easily, thereby causing pollution and
many major side effects. The main cause of environmental pollution is indus-
trial effluents, automobiles, atomic energy plants, and other harmful human
activities. In order to control the environmental pollution it is necessary to
understand the root of pollutants, their side effects, and their persistency in
the environment. Removal of harmful pollutants from the surroundings is an
important criterion and many attempts with chemicals as well as biological
mediators have been performed in order to attain pollution free environment.
The application of fungal genetic resources to remove heavy metals and ra-
dioactive wastes are proven to be better when compared to other techniques.
Fungi are prominent group of microorganisms which occurs ubiquitously in
natural habitats, with their unique property of degrading organic and inorgan-
ic substances which are categorized as good pollution control agents. Using
fungi as bio-remediating agent have much importance because it requires less
cost, less work force, and least maintenance as well as negligible side effects.
Arrays of sources are available to study the application and technology
of using fungi for bioremediation. The mycotechnologies such as mycoac-
cumulation, mycosorption, mycofiltration, and mycodegradation have got
more importance and hence, these qualities facilitate the use of fungi in con-
trol of environmental pollution. The biosorption of heavy metals have got
more importance since, it is possible to re-collect the metal from the fungi.
AUTHOR COPY
Many techniques with respect to the fungal applications in soil remedia-

tion are discussed above in this chapter. There are possibilities of removal
of numerous toxicants from the environment by the implementation of the
fungal resources. Furthermore, many advancements are possible by inter-
mediate use of biotechnological approaches. There is a possibility of con-
structing a genetically modified fungal species by inserting a gene coding for
particular type of protein which take part in enzymatic degradation of pollut-
9781771883627
ants. Biotechnology also helpful to get a heavy metal and radioactive waste
resistant strains of microorganisms. By repeated experimental approaches, it
is possible to produce such type of fungal strains with better quality of soil
remediation and ecological restoration.
KEYWORDS
Acute somatic effects

Bioindicators
Bioleaching
Biomagnifications
Bioremediation
Biosorption
Comparative genomics
Detoxification
Ecological restoration
Epigenetic modification
Fungal genetic resources
Fungal genomic resources
Heavy metal tolerance
Heavy metal toxicity
Hyperaccumulation
Metal remediation
Metallothioneins
Mycoaccumulation
Mycodegradation
Mycofiltration
AUTHOR COPY
Mycoremediation
Mycorrhizal colonization
Mycosorbents
Mycosorption
Mycotechnologies
9781771883627
Nuclear residual waste
Phytochelatins
Radioactive isotopes
Radioactive nuclides
Radioactive waste materials
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CHAPTER 14
A MULTIFACETED BIOREMEDIATION
OF XENOBIOTICS USING FUNGI
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DEVIPRIYA RABIN MAJUMDER
Department of Microbiology, Abeda Inamdar Senior College,
2390-KB Hidayatullah Road, Azam Campus, Camp, Pune 411001,
Maharashtra, India
CONTENTS
14.1 Introduction....................................................................................364
14.2 Recalcitrant Organic Compounds..................................................365
14.3 Fate of Xenobiotics .......................................................................367
14.4 Factors Influencing Rate of Biodegradation..................................369
14.5 Role of Fungi as Efficient Bioremediator......................................370
14.6 Conclusion and Future Direction...................................................390
Keywords..................................................................................................390
References.................................................................................................391
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14.1INTRODUCTION
14.1.1 WHAT ARE XENOBIOTICS?
Xenobiotics (Greek Xenos: Strange, foreign, foreigner) are chemically syn-

thesized compounds that are not found in nature and are therefore branded
as Foreign to Biosphere. Microorganisms have not been exposed to such
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strange structures during evolution. This leads to resistance to biodegrada-
tion of xenobiotics by microorganisms. These foreign compounds with un-
natural structures are mostly toxic and are indeed harmful to life in general.
Some of the core anthropogenic compounds (pollutants) present in the envi-
ronment are petroleum hydrocarbons, halogenated solvents, endocrine dis-
rupting drugs, explosives, agricultural chemicals, heavy metals, metalloids,
and radionuclides. Pollutants with xenobiotic structural features like poly-
cyclic aromatic hydrocarbons, halogenated aliphatic and aromatic hydrocar-
bons, nitroaromatic compounds, azo compounds, s-triazins, organic sulphonic
acids, and synthetic polymers are also recalcitrant in the environment.
14.1.2MYCOREMEDIATION
Fungi are chemoheterotrophic, ubiquitous in sub-aerial and subsoil environ-

ments, and are infallible decomposers. The study of fungi in fundamental
geological processes can be termed geomycology. These include organic
and inorganic transformations, element cycling, mineral transformations,
mycogenic bioweathering, metal-fungal interactions, and the significance
and relevance of such processes in the environmental biotechnology such
as bioremediation. A fungal role in biogeochemical cycling of the elements
is interlinked with the ability to adopt a variety of growth, metabolic and
morphological strategies, their adaptive capabilities to environmental ex-
tremes and, their mutualistic associations with animals, plants, algae, and
cyanobacteria. Fungal polymorphism and reproduction by spores help suc-
cessful colonization of many different environments. Most fungi exhibit
filamentous growth, which has the ability to adopt both explorative or ex-
ploitative growth strategies, and the formation of aggregated hyphae for
protected fungal translocation. Some fungi are polymorphic, occurring as
both filamentous mycelium and unicellular yeasts or yeast-like cells, as in
microcolonial fungi colonizing rocks. The capability of fungi to translocate
nutrients through the mycelial network is an important feature for exploring
heterogeneous environments. The ability of fungi to form extended mycelial
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Bioremediation of Xenobiotics Using Fungi 365
networks, the low specificity of their catabolic enzymes, and their capacity
to use pollutants as a growth substrate make fungi most coveted for bioreme-
diation processes. Bioremediation and waste treatment by a variety of fungi
through an array of processes have been discussed in this chapter.
Fungi are intimately involved in biogeochemical transformations at both
aquatic and terrestrial habitats. The geochemical transformations by fungi in-
fluence plant productivity, mobility of toxic elements, and are therefore of
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considerable socioeconomic relevance, specially the mutualistic symbioses,
lichens, and mycorrhizas. Fungal transformations from bioremediation point
of view have beneficial applications in environmental biotechnology, e.g., in
metal leaching, recovery and detoxification, and xenobiotic and organic pollut-
ant degradation. Thus, a multidisciplinary approach is essential to understand
fully all the phenomena encompassed within mycoremediation of xenobiotics
and it is hoped that this chapter will serve to stimulate interest in an area of
mycoremediation of global significance (Gadd, 2007; Harms et al., 2011).
14.2 RECALCITRANT ORGANIC COMPOUNDS
Xenobiotics may persist in the environment for months and years, but most
biogenic compounds are biodegraded rapidly by omnipotent microorgan-
isms. Recalcitrance (i.e., the structure-immanent stability) of a xenobiotic
compound is due to its unphysiological chemical bonds and/or substitu-
ents, which resist the attack by microbial catabolic enzymes. Type, number
and position of bonds, and substituents affect the xenobiotic character.
14.2.1 CHARACTERISTICS OF RECALCITRANT XENOBIOTICS
Typical features of recalcitrant organic compounds are as follows: high mo-

lecular mass, low solubility in water, condensed benzene and pyridine rings,
especially polycyclic structures, three-fold substituted N atoms, quarternary
C atoms, unphysiological bonds and substituents RX, especially polysub-
stitution, where X = OR, N=N, F, Cl, Br, NO2, CF3, or SO3H.
Figure 14.1 gives the examples of relatively persistent xenobiotics which are
dangerous recalcitrant environmental pollutants.
D.T. Gibson in 1980 had said, Many of these compounds bear little rela-
tionship to the biological products from which they were originally derived.
For example, soils and young sediments contain thousands of substituted
polycyclic aromatic hydrocarbons. These molecules, formed by the thermal
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alteration of cellular material, have been in contact with living organisms

throughout evolutionary periods of time. Consequently, one would predict
the existence of microorganisms that will degrade them, and organisms that
metabolize aromatic hydrocarbons ranging in size from benzene to benzo[a]
pyrene have been described.
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FIGURE 14.1 Examples of relatively persistent xenobiotics, which are dangerous

recalcitrant environmental pollutants.
Organic chemicals of anthropogenic origin are not necessarily recalci-

trant. There are several industrial products that are degraded by microor-
ganisms. These compounds are readily recognized by microbial catabolic
enzymes. Extensive research in biodegradation has demonstrated that a
number of xenobiotic compounds such as polychlorinated biphenyls (PCBs)
and nitroaromatics which once were thought to be recalcitrant are actually
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degraded by microbial enzymes. Moreover, microorganisms throughout

geological time have been exposed to a variety of xenobiotic chemicals pro-
duced by abiotic natural processes (Fetzner, 2002).
14.3 FATE OF XENOBIOTICS
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Xenobiotic compounds are present in the biosphere: air, water, soil, sedi-
ment. These pollutants are exposed to microorganisms in different envi-
ronmental compartments. The most competent and efficient mechanism of
bioremediation of xenobiotic compounds lies with microorganisms on the
face of the Earth. The different physiochemical properties of the environ-
ment may affect the bioremediation efficiency.
14.3.1 ENVIRONMENTAL FRIENDLY BIOREMEDIATION BY

NATURAL ATTENUATION
Today, bioremediation is regarded as the default method for the rehabilita-
tion of pollutants due to its cost-effectiveness and environmental friendli-
ness. But the extent to which such advantage can be exploited depends on
the degree of technical expertise. The efficiency with which bioremediation
is achieved ranges from the most intensive ex situ mechanism with special-
ized organisms, mechanical forces, heat, solvents, detergents through the
stimulation of indigenous microbial communities with nutrients or oxygen
to the completely passive in situ natural attenuation. Since 1990s, passive
methods have been adopted by implementing risk-based remediation of tar-
gets, which lead to physical or chemical stabilization of pollutants instead of
their elimination. Thus, to attain a better ecological status, risk reductions by
stabilization become the only option. Depending on the fate of xenobiotics
in air, water, soil, or sediment, they may become available to microorgan-
isms in different environmental compartments.
14.3.2 MICROBIAL DEGRADATION OF XENOBIOTICS

Eco-friendly bioremediation involve four types of mechanisms, viz. bio-
degradation, biotransformation, co-metabolism, and biosorption. The sub-
stances transformed or degraded by microorganisms are used as a source of
energy, carbon, nitrogen, or other nutrient, or as final electron acceptor of a
respiratory (ETC, Electron Transport Chain) process.
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Biodegradation involves the breakdown of organic compounds, usu-

ally by microbial enzymes, into less complex compounds, like water, carbon
dioxide, and the oxides or mineral salts of elements. Biotransformation is
the metabolic modification of the molecular structure of a compound, result-
ing in the loss or alteration of some characteristic properties (toxicity) of the
original compound, with no loss of molecular complexity. Co-metabolism
is a process where a microbial population growing on one compound may
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fortuitously transform a contaminating chemical that cannot be used as
carbon and energy source. This phenomenon has been designated co-ox-
idation and gratuitous or fortuitous metabolism. Usually, the primary
substrate induces production of (an) enzyme(s) that fortuitously alter(s) the
molecular structure of another compound. The organisms do not benefit in
any way from the co-metabolic process. Co-metabolic transformation may
result in a minor modification of the molecule, or it may lead to incomplete
or complete degradation. Biosorption is bioengineering where metabolism
independent adsorption of xenobiotics to living or dead cells takes place.
Microorganisms dead or alive are successfully exploited for bioremediation
of xenobiotics by biosorption.
In the biosphere, the products of bioconversion processes may be further
transformed or degraded by other microorganisms and eventually leading to
complete degradation by the microbial consortium. Co-metabolic processes
and biodegradation by microbial consortia are of enormous ecological im-
portance. However, persistent xenobiotics and metabolic dead-end products
accumulating in the environment, become part of the soil humus, enter the
food chain leading to biomagnification. Fungal bioremediation of xenobiotic
compounds is depicted in Figure 14.2.
FIGURE 14.2 Fungal bioremediation of xenobiotic compounds.

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14.4 FACTORS INFLUENCING RATE OF BIODEGRADATION
The extent and the rate of biodegradation depend on the chemical structure
and concentration of the xenobiotic compound being degraded, the type and
number of microorganisms present, and the physico-chemical properties of
the environment. Bioavailability of the pollutant is controlled by several pa-
rameters such as the physical state of the pollutant (solid, liquid, gaseous),
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its solubility in water, and its tendency to adsorb or bind to soil or sediment
particles. Sorption, immobilization, and micropore entrapment are major
causes for the persistence of xenobiotics. Aging, is the length of time a soil
or sediment has been exposed to contamination, thereby affecting bioavail-
ability. Pollutants may form stable chemical bonds with soil and sediment
material, becoming increasingly unavailable to microorganisms with the
passage of time. Many xenobiotics, like the polycyclic aromatic hydrocar-
bons and the polychlorinated biphenyls, are poorly soluble in aqueous phase,
and tend to adsorb to and be immobilized by the soil matrix and sediment
material which impede or even prevent biodegradation. Concentrations of
the xenobiotic compound affect biodegradation. In high concentrations,
many xenobiotics are toxic to degradative bacteria. On the other hand, there
is a minimum concentration below which a compound is not degraded as
synthesis of catabolic enzymes may not occur when the concentration of a
chemical is below a level that is effective for induction of the corresponding
catabolic genes. The minimal threshold concentration depends mainly on
the kinetic parameters of growth and metabolism, and on the thermodynam-
ics of the overall transformation reaction. The substrate affinity constant is
the important parameter with respect to the biodegradation of contaminants
to very low concentrations. Minimal substrate concentrations for aerobic
systems are in the range of 0.1 to 1.0mg/L, and the desired end concentra-
tions in environmental systems often are 1g/ L or less. Other factors that
influence biodegradation are environmental conditions such as temperature,
pH, water content and salinity, presence of inhibitory chemicals, availability
of electron donors and nutrients, and availability of oxygen or other electron
acceptors. In soil, oxygen availability is very often the limiting factor for
aerobic biodegradation processes. An observed disappearance of a xenobi-
otic from an ecosystem does not necessarily mean that it was biodegraded,
since loss can occur by partial degradation, biotransformation, by volatil-
ization, leaching, and chemical conversion (polymerization, modification,
and breakdown). While the environmental fate of a chemical, one should
monitor the products formed, not only the disappearance of the parent com-
pound. The time required for xenobiotic biodegradation in the environment
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may range from days to weeks to years and decades. The organophosphate
insecticide malathion disappears from soil within approximately one week,
and the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid) is degraded within
four to six weeks in soil. Simple structural changes of a molecule, such as
the addition of a chlorine substituent, can convert a readily biodegradable
compound such as 2,4-D into a more persistent substance such as 2,4,5-T
(2,4,5-trichlorophenoxyacetic acid) which is degraded in soil within approx-
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imately 612 months. A very persistent xenobiotic is the insecticide DDT
(1,1,1-trichloro-2,2-bis[-chlorophenyl]ethane), which was used extensive-
ly from the 1930s until its ban in 1979. DDT persists with an average half-
life of 4.5 years in field soils, and a half-life in anoxic soils of about 700d.
Stable metabolites of DDT have been detected in soil, groundwater, and in
the tissue of organisms (Fetzner, 2002).
14.5 ROLE OF FUNGI AS EFFICIENT BIOREMEDIATOR
This chapter focuses on mechanisms and pathways of fungal bioremediation

of the previously mentioned xenobiotic pollutants. Fungi possess the most
efficient ecological and biochemical capability to bioremediate environmen-
tal xenobiotic pollutants thereby decreasing the risk associated especially
with metals, metalloids, and radionuclides by chemical or physical modi-
fication. Fungi form extended mycelial networks and have low specificity
of their catabolic enzymes. Furthermore, a fungus does not always use the
pollutants as their growth substrates thus making them ideal for bioremedia-
tion process. Fungi dominate the living biomass in soil and are also abundant
in aqueous systems, thus it becomes the obvious choice for exploitation for
bioremediation of xenobiotic pollutants.
Fungal action on either naturally-occurring or anthropogenically-derived
organic and inorganic pollutants is depicted in Figure 14.3. There are aquatic
and terrestrial habitats of fungi. They exist as aerial spores, yeasts, aquatic
saprobes, symbiotic partners of lichens and mycorrhizal symbionts. They
can form endophytic, ecto-mycorrhizal, and endo-mycorrhizal associations.
They are exposed to anthropogenic chemicals which they act upon. We need
to understand in detail the fungus as a unique bio-system for bioremediation
(Table 14.1). Knowledge on fungal biogeochemistry in freshwater and ma-
rine systems, sediments, and the deep subsurface is required. Fungal roles
have been categorized based on growth, organic and inorganic metabolism,
physico-chemical attributes, and symbiotic relationships. However, many if
not all of these are inter-linked, and almost all directly or indirectly depend
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on the mode of fungal growth (symbiotic relationships). Figure 14.4 shows

the biogeochemical action of free-living and mycorrhizal fungi for metal
mineral transformations.
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FIGURE 14.3 Fungal action on naturally-occurring and/or anthropogenically-derived
organic and inorganic pollutants.
TABLE 14.1 Activities of Fungi in Biogeochemical Processes.

Fungal Activity Biogeochemical Processes
Growth and Mineral tunneling; plant colonization (mycorrhizas); animal colonization
mycelium (symbiotic); translocation of inorganic and organic nutrients; produc-
development tion of exopolymeric substances (nutrient resource for other organisms);
mycelium acting as a reservoir of nitrogen and/or other elements (e.g.
wood decay fungi)
Carbon and en- Organic matter decomposition; cycling and transformations of organic
ergy metabolism compounds and biomass: carbon, hydrogen, oxygen, nitrogen, phos-
phorus, sulphur, metals, metalloids, radionuclides (natural and accumu-
lated from anthropogenic sources); breakdown of polymers; changes in
redox, oxygen, pH; production of inorganic and organic metabolites, for
example, protons, carbon dioxide, organic acids; extracellular enzyme
production; oxalate formation; metalloid methylation (e.g., arsenic, sele-
nium); xenobiotic degradation (e.g. polynuclear aromatic hydrocarbons);
organometal formation and degradation
Inorganic Cycling of inorganic nutrient, for example, nitrogen, sulphur, phospho-
nutrition rus, essential and inessential metals, by transport and accumulation;
transformation and incorporation of inorganic elements into macro-
molecules; alterations in oxidation state; metal(loid) oxido-reductions;
heterotrophic nitrification; siderophore production for iron(III) capture;
translocation of nitrogen, phosphorus, calcium, magnesium, sodium,
potassium through mycelium to plant hosts; degradation of organic and
inorganic sulphur compounds
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Fungal Activity Biogeochemical Processes

Mineral Bioweathering including carbonates, silicates, phosphates and sulphides;
dissolution bioleaching of metals and other components; manganese dioxide (MnO2)
reduction; bioavailability of metals, phosphorus, sulphur, silicon,
aluminum
Mineral Element immobilization including metals, radionuclides, carbon, phos-
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formation phorus, and sulphur; mycogenic carbonate formation; mycogenic metal
oxalate formation; soil storage of carbon and other elements
Sorption of Metal distribution and bioavailability; leads to secondary mineral
soluble and par- formation
ticulate metal
Exo-polysaccha- Complexation of cations; chemical interactions of exopolysaccharide
ride production with mineral substrates
Mycorrhizas Bioavailability of nutrient, nitrogen, phosphorus, sulphur; carbon flow
and transfer between plant and fungus; mineral dissolution and metal
and nutrient release from bound and mineral sources
Lichens Bioweathering; mineral dissolution by lichen acids; metal accumula-
tion; metal sorption
FIGURE 14.4 Biogeochemical action of free-living and mycorrhizal fungi for metal
mineral transformations.
We should identify the unique characteristics of fungi that qualify them

for pollutant degradation, bioremediation, the fungal way of life and its
function in ecosystems where important groups of fungi act on environmen-
tal chemicals. One of the unique characteristic of fungi is their ability to at-
tack organic compounds using a range of extracellular oxido-reductases with
relatively nonspecific activities. Figure 14.5 explains the fungal degradation
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of organic pollutants. Detailed knowledge of enzymatic and genetic mecha-

nisms would propose several fields of environmental biotechnology in which
the use of fungi promises to be particularly effective. The list of fungal en-
zymes for catabolism of organic pollutants is given in Table 14.2.
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FIGURE 14.5 Fungal degradation of organic pollutants.
TABLE 14.2 Fungal Enzymes Involved in the Catabolism of Organic Pollutants.

Fungal Phylum Enzymes Reaction Mechanism
Ascomycota Laccases Direct oxidation of organic compounds like phenols,
Basidiomycota Extracellular aromatic amines, and anthraquinone dyes; Activity in
the acidic pH range
Ascomycota Tyrosinases Hydroxylation of monophenols to odiphenols (creso-
Basidiomycota extracellular/ lase activity); Oxidation of o-diphenols to catechols
Mucoromycotina intracellular (catecholase activity); Oxidation of various highly
chlorinated phenols; Activity from the acidic to the
alkaline pH range
Basiiomycota Lignin Direct oxidation of aromatic compounds like PAHs
peroxidases (polycyclic aromatic hydrocarbon), dyes, phenols;
Extracellular Activity in the acidic pH range
Basidiomycota Manganese H2O2-dependent one-electronoxidation of Mn2+ to
peroxidases Mn3+, which subsequently oxidizes various phenols
Extracellular and aromatic amines; Extended substrate range in the
presence of co-oxidants (organic SH-containing com-
pounds, unsaturated fatty acids and their derivatives);
Activity in the acidic pH range
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Basidiomycota Coprinop- H2O2-dependent one-electron oxidation of aromatic
siscinerea compounds; Direct oxidation of phenols and dyes;
peroxidase Activity from the acidic to the alkaline pH range
Extracellular
Basidiomycota Versatile H2O2-dependent direct one-electron oxidation of phe-
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Peroxidases nols, dyes and aromatic compounds; H2O2-dependent
Extracellular one-electron oxidation of Mn2+ to Mn3+, which subse-
quently oxidizes organic compounds; Mn3+-dependent
reactions as for manganese peroxidase; Activity in the
acidic pH range
Basidiomycota Dye-decoloriz- H2O2-dependent one-electron oxidation of anthraqui-
ing Peroxidases none dyes (only rarely oxidized by other peroxidases);
Extracellular Additional hydrolysing activity; Highly stable at high
pressure, high temperature and very low pH; Activity
in the acidic pH range
Ascomycota Caldariomyces H2O2-dependent halogenations of organic compounds
fumago heme in the presence of halides (one-electron transfer);
thiolate chloro- H2O2-dependent one-electron oxidations of phenols
peroxidase and anilines in the absence of halides; H2O2-dependent
Extracellular peroxygenation (two-electron oxidation), leading to ep-
oxidation of (cyclo)alkenes, hydroxylation of benzylic
carbon and sulphoxidation of S-containing organic
compounds; No activity on non-substituted aromatic
rings and n-alkanes; Activity in the acidic pH range
Basidiomycota Hemethiolate H2O2-dependent peroxygenation of aromatic, aliphatic
peroxygenases and heterocyclic compounds, leading to aromatic and
Extracellular alkylic carbon hydroxylation, double-bond epoxida-
tion, ether cleavage, sulphoxidation or N-oxidation
reactions (depending on the substrate); H2O2-
dependent one-electron abstractions from phenols;
H2O2-dependent bromination of organic substrates;
Peroxygenation of various monoaromatic to polyaro-
matic pollutants, including PAHs, dibenzofuran, and
mono-hydroxylated and polyhydroxylated products;
Ether bond cleavage between aromatic and aliphatic
parts of molecules and in alicyclic and aliphatic ethers
(for example, MTBE: methyl-tert-butylether); Activ-
ity from the acidic to the alkaline pH range
Ascomycota Cytochrome- Incorporation of a single atom from O2into a sub-
Basidiomycota P450 Monooxy- strate molecule, with concomitant reduction of the
Mucoromycotina genases other atom to H2O; Epoxidation and hydroxylation of
Chytridiomycota Cell bound aromatic or aliphatic structures of many pollutants,
including PAHs, PCDDs (polychlorinated dibenzo-p-
dioxin), alkanes and alkyl-substituted aromatics
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Ascomycota Phenol-2-mono- Incorporation of a single atom from O2into a substrate
Basidiomycota oxygenases molecule, with concomitant reduction of theother
Cell bound atom to H2O; Ortho-hydroxlyation of various (halo)
phenols to the corresponding catechols
Ascomycota Nitroreductases NAD(P)H-dependent reduction of nitro-aromatics to
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Basidiomycota Cell bound hydroxyl-amino and amino(nitro) compounds, and
Mucoromycotina of nitro functional groups of N-containing hetero-
cycles; Reduction of TNT (2,4,6-trinitrotoluene) to
hydroxylamino-dinitrotoluene and amino-dinitrotolu-
enes; Formation of mononitroso derivatives and ring
cleavage products from cyclic nitramine explosives;
Widespread among fungi
Basidiomycota Quinone reduc- NAD(P)H-dependent reduction of quinones; Func-
tases tions in quinone detoxification, in the conversion of
Cell bound quinones arising from extracellular pollutant oxida-
tion into substrates for extracellular and intracellular
oxido-reductases, and in pollutant attack by hydroxyl
radicals arising from quinine redox cycling; Occur-
rence in white-rot and brown-rot basidiomycetes
Basidiomycota Reductive deha- Two-component system comprising a membrane-
Ascomycota logenases bound glutathione S-transferase that produces gluta-
(perhaps) Cell bound thionyl conjugates with concomitant chlorine removal,
and a soluble glutathione conjugate reductase that
releases reductively de-chlorinated compounds; Re-
ductive dechlorination of chloro-hydroquinones arising
from chlorophenol degradation and of diphenylether
herbicides (basiodiomycetes); Perhaps responsible for
reductive de-chlorination of chloro-catechols arising
from PCDD degradation (ascomycetes)
Ascomycota Miscellaneous Formation of glucoside, glucuronide, xyloside,
Basidiomycota transferases sulphate or methyl conjugates from hydroxylated
Mucoromycotina Cell bound compounds; Phase II enzymes are prominent in fungal
PAH metabolism but also act on other pollutants;
Widespread among fungi
Bioremediation by fungi is due to their unique characteristics coupled

with environmental circumstances, which make fungi particularly suitable
for application in environmental biotechnology. Fungi should be considered
for pollutant classes that are inefficiently degraded by bacteria. Bacteria
might be disadvantaged if substrates contain rare structural elements, have
a low bioavailability, contain little energy or occur permanently at min-
ute concentrations. With disturbingly high biological activities, human and
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veterinary drugs (including synthetic hormones or antibiotics) and chemi-

cals with low environmental concentration together with active ingredients
are now found in environmental matrices (water, aquatic sediments and
soil), as they are not retained in wastewater treatment plants. Ligninolytic
basidiomycetes, mitosporic ascomycetes and aquatic fungi, are known to
degrade endocrine disrupting chemical (EDC) (nonylphenol, bisphenol A
and 17--ethinylestradiol); analgesic, anti-epileptic and non-steroidal anti-
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inflammatory drugs; X-ray contrast agents; polycyclic musk fragrances;
and ingredients of personal care products. The use of filamentous fungi
is advantageous for the long-range transport or translocation of essential
factors (nutrients, water, and pollutants). They are required for the trans-
formation or detoxification of environmental chemicals. Fungi in their nat-
ural habitat cope with resource heterogeneity by translocating resources
between different parts of their mycelium. Time-lapse recordings demon-
strate the immense traffic of resources in fungal hyphae, in the direction
of growth, for example hyphal transport of autofluorescent polyaromatic
hydrocarbons (PAHs) particularly. Resource translocation is the recycling
of hyphal biomass located in substrate-depleted regions for the benefit of
exploration for food in other regions. Owing to growing fungal hyphae they
have wedge-shaped, hydrophobic tips which penetrate airwater interfaces
and soil aggregates.
14.5.1 ECOLOGICAL FEATURES OF FUNGI
The kingdom fungi include moulds, mushrooms, lichens, rusts, smuts, and
yeasts eukaryotes with remarkably diverse life cycles. Fungi exist as sap-
robes, parasites, mutualists of plants (mycorrhizae), or algae (lichens). Fungi
have been defined as eukaryotic, heterotrophic, absorptive organisms that
typically develop a branched, tubular body called a mycelium and reproduce
by means of sporulation. The microscopic diameters of these fungal hyphae
are between 2 and 10m. Some of the largest living organisms on earth are
fungi with networks extending over hundreds of hectares. Therefore, fungi
can be regarded as macro-organisms packaged in microscopic units that
is, they exhibit a unique lifestyle that is adapted to heterogeneous environ-
ments. Table 14.3 lists the major organic pollutants degraded by diverse fun-
gal phyla and subphyla.
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TABLE 14.3 Organic Pollutants Degraded by Various Fungal Phyla and Subphyla.
Phylum or Subphylum Organic Chemicals Degraded
Microsporidia, Kickxellomy- PAHs
cotina, Zoopagomycotina,
Entomophthoromycotina, Blasto-
cladiomycota, Chytridiomycota
Mucoromycotina, Benzoquinoline, biphenyl, synthetic dyes, TNT,
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Neocallimastigomycota pesticides
Glomeromycota PAHs, Pesticides
Ascomycetes
Pezizomycotina Alkanes, alkylbenzenes, biphenyl, chlorophenols,
coal tar oil, crude oil, diesel, EDCs, fragrances, PAHs,
PCDDS, pesticides, synthetic dyes, TNT, toluene
Saccharomycotina Alkanes, alkylbenzenes, biphenyl, crude oil, EDCs,
PAHs, TNT
Other ascomycetes Alkanes, diesel, coal tar oil, crude oil, MTBE, PAHs,
pesticides, RDX, toluene, synthetic dyes
Basidiomycota
Agaricomycotina Alkanes, BTEX compounds, chloroaliphatics, lignols,
phenols, crude oil, coal tar, EDCs, PAHs, PCBs,
PCDDs, PCDFs, personal care product ingredients,
pesticides, pharmaceuticals, drugs, RDX, synthetic
dyes, synthetic polymers, TNT, other nitroaromatics
Pucciniomycotina Cresols, crude oil, dibenzothiophene, PAHs, RDX
14.5.2 CATABOLISM OF RECALCITRANT ORGANIC

POLLUTANTS
Both polychlorinated dibenzo--dioxins (PCDDs) and polychlorinated

dibenzofurans (PCDFs) are highly oxidized owing to the electronegativity
of the chlorine atoms which withdraw electrons from the aromatic rings.
These compounds are poor electron donors and always occur at very low
environmental concentrations. The white-rot fungus Phanerochaete sor-
dida transforms 2,3,7,8-tetrachlorodibenzo--dioxin (TCDD) into chloro-
catechols in 10d and Phanerochaete chrysosporium even mineralizes the
compound. PCDDs with 68 chlorines and PCDFs with 48 chlorines were
degraded by Phanerochaete sordida. White-rot and litter-decaying basidio-
mycetes mineralize TNT (2,4,6-trinitrotoluene), the nerve gases VX and sa-
rin rapidly. Fungi of the Ascomycota, Basidiomycota and Mucoromycotina
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hydroxylate PAHs intracellularly and convert them to water-soluble prod-

ucts which are then excreted. This is a nonspecific mechanism for their
detoxification. Various fungi use extracellular oxido-reductases in PAH
degradation and mineralize even high-molecular-mass PAH, such as the
highly carcinogenic benzo[a]pyrene. Benzo[a]pyrene is converted to qui-
nones, carbon dioxides, gluconic acids, and sulphate conjugates. Figure 14.6
shows the fungalmetal interactions for bioremediation. Fungi can also pre-
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cipitate a variety of uranium-containing minerals after growth on uranium
oxides (Gadd, 2007; Harms et al., 2011). Several fungi and oomycotes like
Phaeosphaeria spartinicola, Mycosphaerella sp., Buergenerula spartinae,
Phaeosphaeria alima, Passeriniella obiones, Phaeosphaeria neomaritima,
Pleospora spartinae, Tremateia halophila, Spartina patens, Phoma sp.,
and Fusarium lateritum have exhibited their bioremediation potential of
Dimethylsulfoniopropionate (DMSP) from marine plants by DMSP lyase
activity (Bacic et al., 1998). Bioremediation of other recalcitrant pollutants
by fungi is listed in Table 14.4.
FIGURE 14.6 Fungal-metal interactions for bioremediation.

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TABLE 14.4 Catabolism of Recalcitrant Pollutants.

Fungi Pollutant Reference
Fusarium oxysporium f. sp. pisi Butyl benzyl Kim et al. (2002)
phthalate
Aspergillus sp. Sulphur in coal Acharya et al.
(2005)
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Aspergillus niger, Phoma sp., Penicillium sp., Geo- Polyurethane Russell et al.
myces pannorum, Candida rugosa (2011); Norton
(2012)
Graphium sp., Fusarium sp., Penicillium sp., Hypo- Hydrocarbon Adekunle and
crea sp., Discophaerina fagi, Aureobasidiumpullu- Oluyode (2005);
lans, Botryodiplodia theobromae, Bipolaris sp., Cun- Rauch et al.
nighamella sp., Drechslera sp., Helminthosporium (2006); Norton
sp., Macrophomina phaseolina, Mucor sp., Rhizopus (2012)
sp., Talariomyces sp., Aspergillus flavus
Trichoderma viride, Aspergillus niger, Aspergillus Municipal solid Gautam et al.
fumigatus, Curvularia sp., Fusarium sp. waste (2011)
Fusarium solani, Penicillium variabile Rubber Linos and Stein-
bchel (2001)
Penicillium frequentans, Kvehneromyces mutabilis TNT Spain (1995)
Mortierella sp. DCP Nakagawa et al.
(2006)
Nematoloma frowardii Aromatic Hofrichter et al.
and Aliphatic (1998)
compounds
Antrodia vaillantii FRLP-14G Cu, Cr, As Sierra-Alvarez
(2009)
Aspergillus niger, Fusarium solani, Fusarium oxy- Nitrites Martnkovet al.
sporium f. sp. melonis (2009)
Rhodotorula, Acremonium spp. RDX Harms et al.
(Royal Demoli- (2011)
tion Explosive)
Penicillum sp. Atrazine Singh et al. (2008)
Aspergillus fumigatus, A. japonicas, A. niger, A. Ochratoxin A Abrunhosa et al.
carbonarius, A. ellipticus, A. foetidus, A. alliaceus, A. (2002, 2010)
clavatus, A. flavus, A. ochraceus, A. terreus, A. ustus,
A. versicolor, A. wentii, Rhizopus homothallicus,
Rhizopus japonicus, R. oryzae, R. stolonifer, Botrytis
cinerea, Alternaria sp., Aureobasidium pullulans,
Emericella nidulans, Mucor sp., Penicillium auran-
tiogriseum, Penicillium spinulosum, Trichoderma sp.,
Trichothecium roseum
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The following fungi were found in domestic sewage effluents treat-

ment plant: nonpigmented yeasts, Geotrichum candidum, Penicillium spp.
(including P. lilacinum), Mucor spp.,Yeast-like fungi, Rhodotorula spp.,
Fusarium aquaeductuum, Fusarium spp., Mycelia sterile, Trichoderma
viride, Phoma spp., Cladosporium cladosporioides, Margarinomyces
heteromorphum, Gliomastix convolute, Aspergillus spp., Isoachlya spp.,
Moniliaceae, Rhizopus spp., Scopulariopsis brevicaulis, Pullularia pullu-
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lans, Cephalosporium spp., Mucorales, Saprolegnia spp., Syncephalastrum
spp., Acrostalagmus cinnabarinus, Aspergillus flavus, Cunninghamella sp.,
Monilia sitophila, Myrothecium verrucaria, Spicaria violacea, Thamnidium
sp., Trichoderma album and Verticillium sp. (Becker et al., 1954). This
proves that these indigenous fungal species are capable of bioremediating
domestic sewage. Different fungal species isolated from endosulfan treated
pine forest soil are Alternaria alternate, A. humicola, Aspergillus candidus,
A. flavus, A. fumigatus, A. niger, A. terreu, Cladosporium cladosporioides,
Fusarium lateritium, F. solani, Gliocladium deliquescen, G. fimbriatum,
Mammaria echinobotryoides, Mucor hiemalis, M. varians, Paecilomyces in-
flatus, P. lilacinus, Penicillium decumbens, P. frequentans, P. lilacinum, P.
notatum, P. restrictum, P. spinulosum, P. striatum, P. tardum, Phoma fimeti,
Trichoderma atroviride, T. aureoviride, T. harzianum, T. longibrachiatum,
T. pseudokoningii, and Verticillium tenuissimum (Bisht et al., 2014). These
fungi are surviving in the soil contaminated with endosulfan, which indi-
cates that they are degrading this pollutant.
14.5.3 BIOSORPTION BY LIVE AND DEAD FUNGI
Biosorption is considered to be an eco-friendly technique. This technique

was first introduced by Ames Crosta Mills and Company, Ltd. in 1973.
Biosorption is defined as the ability of biological materials to accumulate
pollutants from waste water through physico-chemical interaction. It can be
also defined as the process that utilizes inexpensive dead biomass to seques-
ter toxic xenobiotics. Biosorption process involving the use of dead biomass
is more faster in comparison to living cells as it is cell surface based binding
and displays high affinity for metal ion removal from aqueous solution
(Mali et al., 2014a, 2014b; Takey et al., 2014). Table 14.5 gives the bio-
sorption capacity of different fungi with different heavy metals. According
to investigations, the possibility of living organism to accumulate metallic
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elements could be toxic. In comparison to living cells the use of dead bio-
mass is an easy and a non-destructive method for recovery of adsorbed metal
ions which allows regeneration and reuse of biosorbents. The process of
bioremediation by live organism has several limitations: (a) only some of the
contaminants are biodegradable; (b) bioaccumulation and biomagnification;
(c) not all contaminants are treated, some heavy metals are not absorbed
by organisms; (d) biological process is highly specific; (e) requires suitable
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environmental growth conditions; (f) appropriate level of nutrients and con-
taminants; and (g) contaminants may be present in all the three phases (solid,
liquid, and gas) which might not be adequately bioremediated. To overcome
these problems the process known as biosorption is applied, which is be-
coming an excellent bioengineering process of heavy metal removal. The
mechanism of biosorption is represented in Figure 14.7 which includes co-
plexation, precipitation, reduction, chelation, and ion exchange. Figure 14.8
gives the schematic representation of advantages of biosorption (Mali et al.,
2014a).
TABLE 14.5 Biosorption Capacity of Various Fungi with Different Heavy Metals (Mali et
al., 2014a).
Heavy Metal Organism % of Sorption
Cd Penicillium 95.27
Aspergillus amari 51.69
Trichoderma species 89
Ni Aspergillus amari 58.74
Trichoderma species 7789.41
Cu Amanita muscaria ~90
Spirulina platensis ~90.6
Zn Aspergillus tamarii 54.3
Pb Mycelial Aspergillus tamarii 74
Sb Agaricus campester ~95
Al Agaricus campester ~95
Cr Trichoderma species 81.5
Mn Aspergillus tamarii 46.99
Fe Mycelial Aspergillus tamarii 46
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FIGURE 14.7 Schematic representation of biosorption mechanism (Mali et al., 2014a).
FIGURE 14.8 Schematic representation of advantages of biosorption (Mali et al., 2014a).

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14.5.4 FUNGI AS BIOINDICATORS FOR BIOPROSPECTING
Extensive research has shown that a correlation exists between the quanti-
ties of metals in a growth substrate and the amounts subsequently found
in fruit bodies of fungi. Therefore, they are bioindicators of metal and ra-
dionuclide contamination (Table 14.6). The concept of bioindicators is in
terms of reaction indicators and accumulation indicators. Reaction indica-
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tors are individual organisms and/or communities that may decline or dis-
appear (sensitive species) or show increases (tolerant species). In case of
accumulation indicators, the indicator organism is analysed for the pollutant
theoretically and technically. Some organisms can serve as both reaction and
accumulation indicators. Laccaria laccata increased in frequency at more
polluted locations. Amanita muscaria and several species of Boletus tolerate
high metal pollution. Hebeloma cylindrosporum exhibited the highest ura-
nium and thorium transfer factors, suggesting that this species was a good
bioindicator of soil radioactive content. H. cylindrosporum and Lycoperdon
perlatum exhibited plutonium-239 (239Pu), plutonium-240 (240Pu) and am-
ericium-241 (241Am) transfer values. These species were therefore pro-
posed as bioindicators for 239Pu, 240Pu and 241Am. Metal concentrations are
species-dependent, and the highest levels were found in Calvatia utrifor-
mis (235.5mgCu/kg), Macrolepiota procera (217.8mgCu/kg), Agaricus
macrosporus (217.7mgCu/kg), C. utrifomis (265.8mgZn/kg), Lactarius
deliciosus (231.0mgZn/kg), and A. macrosporus (221.3mgZn/kg) for cop-
per and zinc, respectively. Mercury concentrations in fungi generally occur
in the range 0.0321.60mg kg D.W.-1 (Dry Weight), although concentrations
greater than 100mg[kgDW]1 have been recorded from polluted sites. The
concentrations of seven metals (lead, cadmium, manganese, copper, nickel,
silver, and chromium) were determined in 32 species of wild mushrooms
from Konya, an Inner Anatolian region of Turkey. The highest metal con-
centrations were given as 39mgkg1Pb and 3.72mgkg1 Cd in Trichaptum
abietinum, 467mgkg1 Mn in Panaeolus sphinctrinus, 326mgkg1 Cu in
Trametes versicolor, 69.4mgkg1 Ni in Helvella spadicea, 6.97mgkg1
Ag in Agaricus campestris, and 84.5mgkg1 Cr in Phellinus igniarius. The
maximum contents were 1.52, 2.22, and 60.2mgkg1 in Pleurotus eryngii
(for lead), Amanita vaginata (for cadmium), and Helvella leucomelana (for
copper), respectively. Environmental factors are of paramount importance in
relation to metal accumulation by macrofungi, and include physico-chemical
soil properties like moisture and temperature, all of which influence metal
availability as well as the physiological activity of the fungus (Gadd, 2007).
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TABLE 14.6 Bioindicators for Metal Pollution.

Metals Fungi
Mercury, cadmium Agaricus arvensis, A. campestris, A. edulis, Mycena pura
Mercury Amanita rubescens, A. strobiliformis, Coprinus comatus, Lycoperdon
perlatum, A. haemorrhoidarius, A. xanthodermus, Marasmius oreades
Lead, zinc, copper Agaricus sp., Lycoperdon sp.
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14.5.5 UNIQUE POTENTIAL OF WHITE-ROT FUNGI FOR
MYCOREMEDIATION
White-rot fungi digest lignin by the secretion of ligninolytic enzymes and

give a bleached appearance to wood, from undissolved cellulose, hence their
name. White-rot fungi are excellent mycoremediators of toxins held together
by hydrogencarbon bonds. Table 14.7 gives the list of xenobiotics bioreme-
diated by white-rot fungi (Hansen, 2012). The prospect of using principally
white-rot fungi for mycoremediation is because of its effective degradation
of a wide range of highly recalcitrant organopollutants due to their release
of extra-cellular lignin-modifying enzymes, with a low substrate-specificity,
which enables them to act upon various molecules that are broadly similar
to lignin. The enzymes of the lignin degradation system of white-rot fungi
being extracellular, they do not need to internalize the pollutants and thus en-
abling the fungi to tolerate a high concentration of pollutants. The enzymes
present in the system employed for degrading lignin include lignin-peroxi-
dase (LiP), manganese peroxidase (MnP), various H2O2producing enzymes
and laccase. White-rot fungi cannot utilize lignin as a source of energy for
growth and instead require cosubstrates as a carbon source. They grow by
hyphal extension and thus can reach pollutants in the soil in ways that other
organisms cannot.
The white-rot fungusPhanerochaete chrysosporiumis an ideal model for
bioremediation by fungi, since it is more efficient than other fungi or micro-
organisms in degrading toxic or insoluble materials like chlorinated organic
compound (chloroaliphatics, chlorolignols, chlorophenols, polychlorinated
biphenyls, and PCDDs), aromatic hydrocarbon (benzene, toluene, ethyl-
benzene, and xylenes BTEX compounds), polyaromatic hydrocarbons,
synthetic dyes, lignocellulosic materials, nitro-substituted compounds (ni-
troaromatic and N-heterocyclic explosives TNT and RDX, respectively),
modified synthetic polymers and pesticides.
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TABLE 14.7 Xenobiotics Bioremediated by White-Rot Fungi.

Type Examples
Polycyclic aromatic Anthracene, 2-methyl anthracene, 9-methyl anthracene, benzo()
hydrocarbon pyrene, fluorene, napthalene, acenapthene, acenapthylene, phenan-
threne, biphenylene, pyrene
Chlorinated aromatic Chlorophenols (e.g. pentachlorophenols [PCP], trichlorophenols
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compounds [TCP], dichlorophenols [DCP]); Chlorolignols, 2,4-dichlorophen-
oxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T),
Polychlorinated biphenyls (PCBs), dioxins, chlorobenzenes
Dyes Azure B, Congo red, Disperse Yellow 3 (DY3), Orange II, Poly R,
Reactive Violet 5, Reactive Black 5, Reactive Orange 96, Reactive
Brilliant Blue R (RBBR), Solvent yellow 14, Tropaeolin
Nitroaromatics TNT (2,4,6-trinitrotoluene); 2,4-dinitrotoluene; 2-amino-4,6-dinitro-
toluene; 1-chloro-2,4-dinitrobenzene; 2,4-dichloro-1-nitrobenzene;
1,3-dinitrobenzene
Pesticides Alachlor, Aldrin, Chlordane, Heptachlor, Lindane, Mirex, Atrazine,
1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane [DDT]
Other environmental Benzene, Toluene, Ethylbenzene, o-, m-, p-xylenes (BTEX com-
pollutants pounds), linear alkylbenzene sulfonate (LAS), trichloroethylene
It presents simultaneous oxidative and reductive mechanisms which per-

mit its use in many different situations, regarding the type of contamina-
tion, its degree, and the nature of the site itself. A number of other white-rot
fungi also can degrade persistent xenobiotic compounds, e.g.,Pleurotus os-
treatus, Trametes versicolor, Bjerkandera adusta, Lentinula edodes, Irpex
lacteus, Agaricus bisporus, Pleurotus tuberregium, andPleurotus pulmo-
narius(Rhodes, 2014).
Seven white-rot fungi, viz. Pleurotus cystidiosus, Pleurotus sajor-caju,
Pleurotus ostreatus, Polystictus sanguineaus, Trametes socotrana, Trametes
versicolor, and Phanerochaete chrysosporium degrade the pesticides like
simazine, trifluralin, and dieldrin (Fragoeiro, 2005; Magan et al., 2010). Ten
different genera of white-rot fungi are capable of phenanthrene degrada-
tion Coriolopsis gallica, Irpex lacteus, Phanerochaete chrysosporium,
Pleurotus ostreatus, Phlebia lindtneri, Phlebia radiate, Trametes versicolor,
Trichaptum biforme, Puntularia sp., Bjerkandera sp. and Phomopsis sp.
(B3) (Dai et al., 2010; Young, 2012). Biodegradation of hexachlorocyclo-
hexane (HCH) isomers by several white-rot fungi after 10d in liquid me-
dium are as follows: Phanerochaete chrysosporium, Phanerochaete sordid,
Bjerkandera adusta, Poliporus ciliates, Phlebia radiate, Lentinus tigrinus,
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Stereum hirsutum, Pleurotus eryngii, Irpex lacteus and Trametes hirsutus

(Quintero et al., 2008). Several white-rot fungi like Pleurotus sp., Trametes
versicolor, Panus tigrinus, Phanerochaete chrysosporium, Trametes villosa,
Poliporus pinsitus, Cladosporium sp., are used in the treatment of wastes
containing phenols, chlorophenols, oligomeric polyphenols coming from
oil, coking and olive mill wastewaters (Tima et al., 2010). Tables 14.8
14.11 prove the unique power of white-rot fungus for bioremediation of the
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most stubborn and diverse xenobiotics (Bogan et al., 1996; Pickard et al.,
1999; Raghukumar, 2000; Mori et al., 2003; Potin et al., 2004; Varasaritha et
al., 2010; Norton, 2012; Singh, 2013).
TABLE 14.8 White-Rot Fungi Used in Decolorization of Industrial Effluents.

White-Rot fungi Industry
Coriolus versicolor Textile
Funalia trogii
Pleurotus ostreatus
Phanerochaete chrysosporium Pulp and paper
Funalia trogii
Pleurotus ostreatus
Phanerochaete crasa
Fomitopsis sp. IMER2
Trametes hirsute
Flavodon flavus
Marasmius quercophilius
Polysporus ostreatus
Pycnoporus cinnabarinus
Pycnoporus coccineus Food
Trametes pubescens MB 89
Trametes versicolor
Pleurotus sajor
Panus tigrinus
Pleurotus ostreatus
Phanerochaete chrysosporium Coke
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TABLE 14.9 White-Rot Fungi Used in Treatment of Polycyclic Aromatic Hydrocarbons

(PAHs).
White-Rot fungi Waste Treated
Pleurotus ostreatus PAHs aged creosote contaminated soil
Bjerkandera adusta, Irpex lacteus, PAHs in forest and salt marsh soils
Lentinus tigrinus
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Bjerkandera adusta Hexachlorocyclohexane (HCH) isomers pres-
ent in a spiked soil
Phanerochaete chrysosporium, Pleurotus Aromatic hydrocarbons in an aged contami-
pulmonarius nated soil containing high concentrations of
heavy metals
Phanerochaete laevis HHB-1625, Phlebia PAH
lindtneri, Coriolopsis gallica UAMH-
8260, Stropharia rugoso-annulata,
Cladosporium sphaerospermum
TABLE 14.10 White-Rot Fungi Used in Treatment of Dyes.

White-Rot fungi Dye(s)
Phanerochaete chrysosporium, Trametes Anthraquinone dyes, Azo dyes, indigo
versicolor, Bjerkandera sp., Clitocybula carmine, Direct Red-80
dusenii, Pleurotus eryngii
Phanerochaete chrysosporium, Coriolus Reactive Blue 4, Reactive Red 2
versicolor, Pleurotus ostreatus, Pleurotus
sajorcaju
Phanerochaete chrysosporium, Phanero- Direct Blue 71, Direct Red 80, Direct Yellow
chaete ostreatus 106, Reactive Blue 222, Reactive Red 195,
Reactive Yellow 145, Reactive Black 5, Acid
Blue 62, Acid Yellow 49, Acid Red 266
Coriolus versicolor Poly S119
Pleurotus ostreatus, Coriolus versicolor, Remazol Brilliant Blue Royal, Drimaren
Funalia trogii Blue
Pleurotus ostreatus, Cladosporium Acid Orange 7, Acid Orange 8, Mordant
cladosporioides Violet 5, Acid Blue 193, Acid Black 210,
Crystal Violet, Reactive Black B(S), Reac-
tive Black BL/LPR
Dichomitus squalens, Daedalea flavida, Coracryl dyes (black, pink, violet, red), Re-
Irpex flavus, Polyporus sanguineus active dyes (yellow, orange, red), Rathiodal
dyes (scarlet)
Geotrichum candidum, Coriolus versicolor, De-colorization of digested molasses spent
Phanerochaete chrysosporium, M. sterile wash (DMSW)
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TABLE 14.11 Other Applications of Bioremediation by White-Rot Fungi.

White-Rot fungi Xenobiotics References
Polysporus sp. S133, Pleurotus tuberregium Crude oil Isikhuemhen et al.
(Fr.) Sing. (2003), Kristanti et al.
(2011)
Phanerochaete velutina, Stropharia rugo- Nitro-aromatic Tuomela et al. (2013)
soannulata, Gymnophilus luteofolius compounds
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Trametes trogii UAMH 8156, Trametes hir- Dibenzyl sulphide Van Hamme et al.
suta UAMH 8165, Trametes versicolor IFO (2003)
30340, Phanerochaete chrysosporium ATCC
24725, Tyromyces palustris IFO 30339
Pleurotus ostreatus Alkaline batteries Calamur (2012)
Cladosporium resinae Polyurethane Russell et al. (2011)
Cladosporium cladosporioides Rubber Linos and Steinbchel
(2001)
Cladosporium sp., Pleurotus ostreatus, Ochratoxin A Abrunhosa et al. (2002,
Phanerochaete chrysosporium 2010)
Cladosporium sp. Hydrocarbon Adekunle and Oluyode
(2005)
Stropharia coronilla Benzo[]pyrene Steffen et al. (2003)
Phanerochaete chrysosporium RDX Harms et al. (2011)
14.5.5.1 PRACTICAL IMPLEMENTATION OF MYCOREMEDIATION

USING WHITE-ROT FUNGI
In order to use white-rot fungi successfully for bioremediation, knowledge

of fungal physiology, biochemistry, enzymology, ecology, genetics, mo-
lecular biology, and engineering, among other cognate subjects is required.
Substrates such as wood chips, wheat straw, peat, corn cobs, sawdust, a nutri-
ent-fortified mixture of grain and sawdust, bark, rice, annual plant stems and
wood, fish oil, alfalfa, spent mushroom compost, sugarcane bagasse, coffee
pulp, sugar beet pulp, okra, canola meal, cyclodextrins, and surfactants can
be used as co-substrates in inoculum production either off-site or on-site, or
as mixed with contaminated soils to improve the processes of degradation. It
is critical to attain the correct nitrogen/carbon ratio in the substrates used, so
to avoid any impeding effect on the efficiency of the fungi in the bioremedi-
ation process. Fungal inocula encapsulated with alginate, gelatin, agarose,
carrageenan, chitosan, in the form of pellets, may offer a better outcome than
with inocula produced using bulk substrates. They preserve the viability of
the inoculum and contribute nutrients to maximally support the degradation
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of pollutants. This, furthermore, increases the survival and effectiveness of

the introduced species (Rhodes, 2014).
14.5.6 MYCOREMEDIATION BY ENDOPHYTES
Endophytes are hyper-diverse microorganisms that live within the inner tis-
9781771883627
sues of plants, without causing any overt disease symptoms. These organ-
isms enter their hosts by penetrating exterior surfaces, and some play a key
role in plant decomposition following host tissue death. These endophytic
fungi contribute to decomposition of lignocellulose polymers and are major
contributors to the carbon cycle. Table 14.12 gives the list of these micro-
organisms which have the ability to degrade a polymer as complex as lig-
nocellulose suggesting that these organisms offer promise for their ability
to degrade other complex polymers, such as those present in plastics, PAH,
leachate, mine-tailings. The unique biological niche of endophytes as endo-
symbionts of tissues rich in complex carbon polymers justifies the investi-
gation of their wider metabolic capabilities. Each of the more than 300,000
land plant species on Earth potentially hosts multiple endophyte species.
Only a small sampling of plants has been examined for their endophytic as-
sociations, yet many of these organisms can be readily cultured. Individual
trees can harbor hundreds of endophytic species, some of which are known
but many of which are yet new to science (Russell et al., 2011).
TABLE 14.12 Mycoremediation of Xenobiotics by Endophytes.

Endophytic Fungi Xenobiotics References
Ceratobasidium stevensi Polyaromatic Dai et al. (2010)
hydrocarbon
Colletotrichum gloeosporiodes Leachate Rashmi et al. (2014)
Alternaria sp., Cladosporium sp., Paecilomyces Mine tailings Garza et al. (2013)
sp., Penicillium sp., Aspergillus sp.
Lasiodiplodia sp., Bionectria sp., Alternaria Synthetic Russell et al. (2011)
dauci, Nectria sp., Pestalotiopsis microspora, polymer
Edenia gomezpompae, Pleosporales sp., Phaeos-
phaeria sp., Plectosphaerella sp., Neonectria sp.
Phomopsis sp. Phenanthrene Tian et al. (2007)
Paecilomyces lilacinus Rubber Linos and Steinbchel
(2001)
Paecilomyces Hydrocarbon Adekunle and
Oluyode (2005)
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14.6 CONCLUSION AND FUTURE DIRECTION
There is always a great demand for sustainable, cheap and tailor made tech-
nologies for bioremediation. There is always a high impetus to translate
powerful ecosystem of fungi into ecology based technologies. The degree
of mechanical intervention in natural attenuation of soil is quite low, which
probably favors the establishment of filamentous fungi. Many fungi produce
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exudates those might serve as auxiliary carbon source for pollutant degrad-
ing bacteria. Therefore, it is crystal clear that fungi have important biochem-
ical roles in the biosphere. Fungi being ubiquitous member of sub-aerial and
sub-soil environments, they become dominant group in metal polluted habi-
tats. Their oligotrophic growth and explorative filamentous growth habit,
flexible growth strategies, and resistance to extreme environmental factors
including metal toxicity, irradiation, and desiccation make fungi successful
colonizers of earth surface. Among the numerous fungal species the White
Rot Fungi deserve a special mention as a highly promising and efficient
bioremediation agent. They have a capacity to produce a specialized group
of peroxidases called The Ligninolytic System which is capable of bio-
degradation of anthropogenic chemicals. An important aspect of passive
bioremediation technologies involving fungi, besides their low cost is the
common acceptance of risk-based clean-up standards such as those currently
present in the legislation of two countries, viz. USA and UK. Thus, there are
important financial, ecological, and legal reasons for a better understanding
of fungal activities and their implementation in environmental technology
leading to fungal bioremediation.
KEYWORDS
Biodegradation
Bioindicator
Bioprospecting
Bioremediation
Biosorption
Biotransformation
Co-metabolism
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Endophytes
Mycoremediation
Natural attenuation
Recalcitrant compounds
Unique lignolytic system
9781771883627
White-rot fungi
Xenobiotics
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INDEX
A commodities, 86, 168
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fields, 68, 69, 268
Abiotic
industry, 288
biogenic, 42
microbiology, 76
biotic processes, 42
practices, 70, 73, 79, 166
factors, 11
production, 69, 70, 73
formation, 45, 47, 53
Agrochemicals, 45, 68, 74, 83, 87
process, 45
Agro-industrial facilities, 157
Abutilon avicennae, 265, 267
Agro-industrial waste, 295, 301
Acacia nilotica, 264, 274
Agronomic variables, 318
Acetaldehyde, 169
Agronomical properties, 269
Acetonedichloromethane, 143
Alcaligenes eutrophus, 293
Acidic
Alcoholic fermentation, 169
aqueous solution, 54
Algae, 144, 154, 157159, 174, 177, 204,
components, 161
266
contents, 328
Algae turf scrubber, 189
soils, 45
Algal
Acidification, 43, 194, 331
biomass, 202
Acidobacteria, 19
cells, 139, 140, 142, 170, 171, 173, 184,
Acidogens, 203
187
Acinetobacter, 240
cultivation/processing, 183
Actinobacteria, 19, 83, 269
growth, 134, 140, 142, 157, 160, 161,
Adsorbable organic halogens, 41, 42,
172, 187, 204
5461, 124
reproduction, 175
Aeolian dust, 1826, 3032, 34, 35
species, 134, 139, 160, 167, 173, 188
characteristics, 18
strain/consortium, 203
carrier of microorganisms, 19
Alkaloids, 50, 120, 242, 309, 314
public health, 20
Alkylation, 121
collection, 22
Allelopathic effects, 50
sample, 25
Allergic bronchopulmonary aspergillosis,
Aerobic
321
anaerobic treatment, 199
Allergic skin reactions, 21
biodegradation processes, 369
Allium sepa, 263
conditions, 159, 195
Alnus, 76
treatment, 154, 193, 194, 199, 200, 204
Alternaria, 20, 308, 349, 379, 380, 389
Aeruginosins, 48, 49
Amanita
Aframomum sceptrum, 314
muscaria, 381, 383
Agaricus
vaginata, 383
bisporus, 347, 385
Amaranthus, 332
macrosporus, 383
Ambrosia, 263
Agricultural
Amino acids, 49, 80, 120, 157, 181, 241,
activity, 73, 74
244
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Aminoglycosides, 237 Aphanothece nageli, 171

Ammensalism, 2, 13 Apocynum, 263
Ammonia toxicity, 188 Arabidopsis thaliana, 276, 277, 279, 341
Ammonification, 69, 81, 82 Arbuscular mycorrhiza, 81
Ammonium concentration, 181 Archaea, 28, 43
Ammonium persulfate, 28 Aromatic
Amplified Ribosomal DNA Restriction compounds, 47, 48, 55, 373, 374, 388
Analysis, 85 hydrocarbons, 158, 276, 364, 366, 371
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Anabaena azollae, 77 rings, 45, 374, 377
Anaerobic cultivation, 200 Arthrinium, 20
Anaerobic/facultative ponds, 191 Arthrobacter, 269271, 273, 274
Analytical methodology, 143 Arthrospira platensis, 167
Ankistrodesmus, 137, 159 Artificial radionuclides, 336
Anthraquinone, 50, 373 Ascomycetes, 122, 313, 348, 377
Anthropogenic, 42, 43, 47, 48, 5260, 330, Ascomycota, 340, 373375, 377
333, 335, 364, 366, 370, 371, 390 Ascorbate peroxidase, 276, 333
action, 52 Aspergillus, 20, 187, 242, 311, 348, 349,
activities, 43, 54, 330, 333 379381, 389
flux, 53 flavus, 321, 343, 380
influences, 55 nidulans, 339, 342
organohalogen compounds, 56 niger, 321
origin, 43, 366 Atrazine, 54, 106, 108, 109, 112, 262, 265,
pollution, 52, 54, 56 268274, 279, 379, 385
sources, 42, 47, 48, 52, 371 Avicennia marina, 277
Antibacterial, 104, 234, 237, 242248 Axenic culture, 159
activities, 104, 234, 242, 247, 248 Axial dispersion coefficient, 179
agents, 104 Azadirachta, 243, 244, 248
antifungal activities, 242, 243 Azadirachta indica, 243, 244
Anti-cancer activities, 244 Azobiofer, 77, 87
Anticoagulant, 244 Azospirullum, 75
Anti-epileptic/non-steroidal antiinflamma- Azotobacter, 75, 77
tory drugs, 376 Azotobacter vinelandii, 80
Antifungal, 242245
Antihelminthic, 243 B
Antimalarial, 243 Bacillus, 2, 13, 19, 20, 75, 242, 293, 295,
Antimicrobial, 100, 105, 112, 239243, 245, 298, 299
246, 314 B. subtilis, 28, 1113
activities, 105, 112, 241, 242, 245, 246, Bacterial
248, 314 adsorption, 106
potency, 100 cell concentrations, 27
Antineoplastic, 243 collagenase, 240
Antioxidants, 332 colonies, 28, 271
Antiprotozoal, 243 contamination, 11, 24, 239
Antipyretic, 244 enzymes, 277
Antituberular, 245 inactivation, 104
Antitumor, 244, 245 phyla, 83
Antiviral, 243 Bacteriology, 239
Antrodia vaillantii, 346, 379 Bacteriostatic, 234, 248
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Index 397
Bacteroidetes, 83 index, 82, 87

Barley, 263 interactions, 68
Basidiomycetes, 122, 348 medium, 237
Batrachochytrium dendrobatidis, 339 methods, 237, 238, 258, 328, 344
Bentonite, 108, 109, 112, 198 molecules, 336
Benzoxazinones, 269 oxygen demand, 156, 191, 193, 194,
Betaproteobacteria, 19 197202, 231, 289
Betulinic acid, 245 processes, 30, 82, 87, 266
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Binaphthoquinone, 245 subfamily, 226
Bioaerosols, 20 toxins, 106
Bioambient preservation, 246, 248 treatment, 289
Bioaugmentation, 52, 258, 275, 280 Biomagnification, 59, 328, 332, 335, 351,
Bio-catalytic mechanism, 126 368, 381
Biochemical Bioremediation process, 128, 329, 370, 388
processes, 79, 81, 82 Bioremediation technology, 258
properties, 79 Biosynthetic pathways, 47, 343
reactions, 121, 193 Biotechnological
Biocompounds, 157, 166, 199, 203 applications, 329
Biodegradable polymer, 288, 293 methods, 52, 238
Biodegradation, 59, 128, 158, 188, 189, Biotransformation, 78, 79, 81, 82, 87, 121,
334, 364369, 390 367369, 390
Biodegradation of hexachlorocyclohexane, Bjerkandera adusta, 385, 387
385 Blumea malcolmii, 265, 274
Biodeterioration, 237, 320, 323 Boletus edulis, 347
Biodiversity, 68, 69, 73, 74, 289, 338 Bordetella bronchiseptica, 246
Bioenergy, 163 Bosenbergia pandurata, 242
biodiesel, 164 Botryococcus braunii, 137, 141, 143, 165,
biohydrogen, 166 185, 188
biomethane, 165 Botryodiplodia theobromae, 311, 379
generation, 156, 163 Bovidae bubalis, 227
Bio-ethanol, 163, 168172, 188, 199, 203, Bradyrhizobium, 80, 81
204 Brassica napus, 265, 275
Biofertilizer, 77 Brassica, 263
Biogas production, 162, 195, 201, 202 Buergenerula spartinae, 378
Biogenic
compounds, 365 C
formation, 53 Caenorhabditis elegans, 341
Biogeochemical Caldariomyces fumago, 126
action, 371 Calocybe indica, 347
cycle, 42, 48, 55, 86, 328, 364 Calvatia
Bioinformatics, 85, 127, 129 excipuliformis, 347
Biological utriformis, 383
activities, 42, 81 Candida, 125, 242, 345, 379
contaminants, 106, 135 albicans, 244, 339
control, 313, 314, 322 guilliermondii, 339
damages, 276 lusitaniae, 339
decomposition, 81 tropicalis, 339
excess, 194 Canna indica, 278
AUTHOR COPY
Cannabis, 263 Chlorinated

Cannibalism, 14, 7, 913 acetic acids, 43
Cannibalism vs predation, 11 compounds cyanobacteria, 48
Carbamazepine, 109 compounds natural processes, 42
Carbon Emission Reduction, 194 dicarboxylic acid, 57
Carbon nanotube membranes, 99 humic acids, 56
Carbon tetrachloride, 42, 45, 48, 52 insole, 120
Carex, 263 natural compounds, 49
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Casamino acids, 29 organic compounds, 61
Casurina, 76 plant products, 61
Catalytic mechanism, 122 prostaglandins, 120
Cellulose nitrate membrane filters, 23 waste products, 54
Cellulosic biomasses, 142 Chlorination, 47, 48, 51, 54, 55, 5760, 109,
Cement flue gas, 162 158, 375
Centrifugation, 162, 183185, 197, 365 Chloroanisole, 55, 57, 59, 60
Ceratophyllum, 263, 268 Chlorofluorocarbons, 42
Chaetoceros Chloroform, 41, 4558, 60, 61, 143, 247
calcitrans, 137 extraction, 143, 247
muelleri, 137 methanol, 143
Chaetomium globosum, 339 Chlorohumus, 43, 47, 61
Chalara, 311, 323 Chloromethane, 42, 45, 48
paradoxa, 311 Chloroperoxidase, 46, 55, 61, 126
Chemical Chlorophenol, 55- 60, 158, 159, 375, 377,
composition, 26, 135, 245 384386
degradation, 273 Chlorophyceae, 156
energy, 159, 167 Chlorophyll reaction, 172
fertilizers, 75 Chlorothalonil, 69, 82
flocculation, 184 Chlorpyrifos/quinalphos, 80
modification, 143, 261 Choricystis minor, 136
oxygen demand, 103, 156, 167, 181, Chromated copper arsenic, 332
188202, 231, 289, 297 Cladosporioides, 338, 380, 387, 388
residues, 313 Cladosporium, 20, 338, 349, 380, 386389
substances, 236, 347 Clean development mechanism, 195, 201
thermal stability, 107 Clofibric acid, 277
Chemopollutants, 259, 263, 279 Clostridium
Chitranone, 244 acetobutylicum, 167
Chlamydomonas reinhardtii, 154, 169 histolyticum, 240
Chlorella, 136, 137, 141144, 154, 156, Coagulationultra-filtration treatment, 197
159169, 172, 179, 181, 185202 Coal gasification, 166
ellipsoidea, 185 Coccidioides immitis, 20, 339
emersonii, 137 Coccidioidomycosis, 20
kessleri, 161 Coenochloris pyrenoidosa, 159
minutissima, 136, 137 Collagenolytic activity, 240
protothecoides, 137, 141, 165, 201 Colletotrichum, 308, 389
pyrenoidosa, 136, 311315 Constructed wetland, 263
sorokiniana, 137 Continuous stirred-tank reactor, 181, 195,
vulgaris, 137, 141, 142, 159, 167, 169, 196
184, 186188, 200, 201 Coprinus cinereus, 339
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Index 399
Coriandrum sativum, 332 primolecta, 137

Coriolopsis gallica, 385, 387 salina, 137, 138, 155, 162, 189
Corn steep liquor, 294, 298230 tertiolecta, 136, 137, 161, 167
Corollospora lacera, 343 Durvillaea, 157
Cosmetics/pharmaceutical industries, 175
Council for Leather Export, 230 E
Crypthecodinium cohnii, 137 Echornia, 262
Cryptococcus neoformans Ecklonia, 157
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Serotype A, 339 Eclipta alba, 242
Serotype B, 339 Ecological
Cryptophycin, 50 disturbance, 73
Crystalline aluminosilicates, 19 interactions, 13
Cupriavidus necator, 293 restoration, 329, 351
Curcouma longa, 242 system, 74
Curvularia, 20, 242, 379 Economic
Cyanophyceae, 154 considerations, 142, 308
Cylindrotheca, 137 development, 288
Cymbopogon citratus, 242 point, 178
Cypermethrin, 80 Ecosystem, 2, 1113, 19, 45, 53, 68, 69, 73,
Cystine, 244 75, 77, 83, 87, 154, 263, 288, 330, 332,
Cytometry, 27 343, 346, 369, 390
Cytosol, 46 Ecto-mycorrhizal, 350, 370
fungus, 342
D Eichhornia crassipes, 265, 276
Dark septate endophyte, 342 Eicosapentaenoic acid, 51, 162, 166, 182,
Days after sowing, 319 197
Dealkylation, 121, 340 Electric/electronic waste process, 330
Dehydrogenase, 82, 241, 245, 246, 293 Electricity generation, 163
Dehydrogenation, 121 Electrodialysis, 198, 348
Demineralization, 164 Electron microscopy, 5
Denitrification, 69, 82 Electron transport system, 104
Department of Energy, 339 Ellipsoidea, 185
Detoxification, 5, 45, 46, 158, 266, 269, Elliptinone, 244
277, 279, 333, 338, 342, 347, 351, 365, Emericella, 20, 379
375378 Empty fruit bunch, 190, 198, 201203
Deuteromycetes, 122 Endocarditis, 19
Diazomethane, 58 Endocrine disrupting chemical, 376
Dichloroaniline, 125, 277, 280 Endocrine disruption, 69, 278, 332
Dichloromethane extraction method, 271 Endosymbiotic microorganism, 77
Dictyostelium discoideum, 341 Endotoxins and mycotoxins, 30
Dissolved organic carbon, 101 Enterovirus, 30
DNA Amplification Fingerprinting, 85, 184 Environmental
Docosahexaenoic acid, 182 conditions, 78, 137, 139, 154, 170, 279,
Dodecanoic, 244 315, 318, 322, 337, 369
Droserone, 244 factors, 77, 79, 80, 138, 312, 322, 340,
Drutnia carotovora, 308 390
Dunaliella hazards, 336
bioculata, 138 human health, 336
AUTHOR COPY
impact, 47, 83, 87, 163 Filtration

management, 189 attractive, 184
microbial population, 83 method, 186
problems, 191, 230, 259, 261 Food and Agricultural Organization, 86,
sustainability, 98, 134, 144, 162, 168 320, 322
Enzyme systems/bioreactor, 121 Forest ecosystem, 47, 5658, 81
halo acid dehalogenase from fungi, 123 Fossil fuels, 134, 163, 168, 178, 183
laccases, 122 Frankia, 76, 87
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p450 system in fungal dehalogenation, Fraxetin, 245
123 Free Fatty Acids, 318, 320
enzymatic cell, 169 Free radical concentration, 332
detoxification, 269 Fresh fruit bunches, 190, 290
hydrolysis, 169 Fucoxanthin, 178
peroxidases, 121 Fulvic acids, 47, 55
Epifluorescence microscopy, 32 Fungal
Epigenetic modification, 333, 351 biodeterioration/discoloration, 320
Epoxidation, 123, 374 cells, 124, 314
Equilibration/oscillations, 10 dehalogenation, 121, 127
Erythrina poeppigiana, 242 enzymes, 373
Escherichia coli, 11, 13, 103, 105, 106, 239, genetic/genomic resources, 337
240, 245, 246, 293, 295, 299, 300, 314, genome project, 339
341 genomics resource, 339, 351
Ethno-botanical information, 242 source, 121, 123, 128, 129
Ettlia oleoabundans, 140 strains, 122, 128, 329, 340345, 351
Eucalyptus maculata, 242 translocation, 364
Euglena gracilis, 138, 141 Fungicides, 70, 79, 81, 82, 264, 269, 313,
Eukarya, 43 314, 322
Eukaryotic studies, 338 Fusarium, 20, 125, 308, 311, 378, 379
Euphorbia fusiformis, 242 aquaeductuum, 380
Eustigmatophyceae, 155 lateritium, 380
Eutrophic water, 109
Eutrophication, 157, 162, 189, 199 G
Excoecaria agallocha, 330 Galdieria partita, 167
Exophiala pisciphila, 342 Gallic acid, 246
Extraction methods, 134, 143 Gammaproteobacteria, 19
Extremotolerant fungi, 337, 338 Ganoderma applanatum, 346
Gas chromatography-mass spectrometry,
F 58, 59, 275
Fatty acid composition, 137, 138, 141, 319 Gas exchange systems, 172
Fatty acids methyl esters, 185 Gas production, 203
Fermentation, 163, 166, 167, 169, 170, 188, Gelatin membrane filter, 34
190, 237, 288, 294300 Gene
Fertilization, 174 microarray analysis, 7
Fertilizers, 74, 157, 166, 189, 190, 330 quantification, 27
Festuca, 263 transcription, 7, 8
Fibrous protein bundles, 227 Genetic
Fibrous-bed column, 198 material, 336
Filamentous mycelium, 364 mechanisms, 373
AUTHOR COPY
Index 401
resources, 338, 350, 351 activity, 123

transformation, 342 enzyme, 123
Genomes, 85, 127, 339, 340 Halocarbons, 48, 50, 53
Genomic Halogenases, 46
composition, 85 Halogenated
studies, 338 compounds, 44, 46, 5056, 120, 124
Genotoxic effect, 246 organic compounds, 51
Geochemical features, 100 pollutants, 124, 128
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Geographic location, 22 substances, 54, 125, 129
Geologic terpenes, 120
conditions, 100 thiophenes, 120
processes, 364 Halogenating
Geomyces destructans, 339 activity, 46
Geomycology, 364 enzymes, 46, 47
Geothermal processes, 42, 53 Halogenation reactions, 47
Geotrichum candidum, 380, 387 Halometabolite synthesis, 47
Germination, 265, 268, 315, 319, 320, 323 Haloperoxidases, 47
Gigaspora margarita, 342 Halophiles, 233, 338
Gliocladium deliquescen, 380 Hamry water
Gliomastix convolute, 380 catchment, 57
Global reservoir, 42, 5557, 59
chlorine cycle, 52 Haptophyceae, 156
perspectives, 230 Helianthus, 263
production capacity, 234 annuus, 275
warming, 134, 162, 163, 189 Helminthosporium solani, 314
Glomus intraradices, 342 Helvella leucomelana, 383
Glutamic acid, 244 Heme-type haloperoxidases, 46
Glutaraldehyde, 27 Hennadiol, 245
Glutathione, 45, 269, 332, 333, 342, 375 Hepatoprotective, 244
Glutathione S-transferases, 45 Hexadecyltrimethylammonium, 109
Glutathionyl hydroquinone reductase, 341 Hexane-isopropanol, 143
Glycolytic enzymes, 241 Hexanortriterpenoid, 243
Glycosyltransferases, 45 Histidine autokinases, 8
Glyricidia, 268 Histoplasma capsulatum, 339
Good Agricultural Practice, 86 Humic
Gravity fulvic acids, 47
infiltration, 100 material, 45
sedimentation, 184 substances, 48, 5560, 82
Greenhouse gases, 156, 162, 192, 203 Hybrid bioreactor, 163, 196
Greenhouse reactors, 204 Hydrilla, 262, 268, 280
Grevillea robusta, 268 Hydrochloric acid, 58, 143
Guaiacol peroxidase, 333 Hydrochlorofluorocarbons, 51
Hydrofluoric acid, 27
H Hydrolytic enzymes, 245, 246
Haematococcus pluvialis, 138 Hydrophobicity, 51, 101, 106
Haloacetic acids, 45, 51, 56 Hydroponic system, 275, 277
Haloacid Hydrosphere, 42
dehalogenase, 123 Hydroxylation, 121, 123, 374
AUTHOR COPY
Hygrophorus virgineus, 347 Keratin, 240

Hypermethylation, 333 Keratinase, 240
Hyphae, 76, 364, 376 Kernel
Hyphal extension, 121, 384 deterioration, 320, 322
Hypochlorous acid, 54 discoloration, 320, 322
Hypoglycemic, 243 moisture, 322
Hypohalous acid, 46 Klebsiella pneumonia, 75, 242, 246, 293,
314
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I
Immobilization method, 124, 204 L
Immunoprecipitation, 7 Laccaria
Immuno-stimulant, 244 bicolor, 339
Indomalayan origin, 243 laccata, 383
Industrial Lacoumarin, 245
production processes, 51 Lamiaria, 157
waste remediation, 156 Large-scale application, 170
gases, 159 factors for optimal productivity, 170
hydrocarbons, 158 gas exchange, 172
liquid wastes, 156 light intensity, 171
solid wastes, 162 mixing, 173
wastes, 204, 332 nutrients, 170
wastewater, 156, 157, 200 salinity and pH, 173
Industrialization, 258, 330, 331 temperature, 172
Inorganic integrated process engineering, 183
anions, 101 harvesting and extraction, 183
chloride, 43, 55 immobilization and recycling, 186
chlorine, 42 waste treatment and bioenergy co-
compounds, 102, 258 generation, 187
elements, 170 reactor engineering, 174
ions, 112 gas injection, 178
organic nutrients, 371 hydraulic retention time, 180
species, 106 hydrodynamic conditions, 179
Insecticide, 70, 73, 370 light penetration, 177
Integrated Microbial Genomes, 85 open and closed system, 174
Intracellular microbial activity, 53 operating conditions, 180
Ionizing radiation, 334 reactor scale and configuration, 182
Iris pseudacorus, 278 Larrea divaricata, 242
Irpex lacteus, 385387 Lauryl sulfate, 103
Isochrysis galbana, 137, 138, 143, 156, 161 Lawsonia, 243246, 248
Isoplumbagin, 245 inermis, 245, 246
Isoshinanolone, 244 Leather
Isozeylinone, 244 deterioration, 248
industry, 231, 246249
J making, 226, 227, 238, 248, 249
Joint Genome Institute, 339 manufacturing, 226, 237
processing, 227232, 238
K products, 230
technology, 228
Kandelia obovata, 277
AUTHOR COPY
Index 403
Leguminosarum, 81 Macronutrients, 170

Lemna minor, 264, 268, 276 Macrosolutes, 24
Length Heterogeneity PCR, 85 Magnesium incorporated bentonite, 108,
Lentinula edodes, 385 123, 386
Lentinus tigrinus, 385, 387 Magnetic
Lepiota rhacodes, 347 nanocomposites, 185
Lepis tanuda, 347 particles, 185
Lessonia, 157 separation, 185
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Leucaena leucocephala, 265, 275 Magnetization, 185
Life cycle Malondialdehyde, 332
assessment, 183, 189 Mammalian collagenase, 240
costing, 189 Mammaria echinobotryoides, 380
Ligninolytic basidiomycetes, 376 Manganese peroxidase, 122, 384
Lignocellulose polymers, 389 Margarinomyces heteromorphum, 380
Liminoids, 243, 244 Matricaria recutita, 333
Lindane-contaminated environments, 269 MaxamGilbert method, 338
Linear alkylbenzene sulfonates, 278 Melia azadirachta, 243
Linoleic acids, 200, 319 Melilotus, 263
Lipid Membrane filtration, 187, 105, 106, 258
accumulation, 137, 139142, 171, 173, Membrane PBR, 11, 187
182, 199 Membrane
biosynthesis, 141 spargers, 183
extraction, 143, 188 technology, 105
oxidation, 2 ultra-filtration, 197
peroxidation, 30, 141 Mesophilic
production environmental parameters, 139 conditions, 202
carbon dioxide, 140 thermophilic, 203
metal ions, 142 Metabolic
nutrient content, 140 active cell count, 2
oxidative stress, 143 capabilities, 158, 389
salinity, 141 products, 173
temperature, 141 Metagenomics, 85
recovery, 143, 144, 186 Metalaxyl stress, 269
synthesis, 141 Metallothioneins, 342
Listeria, 242 Methane production, 162, 181, 188, 189,
Lithosphere, 42 202, 203
Lodderomyces elongisporus, 339 Methanogenesis, 203
Logistic regression, 30 Methyl tert-butyl ether, 143
Lolium multiflorum, 269, 270 Methylene blue dye reduction test, 11, 14
Luciferin, 28 Microalgae
Lupinus, 263 cells, 186
Lycoperdon perlatum, 383, 384 species, 137, 155, 165
Microalgal
M biofuel, 165
Macroalgae, 45, 53, 154, 181 production, 134
Macrocystis, 157 cells, 135, 136, 139, 140, 186
Macroflora, 68 cultivation, 136
Macrolepiota procera, 383 culture system, 134
AUTHOR COPY
growth, 162 Montmorillonite clays, 107109

production, 135 Moringa, 263
species, 161 Morphotypes, 241
Microbacterium, 19 Mucor hiemalis, 380
Microbial Mucor rouxii, 343
activity, 79, 165, 203, 259, 262, 267, 276 Multidrug resistant bacteria, 245
biodegradability, 202 Mutualistic symbioses, 365
biomass, 79, 80 Mycelia sterile, 380
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community, 7880, 86, 87, 128, 158, 276, Myceliophthora thermophila, 339
367 Mycodegradation, 350, 351
community structures, 78 Mycofiltration, 329, 350
contamination, 240, 321 Mycoremediation, 329, 337, 348, 352, 364,
degradation, 249 365, 384, 391
degradation xenobiotics, 367 Mycorrhizal colonization, 344, 352
diversity, 23, 68, 79, 83, 84, 87 Mycorrhizal
existence, 75 fungus, 350
fermentation, 301 symbionts, 370
flora, 239, 241 Mycosphaerella, 378
growth, 23, 33, 180 Mycotechnologies, 350, 352
indicators, 87 Mycotoxins, 308, 321, 323
inoculants, 186 Myriophyllum aquaticum, 265, 266
interaction, 2, 14, 270274 Myrothecium verrucaria, 380
succession, 249
systems, 85 N
Micrococcus, 242 Nannochloris, 137
Micrococcus luteus, 246 Nannochloropsis, 137141, 154, 155, 161,
Microcoulometric titration, 54 167, 179, 181
Microcystis aeruginosa, 165 Nannochloropsis oculata, 138, 140, 171,
Microspectrophotometer, 26 173, 182, 199, 200, 201
Mineralization, 46 Nanocomposites, 100, 107, 108, 112, 185,
Minimal medium, 269 186
Minimum inhibitory concentrations, 346 Nanofilters, 99
Mitosporic ascomycetes, 376 Nanomaterials, 99, 100, 107, 110112
Molasses, 142, 291, 292295, 298300, 387 water remediation, 100
Molecular nanoscale zero-valent iron, 100
chronometer, 27 titanium dioxide, 102
level, 31, 120, 122, 126, 127 silver nanoparticles, 104
regulation, 127 nanofilters/nanomembranes/nano-
techniques, 83 tubes, 105
Molluscicide, 70 nanoclays and nanocomposites, 107
Monallanthus salina, 138 Nanomembranes, 100, 105, 106, 110, 112
Monilia sitophila, 380 Nanoparticles, 99, 100, 101105, 108, 110,
Moniliaceae, 380 185, 186
Monocrotophos, 80 Nanoremediation, 99
Monodictys pelagica, 343 Nanoscale, 98, 100, 108, 111, 186
Monodus subterraneus, 138, 139 Nanoscale zero-valent iron, 100
Monoraphidium, 179 Nanosensors, 99
Monounsaturated fatty acids (MUFA), 182, Nanotechnology, 98, 100, 107, 111, 112
187
AUTHOR COPY
Index 405
Nanotitanium, 99 values, 155, 332

Nanotubes, 100, 105, 106, 110, 112
Naphthalenone, 244 O
Naphthoquinone derivatives, 244, 247, 249 Ochromonas danica, 159
National Human Genome Research Ocimum
Institute, 339 basilicum, 242
Natural gratissimum, 242
attenuation, 367, 390, 391 Oidiodendron maius, 342
9781771883627
bioactive compounds, 61 Olive mill solid waste, 162
chlorination, 61 Oocystis pusilla, 138
environment, 42, 51 Open-pond algae cultivation system, 189
gas, 166 Organic
organic matters, 106 acids, 43, 78, 296, 297, 329, 371
plant physiological processes, 258 inorganic substances, 156
processes, 42 loading rates, 163, 180, 188, 197, 201,
products, 50, 249 203
radiation, 334 materials, 51, 156, 157, 166
resources, 259, 260, 330, 333 matter, 48, 54, 57, 69, 79, 80, 81, 121,
Nematicide, 70, 243 154, 162, 169, 180, 189, 199, 290, 291,
Nematode pathogens, 319 347
Neochloris, 136, 137, 184 molecules, 48, 58
Neochloris oleoabundans, 136, 138, 184 solvents, 100, 143, 242, 247
Net present values, 189 wastes, 102, 166, 169, 288, 329
Neurospora crassa, 339, 348 Organochlorine, 42, 44, 51, 56, 59, 70, 100,
Neutral lipid and polyunsaturated fatty 269
acids, 140 compounds, 42, 44, 59
Nigrospora, 20 pesticide, 269
Nimbosterol, 244 Organo-halide compounds, 120
Nitrification, 69, 82, 371 Organohalogen compounds, 48
Nitrification/denitrification processes, 69 Organophosphate, 70, 370
Nitroaromatics, 264, 366, 377 Osmosis, 106, 109, 183, 197, 198, 258
Nitrogen fixation, 69, 7678, 81, 87 Osteomyelitis, 321
Nitrogenase, 7678, 81 Otomycosis, 321
Nitrophenols, 158 Oxido-reductase activity, 82, 121, 122, 372,
Nitzschia, 137, 138, 158 375, 378
Nostoc linckia, 50 Oxyluciferin, 28
Nostocyclophane, 50 Ozoflotation, 185
Novel compounds, 51, 338 Ozone, 42, 43, 48, 185
Nuclear
explosion tests, 334, 335 P
industries, 334, 335
radiation, 334 Paecilomyces, 20, 124, 389
residual waste, 334, 352 inflatus, 380
Nutrient management systems, 189 lilacinus, 338, 380
Nutritional Palm oil mill sludge, 166, 190203,
conditions, 4, 13 287290, 294297, 300
properties, 156 Palmitic acid, 141, 171, 182, 200
source, 347 Panus tigrinus, 386
Parathion, 280
AUTHOR COPY
Passeriniella obiones, 378 Phosphorelay, 8

Pavlova Phosphorus components, 104
lutheri, 138, 182 Phosphoryl group, 8
salina, 138 Phosphorylation, 8, 14
Paxillus involutus, 342 Photobioreactor, 135, 145, 175, 177, 203
Pelletization, 187 Photocatalysis
Penicillium, 20, 308, 311, 343, 344, 348, activity, 103
349, 379, 380, 381, 389 potency, 102
9781771883627
camemberti, 125 process, 107
frequentans, 125 Photolysis, 166
miczynskii, 126 Photo-oxidative damage, 172
Pentachlorophenol, 69, 125 Photosynthesis, 78, 139, 157160, 167, 171,
Pentacytic triterpenes, 245 172, 176, 177, 263
Pentadecanoic acid, 200 Photosynthetic
Pentanortriterpenoids, 243 function, 160
Perchlorate, 101 microorganisms, 167
Perchlorobenzenes, 124 Photosynthetically active radiation, 177
Perchloroethylene, 54, 125 Phototrophic biofilms, 78
Perfluorobenzenes, 124 Phragmites, 263
Peroxidases enzymes, 121 Phragmites australis, 278
Peroxide value, 318 Phyllosphere, 74
Pesticide Physical
application, 73, 79, 81, 82, 86, 87 chemical method, 239
poisoning, 73 chemical stabilization, 367
soil bioprocesses, 81 factors, 271, 345
toxicity, 80 methods, 233, 238, 247
Phaeodactylum, 137, 161 curing, 233
tricornutum, 138, 144, 184 Physico-chemical
Phaeosphaeria biological method, 347
alima, 378 biological treatment, 191
neomaritima, 378 methods, 258
spartinicola, 378 microbial factors, 271
Phalaris, 263 process, 347
Phanerochaete properties, 51, 68, 369
chrysosporium, 125, 126, 348, 377, Physiological processes, 4, 259, 261
384388 Phytoaccumulation, 280
sordid, 125, 377, 385 Phytochelatin synthase, 333, 340, 341
Phlebia Phytochemical agents, 309, 314
brevispora, 126 Phytodegradation, 262, 280
lindtneri, 385, 387 Phytoextraction, 261, 270, 280
radiate, 385 Phytoinhalation, 333
Phoma, 20, 379, 380 Phytomining, 260, 261
fimeti, 380 Phytoplankton composition, 50
Phosphate Phytoremediation, 52, 258275, 277280
buffered saline, 11 organic pollutants, 264
solubilizing bacteria, 68 process, 259
starvation, 7 Phytoscreening, 279, 280
Phospholipids, 143, 319, 333 Phytostabilization, 261, 280
AUTHOR COPY
Index 407
Phytostimulation, 262, 269, 280 Polyhydroxyalkanoate, 301

Phytotechnologies, 258 Polyhydroxyalkanote, 293
Phytotoxicity, 275, 276, 280 Polyhydroxybutyrate, 294
Phytotransformation, 261, 262, 267, 280 Polymerase chain reaction, 27, 28, 84, 85,
Phytovolatilization, 262, 280 276
Pichia, 242 Polymerization, 369
Piezoelectronic, 293 Polypeptides, 242, 341
Pisolithus tinctorius, 341 Polyphenols, 241, 386
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Pistia, 262, 265, 268, 277 Polypogon, 263
Pithomyces, 20 Polysaccharides, 186, 340
Plant growth Polysulfone membranes, 105
promoting rhizobacteria, 74, 75 Polyunsaturated fatty acids, 154
regulators, 70, 87 POME treatment, 190192, 195, 197199,
Plant microbe interaction, 280 296
Plant microbial treatment, 274 Populus salicaceae, 268
Plant species, 75, 241, 242, 248, 259, Porphyridium, 137
263269, 274, 277279, 389 Porphyridium cruentum, 138
Plant substances, 242248 Postharvest
Plantation-style farming, 73 deterioration, 320
Plant-based materials, 249 diseases, 308314, 322
Pleospora spartinae, 378 quality, 323
Pleurotus Potamogeton, 263
ostreatus, 125, 346348, 385388 Pricey method, 234
platypus, 347 Prostate epithelial malignant transformation,
pulmonarious, 125, 385, 387 333
sajor-caju, 347, 385 Prosulfuron, 82
tuberregium, 385, 388 Proteobacteria, 19, 83
Plumbagic acid, 244, 245 Prototheca zopfi, 158
Plumbagin, 244, 245, 247, 249 Psalliota campestris, 347
Plumbago, 243245, 248, 249 Pseudomonas, 19, 46, 105, 242, 244, 293,
rosea, 245 295
zeylanica, 244, 245 Pseudomonas putida, 329
Plumbazeylanone, 244 Puerariae radix, 242
Pneumocystis, 339 Pullularia pullulans, 380
Poliporus Pulmonary infections, 321
ciliates, 385 Putrefaction, 232234, 239241
pinsitus, 386 Pyrenoidosa, 136
Polyacrylamide, 108, 186 Pyrethroids, 70
Polyaromatic hydrocarbons, 158, 258, 273, Pyrophosphate, 28
276, 373378, 387 Pyruvate decarboxylase, 169
Polychlorinated biphenyls, 19, 31, 100, 126,
264, 277, 366, 369, 377, 384, 385 Q
Polycyclic Quantifying bacteria, 31
aromatic hydrocarbons, 19, 31, 112, 364, Quantitative composition, 26
365, 369 Quaternary treatments, 157
musk fragrances, 376 Quercetin (flavonoid), 244
structures, 365 Quinalphos, 80
Polyethylenimine, 185 Quinones, 48, 241, 309, 375, 378
AUTHOR COPY
R Rhizospheric
bacteria, 276
R. hookeri, 307316
microorganisms, 277
Radiation pollution, 335
soil, 270
Radioactive
Rhodobacter sphaeroides, 296, 297
bioremediation of radioactive wastes,
Ribosomal, 27
336
Ribosomal Intergenic Spacer Analysis
elements, 30, 335
(RISA), 85
9781771883627
isotopes, 352
Rodenticide, 70
nuclides, 352
Russula delica, 347
potential causes of radioactive
wastes, 335
S
potential soil, air and water pollut-
ant, 334 Saccharomyces, 169, 242, 338, 342, 348,
radiation pollution and its toxic 349
effects, 336 Saccharomyces cerevisiae, 169, 338342,
radiations, 336 348
soil pollution, 335 Saline soils, 243
waste, 328, 329, 334337, 344, 350 Salinization, 164
Radionuclides, 335, 364, 370372 Salix, 263, 275
Radiotracer Salmonella, 239242, 245, 314
methods, 46 Salt concentration, 124
studies, 61 Salvinea, 263
Ralstonia eutropha, 293 Sargassum, 157
Raphia, 307312 Scanning electron microscopy, 26, 31, 35
Raphia palm, 309, 323 Scenedesmus, 136, 137, 141, 145, 154, 161,
Reactive oxygen species, 142 168, 171, 181, 202
Reactor scale and configuration, 182 dimorphus, 138, 179, 200
Recalcitrance, 365 obliquus, 138, 159
Recalcitrant quadricauda, 138
compounds, 277, 391 Schizochytriu, 137
organic compounds, 365 Schizophyllum commune, 346
pollutants, 344, 378 Schizosaccharomyces pombe, 339, 341
properties, 322 Scirpus grossus, 275, 276
Rennin angiotensin system, 332 Scopoletin, 245
Resorcinolic structures, 45 Scopulariopsis brevicaulis, 380
Respiratory diseases, 30, 35 Sedimentation, 33, 173, 175, 183, 184, 197
Restriction fragment length polymorphism, Separate hydrolysis and fermentation, 169
28 Serradella plants, 81
Reverse osmosis, 106, 109 Serratia marcescens, 246
Rhinitis, 321 Sesbania cannabina, 275
Rhizobia, 76, 77 Sewage sludge, 188, 202
Rhizobia-legume, 76 Shea fruit germination, 319
Rhizobium, 75, 76, 80, 81, 87 Shea nut drying/storage, 320
Rhizodegradation, 262, 275, 280 Shea tree, 323
Rhizofiltration, 52, 262, 280 Shigella dysenteriae, 245
Rhizopus stolonifer, 308 Silver nanoparticles, 100, 104, 112
Rhizosphere, 68, 69, 74, 86, 87, 259, 262, Simplicissimum, 343, 344
269, 270, 275, 276
AUTHOR COPY
Index 409
Simultaneous saccharification and fermen- Sphingomonas, 19

tation, 169 Spicaria violacea, 380
Single-Strand Conformation Polymorphism, Spinacia oleracea, 332
85 Spirodela polyrhiza, 274
Sinorhizobium meliloti, 81 Spirulina, 138, 161, 162, 381
Skeletonema costatum, 138, 158 maxima, 138, 181
Sodium platensis, 138, 162, 169, 381
acetate, 29 Sporobolomyces, 341
9781771883627
chloride, 47, 61, 106, 232, 239 Sporulation, 213, 14, 315, 376
dodecyl sulfate, 237 sporulation stages, 5
montmorillonite, 108 methodology to study sporulation, 6
Soil agar method, 7
bacterial communities, 83 delaying protein, 2
biological activities, 68 mechanism, 5
column, 267, 272 process, 5
controls, 271 sporulation killing factor regulation,
ecosystem function, 68 7
environment, 69 stages, 5
enzymatic activities, 82 Staining method, 6
erosion, 78, 164, 331 Staphylococcus, 20, 242, 243
fertility, 68, 78, 79, 82, 86 Staphylococcus aureus, 246
living bacterium, 3 Stearic acid, 200
microbes, 79, 82, 344 Steel production, 331
microbial community, 79 Stenotaphrum secundatum, 278
microbial diversity, 67, 87 Stereum hirsutum, 346, 386
microbial population, 79 Stochastic phenotype, 9
micropopulation, 82 Streptococcus, 242
organic matter, 43, 47, 5561 Streptomyces, 19, 46, 269
organisms, 68, 79 Stropharia rugoso-annulata, 349, 387
processes, 48, 56 Sugar cane molasses, 298, 301
samples, 56, 83, 84 Sulfur and phosphorous solubilization, 69
Solanum nigrum, 269 Super oxide dismutase, 276, 277, 332, 333
Solar
collector, 176 T
energy, 159, 160, 176 Tannery, 249, 265
irradiance, 175 Taxonomic group, 83
radiation, 178 Terminal Restriction Fragment Length
Solid retention time, 180 Polymorphism, 85
Solid wastes, 162 Terpenoids, 242
Solidliquid separation technique, 183 Tetrachloroethylene, 42, 50
Solid-phase cytometry, 32 Tetracyclic
Solids retention times, 180, 203 triterpenoids, 244
Solubilization, 74, 78, 82, 181 type antibiotics, 237
Spartina patens, 378 Tetradecanoic, 244
Spasmogenic, 243 Tetraselmis suecica, 137, 138, 143, 155,
Spectrophotometric and fluorimetric assay, 156, 171, 173, 182, 199, 200, 201
240 Thalassiosira
Sphingobacteria, 19 pseudonana, 138
AUTHOR COPY
weissflogii, 161 Tubular

Thermal array, 176
decomposition, 46, 53 bioreactor, 145
degradation, 53 Turgor pressure, 140
stratification, 18 Typha latifolia, 269, 278
Thermophilic Typhimurium, 239
condition, 203 Tyrosine, 105, 244
plant, 180
9781771883627
Thermoplastics, 293 U
Thespesia populnea, 242 Ulocladium, 20
Thlaspi, 263 Ultra-filtration, 183, 197, 198
Thymus vulgaris, 242 Ultrafiltration membranes, 106
Titanium dioxide, 100, 102104, 110, 112 Ulva, 157
Tonoplast, 46 Unicinocarpus reesii, 340
Torula, 20 Urbanization, 330, 347
Total dissolved solids, 231, 232, 236, 238, Urbanization/industrialization, 328, 330
246, 247 Ustilago maydis, 340
Toxaphene, 69 UV radiation, 102, 109
Toxic industrial pollutants, 158
Toxicological properties, 51 V
Tracheobronchitis, 321
Tradescantia, 263 Vaccinium myrtillus, 342
Trametes Vacuum pump, 23
pubescence, 123 Vallisneria, 263, 268
versicolor, 124, 125, 383388 Valorization, 187
villosa, 125, 386 Vanadate-dependent peroxidases, 46
Tremateia halophila, 378 Vanadium, 46, 47
Triacyl glycerol, 136, 139, 145 Vegetative
Trichloro ethane, 101, 126 cells, 5, 7, 12, 159
Trichloroacetic acid, 45, 54 growth, 6, 13, 309
Trichloromethanide, 54 Verrucomicrobia, 19
Trichloromethyl compounds, 46 Versatile peroxidase, 122
Trichoderma, 125, 126, 242, 311, 349, 379, Vertical column, 177
381 Vertical flow constructed wetland, 278
album, 380 Verticillium, 380
atroviride, 380 Verticillium tenuissimum, 380
viride, 380 Vesicular-arbuscular mycorrhiza, 75
Trichophyton, 20 Vetiver (Vetiveria zizanioides), 267274,
Trihalogenated methanes, 51, 52 279, 280
Trihalomethanes, 61 plants, 270273
Trilinolein, 245 Arthrobacter, 269
Trimethylphenylammonium, 109 Vetiveria zizanioides, 265, 269
Trinitrotoluene, 262267, 279, 375379, Vetiver microbial interaction, 272
384, 385 Viable but non-culturable, 84
Tripeptide glutathione, 45 Vibrio alginolyticus, 237
Triterpenoids of protolimonoids, 243 Vicia/Vetiver, 263
Tropospheric samples, 19 Vinyl chloride, 48
Tuber melanosporum, 339 Viruses, 20, 32
Vitamin C, 244
AUTHOR COPY
Index 411
Vitellaria paradoxa, 317 World Health Organization, 73, 330

Volatile
chlorinated hydrocarbons, 42, 47, 53 X
compounds, 58, 60, 262 Xanthomonas, 244, 246
fatty acids, 166, 170, 203, 296, 297 Xenobiotic
non-volatile halogenated compounds, 47 biodegradation, 369
organochlorines, 4246, 52, 61 compounds, 277, 365369, 385
solid, 109, 162, 163, 167, 172, 180182, detoxification, 123
9781771883627
188, 202, 203 pollutants, 370
Volatilization/mineralization, 48 substances, 276
Volcanic gases, 42 substrates, 52
Volumetric Xenobiotics, 45, 55, 128, 129, 264, 267,
mass transfer rates, 177 268, 277, 330, 342, 363375, 377,
productivity, 168 379391
reactor productivity, 163 Xerophilous, 311
X-ray
W analysis, 26
Wallemia ichthyophaga, 338 analyzer, 26
Waste activated sludge, 162 contrast agents, 376
Wastewater, 51, 104106, 154159, 168, diagnostics, 334
181, 185204, 268, 274279, 288, 289, Xylaria, 311, 323
294, 297, 376 Xylaria feejeensis, 311, 312
Wastewater treatment, 105, 154159, 168, Xylariaceae, 311
181, 195, 199, 203, 204, 278, 297, 376 Xylariales, 311
Water catchments, 55
Water remediation, 97, 100, 102, 106, 107, Z
111, 112, 153, 204 Zeaxanthin, 178
Water source area, 42, 164 Zero-valent
Water treatment strategies, 110 iron, 99101, 110
Waterborne contaminants, 99 metals, 101
Water-soluble Zeylinone, 244
chlorinated compounds, 44 Zinc, 19, 108, 112, 141, 157, 170, 235, 236,
organohalogen compounds, 54 328331, 341, 342, 347, 383, 384
Wautersia eutropha, 293 Zinc dimethyldithiocarbamate, 235, 236
White-rot fungi, 384, 391 Zingiber officinale, 242
Withania somnifera, 242 Zn fingers, 332
Wolffia, 262, 264 Zygophyllum, 333
Wolffia arrhiza, 274
AUTHOR COPY
9781771883627

Environmental Biotechnology Biodegradation - Bioremediation - and Bioconversion of Xenobiotics For Sustainable Development

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Library of Congress Cataloging-in-Publication Data

ABOUT THE EDITORS

Jeyabalan Sangeetha, PhD, is an Assistant Professor in Central University

Devarajan Thangadurai, PhD, is Senior Assistant Professor at Karnatak

Muniswamy David, PhD, joined Karnatak University, Dharwad, South

Mohd Azmuddin Abdullah, PhD, is an Associate Professor of Bioprocess

1. The Role of General Methods and Mathematical Models on Microbial

2. Identification of Microorganisms Carried by Aeolian Dust to

3. Chlorinated Compounds in Natural and Biotechnological

4. Environmental Impact of Pesticide Use on Microbial

5. Recent Advances in Applications of Nanomaterials for

6. Fungal Dehalogenation: An Overview....................................................... 119

7. Insight of Biofuel Prospects from Microalgae as Renewable

8. Integrated Algal Industrial Waste Treatment and

9. Bioambiant Preservation of Raw Hides Using Plant-Based

10. Phytoremediation of Organic Chemopollutants.......................................257

11. Bioconversion of Palm Oil and Sugar Industry Wastes into

12. Influence of Environmental Factors on the Prevalence of

13. Soil Remediation and Ecological Restoration from

14. A Multifaceted Bioremediation of Xenobiotics Using Fungi....................363

Mohd Azmuddin Abdullah

Esiegbuya Ofeoritse Daniel

Swapna Kishor Deshpande

Prashant Kamalakar Dhakephalkar

Etigemane Ramappa Harish

Okungbowa Francisca Iziegbe

Pradnya Pralhad Kanekar

Venkata Sai Badireenath Konkimalla

Paidi Murali Krishna

Devipriya Rabin Majumder

Abhishek Channayya Mundaragi

Subir Kumar Nandy

Ashish Vasantrao Polkade

Noor Fazielawanie Mohd Rashid

Seema Shreepad Sarnaik

Prafulla Namdeo Shede

Parag Avinash Vaishampayan

OOH Peroxide radicals

g m2 d1 Gram per meter square per day

NH4Cl Ammonium chloride

SFA Saturated fatty acids

Every fraction in our close proximities harbors inestimable living organisms

interactions, exploration of microbes in extreme environments, applications

Jeyabalan Sangeetha, PhD

THE ROLE OF GENERAL METHODS

Bacillus subtilis is a spore-forming bacterium that can persist for years

1.2 SURVIVAL STRATEGY

Microorganisms have evolved strategies to adapt in various stress environ-

Froelich et al. (1979) have shown in anaerobic marine sediments that a

B. subtilis is a Gram-positive soil-living bacterium, which adapts to differ-

Cannibalism is a process in which bacteria produce a killing factor to kill

1.4.1 SPORULATION STAGES

This process involves a number of steps which can be monitored by light

1.4.2 METHODOLOGY TO STUDY SPORULATION

1.4.2.1 STAINING METHOD

1.4.2.2 AGAR METHOD