Dipanwita Das.pdf · Certificate TO WHOM IT MAY CONCERN This is to certify that Miss. Dipanwita Das...

COMPREHENSIVE STUDY ON GAMMA RADIATION INDUCED METAL TOLERANCE IN

SOME FUNGAL STRAINS

THESIS SUBMITTED TO UNIVERSITY OF KALYANI FOR THE AWARD OF DEGREE OF DOCTOR OF PHILOSOPHY (Ph.D.)

IN SCIENCE

Dipanwita Das, M.Phil

Department of Environmental Science

University of Kalyani

Kalyani-741235

West Bengal

INDIA

2015

DeDicateD

to my

Parents

Certificate

TO WHOM IT MAY CONCERN

This is to certify that Miss. Dipanwita Das has completed her Ph.D. work

entitled "COMPREHENSIVE STUDY ON GAMMA RADIATION

INDUCED METAL TOLERANCE IN SOME FUNGAL STRAINS" under

the joint supervision of Prof. S. C. Santra, Department of Environmental

Science, University of Kalyani, Nadia, West Bengal and Dr. A. Chakraborty,

Scientist F, University Grants Commission- Department of Atomic Energy

Consortium for Scientific Research (UGC-DAE CSR), Kolkata Centre. Miss.

Das has completed her work through repeated laboratory analysis at Department

of Environmental Science, University of Kalyani and UGC-DAE CSR, Kolkata

centre. This work is original and has not been submitted earlier for any degree

or award.

---------------------------------------- -----------------------------------------

Dr. A. Chakraborty Prof. S. C. Santra

(Supervisor) (Supervisor)

PREFACE

The thesis describes a study on employing gamma radiation in imparting tolerance in

some fungi towards heavy metals thus reflecting the possibility of utilising such fungi for

heavy metal bioremediation. The study also describes the potential of gamma rays in

modulating lignocellulosic enzyme function of the fungi under metal stress, thus implying

prospective use of fungi in bio-waste and plastic polymer degradation.

Divided into seven different chapters, the background perspective and introduction of

the work related to the recent scenario of environmental pollution, especially heavy metal

pollution and scientific motivation behind considering fungi as a suitable candidate for heavy

metal abatement, are described in the first chapter (Chapter 1). This chapter also entails

recent biotechnological development relevant to use of irradiation technique in improving

microbial strains to be used in diverse environmental and industrial field. Chapter 2 deals

with Literature review wherein, reports of works of earlier researchers in the relevant fields

accounting potential of fungi in metal bioremediation and testimonia of various investigations

since decades till date, documenting use of irradiation on microbes and applications of such

in various applied fields are stated. The focus of the study highlighting the precise objectives

of the work is presented in Chapter 3. Chapter 4 describes the methodologies and the

experimental procedures adopted for carrying out the work. The observations and findings of

all the experiments are narrated in four successive sections under Chapter 5 and the data are

presented in Figures and Tables numbered in accordance with the Chapter 5.

Chapter 6 presents the discussion part of the whole study. In this chapter plausible

explanations of the findings are put forth. Major highlights of this study are presented in

concluding chapter (Chapter 7). References used in all chapters are identified by authors and

years and all are collected together in a single bibliography (Chapter 8). Published papers

along with a list of papers presented in conferences are listed at the end of the thesis.

Acknowledgement Ph.D, the journey of more than five years is not just to earn a degree for a better step of

furnished future ; but it is a lesson of life to be calm, have patience , silence tolerance against

every odd and above all acceptance. Thanks to the supreme power for giving me such inner

strength.

I wish to express my sincere gratitude to my supervisor, Prof. Subhash Chandra

Santra, Professor, Department of Environmental Science, Kalyani University, Nadia, West

Bengal, whose guidance, thoughts and suggestions have enlightened me to carry on my work.

His constant encouragement and problem solving attitude assisted me in bringing this work to

fruition. His valuable suggestions and critical analysis helped me to upgrade the standard of my

work.

I like to convey my sincere gratitude to my joint supervisor Dr. Anindita Chakraborty,

Scientist F, University Grants Commission- Department of Atomic Energy Consortium for

Scientific Research (UGC-DAE CSR), Kolkata Centre, for her valuable important suggestions and

critics over my work. Her enormous energy and work oriented mentality encourages my entire

work. I am also grateful to her, for giving me a chance in her laboratory providing necessary

instruments and chemicals.

I owe my deep sense of gratitude to Dr A. K. Sinha, Centre Director, UGC-DAE-

Consortium for Scientific Research (UGC-DAE CSR) Kolkata Centre, for allowing me to carry out

my work in the Consortium and supporting me throughout my work tenure.

My due regards and thanks to Dr. R. Bhattacharya, HOD, Department of Environmental

Science, Kalyani University, Nadia, West Bengal, for her cooperation and all possible helps she

provided to me. I like to convey my thanks also to Prof. D.K. Khan, Dr. Debasis Das and Dr. S,

Mukherjee, my teachers in Dept. of Environmental Science, University of Kalyani.

My sincere regards and thanks to and Dr. A. Saha , Scientist D, for providing me gamma

radiation facility and Dr M. Sudarshan, Scientist E, UGC-DAE CSR, Kolkata Centre, for his

support in every step of my work in particular and providing me the possible helps in his

laboratory during freeze drying my samples.

I like to thank Dr. A.Samal of Dept.of Environmental Sc. University of Kalyani for

running my samples in AAS and guiding me in necessary steps.

I like to thank Dr. B.Chattopadhyay, Principal of Govt. College of Engineering and

Leather Technology (GCELT), Kolkata, for giving me permission to work in his college and Dr.

Anjan Biswas, faculty of that college, for using the UV-VIS spectrophotometer for running my

experiments, without their laboratory it was not possible for me to complete my work.

I take this opportunity to express my sincere gratitude to Dr. Srikanta Bhattachrya,

Central Instrumentation Department, Burdwan University, Burdwan, for his sincere and

constant helping during analysis of my samples in SEM.

I am also thankful to Dr.Prasun Mukherjee of CRNN (Centre for research in Nano-

science and Nano-technology) Calcutta for using the FTIR facility.

I feel personally thankful to Mr. Selvaraj, Dr. A. Datta Scientific Assistant, UGC-DAE

Consortium for Scientific Research, Kolkata Centre, for extending their helping hands all the

times during the entire course of my research, my thanks to Kapilda for helping me making

pellets for FTIR.

I convey my thanks to my seniors Dr.D.Mishra, Dr. S.Majumder of UGC-DAE-CSR, and Dr.

D.chakraborty, Dr.P.Bhattacharya, Dr.J.Majumder, Dr.A.Biswas of Kalyani Env.Sc. Dept. My great

admiration for both of my friends Dr. Shidarth (UGC-DAE CSR)and Dr. Anulipi (GCELT) for their

constant mental support and helping attitude; I can’t express my feelings for lending their hands

till last day. My special appreciation to my labmate Suvojit and thanks to Dipanwita, Shamayita,

Dr.Satabdi, Dr.Amrita for their assistance.

My mother’s constant inspiring-support and father’s keen interest in my research work

motivated me always. The little word ‘thanks’ is too small to express my gratitude to them.

I also convey my thanks to them whom I missed to acknowledge.

--------------------------------------------

( DIPANWITA DAS )

List of Tables Chapter

No Table Caption Page

No

2 Table 2.1 Some Heavy metals of environment and their concentration limits

2 Table 2.2 Biosorption capacity of some fungal strains

2 Table 2.3 Examples of hydrolytic fungi, their substrates, enzymes and applications

2 Table 2.4 An overview of ligninolytic fungi, their substrates, enzymes and applications

5 Table 5.1: Physico-chemical characteristics of soil samples

5 Table 5.2 - Morphological features of selected strains

Table 5.3. Heavy metal tolerance of isolated fungal strains

5 Table 5.4 Sensitive and tolerant fungal species among isolated strains against each metal

5 Table 5.5 Morphological and Physiological features of the selected strains

5 Table -5.6 Cross Metal Tolerance (in terms of MIC) of selected fungal strains

5 Table-5.7 Antibiotic Sensitivity of selected fungal strains

5 Table 5.8 Effect of different pH on growth of selected fungal strains

5 Table 5.9 Effect of different temperatures on growth of selected fungal strains

5 Table: 5.10 Uptake and removal of Cd by P.cyclopium with or without exposure to gamma irradiation

5 Table 5.11 Uptake and removal of Zn by A.terreus with or without exposure to gamma irradiation

5 Table 5.12. Uptake and removal of Pb by P.cyclopium with or without exposure to gamma irradiation

5 Table 5.13 Effect of pH on removal of Zn (100ppm) by Gamma irradiated and unirradiated fungal strains

5 Table 5.14 Effect of temperature on removal of Zn (100ppm) by Gamma irradiated and unirradiated fungal strains

5 Table 5.15 Effect of pH on removal of Cd by Gamma irradiated and unirradiated fungal strains

5 Table 5.16 Effect of temperature on removal of Cd by Gamma irradiated and unirradiated fungal strains

Chapter No

Table Caption Page No

5 Table 5.17 Effect of pH on removal of Pb (2000ppm) by Gamma irradiated and unirradiated fungal strains

5 Table 5.18 Effect of temperature on removal of Pb (2000ppm) by Gamma irradiated and unirradiated fungal strains

5 Table 5.19 CMCase (IU/ml) and α amylase activity (IU/ml) of A.terreus with or without exposure to gamma under Zn stress

5 Table 5.20 Comparative analysis of ultratructural changes in A.terreus with or without exposure to Cd , Zn and gamma

5 Table 5.21 Comparative analysis of ultrastructural changes in A.niger with or without exposure to Pb, Zn and gamma

5 Table: 5.22 CMCase (IU/ml) and α amylase activity (IU/ml) of P.cyclopium with or without exposure to gamma under Cd and Pb stress

5 Table 5.23 Comparative analysis of ultrastructural changes in P.cyclopium with or without exposure to Cd ,Pb and gamma

5 Table 5.24 Metallothionein (µmole/mg of protein) in A.niger

5 Table 5.25 Metallothionein (µmole/mg of protein) in A.terreus

5 Table 5.26 Metallothionein (µmole/mg of protein) in P.cyclopium

List of Figures Chapter

No Figure Caption Page

No

1 Fig 1.1 : Sources and sink of heavy metals in environment

1 Fig:1.2 Schematic representation of cellular mechanisms potentially involved in metal tolerance in ectomycorrhizal fungi

2 Figure 2.1 Processes of a conventional metals precipitation treatment plant

2 Fig 2.2 Metal-processing features adopted by microbes to be utilized in metal bioremediation processes

2 Fig. 2.3 Diagrammatic representation of metal interactions with fungi

5 Fig-5.1 Colony Forming Unit of A.terreus with or without exposure to gamma irradiation grown in Cd supplemented media (a) 90ppm Cd (b)150ppm Cd

5 Fig 5.2 Uptake and removal of Cd by Aspergillus terreus exposed to different absorbed doses of gamma irradiation (20-100 Gy) and grown in media supplemented with different concentrations of Cd. (a) 90ppm Cd (b) 150ppm Cd

5 Fig 5.3 FTIR spectra of A.terreus biomass(a) Control biomass (b) 90ppm Cd treated biomass (c) 100Gy gamma exposed biomass grown in 90ppm Cd enriched media

5 Fig-5.4 Colony Forming Unit of P.cyclopium with or without exposure to gamma irradiation grown in Cd supplemented media (a) 300ppm -650ppm Cd (b) 750ppm Cd

5 Fig 5.5 FTIR spectra of P.cyclopium biomass

(a) Control biomass (b) 300ppm Cd treated biomass (c) 80Gy gamma exposed biomass grown in 300ppm Cd enriched media

5 Fig-5.6 Colony Forming Unit of A.niger with or without exposure to gamma irradiation grown in Cd supplemented media (a) 70-85ppm (b) 100ppm

5 Fig-5.7 Uptake and removal of Cd by Aspergillus niger exposed to different absorbed doses of gamma irradiation (20-100 Gy) and grown in media supplemented with different concentrations of Cd

5 Fig-5.8 Colony Forming Unit of A.niger with or without exposure to gamma irradiation grown in Zn supplemented media (A) 250ppm Zn (B) 500ppZn (C) 1000ppm Zn (D) 1500ppm Zn (E) 2000ppm Zn

5 Fig-5.9 Uptake and removal of Zn by Aspergillus niger exposed to different absorbed doses of gamma irradiation (20-100 Gy) and grown in media supplemented with different concentrations of Cd (a) 250ppm (b) 500ppm (c) 1000ppm (d) 1500ppm (e) 2000ppm

5 Fig 5.10 FTIR spectra of A.niger biomass.(a) Control biomass (b) 250ppm Zn treated biomass (c) 100Gy exposed biomass grown in 250ppm Zn enriched media

5 Fig 5.11 Colony Forming Unit of A.terreus with or without exposure to gamma irradiation grown in Zn supplemented media (a) 7000-9000ppm Zn (b)10250ppm Zn

Chapter No

Figure Caption Page No

5 Fig5.12. FTIR spectra of A.terreus biomass.(a) Control (b) 7000ppm Zn loaded biomass (c) 80Gy gamma exposed biomass grown in 7000ppm Zn enriched media

5 Fig 5.13 Colony Forming Unit of A.niger with or without exposure to gamma irradiation grown in Pb supplemented media

5 Fig-5.14 Uptake and removal of Pb by Aspergillus niger exposed to different absorbed doses of gamma irradiation (20-100 Gy) and grown in media supplemented with different concentrations of Pb

5 Fig 5.15 FTIR spectra of A.niger biomass.(a) Control biomass (b) 2000ppm Pb loaded biomass (c) 100Gy gamma exposed biomass grown in 2000ppm Pb enriched media

5 Fig 5.16 Colony Forming Unit of P.cyclopium with or without exposure to gamma irradiation grown in Pb supplemented media

5 Fig 5.17 FTIR spectra of P.cyclopium biomass.(a) Control biomass (b) 2000ppm Pb loaded biomass (c) 80Gy gamma exposed biomass grown in 2000ppm Pb enriched media

5 Fig 5.18A FTIR analysis of A.niger biomass exposed to different gamma absorbed doses (a)Control biomass (b) 60Gy treated biomass (c) 100Gy treated biomass

5 Fig 5.18B FTIR analysis of P.cyclopium biomass exposed to different gamma absorbed doses (a)Control biomass (b) 60Gy treated biomass (c) 100Gy treated biomass

5 Fig 5.18C FTIR analysis of A.terreus biomass exposed to different gamma absorbed doses (a) Control biomass (b) 80Gy treated biomass (c) 100Gy treated biomass

5 Fig-5.19 α-Amylase and CMCase activity of Aspergillus terreus (a) A. terreus exposed to different absorbed doses of gamma rays and then grown in media containing 90ppm Cd (b) A. terreus exposed to different absorbed doses of gamma rays and then grown in media containing 150 ppm Cd

5 Fig 5.20 CMCase and α-Amylase activity of Aspergillus niger

(a) A. niger grown in medium supplemented with 250-1000ppm Zn (b) A. niger exposed to different absorbed doses of gamma rays and then grown in media containing 250ppm Zn (c) A. niger exposed to different absorbed doses of gamma rays and then grown in media containing 500ppm Zn (d) A. niger exposed to different absorbed doses of gamma rays and then grown in media containing 1000ppm Zn

5 Fig 5.21 CMCase and α-Amylase activity of Aspergillus niger

(a) A. niger grown in medium supplemented with 2000-3000 ppm Pb (b) A. niger exposed to different absorbed doses of gamma rays and then grown in media containing 2000ppm Pb (c) A. niger exposed to different absorbed doses of gamma rays and then grown in media containing 3000ppm Pb

Chapter No


5 Fig 5.22 CMCase and α-Amylase activity of Aspergillus niger (a) A. niger grown in medium supplemented with 70 -85 ppm Cd (b) A. niger exposed to different absorbed doses of gamma rays and then grown in media containing 70ppm Cd (c) A. niger exposed to different absorbed doses of gamma rays and then grown in media containing 85ppm Cd

5 Fig 5.23 CMCase and α-Amylase activity of different fungi exposed to different doses of gamma rays (20-100Gy)

5 Fig 5.24 Antioxidative response of Aspergillus niger

(a) A.niger grown in medium supplemented with 250-1000 ppm Zn (b) SOD and CAT response in A.niger exposed to different absorbed doses of gamma rays and then grown in media containing 250-1000 ppm Zn (c) GSH content in A.niger exposed to different absorbed doses of gamma rays and then grown in media containing 250-1000 ppm Zn

5 Fig 5.25 Antioxidative response of Aspergillus terreus

(a) A.terreus grown in medium supplemented with 8000-9000 ppm Zn ( b) SOD and CAT response in A.terreus exposed to different absorbed doses of gamma rays and then grown in media containing 8000-9000 ppm Zn (c) GSH content in A.terreus exposed to different absorbed doses of gamma rays and then grown in media containing 8000-9000 ppm Zn

5 Fig 5.26 Antioxidative response of Aspergillus terreus (a) SOD and CAT response in A.terreus exposed to effective absorbed dose of gamma rays and then grown in media containing 90ppm Cd

(b) GSH content in A.terreus exposed to effective absorbed dose of gamma rays and then grown in media containing 90ppm Cd

5 Fig 5.27Antioxidative response of Aspergillus niger

(a) A.niger grown in medium supplemented with 70-85ppm Cd (b) SOD and CAT response in A.niger exposed to different absorbed doses of gamma rays and then grown in media containing 70-85ppm Cd (c) GSH content in A.niger exposed to different absorbed doses of gamma rays and then grown in media containing 70-85ppm Cd

5 Fig 5.28 Antioxidative response of Penicillium cyclopium

(a) P.cyclopium grown in medium supplemented with 300-650 ppm Cd (b)SOD and CAT response in P.cyclopium exposed to different absorbed doses of gamma rays and then grown in media containing 300-650 ppm Cd (c) GSH content in P.cyclopium exposed to different absorbed doses of gamma rays and then grown in media containing 300-650 ppm Cd

Chapter No


5 Fig 5.29 Antioxidative response of Aspergillus niger

(a) A.niger grown in medium supplemented with 2000-3000ppm Pb

(b) SOD and CAT response in A.nigrt exposed to different absorbed doses of gamma rays and then grown in media containing 2000-3000ppm Pb (c) GSH content in A.niger exposed to different absorbed doses of gamma rays and then grown in media containing 2000-3000 ppm Pb

5 Fig 5.30 Antioxidative response of Penicillium cyclopium (a) P.cyclopium grown in medium supplemented with 2000-3000ppm Pb (b) SOD and CAT response in P.cyclopium exposed to different absorbed doses of gamma rays and then grown in media containing 2000-3000ppm Pb (c) GSH content in P.cyclopium exposed to different absorbed doses of gamma rays and then grown in media containing 2000-3000 ppm Pb

5 Fig 5.31 Antioxidative response in fungi exposed to different absorbed doses of gamma (a)Aspergillus niger (b) Aspergillus terreus

(c) Penicillium cyclopium

5 Fig 5.32 Metallothionein (MT) expression (Mean Fluorescence Intensity) of A.niger, A.terreus and P.cyclopium in Flow cytometer. (a) A.niger exposed to Zn, Cd and gamma absorbed doses (b) A.terreus exposed to Zn, Cd and gamma absorbed doses (c) P.cyclopium exposed to Cd and gamma absorbed dose

List of Plates

Chapter Plate caption Chapter V (Section III) Plate I Ultrastructure (SEM) of A.terreus (1500X)

[ A : Control A.terreus B: 90ppm Cd treated A.terreus C: 100Gy exposed A.terreus grown in 90ppm Cd D :80Gy exposed A.terreus grown in 150ppm Cd E : 100Gy exposed A.terreus ]

Chapter V (Section III) Plate II Ultrastructure (SEM) of A.terreus (1500X) [A : Control A.terreus B: 7000ppm Zn treated A.terreus C: 80Gy exposed A.terreus grown in 7000ppm Zn]

Chapter V (Section III) Plate III Ultrastructure (SEM) of A.niger (1000-3000X) [A :Control A.niger, B: 250ppm Zn treated A.niger, C: 100Gy exposed A.niger grown in 250ppm Zn, D : 500ppm Zn treated A.niger, E : 60Gy exposed A.niger grown in 500ppm Zn]

Chapter V (Section III) Plate IV Ultrastructure (SEM) of A.niger (1000-1500X) [ A : Contol A.niger ,B : 2000ppm Pb treated A.niger, C : 100Gy exposed A.niger grown in 2000ppm Pb]

Chapter V (Section III) Plate V Ultrastructure (SEM) of P.cyclopium (1500-2000X) [ A : Control P.cyclopium, B : 300ppm Cd treated P.cyclopium, C : 80Gy exposed P.cyclopium grown in 300ppm Cd, D : 2000ppm Pb treated P.cyclopium, E : 80Gy exposed P.cyclopium grown in 2000ppm Pb]

Chapter V (Section III) Plate VI Ultrastructure (SEM) of A.terreus, A.niger and P.cyclopium exposed to gamma absorbed dose (1000-3000X) [A : Control A.terreus, B : 100Gy exposed A.terreus, C : Control A.niger, D : 100Gy exposed A.niger, E : Control P.cyclopium, F : 80Gy exposed P.cyclopium]

Abbreviations

AAS : Atomic Absorbtion Spectrophotometer

ADS : Antioxidative Defense System

BDATs : Best Demonstrated Accessible Technologies

BSA : Bovine Serum Albumin

CAT : Catalase

CD : Czapek Dox Media

CFU : Colony Forming Unit

CMCase : Carboxymethyl cellulose

DNS : 3,5 Dinitro-salicylic acid

DRI : Dietary Reference Intake

DTNB : 5,5-dithiobis-2- nitrobenzoic acid

EC : Electric conductivity

EMS : Ethyl Methane Sulphonate

FACS : Fluorescence Activated Cell Sorter

FTIR : Fourier Transform Infrared Spectrometer

GPx : Glutathione peroxidase

GR : Glutathione reductase

GSH : Reduced Glutathione

GST : Glutathione S-transferase

GWRTAC : Ground Water Remediation Technology Analysis Centre

Gy : Gray ( Unit of absorbed dose of gamma)

Krad : Kilo Rad

Lac : Laccase

LiP : Lignin peroxidase

MCL : Maximum Contaminant Level

MIC : Minimum Inhibitory Concentration

MNNG : N-methyl-N-nitro-N-nitrosoguanidine

MnP : Manganese peroxidase

MSW : Municipal Solid Waste

MT : Metallothionein

NAD (P)H : Reduced Nicotinamide Adenine Dinucleotide Phosphate

PBS : Phosphate Buffer saline

PDA : Potato Dextrose Agar

ROS : Reactive Oxygen species

RSPM: Respirable Suspended Particulate matter

SD : Standard deviation

SEM : Scanning Electron Microscope

SOD : Superoxide- dis-mutase

SPM : Suspended Particulate matter

UNIDO : United nations industrial development organization

USEPA: US Environmental Protection Agency

WHO : World Health Organisation

α-Amylase: Alpha- amylase

Contents

Chapter Title of the Chapter Page

1. Introduction ....................................................................................................................... 1.1. Environmental pollution, a global scenario ................................................................... 1.2. Heavy Metal Pollution .................................................................................................

1.3. Lignocellulosic waste pollution .................................................................................... 1.4. Soil heavy metal pollution ............................................................................................

1.4.1 Soil microbes in restoring ecosystem................................................................... 1.5 Role of fungi in Pollution Abatement ............................................................................

1.5.1 Heavy metal pollution abatement: contribution of fungi ........................................ 1.5.1.1 Inherent mechanism adapted by fungi for being metal resistant .................

1.5.2 Fungi in Lignocellulosic waste management .........................................................

1.6 Recent trend in biotechnology for making microbes more potential ...............................

1.6.1 Ionising radiation in modulating properties of fungi.............................................. 1.7 Focus of this work .........................................................................................................

2 Review of Literature .......................................................................................................... 2.1 Heavy metals – the problem and its control ...................................................................

2.1.1 Physico-chemical methods of Heavy metal removal from environment ................ 2.1.2 Biological methods for heavy metal removal ........................................................

2.1.2.1 Microbes in heavy metal removal ...................................................................... 2.1.2.1.1 Fungi – an alternative for heavy metal removal ...................................

2.2 Fungi in lignocelulosic waste degradation ..................................................................... 2.3 Fungal strain improvement techniques and application ..................................................

2.3.1 Application of gamma radiation on fungi as strain improving agent ...................... 2.3.1.1 Enzyme production ...................................................................................

2.3.1.2 Improvement of mushroom quality ........................................................... 2.3.1.3 Annihilation of Aflatoxin production capacity of fungus ...........................

2.3.1.4 Gamma induced enhancement in survivability and growth in fungi ........... 2.3.1.5 Gamma induced enhancement of essential metabolites ..............................

2.3.1.6 Gamma induced enhancement in biocontrol potential / biodegradation of pesticides by fungi ........................................................

2.3.2 Mechanism of Radioprotection in fungi ................................................................

2.3.3 Modulation of metal tolerance in gamma induced fungi ........................................

3 Aims and Objectives ........................................................................................................... 4 Materials and Methods ......................................................................................................

4.1 Selection of site ..............................................................................................................

4.1.1 Sampling of soil .....................................................................................................

4.1.2 Treatment of soil ....................................................................................................

4.1.3 Physico-chemical Characterisation of the soil (pH, conductivity, organic carbon content, available NPK, Pb, Cd, Zn concentration) .....................................

4.2 Isolation of fungal strains ............................................................................................... 4.2.1 Media compositions ...............................................................................................

4.2.2 Isolation of pure cultures ........................................................................................ 4.2.3 Maintenance of culture...........................................................................................

4.2.4 Identification of fungal population ......................................................................... 4.3 Assessment of metal tolerance of isolated strains ............................................................

4.3.1 Selection of fungal strains for further experiments .................................................

4.3.2 Species Identification of the strains ........................................................................

4.3.3 Cross metal resistance assay................................................................................... 4.3.4 Antibiotic sensitivity assay.....................................................................................

4.3.5 pH and temperature optimisation for growth .......................................................... 4.3.6 Preparation of Spore suspension.............................................................................

4.4 Selection of gamma dose ................................................................................................ 4.4.1 Gamma exposure to the prepared spore suspension ................................................

4.5 Assessment of metal tolerance in terms of Colony Forming Unit (CFU) ........................ 4.6 Estimation of metal uptake and removal potential ..........................................................

4.6.1 Analysis of functional groups responsible for metal absorption by Fourier Transform Infrared Spectroscopic (FTIR) study .....................................................

4.6.2 Optimisation of pH and temperature for removal ................................................... 4.7 Fungal sample preparation for Scanning Electron Microscope (SEM) .............................

4.8 Estimation of extracellular metabolic/ lignocellulosic enzyme and antioxidant assay...............................................................................................................................

4.8.1 Metabolic/ lignocellulosic Enzyme activity ............................................................ 4.8.1.1 CMCase assay [EC 3.2.1.4] ........................................................................

4.8.1.2 α- Amylase assay [EC 3.2.1.1].................................................................... 4.8.2 Antioxidant activity ...............................................................................................

4.8.2.1 Super oxide dismutase (SOD) [EC: 1.15.1.1] ..............................................

4.8.2.2 Catalase assay (CAT) [EC: 1.11.1.6] ..........................................................

4.8.2.3 Total reduced glutathione assay (GSH) [EC: 1.8.1.7] .................................. 4.8.2.4 Total protein estimation ..............................................................................

4.9 Metallothionein protein assay ......................................................................................... 4.9.1 Estimation of total Metallothionein using spectrophotometric method ....................

4.9.2 Expression of Metallothionein in Fluorecence activated cell sorter (FACS) ...........

4.10 Statistical Analysis .......................................................................................................

5. Results ................................................................................................................................ Section I Heavy metal load in soil parameter and diversity of fungal population

in waste dumping site of Kolkata .......................................................................... 5.1 Soil parameter analysis ...................................................................................................

5.2 Fungal population dominating the Dhapa soil ................................................................. 5.3 Metal tolerance of the isolated fungal strains ..................................................................

5.4 Species identification of the selected strains ................................................................... 5.5 Biochemical characterisation of the selected strains ........................................................

5.5.1 Cross Metal tolerance ............................................................................................

5.5.2 Antibiotic sensitivity test........................................................................................

5.5.3 Optimization of pH and Temperature for growth of selected fungal strains ............

Section II Metal tolerance of selected fungal strains before and after gamma exposure ...............................................................................................................

5.6 Cd tolerance efficacies of gamma exposed fungi.............................................................

5.7 Zn tolerance efficacies of gamma exposed fungi ............................................................. 5.8 Pb tolerance efficacies of gamma exposed fungi .............................................................

5.9 pH and temperature optimisation for removal of metals by gamma exposed and unexposed fungal strains .................................................................................................

Section III Alteration in metabolic / lignocellulosic enzyme activity and ultrastructure of fungi after gamma exposure ..................................................

5.10 Effect of gamma on metabolic/lignocellulosic enzyme activities and ultrastructure of Aspergillus terreus stressed with Cd and Zn .......................................

5.11 Effect of gamma on metabolic/lignocellulosic enzyme activities and ultrastructure of Aspergillus niger stressed with Cd ,Zn and Pb .............................................................

5.12 Effect of gamma on metabolic/ lignocellulosic enzyme activities and ultrastructure of P.cyclopium grown in Cd and Pb riched media ..................................

5.13 Effect of gamma irradiation on metabolic/lignocellulosic enzyme activities and ultrastructure of A.terreus, A.niger and P.cyclopium ....................................................

Section IV Possible mechanism for metallo-resistance in fungi in response to gamma exposure .................................................................................................

5.14 Antioxidative enzymes (SOD ,CAT) and antioxidant protein (GSH) response in gamma exposed Aspergillus niger and A.terreus grown in Zn enriched growth media ...............................................................................................................

5.15 Antioxidative enzymes (SOD ,CAT) and antioxidant protein (GSH) response in gamma exposed Aspergillus terreus, A .niger and Penicillium cyclopium grown in Cd enriched growth media ............................................................................

5.16 Antioxidative enzymes (SOD ,CAT) and antioxidant protein (GSH) response in gamma exposed A .niger and P. cyclopium grown in Pb enriched growth media ...........................................................................................................................

5.17 Antioxidative enzymes (SOD ,CAT) and antioxidant protein (GSH) response in gamma exposed A .niger and P. cyclopium ..............................................................

5.18 Gamma radiation induced modulation of Metallothionein in A.terreus, A.niger and P.cyclopium with or without metal stress ..............................................................

6 General Discussion ............................................................................................................

7 Conclusion and major highlights ...................................................................................... 8 Bibliography ...................................................................................................................... Publications & List of papers presented in conferences ..................................................

1. Introduction

1.1 Environmental pollution, a global scenario

Substantial increase in agricultural, industrial and technological growth, in addition to

population escalation resulted in deterioration of environmental quality in a global scale.

Rapidly growing cities, more traffic on roads, growing energy consumption and waste

production together with lack of strict implementation of environmental regulation are

increasing the discharge of pollutants into air, water, and soil (Agarwal, 2005). Air pollution

is the result of emissions from a variety of sources, mainly stationary, industrial and domestic

fossil fuel combustion, and petrol and diesel vehicle emissions (Brulfert et al., 2005; Parra et

al., 2006). Fossil fuels, the primary source of energy consumption, are the greatest source of

ambient air pollution, producing nitrogen oxides, sulphur-oxides, dust, soot, smoke, and other

suspended particulate matter. Most Asian cities cannot meet the terms with the World Health

Organisation (WHO) air quality guidelines or the US Environmental Protection Agency

standards (USEPA); except some cities of developed countries such as Singapore, Taiwan,

and Japan. Several Asian cities in China, India, and Vietnam have the highest levels of

outdoor air pollution in the world (Ta-chen et al., 2011). Developing nations are particularly

affected by air pollution; as many as two thirds of the deaths and lost of life years associated

with air pollution on a global scale occur in Asia (Cohen et al., 2004).High growth has been

achieved with severe environmental damages such as deforestation, widespread acid rain and

deteriorating ambient air quality. These consequences threaten human living space and

health, and are costly to deal with. In 2010 terms, with population increased to about 1.2

billion in India, and it is estimated that the damage and degradation of natural resources is

equivalent to about 10% of the country’s Gross Domestic Productivity (GDP) (Gatdula et al.,

2011). Today India is one of the top ten industrialized country of the world (UNIDO, 2011)

which facilitate India to be the third largest emitter of carbon dioxide in 2009. Of the four

major Indian cities, air quality was consistently worst in Delhi, Kolkata holds a second

position, followed by Mumbai and Chennai. Most Indian cities continue to violate India's and

world air quality PM10 (particulate matter having diameter <10nm) targets. Respirable

particulate matter (RSPM) pollution remains a key challenge for India. Most Indian cities

greatly exceed acceptable levels of suspended particulate matter (SPM). A decreasing trend

has been observed in sulphur dioxide and nitrogen dioxide levels in residential areas of many

cities such as Delhi, Mumbai, Lucknow, Bhopal during last few years. The conservative

estimates of IISC (Indian Institute of Soil Sciences, 2011) showed that the demand for food

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2 | P a g e

grains would increase from 192 million tonnes in 2000 to 355 million tonnes in 2030. In

contrary to increasing food demands, the factor productivity and rate of response of crops to

applied fertilizers under intensive cropping systems are declining year after year; which

directly and indirectly affect soil health. Simultaneously water pollution is an appalling

problem, powerful enough to lead the World towards destruction. Becoming an easy solvent

most of the pollutants get dissolved in water and contaminate the same. Increasing

population load, demand of food, increasing industrialisation and agricultural activities

concurrently welcome water scarcity and obviously deterioration of water quality. It is

projected that most irrigated areas in India would require more water by the year 2030 and

global net irrigation requirements would increase relative to the situation without climate

change by 3.5–5% by 2025, and 6–8% by 2075 (IISC report,2011). Specifically, to meet

needs and well being of mankind, the overall process of development, especially urbanization

and industrialization have contributed to an ever-increasing deterioration of quality of vital

natural resources, namely air, soil and water.

1.2 Heavy Metal Pollution

Environmental contamination through discharge of hazardous effluents from various

industries has become a serious global issue. Heavy metals constitute a major component of

such unwanted industrial effluents. Heavy metals are natural constituents of the earth's crust,

but indiscriminate human activities have drastically altered their geochemical cycles and

biochemical balance. Effluents from industries such as electroplating, paints, plastics, tannery

and battery are important sources of heavy metals. Mining and electroplating industries

discharged aqueous effluents containing high levels of heavy metals like uranium, cadmium,

mercury and copper (Scott et al.,1986; Mullen et al.,1989; El-Shafey, 2010). Pollution

caused by these heavy metals has created an alarming situation in recent years. Increase in

industrial activities and not applying to modern methods of effluent discharge has seriously

augmented heavy metal release. Unlike organic chemicals /contaminants which can be

degraded to harmless chemical species, heavy metals cannot be destroyed. Being persistent in

nature they easily get accumulated in food chain causing critical concern to human health and

environmental issues.

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Fig 1.1 : Sources and sink of heavy metals in environment

Most of them are contaminants in nature even at low concentrations and can be present in

soluble form that are extremely toxic to biological systems and most of them can be treated as

carcinogenic and mutagenic factor (Alloway,1995; Diels et al., 2002). Contamination of

agricultural soil with heavy metals is a major problem all over the world. Heavy metals like

cadmium, lead, chromium, copper and nickel contaminate the soil, ground water, sediments

and surface waters are extremely toxic to biological and ecological system. Irrespective of the

origin of the metals in the soil, excessive levels of many metals can result in soil quality

degradation, crop yield reduction, and poor quality of agricultural products, posing significant

hazards to human, animal, and ecosystem health. Prolonged exposure to heavy metals can

cause deleterious health effects in humans. Cadmium (Cd) a common environmental

contaminant even at very low concentration, known to increase oxidative stress by being a

catalyst in the formation of reactive oxygen species. At the molecular level, cadmium binds

to the sulfur (-SH) groups of proteins, cysteine, and glutathione and inhibits the function of

these bio-molecules (Waisberg et al. 2003). Zinc (Zn) occupies an important position in the

series of heavy metal pollutants that gets its source from various factories and industries like

those involved in strip mines, coal burning power plant, smelters etc. Zinc is an essential

divalent metal ion in all life forms where it serves catalytic and structural roles (Pikalova et

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4 | P a g e

al., 2011). However, at high concentrations, zinc becomes strongly poisonous causing

inhibition of growth and metabolism and even death of organisms (Zenk, 1996; Vaillant et

al., 2005). Incineration of municipal and medical waste and emissions from coal-using power

plants contribute to high levels of mercury. The toxic effects of mercury depend on its

chemical form and the route of exposure. Methyl mercury [CH3Hg] is the most toxic form. It

affects the immune system, alters genetic and enzyme systems, and damages the nervous

system, including coordination and the senses of touch, taste, and sight (Rodriguez et al.,

2005; Chang et al., 2009).The main sources of lead entering an ecosystem are atmospheric

lead (primarily from automobile emissions), paint chips, used ammunition, fertilisers and

pesticides and lead-acid batteries or other industrial products. Lead affects the central nervous

system of animals and inhibits their ability to synthesize red blood cells (Barbosa et al., 2005;

Patrick, 2006).

1.3 Lignocellulosic waste pollution

Rising energy consumption, depletion of fossil fuels and increased environmental

concerns have shifted the focus on biofuel based energy generation. The term biofuel is

referred to as liquid or gaseous fuels that are predominantly produced from biomass. Biomass is organic material which has stored sunlight in the form of chemical energy.

Biomass includes crops, crop wastes, trees, wood waste and animal waste. Some examples of

biomass also include wood chips, corn, corn stalks, soybeans, switch grass, straw, animal

waste and food-processing by-products. Plants are the major sources of biowastes. As

through the process of photosynthesis, chlorophyll in plants captures the sun's energy by

converting carbon dioxide from the air and water from the ground into carbohydrates,

complex compounds composed of carbon, hydrogen, and oxygen. Lignocellulose is a

renewable organic material and is the major structural component of all plants. In addition to

heavy metal pollution, lignocellulosic materials produced by the agricultural industry,

forestry stations and different wastes from municipal solid waste (MSW) are main portions of

lignocellulosic wastes (Kim and Dale, 2004; Kalogo et al., 2007). The major constituents of

lignocellulose are cellulose, hemicellulose, and lignin, polymers those are closely associated

with each other constituting the cellular complex of the vegetal biomass.

However, due to their chemical composition based on sugars and other compounds of

interest, lignocellulosic materials could be utilized for the production of a number of value

added products, such as ethanol, food additives and organic acids others. These precious

materials were treated as waste in many countries in the past and still are today in some

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5 | P a g e

developing countries causing environmental pollution as they are accumulated every year in

large quantities (Levine,1996; Palacios et al., 2005). Therefore, besides the environmental

problems caused by their accumulation in the nature, the non-use of these materials

constitutes a loss of potentially valuable sources. These materials if found in the environment

together with the toxic heavy metals pose serious threat and make the situation more alarming

in terms of increase in pollution load of the concerned ecosystem. Therefore developing

strategies for proper degradation and detoxification of the biowastes together with metal

contaminants is now being perceived as the need of the hour.

1.4 Soil heavy metal pollution

Soil of the cultivable or agricultural land has undergone severe degradation in quality

due to indiscriminate emission of effluents and solid discharge from industries, use of

insecticides or pesticides for controlling parasites and pests to have better yield of the edible

crops. Electroplating, mining industries, agrochemicals and sewage sludges mainly release

effluents containing heavy metals. Apart from having heavy metals as its natural constituent,

soil acts as the major sink for various heavy metals released into the environment by several

anthropogenic activities. Heavy metals are a group of inorganic chemical hazards. According

to (GWRTAC) reports (1997) several heavy metals like Lead (Pb), chromium (Cr), arsenic

(As), zinc (Zn), cadmium (Cd), copper (Cu), mercury (Hg), and nickel (Ni) are commonly

found in all contaminated areas. As such being an important part of ecosystem, soil heavy

metal pollution constitutes to a major share of environmental pollution resulting into

distribution in various trophic levels through food chain transfer (Mclaughlin et al., 2000,

Ling et al., 2007).

To control heavy metal deposition in layers of soil various techniques and strategies have

been into operation. Immobilization, soil washing, and phyto-remediation techniques are

frequently planned among the best-demonstrated accessible technologies (BDATs) for

remediation of heavy metal-contaminated soils (GWRTAC reports, 1997). Due to inadequate

awareness and lack of principles of operation, the instrument based techniques of soil

remediation are used mainly in developed countries. In developing countries low cost and

ecologically sustainable remedial options are mainly adopted to renovate contaminated land

and to reduce the associated risks.

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6 | P a g e

1.4.1. Soil microbes in restoring ecosystem

Soil, in addition to maintaining environmental restoration, plant health, has an

important role in the ecosystem for being habitat of microorganisms. These microbes exist in

large numbers in the soil as long as there is a carbon source for energy. The physical

structure, aeration, water holding capacity and availability of nutrients are determined by the

components (inorganic as well as organic) of soil.Formation of these components are

dependent on metabolic activities of the soil microorganisms and weathering of rock. At the

same time microbes are an important part of the soil as they have the capability in

maintaining soil fertility, cycling of nutrient elements in the biosphere .In addition to this soil

micro-organisms play a major role in maintaining heavy metal homeostasis of soil. The most

diverse group of microorganisms living in soil are bacteria followed by fungi and archaea.

Since bacteria are very tiny in size, they are able to move short distances by actively

swimming, but most move passively with moving soil water as they prefer a semi-aquatic

habitat. Whereas fungi can move more actively through soil, by growth of their thread-like

hyphae. Actinomycetes are a factor of 10 times lesser in number but are larger in size so they

are similar in biomass to bacteria. In the uppermost layer of soil, fungi are more abundant in

terms of biomass. Along with bacteria, fungi are important as decomposers in the soil food

web, converting hard to digest organic material into usable forms. Fungi have 40–55%

carbon utilization efficiency so they store and recycle more carbon (C) compared to bacteria.

Bacteria are generally less efficient in converting organic carbon to new cells. Aerobic

bacteria assimilate about 5-10% of the carbon while anaerobic bacteria only assimilate 2-5%,

leaving behind many waste carbon compounds and inefficiently using energy stored in the

soil organic matter (Hoormann et al., 2010). Bacteria are less efficient at retaining C and

release more into the air as carbon dioxide (Magdoff et al., 2001).

Fungi can survive in the soil for long periods even through periods of water deficit by living

in dead plant roots as spores or fragments of hyphae. Fungi in general easy to grow and

produce high yields of biomass and are agreeable to genetical and morphological

manipulation affected by changes in soil characteristics. Precisely, thus diversity of fungal

population in different soil regime may serve as an indicator not only for physico-chemical

parameters of soil of the particular region but also the adaptability of the concerned fungi.

Earlier researchers reported the capability of a range of fungi to survive and grow in the

presence of potentially toxic concentrations of metals (Ross, 1975; Gadd, 1986a; Baldi, et al.,

1990; Turnau, 1991). In general terms, toxic metals are believed to affect fungal populations

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7 | P a g e

by reducing abundance and species diversity and selecting a resistant/tolerant population

(Babich & Stotzky, 1985; Duxbury, 1985). However, the effect of toxic metals on microbial

abundance in natural habitats varies with the metal species and organisms present and with

environmental factors (Gadd & Griffiths, 1978; Duxbury, 1985).

A diverse spectrum of lignocellulolytic microorganisms, mainly fungi (Baldrian and Gabriel,

2003; Falcón et al., 1995) and bacteria (McCarthy, 1987; Vicuna, 1988; Zimmermann, 1990)

have been isolated and identified over the years and this list still continues to grow rapidly.

Soil fungi seem to be the most important players in lignocellulose transformation processes

by means of extracellular enzymes (lignocellulosic enzymes; biocatalysts for degrading

cellulosic and lignin material) due to their ability to attack both polysaccharides and

polyphenols in the soil organic matter. Unlike bacteria, fungi do not assimilate contaminants

as a single source of carbon and energy; thus, fungi require an additional carbon source to

support their growth. During transformation, lignocellulose is incorporated into fungal

biomass or serves as an energy source while its remains are transformed into humic

substances. Throughout transformation in soils, humic substances (humus, humic and fulvic

acids) are formed from both lignocellulose and structural components of microbial

decomposers. This is achieved through the intensive action of lignocellulose-degrading

enzymes, whose activity is regulated by their microbial producers, soil properties and land

use. Lignocellulolytic fungi are often divided into three groups namely white rots, brown rots,

and soft rots. White rots break down lignin and cellulose and brown rots primarily decay the

cellulose and hemicellulose (carbohydrates) in wood. They decay cellulose, hemicellulose,

and lignin but only in areas directly adjacent to their growth (Mtui, 2012). Soft rots grow

more slowly than brown and white rots and usually do not cause extensive structural damage

to wood of living trees (Hickman and Perry, 2010).

1.5. Role of fungi in Pollution Abatement

For proper abatement of heavy metal pollution, strategies are being planned and

various methods are being implemented by Government and other public sectors. Traditional

processes for removing metals are in general costly and risky for its possibility of generating

hazardous by-products. Furthermore, those treatment procedures (mostly chemical and

physical viz. ion exchange, chemical precipitation, reverse osmosis and evaporative recovery)

are mostly expensive and less effective. As such, bioremediation is now attaining more focus

and considered as an effective tool for abatement of metal pollution for its low cost and high

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8 | P a g e

efficacy. Use of microbiological methods for environmental remediation has come up as a

appreciable concept which often acts as an eco-friendly approach. For controlling metal

pollution through bioremediation, different micro organisms like bacteria, fungi, algae and

yeasts are usually utilized as they have the potential to internally accumulate different heavy

metals. Microbial population in metal polluted environments adapt in different way to

become metal resistant (Prasenjit and Sumathi, 2005).

1.5.1. Heavy metal pollution abatement: contribution of fungi

Bioremediation can be an effective method for controlling metal pollution from

environment. Fungi are being considered as an important agent for bioremediation because

of some unique natural characteristics inherent within the fungi. Firstly they have a wide

range of morphological types (unicellular to filamentous forms) secondly they have higher

surface to volume ratio and extensive hyphal reach in soil. Survivability in higher

concentration of metals, large scale availability and ease to harvest make them better

candidate with potential for bioremediation. It has also been known that they can survive in

higher concentration of metals. However, it is interesting to note that involvement of soil

fungi in the process of bioremediation can be considered as an example of mutual benefit

oriented relationship between the two components (soil: abiotic and fungi : the biotic

component) of the ecosystem. While the metal burden of the soil is diminished by the

process, the fungi in turn utilises the metal for its normal growth and metabolism as

evidenced in natural, laboratory and industrial environments (Gadd, 1993). Fungal

technologies to treat contaminated soil thus have become a better alternative provision over

conventional technologies like stabilization and combustion. Majority of fungi belongs to the

filamentous growth form which helps them to colonize on substrates. Several fungal biomass

have been extensively used for biosorption of metal ions and radio nuclides from

contaminated soils (Kapoor and Viraraghavan, 1995). Potential of filamentous fungi in

bioremediation of heavy metals containing industrial effluents and waste waters has been

increasingly reported from different parts of the world for their capability of long hyphal

reach in depth of soil (Gadd, 1993; Pal and Das, 2005; Wang and Chen, 2009). A. niger has

been used to remove metals from the environment by either adsorption of the metals to fungal

cell wall components, or complexation of the metals with organic acids produced by the

fungus (Akthar and Mohan, 1995; Bosshard et al., 1996). de Rome and Gadd (1987) reported

Cu removal efficacies of Rhizopus arrhizus ,Cladosporium resinae and Penicillium italicum.

Whereas Zhou and Kiff (1991) accounted about the Cu removal efficacies by immobilized

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9 | P a g e

fungal biomass, simultaneously A. carbonarius was capable of removing Cu and Cr was

reported by Al-Asheh and Duvnjak (1995). Heavy metal biosorption capacity by soil fungi

A.niger and Mucor rouxii were reported by Mullen et al., (1992). Other fungi like

Saccharomyces cerevisiae and R. nigricans were also capable of removing metal cations and

lead (Brady and Duncan,1994 and Zhang et al., 1998). Specifically, Fusarium species has

been used as biosorbents, and Penicillium simplicissimum has been used to precipitate metals

from solution (Burgstaller and Schinner, 1993; Gadd, 1993; White et al., 1997). Fungi also

have been utilized for removal of eutrophication agents and bioremediation of metal

contaminated waste streams (Thanh and Simard, 1973; Akthar and Ghafar, 1986; Akthar and

Mohan, 1995; Bosshard et al., 1996).

1.5.1.1. Inherent mechanism adapted by fungi for being metal resistant

Fungi are known to detoxify metals by several mechanisms including ion exchange,

chelation, adsorption, crystallization, valence transformation, extra and intracellular

precipitation and active uptake. Most important part of this mechanism is metal uptake. Metal

uptake by fungi mainly characterized in three sectors: (1) biosorption of metal ions on the

surface of fungi, (2) intracellular uptake of metal ions, and (3) chemical transformation of

metal ions by fungi (Ashida,1965; Gadd, 1993). Biosorption can use both living and non-

living biomass in the processes as it frequently exhibits marked tolerance towards adverse

conditions like heavy metals. The contribution of cell-wall binding to metal tolerance in fungi

has been extensively reviewed (Meharg, 2003). The fungal cell wall is the first site of direct

interaction (there could be excreted substances ahead) between the fungus and the metal. Its

composition implies glucan-, chitin- and galactosamine-containing polymers, and a minor

amount of proteins. Thus a large number of potential-binding sites are exhibited by free

carboxyl, amino, hydroxyl, phosphate and mercapto groups (Strandberg et al., 1981).

Different organic molecules, and in particular di- and tricarboxylic acids that do not belong to

the matrix of the cell wall, are excreted by fungal cells to chelate metal ions .The induction of

oxalic acid efflux correlated closely with Cu tolerance in brown rot fungi (Green & Clausen,

2003), and over excretion of oxalic acid probably contributed to the metal tolerance exhibited

by Beauveria caledonica (Fomina et al., 2005).

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10 | P a g e

Fig: 1.2 Schematic representation of cellular mechanisms potentially involved in metal

tolerance in ectomycorrhizal fungi (Ref: Bellion et al., 2006). M:metal-ion.

[1 Extracellular chelation by excreted ligands (L); 2 Cell-wall binding; 3 Enhanced efflux;

4 Intracellular chelation by metallothionein (MT);5 Intracellular chelation by gluthathione (GSH); 6

Subcellular compartmentation (vacuole or other internal compartments); 7 Vacuolar

compartmentation of GSH-M complex]

Different potential extracellular and cellular mechanisms are involved in metal

tolerance of fungi in environment. These includes mechanisms that reduce uptake into the

cytosol by extracellular chelation or binding onto cell wall components, intracellular

chelation of metals in the cytosol by array of ligands or efflux from the cytosol into

sequestering compartments. In the developmental process fungi need to contact with the

environment constantly, which subjected them to face different physical and chemical

factors. Any chemical or physiological stress (metal and gamma exposure) may lead to the

production of Reactive Oxygen Species (ROS) such as superoxide anion radical (O2·-),

hydrogen peroxide, and hydroxyl radical (OH·) (Fridovich, 1995). Over production of ROS

can cause damage to DNA, lipids and proteins (Fridovich, 1983; Stadtman and Levine, 2000;

Halliwell and Gutteridge, 2007). The aerobic cells are able to cope with ROS toxicity by

virtue of a unique set of antioxidant enzymes that scavenge O2 and H2O2, and prevent the

formation of OH·. A firstline of defense against ROS includes the superoxide dismutase

(SOD) enzymes that catalyze dismutation of superoxide to H2O2 and oxygen (Hassan and

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11 | P a g e

Scandalios, 1998). SOD helps to shunt the formation of lipid hydroperoxides .This shunting

activity is beneficial for cell, since cells have many defence systems to deal with peroxides,

often overlapping or reductant in specificity. There are four known classes of SODs: Mn, Fe,

Ni, and Cu, Zn. They not only showed their expressional variation but also localisation in the

cell. In Saccharomyces cerevisiae (having two of these SODs, Cu, Zn enzymes; SOD1) is

localised in the cytoplasm and the mitochondria, while Mn enzyme is located in

mitochondrial matrix. Secondly Catalase (CAT) an antioxidant metalloenzymes, which

triggers to detoxify H2O2 to oxygen and hydrogen molecule and peroxidases are also

involved in antioxidative defense system (Scandalios, 2005).

Apart from enzymatic defense, there are different metabolites that are important scavengers

of ROS named as ascorbic acid and its derivatives, glutathione, metallothioneins, proline,

trehalose, polyols as well as pigments such as carotenoids and melanins are present in fungal

cell acting against ROS. Glutathione (GSH), a tripeptide (L--glutamyl-L-cysteinyl-glycine)

with a carrier of an active thiol group in the form of a cysteine residue, acts as an antioxidant

either directly by interacting with reactive oxygen/nitrogen species (ROS and RNS, resp.)

The reduced and oxidized forms of glutathione (GSH and GSSG) act in concert with other

redox-active compounds (e.g., NAD (P)H) to regulate and maintain cellular redox status (

Gessler et al., 2007)

Metallothionein (MT) acts as a metal storage , transport and metal detoxification (Peterson et

al.,1996) , it has been documented to bind a wide range of metals including cadmium, zinc,

mercury, copper, arsenic, silver, etc (Freisinger, 2013).

1.5.2 Fungi in Lignocellulosic waste management

In addition to heavy metal pollution, lignocellulosic materials, produced by the

agricultural industry and forestry stations, are main portions of biowastes, causing

environmental pollution as they are accumulated every year in large quantities (Musatto and

Teixeria, 2010). However, due to their chemical composition based on sugars and other

compounds of interest, they could be utilized for the production of a number of value added

products, such as ethanol, food additives, organic acids, enzymes (cellulose, amylase), and

others. Therefore, besides the environmental problems caused by their accumulation in the

nature, the non-use of these materials constitutes a loss of potentially valuable sources. These

materials if found in the environment together with the toxic heavy metals pose serious threat

and make the situation more alarming in terms of increase in pollution load of the concerned

ecosystem. Therefore developing strategies for proper degradation and detoxification of the

biowastes together with metal contaminants is now being perceived as the need of the hour.

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12 | P a g e

Industrially important enzymes, namely amylases, cellulase, lipase, xylanase, laccase have

their inherent characteristics which ensure the significant role of these enzymes in pollution

management. Amylases hydrolyze glycosidic bonds in starch and convert to glucose, maltose,

maltotriose, dextrin, while cellulase catalyses formation of glucose from cellulose of

lignocellulosic compounds. Thus, these enzymes not only serve industrial purposes, but can

also help to control biowastes (cellulosic wastes) and plastic polymers. Ligninolytic enzymes

also responsible for degrading different pesticides and herbicides. Like lignocellulosic

enzymes from Phenerochaete chrysosporium are efficient biodegraders of wastewater from a

coke plant. It has been reported by Lu et al., (2009) that Phenerochaete chrysosporium could

remove about 84% phenolic compounds and 80% COD from the wastewater at pH and

temperature ranges of 4-6 and 28 to 37°C, respectively after only 3 days of incubation

.Moreover, laccases ,ligninase and peroxidases from white rot fungi such as Trametes

versicolor., Pleurotus sp., Polystictus sp. and P. chrysosporiun are also able to degrade a

wide range of xenobiotic compounds including agricultural chemicals (pesticides and

herbicides) such as Dieldrin, Simazine, DDT, Triazines, Trifluralin, Diuron, Diazinon

(Magan et al., 2010; Purnomo et al., 2010).

1.6 Recent trend in biotechnology for making microbes more potential

Research and development in biotechnology has been increasingly adding benefits in

all fields relevant to the needs and wellness of mankind. Application of genetic engineering

and biotechnology are being employed for extracting more usability and to gain better

economic output. Though the natural process of phyto-remediation has become a

considerable idea and has been taken as green technology for heavy metal pollution control

(Rupassara et al., 2002; Marchiol et al., 2007). Bioremediation using microbial biomass is

also becoming a major scientific approach for combating environmental pollution as

microbes grasp distinct advantages over other biosorbents due to its inherent properties

detailed elsewhere. Utilising the knowledge pertaining to normal production of

lignocellulosic enzymes at a very low concentration (Szengyel et al., 2000; Chand et al.,

2005; Li et al., 2009; Pradeep and Narasimha, 2011) by several microbes, a drive towards

qualitative and quantitative enhancement in enzymes has been experienced amongst recent

day researchers. To accomplish such enhancement in qualitative and quantitative manner in

various Industrial as well as Environmental application microbial strain improvement has

been found to be taken up as a major research focus .Normally research of strain

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13 | P a g e

improvements are carried out and or by mutation and genetic recombination. In case of the

former induced mutation deserves mention which could be preferred by different methods

such as chemical and physical mutagen, sexual hybrids, homocaryons, directed mutagenesis,

protoplast fusion, recombination and transformation (Stasz, 1990; Nevalainen, 2001; Haggag

and Mohamed, 2007). Currently the use of mutagens (physical and chemical) is attaining an

important arrangement in fungal strain improvement for its wide application in different

sectors. Various researchers (Zaldivar et al., 2001; Nakkeeran et al., 2005) reported the use of

chemical mutagens (1- Dimethoite (D); N-methyl-N-nitro-N-nitrosoguanidine (MNNG) on

microbes specially on fungi for its better output with reference to enzyme production or to

modulate the biocontrol potential of the concerned fungi.

1.6.1 Ionising radiation in modulating properties of fungi

It is being established that microbial strain improvement can possibly be done by

inducing mutation using physical stress effectors. Exposure to ionizing radiation is one such

physical stress effector known to be used for strain improvement of microbes. Fungi in

general and specifically melanised ones are highly radio resistant when subjected to high

doses of ionising radiation under experimental condition (Saleh et al., 1988; Dadachova et al.,

2004). The energy and penetration power of gamma rays might be used effectively to kill

pathogenic microorganisms and for sterilization of microorganisms infected foods. Such

treatments do not leave toxic residues and can be conducted at ambient temperature. Higher

doses of radiation is mainly used as an excellent tool for food or pharmaceutical sterilisation,

food preservation and different food engineering process, which ultimately benefit for the

society (Dusan, 2004; Hyun-Pa et al., 2006; Sameh et al, 2006). Earlier reports showed that

high doses of gamma radiation cause dose-dependent inhibitory effects on fungal colony

growth infecting seed storage life (Maity et al., 2009). Similar findings with respect to

inhibition of colony growth of Alternaria sp in response to gamma exposure was reported by

Aziz et al., (2001), where they informed 4KGy of gamma inhibited the fungal growth and

toxin production in tomato paste and juice. Sterilization by gamma irradiation also helps in

preserving the quality of processed food /seed as no or very negligible degradation is

observed compared to other techniques, thus reducing the risk for consumers (WHO/FAO,

1988; WHO, 1999; Maity et al., 2009).There are many reports supporting the use of gamma

irradiation as a fungicidal agent (Maity et al., 2008, 2009; Aziz et al., 2007) where gamma

was applied at higher doses.

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14 | P a g e

In contrary to the gamma induced microbial growth control at higher doses, low dose

of irradiation causes stimulatory effect in fungi. Cordeiro et al., (1995) reported that exposure

to gamma showed maximum potential to induce mutation in fungi (Metarhizium anisopliae)

than that of UV or other chemical mutagens. It may be conjectured from earlier reports that

gamma irradiation is an effective mutagenic agent for fungi (Mutwakil, 2011). Low

experimental doses of gamma radiation (5- 100Gy) was observed by earlier researchers to

boost up spore germination of Botrytis cinerea and Penicillium expansum (Geweely and

Nawar, 2006)., In case of Aspergillus tenuissima and S. boryosum stimulatory effect of

gamma was observed at dose upto 250Gy, with further increase of absorbed dose percentage

of germination decreased with an inhibition at an optimum dose (Geweely and Nawar, 2006),

Stimulating effects of radiation on fungal growth and other physiological parameters have

been reported earlier, they observed higher CFUs, increased biomass and faster accumulation

of acetate in melanin containing fungi isolated from higher radiation levelled environment

(Dadachova et al., 2006). Abostate et al., reported higher production of cellulases (CMCase,

Avicelase) by gamma irradiated (0.5 K Gy) Aspergillus than that of the parental non-

irradiated counterparts (Abostate et al., 2010). Fawzi and Hamdy, (2011) observed different

doses of gamma irradiation could induce enhanced production of CMCase in Chaetomium

cellulolyticum.

1.7 Focus of this work

However, no reports are available where gamma is used to improve metal tolerance in

fungi, while some groups have worked on use of UV radiation to induce metal resistance in

bacteria (Ling et al., 2007; Dib et al., 2008). Some groups have reported about developing

metal resistant fungi by chemical applications only (Levine and Marzluf, 1989; Pali et al.,

2007). Therefore, this work is focused on exploring the possibility to utilize gamma induced

enhancement of metal tolerance in fungi for its prospect to be used in bioremediation as well

as to describe the potential of gamma in lignocellulosic enzyme function against metal stress.

Simultaneously it has been tried to evaluate some possible mechanism (antioxidant defence

system) adopted by the gamma exposed fungi for being more metal resistant with respect to

their un-irradiated counterparts.

2. Review of Literature

2.1. Heavy metals – the problem and its control

“Heavy metal” is a general collective term, which applies to the group of metals and

metalloids with atomic density greater than 4000 kg m-3, or 5 times more than water

(Garbarino et al., 1995) and they are natural components of the earth’s crust. Although

some of them act as essential micro nutrients for living beings, at higher concentrations they

can lead to severe poisoning (Lenntech, 2004). The most toxic forms of these metals in their

ionic species are the most stable oxidation states e.g. Cd2+, Pb2+ , Hg2+ , Ag+ and As3+ in

which, they react with the body’s bio-molecules to form extremely stable biotoxic

compounds which are difficult to dissociate (Duruibe et al., 2007). Biosphere pollution by

chemicals and heavy metals such as cadmium (Cd), nickel (Ni), zinc (Zn), lead (Pb), copper

(Cu), mercury (Hg) etc., accelerated dramatically during the last few decades due to mining,

smelting, manufacturing, use of agricultural fertilizers, pesticides, municipal wastes, traffic

emissions, industrial effluents and industrial chemicals etc. The problem of environmental

pollution due to toxic metals has begun to cause concern now in most major metropolitan

cities. Toxic heavy metals entering the ecosystem may lead to geoaccumulation,

bioaccumulation and biomagnification. Environmental pollution with heavy metals is a

global issue. It is present everywhere, though to different degrees and is specific to certain

parts of the biogeosphere. Pollution by heavy metal ions, including mercury, lead and copper

has become a major hazard issue due to their possible toxic effects (Aydin et al., 2008). The

risks of Hg (II) exposure, for instance, may contribute to adverse effects on central nervous

system, pulmonary, kidney functions and chromosomes (Rao et al., 2009). Pb(II) on the other

hand can bioaccumulate through the food chain, while prolonged inhalation of Cu(II) spray is

claimed to cause an increase in the risk of lung cancer (Aydin et al., 2008).Numerous organic

and inorganic compounds, heavy metals, pollute the environment in particular. Living

organisms are not able to prepare and adapt rapidly to a sudden and huge environmental load

with different toxic substances, and therefore, the accumulation of certain elements,

especially of heavy metals with mainly toxic effect, can cause undesirable changes in the

biosphere with unforeseeable consequences (Djukic & Mandic, 2000). In the past decade

many countries have spent billions of dollars trying to clean up contaminated ground water

and soil.

Review of Literature

16 | P a g e

Table 2.1 Some Heavy metals of environment and their concentration limits

Heavy

Metals

Speciation and Chemistry

Concentration

limit

Reference

Lead Pb occurs in 0 and +2 oxidation states. Pb(II) is the more common and reactive form of Pb. Low solubility compounds are formed by complexation with inorganic and organic ligands (humic and fulvic acids, EDTA, amino acids). The primary processes influencing the fate of Pb in soil include adsorption, ion exchange, precipitation and complexation with sorbed organic matter

Surface agricultural soil: 7-20 ppm, Soil levels: up to 300 ppm USEPA, Maximum Contaminant Level (MCL) in drinking water: 0.015

(Smith et al., 1995; Evanko and Dzombak, 1997; WHO, 2000; Hammer and Hammer, 2004)

Chromium Cr occurs in 0, +3 and +6 oxidation states. Cr(VI) is the dominant and toxic form of Cr at shallow aquifers. Major Cr(VI) species include chromate (CrO2

-4) and dichromate (Cr2O2

-7) (especially Ba2+, Pb2+ and Ag+). Cr (III) is the dominant form of Cr at low pH (<4). Cr(VI) can be reduced to Cr(III) by soil organic matter, S2- and Fe2+ ions under anaerobic conditions. The leachability of Cr(VI) increases as soil pH increases

Normal groundwater concentration: < 0.001 ppm MCL of USEPA in drinking water: 0.1 ppm

(Smith et al., 1995; Lenntech, 2004;)

Zinc Zn occurs in 0 and +2 oxidation states. It forms complexes with a anions, amino acids and organic acids. At high pH, Zn is bioavailable. Zn hydrolyzes at pH 7.0-7.5, forming Zn(OH)2. It readily precipitates under reducing conditions and may co-precipitate with hydrous oxides of Fe or manganese

Natural concentration of Zn in soils: 30 - 150 ppm Concentration in plant: 10- 150 ppm Plant toxicity: 400 ppm WHO limit in water: 5 ppm

(Smith et al., 1995; Evanko and Dzombak, 1997; Lenntech, 2004)


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Cadmium Cd occurs in 0 and + 2 oxidation states. Hydroxide (Cd(OH)2) and carbonate (CdCO3) dominate at high pH whereas Cd2+ and aqueous sulphate species dominate at lower pH (<8). It precipitates in the presence of phosphate, arsenate, chromate, sulphide, etc. Shows mobility at pH range 4.5- 5.5

Soil natural conc.: >1 ppm Plant conc.: 0.005- 0.02 ppm Plant toxicity level: 5-30 ppm USEPA MCL in water: 0.005 ppm

(Matthews and Davis, 1984; Smith et al., 1995)

Arsenic As occurs in -3, 0, +3, +5 oxidation states. In aerobic environments, As (V) is dominant, usually in the form of arsenate (AsO4)3-. It behaves as chelate and can co-precipitate with or adsorb into Fe oxy-hydroxides under acidic conditions. Under reducing conditions, As(III) dominates, existing as arsenite (AsO3)3- which is water soluble and can be adsorbed/co-precipitated with metal sulphides.

MCL in drinking water- USEPA: 0.01 ppm WHO: 0.01 ppm

(Bodek et al., 1988; Smith et al., 1995)

Iron Fe occurs in 0, +2, +3 and +6 oxidation states. Organometallic compounds contain oxidation states of +1, 0, -1 and - 2. Fe(IV) is a common intermediate in many biochemical oxidation reactions. Many mixed valence compounds contain both Fe(II) and Fe(III) centers, e.g. magnetite and prussian blue

Dietary Reference Intake (DRI): For adults: 45 mg per day For minors: 40 mg per day

(Holleman et al., 1985)

Mercury Hg occurs in 0, +1 and +2 oxidation states. It may occur in alkylated form (methyl/ethyl mercury) depending upon the pH of the system. Sorption to soils, sediments and humic materials is pH-dependent and increases with pH

Groundwater natural conc: >0.0002 ppm USEPA regulatory limit in drinking water: 0.002 ppm

(Bodek et al., 1988; Smith et al., 1995)


18 | P a g e

Cupper Cu occurs in 0, +1 and +2 oxidation states. The cupric ion (Cu2+) is the most toxic species of Cu e.g. Cu(OH)+ and Cu2(OH)+ .In aerobic alkaline

systems, CuCO3 is the dominant soluble species. In anaerobic environments CuS will form in presence of sulphur. Cu forms strong solution complexes with humic acids

Soil natural conc.: 2- 100 ppm. Normal range in plants: 5- 30 ppm; Plant toxicity level: 30- 100 ppm; USEPA MCL in water: 1.3 ppm.

(Dzombak and Morel, 1990; LaGrega et al., 1994)

2.1.1 Physico-chemical methods of Heavy metal removal from environment

Heavy metal removal from inorganic effluent can be achieved by conventional

treatment processes such as chemical precipitation, ion exchange, and electrochemical

removal.In industry, by far the most widely used process for removal of heavy metals from

solution is that of chemical precipitation ; approximately 75% of the electroplating facilities

employ precipitation treatment using either hydroxide, carbonate, or sulfide treatment, or

some combination of these treatments to treat their wastewaters. The most commonly used

precipitation technique is hydroxide treatment due to its simplicity, low cost of precipitant

(lime), and ease of automatic pH control.

Gopalratnam et al. (1988) found 80% removal of Zn, Cu, and Pb, and up to 96.2% removal of

oil from industrial wastewaters by using a joint hydroxide precipitation and air floatation

system.Lime and limestone are the most commonly employed precipitant agents due to their

availability and low-cost in most countries (Mirbagherp and Hosseini, 2004; Aziz et al.,

2008). However, chemical precipitation requires a large amount of chemicals to reduce

metals to an acceptable level for discharge. Other drawbacks are its excessive sludge

production that requires further treatment, slow metal precipitation, poor settling, the

aggregation of metal precipitates, and the long-term environmental impacts of sludge disposal

(Aziz et al., 2008).


19 | P a g e

Figure 2.1 Processes of a conventional metals precipitation treatment plant (Wang et al.,

2004).

Coagulation / flocculation has been known to be capable of removing heavy metals from

solution. Coagulation refers to the charge neutralization of the particles. Flocculation

involves slow mixing to promote the agglomeration of the destabilized particles. Electro

coagulation is an emerging water treatment technology that has been applied successfully to

treat various wastewaters. It has been applied for treatment of potable water (Vic et al., 1984;

Holt et al., 2002), urban wastewater (Poulet and Grasmick,1995), heavy metal laden

wastewater (Mills,2000).

Flotation depends on the use of a surfactant that causes a non surface active material to

become surface active forming a product that is removed by bubbling a gas through the bulk

solution to form a foam. The use of foam flotation techniques for removal of heavy metals

has been well studied.


20 | P a g e

Ion exchange is an effective means of removing heavy metals from waste waters. It is a

reversible chemical reaction, where the removal of heavy metals is accomplished by the

exchange of ions on the resin for those in wastewater. Recently research has been performed

on the use of liquid ion exchange for removal of heavy metals from plating wastes. The

disadvantage of this method is that it cannot handle concentrated metal solution as the matrix

gets easily fouled by organics and other solids in the wastewater. Moreover ion exchange is

nonselective and is highly sensitive to the pH of the solution. A noticeable disadvantage was

that corrosion could become a significant limiting factor, where electrodes would frequently

have to be replaced.

Cementation is a metal-replacement process in which a solution containing the dissolved

metallic ions comes in contact with a more active metal such as iron. Cementation is thus the

recovery of an ionized metal from solution by spontaneous electrochemical reduction .To the

elemental metallic state with subsequent oxidation of a sacrificial metal (such as iron).

Complexation involves the formation of a complex compound through a complexing or

chelating agent. Sequestration involves the removal of a metal ion from solution by formation

of a complex ion that does not have the chemical reactions of the ion that is removed, in other

words that metal ion is tied up or complexed. However these methods have several

disadvantages such as high reagent requirement, unpredictable metal ion removal, generation

of toxic sludge etc.

Adsorption process being very simple, economical, effective and versatile has become the

most preferred method for removal of toxic contaminants from wastewater. Adsorption is a

process that occurs when a gas or liquid solute accumulates on the surface of a solid or a

liquid (adsorbent), forming a molecular or atomic film (the adsorbate). Activated carbon is

the most widely used adsorbent. It is a highly porous, amorphous solid consisting of micro

crystallites with a graphite lattice, usually prepared in small pellets or a powder. It can

remove a wide variety of toxic metals. Some widely used adsorbents for adsorption of metal

ions include activated carbon (Pollard et al., 1992, Satapathy et al., 2006), clay minerals

(Wilson et al. 2006), biomaterials, industrial solid wastes and zeolites (Wang et al, 2008).

2.1.2. Biological methods for heavy metal removal

Conventional method for heavy metal removal are mostly costly and with inadequate

efficiencies at low metal concentration, particularly in a range of 1-100mg/l (Kapoor and

Viraraghavan ,1995) . Some of this methods generate toxic sludges, the disposal of which is

an additional burden on the techno economic feasibility of treatment procedures. These


21 | P a g e

constrains have caused the search for alternative methods that would be effective for metal

sequestering. Such a possibility offers sorbents of biological methods (Bailey et al.,1999;

Low et al., 2000; Babel and Kurniawan,2003). A definite need was felt to develop a low cost

and eco-friendly technology to remove pollutants particularly heavy metals. Therefore

researchers develop a feasible method to accelerate the process of decay and removal of

heavy metals by encouraging the microbial and associated biota (flora and fauna) within the

ecosystem to degrade, accumulate and/or remove the pollutants from the identified sites. As

such bioremediation offers an attractive alternative. Some examples of bioremediation related

technologies are phytoremediation, bioventing, bioleaching, land farming, bioreactor,

composting, bioaugmentation, rhizofiltration, and biostimulation.

Algae has also been used for removal of heavy metals from wastewaters and for

concentration of valuable metals from dilute solutions (Filip et al., 1979; Hasset et

al.,1980).Mane et al., (2011) have used pretreated algal biomass to remove one of the heavy

metal namely selenium.They used algae isolates Viz., Spirogyra sp and Nostoc community

for this experiment. They observed that the Spirogyra sp and Nostoc community, when

treated physically or chemically are able to remove selenium to considerable extent. They

also observed that pretreatment of biomass with NaOH showed increase in biosorption of

selenium in comparison with living biomass. Wang and Chen (2009) have carried out survey

of the biosorbents used for heavy metal removal . Their study emphasizes the potential of

biomass in wastewater treatment application, especially heavy metal removal. Sloan et al .,(

1984) studied the removal of four different metals (Cd, Cu, Pb, and Zn) at different

concentrations using three different algal species. Cadmium, copper, and lead could be

removed in a two stage process with the first stage being either ion exchange or passive

adsorption and the second stage being removal from solution by passive diffusion through the

cell membrane.

Different macrophytes (spermatophytes, pteridophytes, bryophytes) are also reported as

efficient heavy metal accumulator. Their ability to hyperaccumulate heavy metals make them

interesting research candidates especially for the treatment of industrial effluents and sewage

waste water (Mkandawire et al. 2004; Upadhyay et al. 2007;Mishra et al. 2009; Rai 2010).

Aravind et al. (2009) reported that supplementation of heavy metal Zn in growth media

containing Cd, resulted in decrease in accumulation of Cd in Ceratophyllum demersum

indicating the existence of metal– metal interactions (Zn and Cd). Azolla, a free-floating, fast

growing, and nitrogen fixing pteridophyte seems to be an excellent candidate for removal,


22 | P a g e

disposal, and recovery of heavy metals from the polluted aquatic ecosystems (Arora et al.

2006; Umali et al. 2006).

2.1.2.1 Microbes in heavy metal removal

Microorganisms are nature’s original recyclers, converting toxic organic compounds

to harmless products, often carbon dioxide and water.Interest in the microbial biodegradation

of pollutants has intensified in recent years as humanity strives to find sustainable ways to

clean up contaminated environments ( Diaz, 2008; Koukkou, 2010) These bioremediation

and biotransformation methods endeavour to harness the astonishing, naturally occurring

ability of microbial xenobiotic metabolism to degrade, transform or accumulate a huge range

of compounds including hydrocarbons (e.g.oil), polychlorinated biphenyls (PCBs),

polyaromatic hydrocarbons (PAHs), heterocyclic compounds (such as pyridine or quinoline),

pharmaceutical substances, radionuclides and metals. For controlling metal pollution through

bioremediation, different microorganisms like bacteria, fungi, algae and yeasts are usually

utilized as they have the potential to internally accumulate different heavy metals. Ashok et

al., (2010), confirmed that different bacterial strains were used to remove heavy metals

present in the aqueous environment. Hanjun et al., (2010) also reported the bioremoval of

cadmium from aqueous environment using endophytic bacterium bacillus species. Many

studies indicated that a number of bacterial species were capable of removing metals from

aqueous environment (Manish et al., 2011). Recently Basha and Rajaganesh (2014) isolated

four heavy metal resistance bacterial strains namely Escherichia coli, Salmonella typhi,

Bacillus licheniformis and Pseudomonas fluorescence isolated from textile industry effluents

were used for the bioremediation of heavy metals from the textile dye effluents.

Fig2.2 Metal-processing features adopted by microbes to be utilized in metal

bioremediation processes


23 | P a g e

2.1.2.1.1 Fungi – an alternative for heavy metal removal

Among microbes, fungi are considered to have better prospective to be used in

bioremediation as they can accumulate more metal due to their high surface to volume ratio

than the other microbes. Fungi are used widely in biotechnology for many processes,

including the production of antibiotics, enzymes, food products, industrial acids, and alcohol.

Fungi also have been utilized for removal of eutrophication agents and bioremediation of

metal contaminated waste streams (Thanh and Simard, 1973; Akthar and Ghafar, 1986;

Akthar and Mohan, 1995; Bosshard et al., 1996). As a result of adaptation to their

environment, fungi have developed unique bioremediation properties. Biological mechanisms

implicated in fungal survival (as distinct from environmental modification of toxicity)

include extracellular precipitation, complexation and crystallization, transformation of metal

species by, e.g. oxidation, reduction, methylation and dealkylation, biosorption to cell walls,

pigments and extracellular polysaccharide, decreased transport or impermeability, efflux,

intracellular compartmentation and precipitation and/or sequestration (Ross, 1975; Gadd &

Griffiths, 1978; Gadd, 1989 a,b,1992; Brown & Hall, 1990; Mehra & Winge,

1991).Biosorption is passive uptake of metals by microbial cells usually results in the

formation of metal-oganic complexes with constituents of microbial cell walls, capsules or

extracellular polymers synthesized and excreted by micro-organisms while bioaccumulation

is energy dependant uptake of metallic cations.


24 | P a g e

Fig. 2.3 Diagrammatic representation of metal interactions with fungi

(The metal species are represented in cationic form (Mn+). All the interactions shown may be involved

in fungal survival and/or metal detoxification)

Metal uptake by living fungi can often be divided into two main phases. The first, which can

also occur with dead cells, is often rapid, metabolism-independent binding to cell walls and

other external surfaces; the second is energy-dependent intracellular uptake across the cell

membrane (Tsezos et al., 1986; Huang et al., 1990). The living cells of

Penicillium,Aspergillus, Trichoderma, Rhizopus, Mucor, Saccharomyces and Fusarium have

been shown to bioabsorb metal ions (Say et al., 2001 ; Tan and Cheng, 2003; Dursun et

al.,2003 (a,b); Ozer and Ozer, 2003) .The living fungal cells of A.niger,M.rouxii and

R.arrhizus have been shown to uptake precious metals such as gold and silver (Mullen et

al.,1992). However, a wide range of accumulation values can occur depending on the species

and location of growth. Caps of Laccaria amethystina exhibited total As concentrations of

100-200 µg g-1 d. wt. (Stijve, et al., 1990; Byrne et al., 1990). In a recent study by Mishra and

Malik (2012) the effectiveness of fungal isolates, Aspergillus lentulus (FJ 172995) for

concurrent removal of heavy metals like chromium, copper and lead from industrial effluent


25 | P a g e

was examined. They inferred that Cr, Cu, Pb, and Ni tolerant Aspergillus lentulus

accumulated a significant amount of each metal. The removal of metals from synthetic

solutions showed the trend like Pb2+ (100%) > Cr 3+ (79%) > Cu2+ (78%) > Ni2+ (42%) after 5

days. Das et al., (2008) made a review on biosorption of heavy metals by various microbes of

which filamentous soil fungi made a crucial role as biosorbent. Being an important metal

accumulator A.niger has been highlighted by a group of researchers. They documented Cr,

Pb, Cu,Cd and Ni uptake capacities of A.niger that ranges from 117.33-1.31 mg/Kg of their

body weight (Mungasavalli et al., 2007, Dursun , 2003(a-b), 2006, Kapoor et al., 1999). It

was reported that A.terreus was capable of uptake 201.1mg/Kg of Pb (Kogej and Pavko,

2001) and 160-180mg/Kg of Cu (Gulati et al., 2002). Fusarium species have been used as

biosorbents, and Penicillium simplicissimum has been used to precipitate metals from

solution (Burgstaller and Schinner, 1993; White et al., 1997). Recently, the high potential of

Penicillium simplicissimum to remove Cd (II), Zn(II) and Pb(II) from aqueous solutions has

been reported (Fan et al., 2008). The initial pH significantly influenced Cd (II), Zn(II) and

Pb(II) uptake. The fungus Penicillium canescence was described to be able of remove the Cd,

Pb, Hg and As ions from aqueous solutions by biosorption. P. canescence showed preference

to binding Pb over Cd, Hg and As ions (Say et al., 2003). The ability of Penicillium

purpurogenum to bind high amounts of Cr (VI) was first reported by Say et al., (2004). Metal

sorption and accumulation depends on diverse factors, such as pH, temperature, organic

matter, ionic speciation and the presence of other ions in solutions (Scheuhammes, 1991;

Fourest and Roux, 1992). Metal uptake capacities of some other fungi are demonstrated in

table 2.1.


26 | P a g e

Table 2.2 Biosorption capacity of some fungal strains

Biomass type Metal Biosorption Reference Candida sp. Cu 4.8 Donmez and Aksu (1999)

Kluyveromyces marxianus Cu 6.44 Donmez and Aksu (1999)

Lentinus sajor caju Cr(VI) 191.24 Bayramoglu et al., (2005)

Mucor hiemalis Cr(VI) 53.5 Tewari et al., (2005)

Mucor rouxii Cd 20.31 Yan and Viraraghavan (2008) Mucor rouxii Zn 53.85 Yan and Viraraghavan (2008)

Mucor rouxii Pb 53.75 Yan and Viraraghavan (2008)

Mucor rouxii Ni 20.49 Yan and Viraraghavan (2008) Neurospora crassa Pb 49.1 Kiran et al., (2005)

Neurospora crassa Cu 12.3 Kiran et al., (2005)

Penicillium simplicissium Cd 52.50 Fan et al., (2008) Penicillium simplicissium Zn 65.60 Fan et al., (2008)

Penicillium simplicissium Pb 76.90 Fan et al., (2008)

Penicillium canescens Cd 102.7 Say et al., (2003b)

Penicillium canescens Pb 213.2 Say et al., (2003b) Penicillium canescens Hg 54.8 Say et al., (2003b)

Penicillium chrysogenum Ni 55 Deng and Ting (2005a)

Penicillium chrysogenum Ni 260 Tan et al., (2004) Penicillium chrysogenum Cr(III) 18.6 Tan and Cheng (2003)

Penicillium chrysogenum Ni 13.2 Tan and Cheng (2003)

Penicillium chrysogenum Zn 6.8 Tan and Cheng (2003)

Penicillium italicum Cu 0.4–2 Ahluwalia and Goyal (2007) Penicillium italicum Zn 0.2 Ahluwalia and Goyal (2007)

Penicillium purpurogenum Cr(VI) 36.5 Say et al., (2004)

Penicillium purpurogenum Cd 110.4 Say et al., (2003a) Penicillium purpurogenum Pb 252.8 Say et al., (2003a)

Penicillium purpurogenum Hg 70.4 Say et al., (2003a)

Penicillium purpurogenum As 35.6 Say et al., (2003a)

Phanerochaete chrysosporium Pb 419.4 Kogej and Pavko (2001) Phanerochaete chrysosporium Cu 20.23 Say et al., (2001)

Rhizopus nigricans Pb 403.2 Say et al.,(2001)

Rhizopus arrhizus Cu 10.8 Dursun et al.,(2003b)


27 | P a g e

Biomass type Metal Biosorption Reference Rhizopus arrhizus Cr(VI) 78 Aksu and Balibek (2007)

Saccharomyces cerevisiae Pb 211.2 Say et al., (2001) Saccharomyces cerevisiae Cu 7.11 Donmez and Aksu (1999)

Saccharomyces cerevisiae Pb 79.2 Al-Saraj et al., (1999)

Saccharomyces cerevisiae Cu 6.4 Al-Saraj et al., (1999) Saccharomyces cerevisiae Zn 23.4 Al-Saraj et al., (1999)

Saccharomyces cerevisiae Hg 64.2 Al-Saraj et al., (1999)

Saccharomyces cerevisiae Co 9.9 Al-Saraj et al., (1999)

Saccharomyces cerevisiae Ni 8 Al-Saraj et al., (1999)

Saccharomyces cerevisiae Cd 35.5–58.4 Park et al., (2003)

Saccharomyces cerevisiae Cr(VI) 32.6 Ozer and Ozer (2003)

Saccharomyces cerevisiae Ni 46.3 Ozer and Ozer (2003) Saccharomyces cerevisiae Pb 270.3 Ozer and Ozer (2003)

2.2 Fungi in lignocelulosic waste degradation

Lignocellulosic biomass comprising forestry, agricultural and agro-industrial wastes are

abundant, renewable and inexpensive energy sources. Lignocellulosic wastes are

accumulated every year in large quantities, causing environmental problems. However, the

huge amounts of residual plant biomass considered as “waste” can potentially be converted

into various different value added products. Fungal lignocellulolytic enzymes are important

commercial bio-products used in many industrial and environmental applications including

chemicals, fuel, food, brewery and wine, animal feed, textile and laundry, pulp and paper,

agriculture and bioremediation (Mtui, 2009). A diverse spectrum of lignocellulolytic

microorganisms, mainly fungi (Baldrian and Gabriel, 2003; Falcón et al., 1995) and bacteria

(McCarthy, 1987; Zimmermann, 1990; Vicuňa, 1988) have been isolated and identified over

the years. Already by the year 1976 an impressive collection of more than 14000 fungi which

were active against cellulose and other insoluble fibres were collected (Mandels and

Sternberg, 1976). Lignocellulosic enzymes are a combination of lignolytic enzymes and

cellulolytic enzymes. Fungal cellulolytic enzymes are a group of hydrolytic enzymes

responsible for cellulolytic and xylanolytic activities. They are mostly extracellular enzymes

produced by a wide range of fungi. Such enzymes include cellulases, hemicellulases,

pectinases, chitinases, amylases, proteases, phytases and mannoses. The white-rot fungi


28 | P a g e

belonging to the basidiomycetes are the most efficient and extensive lignin degraders (Akin

et al., 1995; Gold and Alic, 1993) with P. chrysosporium being the best-studied lignin-

degrading fungus producing copious amounts of a unique set of lignocellulytic enzymes. It is

conventional to consider lignocellulose-degrading enzymes according to the three

components of lignocellulose (lignin, cellulose and hemicellulose) which they attack.

However such divisions are convenient classifications since some cross activity for these

enzymes have been reported (Kumar and Deobagkar, 1996). Production of xylanases by

Aspergillus terreus and Aspergillus niger was performed in different lignocellulosic wastes,

including sugarcane bagasse, rice straw and soya bean hulls (Gawande and Kamat, 1999).

Rice straw, rice bran, red gram husk, jowar straw, jowar spathe and wheat bran were used as

substrates for the production of α-amylase by the fungus Gibberella fujikuroi (Mulimani and

Ramalingam, 2000) and spent brewing grains were used for α-amylase production by

Aspergillus oryzae (Francies et al.,2003). Protease production has also been studied using

different lignocellulosic wastes as solid substrate. In a study on the production of this enzyme

by Aspergillus oryzae from wheat bran, rice husk, rice bran and spent brewing grains, wheat

bran has provided the best production results (Sandhya et al., 2005).Some hydrolytic and

lignolytic enzyme producing fungi and application of those fungi in environmental

detoxification are described in Table 2.2 and Table 2.3.

Table 2.3 Examples of hydrolytic fungi, their substrates, enzymes and applications

(Source: Mtui et al., 2012)

Fungal strain Substrate Enzyme Application Reference

Aspergillus niger, A. japonicus, A. terreus

Municipal solid waste, rice straw

CMcases, beta-glucosidases, xylanases

Wastewater treatment, animal feed.

Gautam et al., 2011; Jahromi et al., 2011.

Daldinia caldariorum Crop residues

Beta-glucosidase, FPase, xylanases

Bioremediation Camassola and Dilon, 2009; Ng et al., 2010.

Lentinus edodes Crop residues CMcase, xylanase

Saccharification,improved animal feed

Elisashvili et al.,2008a, 2008b.

Melanocarpus albomyces

Wheat straw, corn husk xylanase Sachharification Biswas et al.,

2010.

Neurospora sitophila

Banan fruit stalks

Endoglucanase, exoglucanase, β-glucosidases

Saccharification, brewing industry

Asad et al., 2006.


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Fungal strain Substrate Enzyme Application Reference

Penicillium echinulatum

Sugarcane bagasse

Cellulases, xylanases

Improved animal feed

Camassola and Dilon 2009.

Phanerochaete chrysosporium paddy straw

Xlylanase, carboxymethyl cellulase

Improved animal feed, saccharification

Sharma and Arora, 2011.

Phlebia brevispora, P. fascicularia, P. floridensis, P. radiata

Paddy straw Xlylanase, carboxymethyl cellulase

Improved animal feed, saccharification

Sharma and Arora, 2011.

Penicillium janthinellum Crop residues Proteases Detergent

industry Oliveira et al., 2006.

Poria subvermispora

Wheat and paddy straw.

Cellulases, xylanases

Glucose production

Salvachua et al., 2011.

Rhizopus microsporus var. rhizopodiformis

Sugar cane bagasse, corn cob, lemon peel

Endo- and exo- polygalacturonase, (pectinases)

Saccharification, beverage industry

Silva et al., 2005; Botella et al., 2007; Damasio et al., 2011.

Rhodotorula glutinis

Castor bean waste

Cellobiohydrolases, CMcases and beta-glucosidases

Brewing industry

Herculano et al.,2011

Fungal ligninolytic enzymes (ligninases) are lignin peroxidase (LiP), manganese peroxidase

(MnP) and laccase (Lac). Among the ligninases, Lac are the mostly studied .

Table 2.4 An overview of ligninolytic fungi, their substrates, enzymes and applications

(Source : Mtui et al.,2012)

Fungi Substrate Enzymes Applied field References Aspergillus niger

Petroleum hydrocarbons, PKC

MnP, LiP, Lac Bioremediation, improved animal feed

Perez-Armendariz et al., 2010; Lawal et al., 2010.

Bjerkandera adusta

Phenolic compounds

LiP MnP, Lac Bioremediation; improved animal feed

Rodrigues et al., 2008; Tripathi et al., 2011.

Cerrena unicolor.

Paper sludge Lac, LiP, MnP De-inking Winquist et al., 2008.

Cladosporium cladosporioides

Petroleum hydrocarbons

MnP, LiP, Lac Bioremediation Perez-Armendariz et al., 2010.

Coriolus versicolor

Wood residues Lip, MnP Biopulping Liew et al., 2011;

Crepidotus Synthetic dyes Lac, LiP, MnP Bioremediation, Mtui and


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Fungi Substrate Enzymes Applied field References variabilis decoloration Nakamura, 2007,

2008. Flavodon flavus Textile

wastewater Lac, LiP, MnP Bioremediation,

decoloration Raghukumar et al, 2006; Mtui and Nakamura, 2008.

Fomes fomentarius

PCPs LiP, MnP, Lac Bioremediation; Improved animal feed

Rodrigues et al., 2008; Ramesh et al., 2009.

Fusarium oxysporum

PAHs Lip, MnP, Lac Bioremediation Silva et al., 2009.

Ganoderma lucidum, G. applanatum

Rice straw, PAHs

Lac Bioremediation, animal feed

Punnapayak et al., 2009.

Irpex lacteus Corn stover Lac, Lip Improved animal feed

Xu et al., 2009.

Laetioporus sulphureus

Synthetic wastewater

Lip, MnP, Lac Laetioporus sulphureus

Mtui and Masalu, 2008.

Lentinus squarrosulus

Nitrophenolic compounds

MnP, Aryl oxidases.

bioremediation Tripathi et al., 2011.

Lentinus crinitus, L. subnudus

Wood residues, maize husks

LiP, MnP, Lac Decolorization. Improved animal feed

Niebisch et al,. 2010; Jonathan et al., 2010.

Mucor mucedo Palm kernel cake

MnP, LiP, Improved animal feed

Lawal et al., 2010.

Paecilomyces farinosus

Olive meal waste

LiP, MnP, Lac Detoxification of phenolics

Sampedro et al., 2009.

Penicillium glabrum

Petroleum hydrocarbons

MnP, LiP, Lac bioremediation Perez-Armendariz et al., 2010.

Phanerochaete chrysosporium

Wastewater, rice straw agro-chemicals

LiP, MnP, Lac Bioremediation, Improved animal feed

Lu et al., 2009; Sharma and Arora, 2010,

Disposal of lignocellulosic wastes and heavy metal dumping into the soil or landfill causes

serious environmental problems, besides to constitute loss of these value added substances

derived from lignocellulosic waste substances. Therefore, the development of processes for

better management is a great concern of today. This thrust for better management welcomes a

new strategy of biotechnology i.e improvement of fungal strains for healthier outcome both in

the field of environment and industry. This fungal strain improvement techniques and applied

field of using these improved fungal species is highlighted in the later part of this review.


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2.3 Fungal strain improvement techniques and application

Microorganisms have proven to be an exceptionally rich in useful products and

enzymatic activities. They are resource of thousands of valuable products with novel

structures and activities. In nature, they only produce tiny amounts of these secondary

metabolic products as a matter of survival. Hence they produce different commercial

products at very low concentration (Szengyel et al., 2000; Chand et al., 2005; Li et al., 2009;

Pradeep and Narasimha, 2011). To fulfill the exponential demand in application of these

microbes in various fields, strain improvement took birth. Nowadays genetic engineering and

biotechnology research are becoming more important for extracting more usability and to

gain better economic output in all fields. The cumulative knowledge of fungal genetics and

their biochemical pathways has been applied for strains improvement.

In the second half of the twentieth century fermentation technologies were used for strain

development that led to the great expansion of the fermentation industries. Recently strain

improvements were carried out by two means, namely mutation and genetic recombination.

In mutation, a gene is modified either unintestinally (‘spontaneous mutation’) or intestinally

(‘induced mutation’). Induced mutation can be carried out by different methods such as

chemical and physical mutation, sexual hybrids, homocaryons, directed mutagenesis,

protoplast fusion, recombination and transformation (Stasz, 1990; Nevalainen, 2001; Haggag

and Mohamed, 2007). Advances within the last decades in molecular genetics of fungi have

provided commercially promising recombinant fungal strains, which led to a new era in

fermentation biotechnology. With the advent of molecular biology, it has been attempted to

improve the expression level of heterologous genes, which encode favourable traits in fungal

strains. Among these strategies, introduction of multicopies of the desired gene, change of

AT-rich sequences, gene fusion with a well expressed gene, the use of strong promoters and

signal sequences, optimization of codon usage, the construction and use of protease-deficient

and chaperones/foldases-overproduced strains and the use of native or artificial intron-

containing genes are well documented (Scorer et al.,1993; Verdoes et al.,1995; Svetina et al.,

2000; Te’O et al., 2000; Xu and Gong , 2003; Penltila et al., 2004; Yu and Keller ,

2005).Application of homologous DNA recombination ( Catlett et al., 2003; Jeong et al.,

2007; Venard et al., 2008; Hoff et al., 2009) and RNA interference ( Lee et al., 2004;

Nakayashiki et al., 2006; Hoff et al., 2009; Janus et al., 2009) to manipulate fungal recipients

for further improvement of physiology and development in regards to biotechnical and

pharmaceutical applications are also reported by a group of researchers. Genetic


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recombination was not much used at first, despite early claims of success (Jarai, 1961;

Mindlin, 1969), mainly due to the absence or the extremely low frequency of genetic

recombination in industrial microorganisms. After 1980, there was a heightened interest in

the application of genetic recombination (by means of protoplast fusion) for the production of

important microbial products such as antibiotics. Inter specific protoplast fusion between the

osmo-tolerant Saccharomyces mellis and the highly fermentative S. cerevisiae yielded stable

hybrids fermenting high concentrations of glucose (49% ww) (Legmann & Margalith, 1983) .

Interspecific protoplast fusion between S. griseus and five other species (Streptomyces

cyaneus, Streptomyces exfoliatus, Streptomyces griseoruber, Streptomyces purpureus and

Streptomyces rochei) yielded recombinants of which 60% produced no antibiotics and 24%

produced antibiotics different from the parent strains (Okanishi et al., 1996).It is being

established that microbial strain improvement can possibly be done through inducing

mutation using physical stress like exposure to ionizing radiation. The first superior penicillin

producing mutant Penicillium chrysogenum (X-1612), was isolated after X-ray mutagenesis

almost 60 years ago. The improvement of penicillin production by conventional strain

improvement resulted both from enhanced gene expression and from gene amplification

(Barredo et al., 1989; Smith et al., 1989). To enhance the production of the biomedically

important enzyme, lipase, from Aspergillus japonicus, Karanam and Medicherla (2008)

treated the strain with random mutagens (UV irradiation, HNO2 and N-methyl-N’- nitro-N-

nitroso guanidine).

The best UVmutant (AUV3) showed 127% higher lipase activity than the parent strain.

Rajeshkumar and IIyas, (2011) isolated certain fungal strains viz., Aspergillus niger,

Aspergillus fumigatus, Penicillium sp from the rhizosphere of paddy fields in Tamilnadu,

India and screened for phosphate solubilization. To enhance Lipase activity of these strains

they were treated through mutagenesis by using UV, sodium azide and ethyl methane

sulphonate (EMS). The mutant ANuv50 (a UV mutant) exhibited 32.72% increased

efficiency for Lipase activity. Ali et al., (2002) found that the UV mutant of Aspergillus

oryzae was found to give 3.72 folds higher production of L-DOPA (useful drug for

Parkinson’s disease) than the parental strain. Veerapagu et al., (2008) tried to increase

penicillin production by inducing P.chrysogenum with different doses of UV radiation. In

contrast to the above mentioned fungal strain improving agent Cordeiro et al., (1995)

reported that exposure to gamma showed maximum potential to induce mutation in fungi

(Metarhizium anisopliae) than that of UV or other chemical mutagens.


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2.3.1 Application of gamma radiation on fungi as strain improving agent

Gamma rays are generally used for the sterilization of gaseous, liquid, solid materials,

homogeneous and heterogeneous systems and medical devices such as syringes, needles,

cannulas etc. Gamma irradiation is a physical means of decontamination, because it kills

bacteria and molds by breaking down DNA and/or inhibiting microbial growth. Such

treatments do not leave toxic residues and can be conducted at ambient temperature. Being

environment friendly in terms of hygiene and economy, these radiation treatment function as

disinfectant at very high doses. Higher doses of radiation is mainly used as an excellent tool

for food or pharmaceutical sterilisation, food preservation and different food engineering

process, which ultimately benefits for the society ( Hyun-Pa et al.,2006; Sameh et al.,2006;

Dusan,2004). Earlier reports from our lab showed that high doses of gamma radiation cause

dose-dependent inhibitory effects in fungi (Maity et al., 2009). Sterilization by gamma

irradiation also helps in preserving the quality of processed food /seed as no or very

negligible degradation is observed compared to other techniques, thus reducing the risk for

consumers (WHO , 1999; Maity et al., 2009).There are many reports supporting the use of

gamma irradiation as a fungicidal agent (Aziz et al., 2007; Maity et al., 2008,2009).In

contrast to higher doses of gamma, low doses of gamma functions as stimulatory factor in

fungi ( Cordeiro et al., 1995; Geweely and Nawar, 2006; Dadachova et al., 2007; Robertson

et al., 2012).

2.3.1.1 Enzyme production

Different fungal enzymes like amylase, cellulase, lipase, xylanase, pectinase etc .have

vast application in industrial field, medical field & simultaneously in environmental point of

view. The production levels of enzymes from parental or wild microorganisms (both fungi

and bacteria) are quite low (Mala et al., 2001). Different techniques are used for improving

fungal strain among them random mutagenesis and fermentation screening have been

reported as an effective way to improve the productivity of industrial microbial cultures

(Parekh et al., 2000). Among all these gamma induced mutagenesis took important part. A

gamma induced mutant of Alternaria alternata excreted enhanced levels of carboxy methyl

cellulase (CMCase) and particularly glucosidase when grown in cellulose liquid media

(Macris , 1984). Aspergillus niger, Rhizopus microsporus and Penicillium atrovenetum

showed enhance production of industrially important enzyme lipase when these were exposed

to various low doses of gamma irradiation (20, 40, 60, 80, 100, 120, 140 and 160 Gy)

(Iftikhar et al., 2010). Gohel et al., (2004) treated Pantoea dispera with two physical mutagen


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namely UV and gamma absorbed dose and one chemical mutagen i.e EMS to observe the

chitinolytic activity. They found that gamma mutant produce better chitinolytic enzyme than

UV mutant. These mutants also better producer of chitinolytic enzyme than their wild strain.

This research group further revealed that these mutants were better producer of protease and

b -1-3 glucans too as compared with their wild type. Abostate et al., (2010) first isolated

potent cellulase producers studying their cellulase productivities on wheat straw, wheat bran,

rice straw and corn cob. Most potent strain (Aspergillus sp) exposed to different doses of

gamma radiation to determine their further enzyme activity. Aspergillus MAM –F-23 (Mutant

4) exposed to 0.5 K Gy of gamma produced higher cellulases (CMCase, F pase, Avicelase)

than parental non-irradiated counterparts; however mutant 1 of Aspergillus MAM F-35 also

exposed to 0.5 K Gy has produced highest cellulase than its parental one. It has produced

26% Avicelase , 24% CMCase and 46% Fpase more than the parental strain. This group

previously have observed that with exposure to gamma irradiation (1K Gy) enhanced

productivity of CMCase ,Fpase, Avicelase, Xylanase, Pectinase , amylase and protease by

8%, 20%, 10%, 4%,31%, 22% and 34% respectively with respect to their non-irradiated

counterparts. Similarly Huma et al.,(2012) developed hyper amylase producing fungus

Phialocephala humicola through gamma ray treatment (100 and 140 krad), which have

diverse industrial applications. Three mutants (M13, M16 and M24) were selected based on

the hyper-production of α-amylases. Three mutants showed greater than two fold

improvement in enzyme production.

2.3.1.2 Improvement of mushroom quality

Mushroom has immense utility in modern daily life. It is used as food, as alternative

of protein mal nutritional gap (due to its high protein content), simultaneously as medicine (

due to its low fat content, low cholesterol, high antioxidant content, presence of iron in

available form). Different biologically active compounds from the mushrooms have

antifungal, antibacterial, antioxidant and antiviral properties, and have been used as

insecticides and nematocides. So storing of mushroom and improving its quality is of prime

importance. Ionizing radiation has been a common practice for extending storage life in

mushrooms especially champignon. Radiation dose at 1.5 – 3 KGy on champignon fruit

bodies will increase storage life from 8 weeks to 12 weeks as has been reported by Maha &

Pangerteni (1989). Ionizing radiation has also been used to generate mutants in several edible

mushrooms. Djajanegara and Harsoy (2008) through their research have revealed higher

antioxidant containing mushrooms which is beneficial for medical and food industry. Besides


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they got a remarkable stimulatory effect of gamma induced mutagenesis when Pleurotus

florida (white oyster mushroom) was subjected to gamma rays (60Co).They got 4 putative

mutants which are PO-2, PO-3, PO-4 and PO-5. There were no differences in the morphology

of the mycelia and fruit bodies between those mutants and control. Among three positive

mutants, only PO-5 showed higher productivity compared to control (PO-K) reflecting higher

number of fruit bodies, higher fresh weight and dry weight yield of three successive flush

periods. Antioxidant analysis of those mutants indicate that mutant PO-4 has significantly

higher antioxidant content compared to control (PO-K) and the two other mutants (PO-3 &

PO-5). Lee (2000) has measured the activities of eight enzymes (namely Exo β- glcosidase,

Exo- amylase, Exo- phosphatase, Exo-chitinase, Endo- chitinase, Exo-cellulase, Endo-

cellulose, Exo- lipase) by MUF-assay method in gamma irradiated Pleurotus ostreatus (PO).

All enzyme activities of the 1-2KGy gamma induced strains were variable and higher than

those of the control ,except those of exo-amylase, exo- and endo- cellulase of PO-6 strain.

The activity of exo-cellulase increased 1.2 to 1.7 times and endo-cellulase was 1.0 to 1.4

times in four strains (PO-5, PO-14, PO-15 and PO- 16) compared to those of the control, but

PO-6 strain showed only 75% and 55% higher than that of the control, respectively. In exo-

b-glucosidase, the activities were increased 1.3 to 5 times in all strains compared to the

control. Among the strains, the PO-5 strain had the highest activities of those three enzymes

related to celulolytic activity. It could be depicted from their observations that gamma-ray

irradiation has the potential to enhance or reduce the activities of extracellular enzymes

related to the hydrolysis of carbohydrates in biowastes depending on the strain.

2.3.1.3 Annihilation of Aflatoxin production capacity of fungus

Some molds have the capacity to produce substances that are toxic and generally

carcinogen, such as aflatoxins, that stand between the most important carcinogenic

substances (class I of the agents which are certainly carcinogenic for human people according

to the “International Agency for Research on Cancer”. Aziz and Youssef, (2002) showed that

a complete destruction of aflatoxin B1 with a dose of 20kGy exposure of gamma. Recently

Aquino et al., (2005) showed that aflatoxins B1 and B2 are reduced to not detectable levels

with a dose of 10kGy . Rogovschi et al., (2007) tried to establish a minimum dose of

radiation that degrades aflatoxins produced by fungi Aspergillus spp. They found that no

aflatoxin (B1 and B2)was detected in samples treated with gamma ray processing at dose of

20kGy, while all the control samples were positive to the presence of aflatoxins B1 and B2.


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2.3.1.4 Gamma induced enhancement in survivability and growth in fungi

Survivability of fungi can be expressed in terms of Spore germination, mycelial

growth. Inhibition of spore germination was found to be directly correlated to gamma

radiation dose (Golan et al., 2002) i.e higher doses of gamma causes decrease in fungal

mortality , whereas, low doses of gamma act as a stimulatory agent for spore germination and

growth of some fungi(Salama et al., 1977) . Dadachova et al., (2007) have isolated three

melanin containing fungi Cladosporium sphaerospemum, Wangiella dermatitidis ,

Cryptococcus neoformans from near about areas of Chernobyl Nuclear power plant. These

fungal strains were with higher CFUs, increased biomass and accumulated acetate faster in

500 times higher radiation leveled environment than in normal environment. Ziombra (1994)

reported ,100Gy and 200Gy of absorbed doses of gamma radiation accelerate spore

germination of Pleurotus sp. Similar findings were revealed by some group of researcher,

who observed that low experimental doses of gamma radiation (5- 100Gy) are significant

stimulatory to spore germination of Botrytis cinerea and Penicillium expansum, while in case

of Aspergillus tenuissima and S. boryosum this stimulatory dose was 250Gy, with further

increase of absorbed dose percentage of germination decreased with an inhibition at an

optimum dose (Geweely and Nawar,2006).Enhanced radial growth of mycelium and fungal

dry weight after gamma induction reflects similar trend as in case of spore germination.

Increased mycelial dry weight after gamma treatment is strain specific. The effective dose

reflecting highest mycelial dry weight varies from strain to strain. According to Hassain

(1987) lower gamma dose have stimulatory effect on growth of Aspergillus flavus while with

3000Gy caused complete inhibition of A.flavus. Abo-zeid, (1987) reported highest dry

weight of Aspergillus koningi was found at 1000Gy and for A. flavus this dose was

1500Gy.While for Auricularia auricular the effective dose was 2000Gy but for A.

fuscosucinea it was 100Gy. Ibrahim, (1986) has also noticed through his experimental results

that dry mycelial weight of Paecilomyces violacea was significantly increased at two lower

doses of gamma (100 and 250Gy) over control at 11.38% and 19.89% respectively, but

63.44% and 70.4% decrease in mycelial weight found when that strain was treated with

2500Gy and 3000Gy respectively. Geweely and Nawar (2006) have reported that lower doses

of gamma radiation significantly increased radial growth rate and dry weight of both

radioresistant strains (Aspergillus teniussima & Stemphylium botryosum) as well as radio

sensitive strains (Botrytis cineara & Penicillium expansum). For radioresistant strains this

effective dose was 250Gy while for radiosensitive strains it was 5Gy.


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2.3.1.5 Gamma induced enhancement of essential metabolites

Nadia et al., (1988) stated that total proteins and some amino acids of gamma

irradiated Paecilomyces violacea were higher than the non-irradiated one .Awny et al., (1982)

reported that new protein was formed to protect the cell against a variety of harmful

environmental stresses including ionizing radiation exposure. Osman et al., (1987) reported

that gamma irradiation caused increase in total protein in Fusarium solani, which suggested

that protein might play an important role in protection against the harmful effect of radiation.

Appearance of some amino acids only after irradiation and their accumulation in amounts

higher than the control values could be explained in the light of their probable roles as radio-

protector. Fungus showed resistance to gamma radiation due to its contents of cystine,

methionine and histidine. It is well demonstrated that organisms containing certain amino

acids specially those containing sulphur bond (cysteine, cystine and methionine) and that

having double bond (histidine) are high resistance to gamma radiation. El –Foulty et

al.,(2012) found a change in amino acid profile between irradiated and unirradiated

Aspergillus niger after inducing with different doses of gamma radiation.

β – glucans are recognized as potent immunological stimulators in humans and some of that

group are now used clinically. Moreover they are also effective for treating cancer, different

microbial infections , hypercholes, terolaemia and diabetes. Dawoud and Taleb (2011)

worked with productivity of β- glucans by Pleurotus ostreatus .This group has shown that

low doses of gamma (1KGy) causes enhancement of protein , carbohydrates, glucans as well

as growth in terms of dry mass of that particular fungi with respect to their non-irradiated

counterparts. Further increase of radiation doses caused linear decrease (2 to 3 k Gy) of

fungal growth and finally failed to grow at 5 to 6 K Gy. So these irradiated glucan

hyperproducing fungi may be used in commercial purposes.

2.3.1.6 Gamma induced enhancement in biocontrol potential /biodegradation of

pesticides by fungi

Trichoderma sp are considered as promising biological control agents against

numerous phytopathogenic fungi . Haggag and Mohamed, (2002) detailed that gamma

induced mutants of Trichoderma sp when naturally infested in soil, it significantly reduced

the onion white rot disease incidence and improved the plant yield with respect to the wild

strain (unirradiated). Gamma exposed Trichoderma sp. were effective in reducing the

pathogen growth in rhizosphere soil with the help of upregulated cellulase, chitinase

enzymes, metabolites including phenolic compounds and antibiotics (those enzymes and


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metabolites cause cellwall degradation and growth inhibition of the pathogen) compared to

the wild type strains . Recently Afity et al., (2013) applied gamma radiation on the same

species to enhance effective hydrolytic enzymes in their bio-control abilities for

biodegradation of oxamyl pesticides.

2.3.2 Mechanism of Radioprotection in fungi

Melanised Wangiella dermatitidis and Cryptococcus neoformans cells exposed to

ionizing radiation showed higher CFUs, increased biomass and faster acetate accumulation

potential (500 times higher than their unirradiated counterparts). They accomplished that

ionizing radiation mainly gamma changed melanin’s electronic properties which possibly

lead increased growth of irradiated melanised fungal strain than unirradiated melanised

counterparts. Cell wall thickness could not be correlated with the radio-protectiveness in

fungi as proposed by Geweely and Nawar, (2006). They have established that Botrytis

cinerea & Penicillium expansum with lighter pigmentation and thin cell walls are more

sensitive to gamma rays than the dark walled Alternaria tenuissima and Stemphylium

botryosum spores and that the effect of radiation on spores diminish as pigmentation

increased. Radioprotective fungi, appears to be able to use melanin as a photosynthetic

pigment that enables them to capture gamma rays and use its energy for growth. Many fungi

especially melanized ones are highly radioresistant. Photochemical properties of melanin

make it an excellent photoprotectant. Electronic complexity of melanins allows them to

scatter /trap photons and electrons which supports the hypothesis of Dadachova et al., (2007)

that melanin harness metabolic energy from ionizing radiation, which reflect that melanised

cells might consume more energy when exposed to radiation. It was postulated by Robertson

et al., (2012) that melanin played two roles upon ionizing radiation, 1) presence of melanin

upregulate different transporter genes which helped melanised cells to take more carbon and

amino acids as nutrition and expel toxic metabolites generated by radiation stress and 2)

Melanin upregulate the gene expression involved in ribosomal biogenesis .These properties

of melanin supports the enhanced growth and survivability of radiation exposed fungi than

unirradiated counterparts. So melanin efficiently plays different roles against different

stresses that involve cell damage such as UV, Gamma radiation, Sun and reactive oxygen

species, protects against damage from high temperature, chemical stress (heavy metals and

oxidizing agent) and biochemical threats.


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It is well known that ionizing radiation activates DNA repair mechanism and anti oxidant

defence systems (Robertson et al., 2012). Bennett et al., (2001) identified the genes

responsible for resistance to ionizing radiation in Sachharomyces cerevisiae and those genes

responsible for various important functions like repair (RAD50 and RAD51), recombination

(HRP1), chromosome stability (CHL1 and CTF4), endocytosis (VID21) and some others.

2.3.3 Modulation of metal tolerance in gamma induced fungi

It is well established from the reports of early researchers that radiation induced

fungal strain can serve better environmental as well as industrial purposes than its non-

irradiated counterparts. Beside keeling microbes as a sterilization agent, radiation can take

part in improving fungi in terms of their physiological and biochemical aspects . Considering

these, our group are trying to evaluate whether gamma induced fungal strain have better

efficacy for heavy metal removal with respect to their non-irradiated counterparts. Utilisation

of low doses of gamma irradiation for developing microbes with higher metal tolerance might

possibly lead us to formulate better strategy for combating heavy metal pollution.

3. Aims and Objectives

Microbial bioremediation is attaining attention as an important procedure for abatement

of metal pollution due to its low cost and high efficacy. Among different microorganisms

used in bioremediation, fungi are considered as most effective species for metal removal

from metal contaminated sites. Explicate report regarding physico chemical nature of soil

of Dhapa, the major waste dumping site in eastern fringe of Kolkata, contaminated with

heavy metals is not available Furthermore, works describing the fungal population which

utilize the same soil habitat for its growth and in turn maintain the soil health, are also

scanty. To gauge the extent of metal tolerance of the soil fungi inhabiting Dhapa, soil was

collected from Dhapa with the intent of isolating dominant fungal strains which can be

made more potent in terms of metal tolerance so as to be used as prospective agents for

bioremediation. Recently a global thrust is felt in utilising biotechnology for microbial

strain improvement so as to extract more usability and gain better economic output in all

fields. Ionising radiation, a mutagenic agent is established as an effective microbial strain

improving agent among different chemical as well as physical mutagens. Various groups

of researchers have worked earlier on radiation exposed fungal strains searching out

better outcome in various environmental and industrial fields (briefly discussed in

Introduction and Literature Review chapter) with respect to their un-irradiated

counterparts. However reports elucidating use of ionising radiation for inducing metal

tolerance in fungal strains has not been put forth so far.

Main objectives of this work are as follows:

Isolation of metal (Cd, Pb, Zn) tolerant and metal sensitive fungal species and

determination of their metal tolerance.

To evaluate the potentiality of gamma radiation in imparting higher metal tolerance in

fungi.

To understand metal uptake and removal potentials of radiation exposed fungal strains

with respect to their unirradiated counterparts and study of functional groups involved

in biosorption of metal both in irradiated and un-irradiated fungal strains.

To observe different morphological and some metabolic changes of fungal strains

exposed to metal as well as gamma radiation.

To understand possible mechanisms adopted by fungi for being metal tolerant with

special reference to antioxidative status of the fungi after gamma exposure.

4. Materials and Methods

4.1. Selection of site

Kolkata, one of the fastest growing metropolises in India has been suffering from

different pollution load due to population load, increasing demand for food and shelters.

Dhapa is an important municipal garbage dumping site of Kolkata which now being

converted to cultivable land just to use the waste land for mankind. Almost 40% of

vegetables supplied to Kolkata and adjacent areas are harvested from Dhapa cultivable land.

The disposal site has served the City of Kolkata as an uncontrolled dumping ground since

1981. Based on site volume and waste density estimates, the site is estimated to have received

approximately 7 million metric tonnes (Mg) of municipal solid waste (MSW) as of the time

of the site visit in October 2009 (KMC,2011). An ample agricultural field is developed

adjacent to the garbage dump site just to reuse these wastes as manure as much as possible.

4.1.1. Sampling of soil

Soil samples were collected from three different sites of garbage dump site of Dhapa,

Kolkata and considered as contaminated sites for the present study. 100gms of surface soil

was collected aseptically in poly bag and were stored at 40C before further analysis.

4.1.2. Treatment of soil

Collected soil was kept in 40C for fungal isolation and rest was dried in hot air oven

at 650C for overnight and then grinded in mortar and pestle and screened through 2.0 mm

pore sized sieve to get uniformly homogenised mixture.

4.1.3. Physico-chemical Characterisation of the soil (pH, conductivity, organic

carbon content, available NPK, Pb, Cd, Zn concentration)

pH of soil sample was measured by Electrometric method with the help of a pH meter

using combination Glass electrode. Organic Carbon was done by Walkley and Black method

(Walkly and Black, 1934). Total potassium and phosphorous were estimated by methods

prescribed by American Public Health Association (APHA 1992). The total nitrogen content

was estimated by Kjeldahl method (APHA 1992) and for heavy metal analysis each soil

sample (1gm) was allowed for digesion according to Heating Block digestion procedure

(Rahman et al., 2007). Soil sample taken in digestion tube was mixed with 10ml of HNO3:

HClO4 (1:2) solution and heated in sand bath until clear solution was developed. The

solutions were filtered through Whatman No1 filter paper and volume was made up to 50ml

by adding distilled water. Each soil sample was digested in triplicate for analysis of

Materials and Methods

42 | P a g e

contaminated heavy metal content separately. Blank (control) samples containing 10ml of

HNO3: HClO4 (1:2) solution were also digested and diluted in the same way for comparison.

All metal analysis (Zinc, Lead and Cadmium) were performed by using Atomic Absorption

Spectrophotometer (Perkin Elmer Analyst 400) (APHA, 1998).

4.2. Isolation of fungal strains

Fungal strains were isolated from soil using conventional dilution plate culture

techniques. One gm of pre-weighted soil was suspended in 10ml of sterilized distilled water

and then serially diluted to 10-3 dilution and 1ml of such dilatants were plated in sterile

petridish containing pre-coated growth media. The soil dilatants were spread over the plate

and incubated at 29-30°C for six days. Three fungal growth media viz. Potato Dextrose Agar

(PDA), Malt agar (MA) and Czapek Dox Agar (CDA) were used for this purpose.

4.2.1. Media compositions

Potato Dextrose Agar (PDA) media was prepared using 200gms of potato boiled in

100ml of distilled water for 30 minutes and the filtrate was mixed with 12.5 gms of dextrose

and 20 gms of agar powder per liter. Malt Agar (MA) medium was prepared with mixture of

malt extract and agar powder 15gms per liter. Identically Czapek Dox (CD) medium was

prepared (glucose -2gm, NaNO3 – 2.5gm, KCl -0.5gm, MgSO4,7H2O -0.5gm, FeSO4 –

0.5gm, ZnSO4, 7H2O – 0.5gm, KH2PO4 – 1gm and agar powder 15gms per liter) (APHA,

1992).

4.2.2. Isolation of pure cultures

By the streak-plate dilution technique i.e. by spreading a loopful of culture over the

surface of an agar (PDA) plate and incubating, distinct single colonies with sufficient

distance from other colonies (Trivedy and Goel, 1984) were obtained. These distinct

colonies of a particular fungal strain were considered as pure culture.

4.2.3 Maintenance of culture

Properly grown fungal cultures (incubated at 29-30°C) were preserved under

refrigeration of 4°C in Potato Dextrose Agar plates as well as in slants and stabs as stock

cultures. Sub-culturing was done at regular intervals to maintain purity of cultures.

4.2.4. Identification of fungal population

All isolated strains were identified upto genus level depending on morphological


43 | P a g e

characters (conidial shape,vesicle shape, sterigmata position, conidiophores shape etc.).

4.3 Assessment of metal tolerance of isolated strains

Assessment of metal (Cd, Pb, Zn) tolerance was done in two steps. Initially

determination of minimum inhibitory concentration (MIC) of selected stains against metals

were done after isolation from soil. Different concentrations of metal solutions were added

separately to PDA medium and inoculated with 100μl of fungal culture to determine MIC

(Minimum Inhibitory Concentration). CdCl2 (Merck), ZnSO4, 7H2O (SRL) and Pb (CH3OO) 2

(SRL) salts were selected for running all experiments.

4.3.1 Selection of fungal strains for further experiments

Depending on MIC (Minimum Inhibitory Concentration) values of each strains

against each metal (Cd, Pb, Zn) one tolerant and one sensitive fungal strain were selected for

further study.

4.3.2 Species Identification of the strains

Selected fungal culture strains were identified on basis of macroscopic (colony

character, morphology, colour, texture, shape, diameter and appearance of colony) and

microscopic (septation in mycelium, presence of specific reproductive structures, shape and

structure of conidia and conidiospore etc) characteristic using fungal identification manuals

(Thorn and Raper, 1945; Domsch et al., 1980).

4.3.3 Cross metal resistance assay

Cross heavy metal resistance of the fungal isolates was determined by conventional

cup assay method (Pujol et al., 1997). Selected metal tolerant fungal strains were grown

separately in Potato Dextrose Agar medium supplemented with diverse concentration (sub-

lethal to lethal) of each heavy metal separately. Then cups were prepared where various other

metallic solutions (10 ppm to 10,000 ppm) were poured. For each test, triplicates were made

and incubated for 6 -8 days. Zone of inhibition of fungal growth around the cup was

examined and measured in terms of MIC in each case separately. CdCl2 (Merck), ZnSO4,

7H2O (SRL) and Pb (CH3OO) 2 (SRL), HgCl2 (Merck), K2CrO4 (SRL) salts were selected for

running all experiments.


44 | P a g e

4.3.4. Antibiotic sensitivity assay

Antibiotic (antifungal) sensitivity of each metal tolerant fungal strains was also

measured using the same cup assay method described earlier (Pujol et al., 1997). The

antibiotics used for this assay were Nystatin, Griseofulvin and Kanamycin.

4.3.5. pH and temperature optimisation for growth

Selected fungal strains were grown in sterilized PD broth medium for establishment

of the optimum temperature, pH that supports the exuberant growth of those species. The pH

were varied from 5 to 9 (5, 7 and 9). The pH of the medium was adjusted using dilute (N/10)

HCl or NaOH. For optimization of temperature against each pH the representative culture

was incubated at different temperatures (25 to 35oC). Equal amount of spore suspensions

were loaded against each pH and temperature and allowed for growth at 290C for 8days.

Total biomass was considered for optimum growth at a particular pH and temperature.

4.3.6. Preparation of Spore suspension

8days old culture (grown in potato dextrose broth media) was agitated fully and then

hyphal mat was removed and the liquid was filtered through a Whatmann filter paper No-

1.Filtrate was centrifuged in10000 rpm for 15mins, supernatant was discarded and the pellet

was suspended in Tween 20 (0.02%v/v) and NaCl (0.85%w/v) solution (Tanaka et al., 1988;

Cordeiro et al., 1995).

4.4. Selection of gamma dose

Spore suspensions were allowed at lower 20Gy of gamma absorbed dose (observing

no significant change in CFU under metal stress with respect to their un-irradiated

counterparts) and higher at 100Gy of gamma absorbed dose (higher metal tolerance in terms

of CFU observed at lower doses of gamma than 100Gy); Previous references (Fadel and

Batal, 2000; Geweely and Nawar, 2006; Iftikhar et al., 2010) also considered for selecting

gamma dose range. Gamma absorbed dose at which maximum number of colonies (in terms

of CFU) were obtained against each metal concentration was considered as effective dose.

4.4.1. Gamma exposure to the prepared spore suspension

Spore suspensions (5 x 105 spores/ml, measured in haemocytometer) were taken in

small centrifuge tubes and then exposed to 20, 40, 60, 80 and 100 Gray (Gy) of absorbed

doses of gamma radiation from a Co60 as gamma source ( GC 1200, BRIT). One set was


45 | P a g e

kept without gamma exposure to observe the differences between gamma irradiated and un-

irradiated group.

4.5 Assessment of metal tolerance in terms of Colony Forming Unit (CFU)

Irradiated as well as un-irradiated spore suspensions were being inoculated in agar

plates containing different metal concentrations with 300μl inoculation of 10-.3 dilution. After

3rd day to 14th days of irradiation colony numbers were considered. In each case 6 replicates

were considered for analysis.

CFU/ml = Number of colonies per ml plated -----------------------------------------------------

Total dilution

4.6. Estimation of metal uptake and removal potential

After 14th days of irradiation, one 8 mm disc (Visoottiviseth and Panviroj , 2001;

Zapotoczny et al., 2007; Yazdoni et al., 2010) from petri plates ( both from unirradiated and

irradiated growth cultures) of fungal strains were transferred to same metal concentrated

liquid medium with a sterilized cork borer and allowed for growth in an orbital shaker at 100

rpm for 14days in incubation at 29-30°C. Uptake of metal (mg/g of tissue) and removal

percentage were carried out following the method of Srivastava and Thakur, (2006) using

Atomic Absorption Spectrophotometer (FI-HG-AAS Perkin Elmer Analyst 400). In most of

the cases the effective dose at which maximum CFU was obtained under metal stress was

considered for evaluating metal uptake and removal potential but in some cases metal uptake

and removal potential of all gamma exposed (20-100Gy) fungal groups were evaluated. In

each cases 3 replicates were considered for analysis.

4.6.1. Analysis of functional groups responsible for metal absorption by Fourier

Transform Infrared Spectroscopic (FTIR) study

The functional groups (present in fungal cell wall) involved in metal adsorption are

studied through FTIR. Samples were prepared considering the methods of Xu et al., (2012)

and Das and Guha, (2009). For Fourier Transform Infra Red (FTIR) spectroscopy

measurement, the lyophilized fungal mycelium (lyophilized at -850C under high vacuum

condition using freeze dryer ;Virtis EL-65,New York) was mixed with overnight oven dried

(1000C) potassium bromide (KBr) in the ratio of 1:100. FTIR spectrum of samples was

recorded in JASCO-6300 FTIR instrument with a diffuse reflectance mode (DRS8000)

attachment. All measurement were carried out in the range of 400-4000´cm-1 at a resolution

of 4cm-1. The effective gamma dose at which maximum CFU were obtained under metal


46 | P a g e

stress was considered for FTIR analysis along with the control and metal treated (in every

cases metal concentration was selected near the MIC value) group.

4.6.2 Optimisation of pH and temperature for removal

Most of the experiments were carried out in pH 7 and temperature 290C, but against

one metal concentration the optimum pH and temperature for removal were evaluated for

each fungal strain both in un-irradiated as well as irradiated (at effective gamma dose

showing maximum CFU) condition. The pH was varied from 5 to 9 (5, 7 and 9). The pH of

the medium was adjusted using dilute (N/10) HCl or NaOH. For optimization of temperature

against each pH the representative culture was incubated at different temperatures (250 to

35oC).

4.7 Fungal sample preparation for Scanning Electron Microscope (SEM)

Samples were prepared according to the methods of Kaminskyj and Dahms (2008),

Mishra and Malik, (2013). Fungal mycelium grown in liquid medium was cut into small

pieces (maximum 2mm in diameter) and these small pieces were fixed in 2.5%

Glutaraldehyde (Sigma Aldrich, USA) for 2hrs.After 2hrs of fixation the samples were

washed in Phosphate buffer ( pH 7.4, 100mM) just to remove extra fixative. Then the

samples were allowed for alcoholic dehydration through 30%, 50%, 70%, 90% and 100%

ethyl alcohol solution simultaneously for 15-20mins in each. Dehydrated samples were

finally placed in amyl acetate (100%) until visualisation. Finally samples were gold coated in

IB2 ION Coater for 30mins and observed in HITACHI S-530 Scanning Electron Microscope.

The voltage was constant at 25KeV and the microscope was focussed at different

magnification depending upon the sample. The effective gamma dose at which maximum

CFU were obtained under metal stress was considered for analysis of ultrastructure using

SEM along with the control and metal (in every cases metal concentration was selected near

MIC value) treated group.

.

4.8 Estimation of extracellular metabolic/ lignocellulosic enzyme and antioxidant

assay

4.8.1 Metabolic/ lignocellulosic Enzyme activity

After 14days of shaking incubation the biomass part and some portion of broth were

separated for determining uptake and removal (%) potential. Remaining broth was

centrifuged at 10,000 rpm for 15 mins to avoid any solid debris. The clarified supernatant

was used for enzyme assay. In most of the cases the effective dose at which maximum CFU


47 | P a g e

was observed against a metal stress was considered for evaluating extracellular enzyme

activity but in some cases enzyme activities of all gamma exposed groups (20-100Gy) under

metal stress were evaluated. In each case 6 replicates were considered for analysis.

4.8.1.1 CMCase assay [EC 3.2.1.4]

CMCase activity was studied according to the method of Denison and koehn (1977).

One tube containing 0.45 ml of 1% CMC [Carboxy methyl cellulose] (prepared in 0.1M

sodium citrate buffer; pH: 5.0) and 0.05ml enzyme extract was allowed for 15mins

incubation at 55°C. After incubation, 0.5ml DNS (3,5 dinitrosalicylic acid) was added and

kept in a boiling water bath for 5mins.Then 1ml 40% Rochelle salt was added , volume made

upto 5ml and the absorbance was taken at 540nm (UV –VIS Spectrophotometer, Perkin

Elmer Lambda-25), keeping a blank ( without sample). CMCase activity was calculated with

the help of glucose standard curve. (1Unit/ml = amount of enzyme which releases 1µ mole

glucose under assay condition).

4.8.1.2 α- Amylase assay [EC 3.2.1.1]

α- Amylase activity was studied according to the method of Bernfield (1955). One

tube containing 1ml of 1% starch (prepared in 0.1M acetate buffer; pH: 4.7) and 1ml enzyme

extract was allowed for 15mins incubation at 27°C.After the incubation period 2ml DNS

solution was added and kept in a boiling water bath for 5mins.Then 1ml 40% Rochelle salt

was added, volume made upto 10ml and the absorbance was taken at 560nm (UV –VIS

Spectrophotometer, Perkin Elmer Lambda-25). α- Amylase activity was calculated with the

help of maltose standard curve (1Unit/ml= amount of enzyme which releases 1µ mole

maltose under assay condition).

4.8.2 Antioxidant activity

Mycelium of tested fungus were washed with cold water, squeezed with filter paper

and finally homogenised in 0.015 M potassium phosphate buffer (pH 7.8) containing 0.5 mM

phenyl methylsulfonyl fluoride (PMSF) that inhibited proteases using liquid nitrogen. Then

centrifuged in 20,000rpm for 20mins at 4°C.The supernatant was taken as enzyme extract.

The enzyme activity was expressed as Unit of enzyme/ mg of protein. Total protein was

estimated according to Lowry et al., (1951).In each cases the effective gamma absorbed dose

at which maximum CFU was obtained under metal stress was considered for evaluating

antioxidant response. In each cases 6 replicates were considered for analysis.


48 | P a g e

4.8.2.1 Super oxide dismutase (SOD) [EC: 1.15.1.1]

SOD activity was measured spectrophotometrically based on the inhibitory action of

the enzyme on the rate of NADH oxidation, following Paoletti et al., (1986). In this assay 1

Unit (U) of SOD is defined as the amount of enzyme required to cause 50% inhibition of the

rate of NADH reduction at 340nm using UV-VIS spectrophotometer (Perkin Elmer Lambda-

25) .The assay mixture contained 100mM Triethanolamine- diethanolamine buffer (pH 7.4),

7.5 mM NADH, 100mM/50mM EDTA/MnCl2 ,10mM β mercaptoethanol and the enzyme

extract. The reaction was started by adding of β mercaptoethanol (10mM), the decrease in

absorbance at 340nm for 10min was noted. Activity was expressed as Units/mg of protein.

4.8.2.2 Catalase assay (CAT) [EC: 1.11.1.6]

CAT activity was measured based on the ability of the enzyme to break down H2O2 to

form water and molecular oxygen following the method of Aebi, (1984). H2O2 absorbs

maximum light at 240nm and as compared by catalase, the absorbance decreases. The

difference in absorbance per unit time is a measure of catalase activity. The assay mixture

contained 100mM phosphate buffer (pH 7.0), 30mM H2O2 and the enzyme extract. The

reaction was started by addition of H2O2. The decrease in absorbance was noted at 25°C

with a recorder at an interval of 30sec for 5mins.The unit of CAT activity is determined by

following expression and expressed as µmoles/sec/mg of protein. Moles of H2O2

consumed/min (units/mg)=2.3/Δt x ln (E initial /E final)

Where, E = optical density at 240nm,

Δ t = time required for a decrease in the absorbance

4.8.2.3 Total reduced glutathione assay (GSH) [EC: 1.8.1.7]

Total reduced glutathione was assayed according to the method of Moron et al.,

(1979).A total 3 ml volume was prepared by adding 2 ml of 0.5 mM DTNB (prepared in 0.2

M phosphate buffer ; pH 8) and 1 ml of the enzyme extract. The GSH reacted with DTNB

(5,5-dithiobis-2- nitrobenzoic acid; SRL chemical) and formed a yellow complex with

DTNB. The absorbance was taken at 412 nm.GSH content was measured according to the

reduced glutathione (GSH, Sigma Aldrich, USA) standard curve.

4.8.2.4 Total protein estimation


49 | P a g e

Protein assay was done by the method of Lowry et al., (1951). The principle behind

the Lowry method of determining protein concentrations lies in the reactivity of the peptide

nitrogen[s] with the copper [II] ions under alkaline conditions and the subsequent reduction

of the Folin Ciocalteau phosphomolybdic phosphotungstic acid to heteropoly molybdenum

blue by the copper-catalyzed oxidation of aromatic acids. For the assay of protein,

appropriate volumes of protein extract (5-20µl) was taken in tube, the final volume was made

to 1ml with 100mM potassium phosphate buffer (pH 7). 2ml of solution containing 2% Na2

CO3 in 0.1 N sodium hydroxide , 1% NaK tartarate and 0.5% CuSO4, 5H2O (49:1:1) were

added to each tube. After 10mins of incubation in room temperature, 0.2 ml of diluted Folin’s

reagent (1 part 2N Folin Phenol : 1 part deionised water ) was added followed by immediate

mixing. After 30mins the absorbance was measured at 600nm using UV-VIS

spectrophotometer (Perkin Elmer Lambda-25) against phosphate buffer (pH 7) as blank. The

protein concentration was calculated using BSA (Bovine Serum albumin ;Sigma Aldrich,

USA) protein standard solution (10-400 µg/ml).

4.9 Metallothionein protein assay

4.9.1 Estimation of total Metallothionein using spectrophotometric method

Quantitative estimation of metallothionein protein was estimated according to the

method of Viarengo et al., (1997).Fungal mycelium grown in liquid medium were

homogenized separately in homogenising buffer (a mixture of 0.5 mol/l sucrose, 0.02 mol/l

Tris-HCl buffer [pH 8.6] containing 6 × 10–6 mol/l leupeptine [Sigma Aldrich, USA ], 5 × 10–

3 mol/l phenyl methyl sulphonyl fluoride [PMSF; SRL chemicals] as antiproteolytic agent

and 0.01% β-mercaptoethanol as reducing agent). The homogenate was centrifuged at 30000

g for 20 min. The supernatant was then treated with ethanol-chloroform as described by

Kimura et al (1979). Cold (– 20°C) absolute ethanol (1.05 ml) and 80 μl of chloroform were

added to aliquot of 1 ml of supernatant; the samples were then centrifuged at 6000 g for 10

min at 0 – 4°C. The supernatant was mixed with 1 mg RNA (SRL chemicals) and 40 μl 37%

HCl and subsequently with three volume of cold ethanol. The sample was maintained at

-20°C for 1 h and centrifuged at 6000 g for 10 min. The pellets containing metallothionein

was then washed with 87% ethanol and 1% chloroforms in homogenizing buffer (mentioned

earlier), and centrifuged at 6000 g for 10 min and dried under nitrogen stream. The pellets

were re-suspended in 150 μl NaCl (0.25 mol/l) and subsequently 150 μl HCl (1 N) containing

EDTA (0.004 mol/l) and 4.2 ml NaCl (2 mol/l) containing 5,5-dithiobis-2- nitrobenzoic acid

(DTNB; SRL chemicals) (4.3 × 10–4 mol/l) buffered with 0.2 mol/l Na-phosphate (pH 8)


50 | P a g e

were added. The mixture was then centrifuged at 3000 g for 5 min. The absorbance of

supernatant was taken at 412 nm. The metallothionein concentration was estimated

considering reduced glutathione (GSH, Sigma Aldrich, USA) standard curve (Viarengo et al.,

1997; Pal et al., 2005).The effective gamma dose at which maximum CFU were obtained

under metal stress was considered for metallothionein estimation along with the control and

metal treated group.

4.9.2 Expression of Metallothionein in Fluorecence activated cell sorter (FACS)

Qualitative estimation of metallothionein in flow cytometer was carried out following

the method of Saha et.al.,(2008). 200mg of homogenised mycelium (homogenized with 1X

PBS; PBS; NaCl 0.136 M [w/v], KCl 0.004 M [w/v], Na2HPO4 0.012 M [w/v], KH2PO4

0.002 M [w/v]; pH 7.4) was treated with 200μl of 0.1% chitinase (Sigma Aldrich, USA),

200μl of 1% cellulose (SRL chemical) and 500μl of 15% D- Mannitol and allowed for 16 hrs

incubation. After incubation the mixture was filtered through 40μm mesh to avoid mycelium

debris (if any) and centrifuged at 2000rpm for 5mins. Supernatant was aspirated and cells

were resuspended in 0.5-1ml of PBS and then for fixation formaldehyde was added to it (1–

2% final concentration) and fixed for 10 min at 37°C, then tubes containing cells were chilled

for 1min. After fixation cells were permeabilized with ice cold 100% methanol and incubated

for 30mins on ice. After permeabilization, 1×106 cells (cells counts using heamocytometer)

were aliquoted into each assay tube and 1ml of suspension from each tube taken in another

.For staining, 2–3 ml of incubation buffer containing PBS (pH 7.4) and BSA (w/v) was added

to each tube and rinsed by centrifugation. The process was repeated twice. One portion was

taken as negative control i.e without any primary as well as secondary antibody and another

portion (taken as positive control) was resuspended in 100μl of incubation buffer and allowed

for10mins blocking.1μl of Metallothionein specific primary antibody ( mouse anti rabbit IgG

with 1:100 dilution, Santa Cruz Biotechnology; Santacruz, CA) was added and kept for 30-60

mins at room temperature. Rinsed as before with incubation buffer by centrifugation and cells

were resuspended in 1μl of fluorochrome conjugated secondary antibody (FITC conjugated

anti mouse with 1:200 dilution; Santa Cruz Biotechnology; santacruz, CA) for 30 mins in

room temperature .Again cells were rinsed in incubation buffer and finally resuspended in

0.5ml PBS .Both positive and negative controls were analysed in Flow cytometer. FITC was

detected in a green channel or FL-1. Finally MT was estimated using FACS Calliber Flow

Cytometer (Becton Dickinson) equipped with an argon ion laser emitting at 488 nm. Finally,


51 | P a g e

the data were analyzed using software Cell Quest. At a time 50,000 cells were analysed for

both positive and negative control. The effective gamma dose at which maximum CFU were

obtained under metal stress was considered for metallothionein estimation along with the

control and metal treated group.

4.10 Statistical Analysis

Statistical analyses were based on the mean values performed on 3-6 replicates for

each set of experiments. Students‘t’ test was analysed to determine the level of significant

differences between control and treated samples. The difference is considered to be

significant in the case of p<0.05. All statistical analysis were done in Origin 6.1 software.

Results

52 | P a g e

Section I Heavy metal load in soil parameter and diversity of fungal population in waste dumping site of Kolkata 5.1. Soil parameter analysis

Analysis of texture of the soil samples of Dhapa site was found to be clay loam.

Physico-chemical nature of soil revealed the basic nature of the soil having pH range 6.1-6.8.

Soil samples showed 600- 430 µS/cm conductivity and contained 2.15-3.37% organic carbon

reflecting good quality of soil. Zinc concentrations were significantly higher in all three soil

samples (180-225ppm) while Cd concentrations were in the range of 2.8-3.6ppm. Pb

concentrations in all soil samples were below the detectable limit (BDL) of AAS (0.015ppm).

Physico chemical properties of the collected soil samples are presented in Table 5.1

Table 5.1. Physico-chemical characteristics of soil samples

Parameters Soil samples S1 S2 S3

Soil type Clay loam Clay loam Clay loam pH 6.8 6.5 6.1 Conductivity 430 µS/cm 550 µS/cm 600µS/cm Percentage of Nitrogen 0.163 0.220 0.190 Percentage of Phosphate 0.036 0.079 0.085 Percentage of Organic 3.37 2.15 2.20 Concentration of Zn 225ppm 210 180 Concentration of Pb BDL BDL BDL Concentration of Cd 3.6ppm 3.0 3.2

5.2. Fungal population dominating the Dhapa soil

Ten different fungal strains were found to be dominant in the soil sample collected

from Dhapa. Morphological and biochemical characterization of the fungi confirmed all the

strains to be filamentous in nature. Morphological characteristics validated classification up

to generic level and amongst the dominant fungi 3 different Aspergillus sp, 3 Penicillium sp,

and 1 each of Curvularia sp, Alternaria sp, Trichoderma sp and Fusarium sp. were obtained.

The strains were deposited in department of environmental science, University of Kalyani

for future preservation. The detailed morphological characters of the dominant fungal strains

isolated from Dhapa soil are given in Table 5.2.

Results

53 | P a g e

Table 5.2. - Morphological features of isolated strains

Sl. No Parameters

Isolated Fungal strains DESFC

10 DESFC

11 DESFC

12 DESFC

13 DESFC

14 DESFC

15 DESFC

16 DESFC

17 DESFC

18 DESFC

19

1 Colony colour Black Brown Green Green Yellowish white Black White Black Yellow green Black

2 Conidial shape Globous Globous Chain of

ovoid conidia

Chain of ovoid

conidia

Large,multicellular, crescent shaped;

microconidis also present

Sympodically formed;

Curved with 3 septa

Bicelled

Multiv-cellular with a

transverse and

longitudinal septa

Globous Chain of

ovoid conidia

3 Vesicle shape Globular/ bulbous

Globular/ bulbous Septate Septate - - - - Globular/bulb

ous Septate

4 Sterigmata number and

position One layer One layer

Conidiophores are

branched into

clusters

Conidio-phores are

un-branched Branched

Compact mass

developed on cushion like

str.

Many, Entire surface

Short, dark colore,

undifferentiated

One layer

Conidiophores are

branched into

clusters

5 Conidiospore

colour /morphology

Black Brown Green Green Dark

Macroscopic with 2-3

transeverse section

White Black Yellow green Green

6 Identification

of fungal strains

Aspergillus Aspergillus Penicillium Penicillium Fusarium Curvularia Tricho-derma Alternaria Aspergillus penicillium

DESFC: Department of Environmental Science Fungal Culture

Results

54 | P a g e

5.3. Metal tolerance of the isolated fungal strains

The different fungal strains isolated from Dhapa showed differential tolerance

towards metals showing different interaction potential of fungi with a particular metal. The

difference in metal tolerance values indicated that such tolerance was metal specific and

dependant on the strain of the fungi. Cd tolerance of the isolated fungal strains range from

90-675ppm and Zn from 1200-9100ppm. Analysis of metal tolerance revealed DESFC10 to

be sensitive for Cd and Zn. (MIC values are 90ppm, and 1200ppm respectively), DESFC11

was found to be tolerant for Zn having MIC of 9100ppm but sensitive for Cd (MIC 95ppm).

DESFC12 was observed to be tolerant towards Cd showing the MIC as 675ppm. Except

DESFC10 and DESFC12 no other fungi could grow in media with Pb .Table 5.3 showed

metal tolerance (Cd, Zn and Pb) of the isolated fungi.

Table 5.3. Heavy metal tolerance of isolated fungal strains

Strains

MIC values

Cd(ppm) Zn(ppm) Pb(ppm)

DESFC10 90 1200 3050

DESFC11 95 9100 -

DESFC12 675 1500 3100

DESFC13 225 1300 -

DESFC14 250 1500 -

DESFC15 300 2000 -

DESFC16 150 2500 -

DESFC17 175 3000 -

DESFC18 125 3500 -

DESFC19 225 2150 -

DESFC20 250 1550 -

Among ten isolated strains some fungal strain is sensitive while some one is tolerant against a

particular metal. Table 5.4 listed the sensitive and tolerant strain against Cd, Pb and Zn.

5.4. Species Identification of the selected strains

Species identification of the selected fungal strains showed that the soil sample

contained Aspergillus niger, Aspergillu terreus and Penicillium cyclopium. Their

characteristic features are represented in Table 5.5.

Results

55 | P a g e

Table 5.4. Sensitive and tolerant fungal species among isolated strains against each

metal

Metal Sensitive strain Tolerant strain

Zn DESFC10 DESFC11

Cd DESFC10, DESFC11 DESFC12

Pb* DESFC10 DESFC12

*No other fungal strains except DESFC10 and DESFC12 could grow properly against Pb

enriched media.

Table 5.5. Morphological and Physiological features of the selected strains

Strain ID Strain Name

Colony Characters

Colony color

Conidiophore structure

Diameter of conidiophores

(μm)

Diameter of

conidia

Sporulation time (hrs)

Wet biomass

(g)

DESFC10 Aspergillus niger

Black Unbranched 10-20 3.5-5.0 um

48-72 9.136

DESFC11 A.terreus Sand brown

Unbranched 10-15 1.5-3.4 μm

48-72 6.697

DESFC12 Penicillium cyclopium

Greenish with white border

Divaricate 44 – 44.95 10.99-11.59μ

>72 7.025

5.5. Biochemical characterisation of the selected strains

5.5.1. Cross Metal tolerance:

Assessment of cross metal tolerance study divulged that one particular fungal strain

might be tolerant for one metal and simultaneously sensitive for another metal and metal

tolerance varies with metal to metal against each strain and vice versa. A.terreus was

observed to be tolerant for Zn (MIC 9100ppm) but sensitive for Hg (MIC 20ppm) and Cd

(MIC 95ppm), P.cyclopium was found to offer highest tolerance towards Pb (MIC 3100ppm)

followed by Zn (MIC 1500ppm) and Cd (MIC 675ppm). This strain, like A terreus is also

sensitive towards Hg and showed growth inhibition at a concentration of 50ppm (MIC) of

Hg. Degree of Zn tolerance of the selected strains showed A.terreus having maximum

tolerance followed by P.cyclopium and then A.niger. The strain A.niger showed its maximum

tolerance for Pb (MIC 3050ppm) followed by Zn (MIC 1200ppm) and Cr (MIC 1000ppm).

The same strain showed very less tolerance towards Hg (MIC 30ppm). However interaction

Results

56 | P a g e

of A.niger towards Hg reflected higher tolerance than that of A.terreus but lower than that of

P.cyclopium. While P.cyclopium could tolerate a high concentration of 675ppm of Cd, both

the Aspergillus sp showed much less tolerance towards Cd. While the degree of tolerance of

A.niger and A.terreus was noted to be similar in case of Cd and Hg the two species showed a

wide difference in their tolerance towards Zn and Cr. A. terreus demonstrated nearly 8 times

more tolerance for Zn and 2 times for Cr. Tolerance of three fungal strains towards Hg, Cd,

Zn, Pb, and Cr is presented in Table 5.6.

Table 5.6. Cross Metal Tolerance (in terms of MIC) of selected fungal strains

Strain Name

MIC in ppm

Hg Cd Zn Pb Cr Aspergillus niger 30 90 1200 3050 1000

A.terreus 20 95 9100 - 500

Penicillium cyclopium 50 675 1500 3100 650

5.5.2. Antibiotic sensitivity test

The antibiotic sensitivity test of the studied fungi showed resistance of one fungi to a

certain concentration of one antibiotic while another fungi was sensitive for same

concentration of that antibiotic. This confirmed response of fungi towards antibiotics is

highly strain or species specific. Nystatin was found to be maximum effective for all strains.

While Griseofolvin was most effective for P.cyclopium than that of A.niger and A.terreus. At

the same time Kanamycin showed its more efficacy towards A.terreus with respect to other

two strains (A.niger and P.cyclopium). Minimum Inhibitory Concentrations (MIC) for tested

fungal strains against three antibiotics were presented in Table 5.7.

Results

57 | P a g e

Table 5.7. Antibiotic Sensitivity of selected fungal strains

Strain Name Antibiotics

Nystatin Griseofolvin Kanamycin

MIC(ppm) MIC(ppm) MIC(ppm)

Aspergillus niger 80 100 120

A.terreus 80 100 80

Penicillium cyclopium 80 80 120

5.5.3. Optimization of pH and Temperature for growth of selected fungal strains

Different pH values (5,7and 9) used to study growth of the tested fungi keeping the

temperature constant at 29 0 –30 0 C, showed the optimum pH to be 7. Hence maximum

growth of all the tested fungal strains were obtained at pH7. With increasing pH from 5 to 7

the biomass yield was found to be increased in all the cases being maximum at pH 7. Further

increase in pH (at pH 9) caused a decrease in biomass. Table 5.8 represents the optimum pH

for fungal growth). While for temperature variation in growth, pH was retained constant at

7.At lower temperature ( 250 C) and at higher temperature (350 C) less growth were noticed

for tested fungal strains than temperature 29 0 –30 0 C.

Table 5.8. Effect of pH on growth of selected fungal strains

Strain name Mycelial weight (mg) (Mean ± SD)

pH 5 pH 7 pH 9

Aspergillus niger 1.8±0.25 2.221±0.15 1.95±0.27

A.terreus 1.25±0.15 1.87±0.35 1.56±0.17

Penicillium

cyclopium

1.36±0.22 1.995±0.25 1.546±0.35

Results

58 | P a g e

Table 5.9.Effect of temperatures on growth of selected fungal strains

Strain name Mycelial weight (mg) (Mean ± SD)

Temperature 25°C Temperature 29-

30°C

Temperature 35°C

Aspergillus niger 1.51±0.18 1.86±0.23 1.36±0.18

A.terreus 1.36±0.19 1.995±0.15 1.546±0.25

Penicillium

cyclopium

1.25±0.16 1.652±0.15 1.211±0.15

Effect of different temperatures on growth of isolated fungi in terms of weight of the

mycelia showed maximum mycelial weight at 29-30°C temperature in cases of all the strains

(Table 5.9). Below that temperature (25°C) and above that temperature (35°C) caused

decrease in mycelial growth.

Results

59 | P a g e

Section II Metal tolerance of selected fungal strains before and after gamma exposure

Results of the present investigation depict that gamma irradiated group of fungi (A

niger, A.terreus,P .cyclopium) had significantly enhanced metal (Cd, Zn, Pb) tolerance in

terms of increase in their Colony Forming Unit (CFU). Data of Atomic absorption

spectroscopic (AAS) analysis reveal significant (p≤0.05) increase in metal uptake in the

mycelium of the gamma irradiated groups of fungi in addition to higher potential of those

fungi for metal removal as compared to the unirradiated counterparts. Interestingly the

absorbed dose of gamma irradiation that was most effective in resulting maximum increase in

CFU was observed to be the same dose manifesting maximum uptake and removal of metal

for each fungal strain against each metal.

FTIR spectra of all the selected species of fungi confirmed the involvement of

different functional groups in adsorption of different metals by the fungi. The functional

groups that were observed to be associated with adsorption of Cd, Zn and Pb by A niger,

A.terreus, P .cyclopium are detailed as follows:

Peak between 3600-3100 Cm-1 regions representing bonded OH stretching vibration

as well as acetamido group of chitin fraction.

Peaks in 2925 Cm-1 and 2855 Cm-1 regions attributed to the asymmetric and

symmetric stretching vibration of CH2 respectively.

C=O stretching vibration and NH deformation (amide I) At 1646 Cm-1.

The peak at 1545 Cm-1 assigned for a motion of combined groups of –NH bending

(amide II) and –CN stretching vibrations of protein.

Peaks at 1230 Cm-1 and 1078 Cm-1 regions reflecting SO3 groups and C-O, C-N

stretching vibration respectively

Peaks at 925 Cm-1 and 821-869 Cm-1 regions representing –OH and aromatic –CH

stretching respectively

The finger print region 500 Cm-1 to 700 Cm-1 represents phosphate or sulphur

functional groups.

Results

60 | P a g e

5.6. Cd tolerance efficacies of gamma exposed fungi

Gamma irradiated group of A.terreus showed 1.6 times more cadmium tolerance than

the unirradiated counterparts.

Fig-5.1 Colony Forming Unit of A.terreus with or without exposure to gamma irradiation grown in Cd supplemented media (a) 90ppm Cd (b)150ppm Cd (Error bars represent standard deviation for n = 6. p≤0.05 was considered significant)

Results reveal gamma irradiation could induce significant increase in Cd tolerance of

A.terreus Gamma irradiated A.terreus was observed to grow in media supplemented with

150ppm of Cd, while the unirradiated group of the same strain could grow upto a maximum

concentration of 90ppm of Cd , showing 1.6 times increase cadmium tolerance after gamma

irradiation than unirradiated counterparts. A.terreus grown in 90ppm Cd following exposure

to 20-100 Gy (absorbed dose) of gamma rays showed radiation dose dependent increase in

Results

61 | P a g e

CFU, manifesting a maximum of more than 3 fold increase in the group exposed to 100Gy

(Fig 5.1a) as compared to the unirradiated but 90ppm Cd stressed counterparts. Gamma

irradiated A terreus grown in 150ppm of Cd in the medium manifested radiation dose-

dependent increase in CFU in the groups exposed at 20 to 80Gy absorbed dose having

maximum increase in CFU in the group of A.terreus exposed at 80Gyabsorbed dose of

gamma radiation. The group exposed at an absorbed dose of 100Gy showed decrease in CFU

in comparison to that observed in case of the group exposed at a 80Gy (absorbed dose) (Fig-

5.1b). Without being exposed to gamma A.terreus could not tolerate 150ppm of Cd so the

change after gamma exposure could not be comparable with respect to their unirradiated

counterparts.

Atomic absorption spectrophotometric (AAS) analysis showed higher concentration

of Cd in the mycelia of gamma irradiated Cd treated A. terreus as compared to that of the

unirradiated but Cd stressed groups. This reflected potential of gamma radiation to induce

higher uptake of Cd. Removal of Cd from the growth media was also observed to be higher in

case of the groups exposed to gamma irradiation followed by growth in Cd supplemented

media in contrast to the unirradiated groups exposed to only Cd. Gamma exposed A.terreus

grown in 90ppm Cd supplemented media showed a dose-dependant enhancement in

efficiency for uptake and removal of Cd. The group exposed at 100Gy absorbed dose of

gamma showed maximum efficiency of Cd uptake (1.8times more) as compared to the

respective control group (Fig 5.2a). Gamma irradiated A.terreus grown in 150ppm Cd

supplemented media showed steep increase in potential for uptake and removal of Cd when

exposed at absorbed dose range higher than 40Gy (Fig 5.2b) and the 80 Gy gamma exposed

group of A.terreus could uptake highest amount of cadmium (11.32mg/g of tissue) and

showed 74% removal in comparison to the other irradiated groups.

Results

62 | P a g e

Fig 5.2 Uptake and removal of Cd by Aspergillus terreus exposed to different absorbed doses of

gamma irradiation (20-100 Gy) and grown in media supplemented with different concentrations

of Cd. (a) 90ppm Cd (b) 150ppm Cd (Error bars represent standard deviation for n = 3; p≤0.05

was considered significant)

Results

63 | P a g e

FTIR spectra of freeze dried biomass of A.terreus treated with Cd with or without

exposure to gamma irradiation and the normal control group is shown in Fig 5.3. Data

showed alterations in amide group (-NH), hydroxyl group (-OH), carboxylate group (-

COO), carbonyl group (-CO) in the experimental groups. Significant shifts in spectral peaks

were observed in fungal biomass from Cd (90ppm) treated group compared to the control

group without any metal treatment. Shifting of bands were noted at 3380 Cm-1; 1459 Cm-1,

1247 Cm-1 in gamma irradiated group of A.terreus grown in media containing 90ppm Cd and

unirradiated group of fungi grown in same Cd concentration as compared to the normal

control group without irradiation or Cd exposure. While in the experimental group of

A.terreus treated with 90ppm of Cd, the peak in 3380 Cm-1 region was found to be shifted to

3375 Cm-1 region, the gamma exposed fungal biomass grown in same concentration of Cd

enriched media showed a much larger shift to 3356 Cm-1 region, all compared to the normal

control group. Shifting of peak in 1459 Cm-1 region however was similar in the two

experimental groups showing a stretch to 1463 Cm-1 region in Cd treated group and to 1464

Cm-1 region in the gamma irradiated group grown in Cd loaded media, all compared to that of

the normal control group. Interestingly the peak in 1372 Cm-1 region was not observed in Cd

treated group.

Results

64 | P a g e

Fig 5.3 FTIR spectra of A.terreus biomass

(a) Control biomass (b) 90ppm Cd treated biomass (c) 100Gy gamma exposed biomass

grown in 90ppm Cd enriched media

P.cyclopium showed 1.1 times more Cd tolerance than that of their unirradiated

counterparts. Gamma exposed P.cyclopium could grow in 750ppm of Cd in growth media

while the unirradiated group had MIC of 675ppm Cd. Increase in Cd tolerance was observed

in terms of increase in CFU of gamma irradiated Penicillium cyclopium grown in Cd

supplemented growth media. Gamma irradiated groups of P. cyclopium grown in 300-650

ppm of Cd in the media, showed a radiation dose dependent enhancement of CFU up to a

certain dose of gamma exposure and the absorbed dose of gamma that produced maximum

enhancement in CFU was dependent on concentration of Cd treatment. Irradiated

P.cyclopium grown in 300ppm of Cd had 20%-73% increase in CFU in groups exposed to

20-60Gy absorbed dose of gamma irradiation (Fig 5.4a). Maximum increase (1.13fold) in

CFU was observed in the group exposed at 80Gy of absorbed doses of gamma. Although a

slight decrease in CFU was noticed in the group having exposed to 100Gy absorbed dose of

gamma rays in comparison to that exposed to 80Gy absorbed dose of gamma rays, it was still

93% more than un-irradiated counterparts

Results

65 | P a g e

Gamma irradiation also stimulated CFU formation in P.cyclopium grown in media

supplemented with 650ppm Cd. However, in contrast to the group of fungi grown in 300ppm

of Cd treated media, the fungi grown in 650ppm of Cd manifested a maximum of 1.2 fold

increase in CFU when exposed to 60Gy absorbed dose of gamma irradiation (Fig 5.4a).

Although CFU in the groups exposed to gamma irradiation at higher absorbed doses (80Gy

and 100Gy) had been lesser than the 60Gy exposed group, however number of CFU was

more than that observed in the unirrdiated control group.

CFU analysis depicted an interesting data which validated the potential of gamma

irradiation in significantly enhancing Cd tolerance of P.cyclopium. In contrast to no growth

of P.cyclopium at 750 ppm Cd, exposure of the fungi to gamma irradiation and then grown in

media supplemented with 750ppm Cd showed significant growth of the fungi having

maximum CFU in the group exposed at 40Gy (absorbed dose) of gamma irradiation.

Data of AAS analysis reveal significant (p≤0.05) potential of gamma irradiation in

modulating uptake as well as removal of Cd by P.cyclopium exposed to different doses of

gamma rays and grown in media supplemented with Cd. 80Gy exposed P.cyclopium could

uptake three times more Cd (which supports 13% more cadmium removal) than its

unirradiated counterparts when grown in 300ppm of cadmium. Similarly from 650ppm of

cadmium enriched medium a 60Gy exposed P.cyclopium could remove 13% more cadmium

than its unirradiated counterparts (Table 5.10). Interestingly 40Gy exposed P.cyclopium

could remove 60% of Cd from 750ppm Cd enriched media, while unirradiated one could not

tolerate such higher concentrations of Cd (Table 5.10).

Results

66 | P a g e

Fig-5.4 Colony Forming Unit of P.cyclopium with or without exposure to gamma irradiation grown in Cd supplemented media (a) 300ppm -650ppm Cd (b) 750ppm Cd (Error bars represent standard deviation for n = 6. p≤0.05 was considered significant)

UI 20Gy 40Gy 60Gy 80Gy 100Gy-2

0

2

4

6

8

10

12

14

16

18

UI- Unirradiated

(b)

CFU

/ml

Dose

750ppm

Results

67 | P a g e

Table: 5.10 Uptake and removal of Cd by P.cyclopium with or without exposure

to gamma irradiation (Mean±SD for n=3)

Cultured condition 300ppm of Cd

Uptake Potential (mg/g of

tissue)

Removal (%)

Unirradiated P. Cyclopium

14.681±1.25 82±3.5

80Gy gamma exposed P.

cyclopium

44.001±1.5 95±3.25

650ppm of Cd

Unirradiated P. Cyclopium

30.62±1.5 85±4.25


cyclopium

65.23±3.25 98±4.25

750ppm of Cd


cyclopium

15.65±1.05 60±2.5

Fig 5.5 shows the FTIR spectra of fungal biomass from control group of P.cyclopium, groups

grown in 300ppm Cd supplemented media and gamma exposed P.cyclopium grown in

300ppm Cd supplemented media. Spectral data analysis showed involvement of amide group

(-NH), hydroxyl group (-OH), carboxylate group ( -COO), carbonyl group (-CO) functional

groups in Cd adsorption. Significant shift in peak regions were found at 3394 Cm-1, 2928 Cm-

1, 1548 Cm-1, 1240 Cm-1 regions in biomass of P.cyclopium grown in Cd supplemented

media with respect to its control (Cd free biomass).

Results

68 | P a g e

Fig 5.5 FTIR spectra of P.cyclopium biomass

(a) Control biomass (b) 300ppm Cd treated biomass (c) 80Gy gamma exposed biomass

grown in 300ppm Cd enriched media

Significant changes in peaks were observed in Cd treated P.cyclopium exposed to

gamma irradiation. The peak at 3394 Cm-1 region observed in the control P.cyclopium

biomass (Fig 5.5 a) was noted to be shifted to 3385 Cm-1 in gamma irradiated and Cd treated

group of P.cyclopium (Fig 5.5 c) while in case of the group that received only Cd

supplementation during growth but was not exposed to gamma rays, the same peak was noted

at 3363 Cm-1 region (Fig-5.5b). Peaks in the regions 1548 Cm-1 and 1240 Cm-1 were also

noted to be shifted in the two experimental groups as compared to the normal control group.

One new peak at 1450 Cm-1 region was noted in Cd treated biomasses with or without gamma

exposure, which was not observed in control one.

4000 3500 3000 2500 2000 1500 1000 5001020304050607080

4000 3500 3000 2500 2000 1500 1000 500102030405060704000 3500 3000 2500 2000 1500 1000 500

30405060708090

1034.57

1080

.80

1153

.43

1240

.04

1376

.114

14.2

1548

.64

1646.22

1746.83

2855.252928.24

3394.51

Wavenumbers (Cm-1)

(a)

1037.9

1078.371151

.36

1235

.22

1373

.21

146.

1214

52.1

215

44.4

2

1647.35

1748.12854.72

2926.85

3363.71

1036.58

1080

.78

1152

.22

1237

.61

1372

.57

1411

.18

1455

.17

1546

.35

1645.20

1748.16

2854.35

2926.803385.43

% T

rans

mitt

ance

(b)

(c)

Results

69 | P a g e

Gamma exposed A.niger showed 1.17 times more Cd tolerance than unirradiated

counterparts. Fig-5.6 reflects change in number of colony forming units (CFU) of Aspergillus

niger in response to different absorbed doses of gamma when grown under different

concentrations of Cd in the medium. The pattern of change in CFU is different in case of

different concentrations of Cd supplemented in the medium (Fig5.6a-b).While gamma

exposed A.niger grown in 70ppm Cd in the medium showed radiation dose dependent

increase in CFU upto 60Gy absorbed dose of gamma irradiation, the group of A.niger grown

in 85ppm of Cd in the medium manifested maximum increase (5 fold) in CFU when exposed

at 40 Gy absorbed dose of gamma rays, all compared to that of the unirradiated but Cd

treated counterparts. Exposure at 80 and 100y Gy absorbed dose of gamma irradiation also

induced higher CFU in A. niger grown in media supplemented with 70 and 85 ppm of Cd as

compared to the unirradiated but Cd stressed group. The group of gamma irrdiated A.niger

grown in 100ppm Cd supplemented medium manifested significant growth in contrast to the

unirradiated group which tolerate 90ppm of Cd only and maximum CFU of the gamma

irradiated group grown in 100ppm Cd enriched media was observed when exposed at an

absorbed dose of 20Gy. This group upon exposure to still higher doses however manifested

gradual decline in CFU (Fig 5.6b).

Results

70 | P a g e

20Gy 40Gy 60Gy 80Gy 100Gy

0

50

100

150

200

250

(a)

70ppm 85ppm

Dose (Gy)

Cha

nge

in C

FU (%

) with

resp

ect t

o un

irrad

iate

d co

unte

rpar

ts

0

50

100

150

200

250

300

350

400

450 Change in C

FU (%

) with respect to unirradiated

counterparts

Fig-5.6 Colony Forming Unit of A.niger with or without exposure to gamma irradiation grown in Cd supplemented media (a) 70-85ppm (b) 100ppm (Error bars represent standard deviation for n = 6. p≤0.05 was considered significant)

The absorbed doses of gamma irradiation, which resulted in maximum CFU in all the

three groups of A.niger grown with three different concentrations of Cd in the medium, also

reflected significant stimulation of potential of the fungi for Cd uptake and removal.

Aspergillus niger exposed to 60Gy absorbed dose of gamma could accumulate 1.73 times

more Cd and could remove 11% more Cd from 70ppm Cd supplemented media with respect

to its unirradiated counterpart. From 85 ppm Cd supplimented media the 40Gy exposed

A.niger showed 1.65 times more accumulation and 10.5 % more removal with respect to

unirradiated counterparts.Without being exposed to gamma irradiation Aspergillus niger

could not tolererate 100ppm Cd but after being irradiated, a 20 Gy exposed Aspergillus sp

could remove 45% Cd from 100 ppm Cd supplemented media (Fig-5.7)

100ppm+20Gy 100ppm+40Gy 100ppm+60Gy 100ppm+80Gy 100ppm+100Gy0

10

20

30

40

50

60

(b)

CFU

/ml

Results

71 | P a g e

70ppm 70ppm+60Gy 85ppm 85ppm+40Gy100ppm+20Gy0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8 Uptake (mg/g of tissue) Removal (%)

Upta

ke (m

g/g

of ti

ssue

)

40

42

44

46

48

50

52

54

56

58

Removal(%

)

Fig-5.7 Uptake and removal of Cd by Aspergillus niger exposed to different absorbed doses of gamma irradiation (20-100 Gy) and grown in media supplemented with different concentrations of Cd. (Error bars represent standard deviation for n = 3; p≤0.05 was considered significant)

5.7. Zn tolerance efficacies of gamma exposed fungi

Analysis of CFU of the selected fungi with or without exposure to gamma irradiation

and having grown in Zn supplemented media reflects significant potential of gamma

irradiation in inducing higher tolerance towards Zn in both the species of Aspergillus sp.

While gamma irradiated A.terreus grown in Zn enriched media showed 1.13times more Zn

tolerance (Fig 5.11), A.niger being exposed to gamma irradiation followed by culture in Zn

supplemented media showed 1.82 times more tolerance (Fig 5.8) as compared to their

respective unirradiated metal treated counterparts. However, the effect of radiation varied

depending upon the concentration of Zn in the media, Gamma irradiated A.niger grown in

250ppm of Zn showed dose dependent increase in CFU when exposed at 40-80Gy absorbed

dose of gamma rays, having maximum number of colonies in groups exposed at 80Gy.

Exposure to 100 Gy absorbed dose of gamma did not produce any further increase in CFU of

A.niger than that observed in the group exposed at 80 Gy(Fig 5.8A). In conditions having

higher concentration of Zn (500 to 2000ppm) in the growth media however, maximum CFU

was observed in groups exposed at 60 Gy absorbed dose of gamma rays (Fig 5.8B-

E).Assessment of uptake and removal potential of Zn by Aspergillus niger through AAS

analysis showed increase in zinc uptake in the gamma irradiated groups of Aspergillus niger

as compared to that of the unirradiated groups. A. niger exposed to 100 Gy absorbed dose of

Results

72 | P a g e

gamma rays, could accumulate 1.5 times more Zn from the medium containing 250ppm of Zn

as compared to that observed in case of the unirradiated group grown in media containing

same concentration of Zn. Efficiency of removal of Zn from the medium was also observed

to be 10% more in irradiated group (100Gy absorbed dose exposed) when compared to that

of the un-irradiated group (F of Fig5.9a). Though in terms of Zn tolerance as measured by

CFU, no difference was observed between the groups exposed to 80 Gy and 100 Gy of

absorbed doses (Fig 5.8A), the latter showed better prospective in terms of modulating Zn

accumulation efficiency simultaneous with the Zn removal potential of the respective group

of the fungi(F of Fig 5.9a). A.niger treated with higher concentration of Zn (500ppm and

1000ppm) showed a maximum of 3 fold increase in Zn uptake in mycelium and 20% more

removal of Zn from the respective media when exposed to 60 Gy absorbed dose of gamma as

compared with the respective unirradiated counterparts (group D of both Fig-5.9b and Fig-

5.9c). Gamma irradiated A.niger grown in media supplemented with 1500ppm and 2000ppm

of Zn in the media showed significant accumulation of Zn in the mycelia and could also

remove substantial Zn efficiently from the media in contrast to the control group of fungi

which could not tolerate that much Zn in the media and showed no growth at all. An

exposure to 60 Gy of absorbed dose of gamma showed maximum Zn uptake and removal in

both the groups treated with 1500ppm and 1000ppm of Zn (group C of both Fig-5.9d and

5.9e ).

Results

73 | P a g e

Fig-5.8 Colony Forming Unit of A.niger with or without exposure to gamma irradiation grown in Zn supplemented media (A) 250ppm Zn (B) 500ppZn (C) 1000ppm Zn (D) 1500ppm Zn (E) 2000ppm Zn (Error bars represent standard deviation for n = 6; p≤0.05 was considered significant)

cont 20Gy 40Gy 60Gy 80Gy 100Gy40

60

80

100

120

140

160

180

200

220

240

*

Aspergillus nigerMetal- 250 ppm ZnAfter 14 days of Incubation

CFU

/ml

Dose(A)

cont 20Gy 40Gy 60Gy 80Gy 100Gy

0

10

20

30

40

50

60

70


CFU

/ml

Dose(C)

cont 20Gy 40Gy 60Gy 80Gy 100Gy-5

0

5

10

15

20

25

30

35


CFU

/ml

Dose(D)

con t 20Gy 40Gy 60Gy 80Gy 100G y

0

20

40

60

80

100

120

140

160

180

*

A sperg illus nigerMetal- 500 ppm ZnA fter 14 days o f Incubation

CFU

/ml

Dose(B)

cont 20Gy 40Gy 60Gy 80Gy 100Gy

0

5

10

15

20

25


CFU/

ml

Dose(E)

Results

74 | P a g e

Results

75 | P a g e

Fig-5.9 Uptake and removal of Zn by Aspergillus niger exposed to different absorbed doses of gamma irradiation (20-100 Gy) and grown in media supplemented with different concentrations of Cd (a) 250ppm (b) 500ppm (c) 1000ppm (d) 1500ppm (e) 2000ppm (Error bars represent standard deviation for n = 3; p≤0.05 was considered significant)

051015202530354045

0123456789

A B C

Upt

ake

(mg/

g o

f tis

sue)

Uptake(mg/g of tissue)Removal(%)

Removal(%

)

(e)

A-2000ppm+20Gy, B- 2000ppm+40Gy, C-2000ppm+60Gy

Dose

Results

76 | P a g e

Fig 5.10 FTIR spectra of A.niger biomass

(a) Control biomass (b) 250ppm Zn treated biomass (c) 100Gy exposed biomass grown

in 250ppm Zn enriched media

FTIR studies of biomass of A.niger grown in 250ppm Zn supplemented media and

100Gy exposed A.niger when grown in 250 ppm Zn supplemented growth media showed

significant changes in the region of 3383Cm-1, 2928 Cm-1, 2856 Cm-1, 1552 Cm-1, 1149 Cm-1,

1033 Cm-1 regions (Fig 5.10) when compared to their normal control counterparts (neither

exposed to gamma nor treated with metal). A peak in 1230 Cm-1 region in control biomass

was not observed in spectra of the two other experimental groups. Spectral characteristics at

regions 3383Cm-1 and 2928 Cm-1 noted in biomass of the control group were observed to be

altered in both the experimental groups. While in case of the former a shift to 3373 - 3374

Cm-1 regions was observed, the latter region showed a shift to 2923 Cm-1 region in Zn treated

group and gamma exposed group grown in Zn enriched media respectively. Peak in 1033

Cm-1 region of control group was shifted to 1025 Cm-1 region in the two experimental

groups.

Results

77 | P a g e

Exposure to gamma irradiation induced 1.13 times more Zn tolerance in A.terreus as

compared to the unirradiated counterparts and in contrast to the unirradiated group which

could tolerate a maximum of 9100ppm of Zn in the medium as expressed by CFU, the

gamma exposed group of the fungi showed growth even when Zn concentration in the

medium was 10,250 ppm (Fig 5.11). Response of gamma irradiated A. niger in terms of its

colony number was observed to be inversely proportional with increase in zinc concentrations

in the media (Fig 5.8). Although an exposure to 80Gy absorbed dose of gamma rays showed

maximum increase in CFU in all the three groups of A.terreus grown in Zn supplemented

media, the group supplemented with 7000ppm was observed to have highest induction in

CFU as compared to the groups grown in 8000ppm and 9000ppm of Zn. Against 7000ppm of

Zn, 80Gy exposed A.terreus manifested 65% increase in CFU than unexposed counterparts,

while against 8000 ppm and 9000ppm of Zn , only 35% and 15% more CFU was noted when

compared to the respective unirradiated counterparts. Still higher concentration of Zn

(10,250ppm) in the media was observed to support growth of A.terreus only if exposed to

gamma irradiation and maximum CFU was noted in the group exposed to an absorbed dose

of 80 Gy.

Results

78 | P a g e

Fig 5.11 Colony Forming Unit of A.terreus with or without exposure to gamma irradiation grown in Zn supplemented media (a) 7000-9000ppm Zn (b)10250ppm Zn (Error bars represent standard deviation for n = 6. p≤0.05 was considered significant).

The effect of gamma irradiation on Zn uptake and removal potential of A.terreus was

observed to be similar to that observed in case of A.niger. The group of A.terreus exposed at

an absorbed dose of 80Gy of gamma rays and showing maximum CFU when grown in 7000-

9000ppm of Zn, manifested 3 – 3.8 times higher uptake potential for Zn from 7000 –

9000ppm zinc enriched media and 6%- 11% more removal of Zn than that of the respective

unirradiated counterparts (Table 5.11). Interestingly 80Gy exposed A.terreus could remove

40% Zn from 10,250ppm Zn enriched media, while unirradiated one could not grow at that

Zn enriched growth media (Table 5.11).

20Gy 40Gy 60Gy 80Gy 100Gy0

10

20

30

40

50

60

70

(a)

Cha

nge

in C

FU(%

) with

resp

ect t

o un

irrad

iate

d co

unte

rpar

ts

Dose

7000ppm 8000ppm 9000ppm

UI 20Gy 40Gy 60Gy 80Gy 100Gy-2 0

2

4

6

8

10

12

14

(b)

UI- Unirradiated

Dose

10250ppm

CFU/ml

Results

79 | P a g e

Table 5.11 Uptake and removal of Zn by A.terreus with or without exposure to gamma irradiation (Mean ±SD for n=3)

Dose Uptake (mg/g tissue) Removal (%)

A.terreus grown in 7000ppm

Zn enriched growth media

26.65±1.5 75.55±5.5

80Gy exposed A.terreus

grown in 7000ppm Zn

102.22±5.5 85.69±5.5



24.85±2.5 65.89±2.5


grown in 8000ppm Zn

83.33±2.5 76.66±2.5



20.22±1.5 56.32±3.5


grown in 9000ppm Zn

65.55±2.5 62.22±2.5


grown in 10250ppm Zn

40.23±1.75 49.5±2.5

Results

80 | P a g e

Fig5.12. FTIR spectra of A.terreus biomass

(a) Control (b) 8000ppm Zn loaded biomass (c) 80Gy gamma exposed biomass grown in

8000ppm Zn enriched media

Fig 5.12 showed the FTIR spectra of different experimental groups of A. terreus. Data

analysis showed involvement of amide group (-NH), hydroxyl group (-OH), carboxylate

group (-COO), carbonyl group (-CO) functional groups in Zn adsorption by A. terreus.

Significant shifting of peaks at regions 3380 Cm-1, 2925 Cm-1, 1746 Cm-1,1247 Cm-1, 1151

Cm-1 and 1079 Cm-1 regions were noted in both gamma unexposed and exposed A.terreus

biomass grown in Zn enriched media (8000ppm) as compared to the normal control group

having no metal supplementation or gamma irradiation. Interestingly no signifcant difference

in spectra was observed between gamma irradiated and unirradiated groups grown media

having same concentration of Zn. One new peak at 1411 Cm-1 region was found in the group

grown in Zn supplemented media.

Results

81 | P a g e

5.8. Pb tolerance efficacies of gamma exposed fungi

Exposure to gamma irradiation imparted similar trend of change in Pb tolerance of

A.niger and P.cyclopium, where gamma exposed groups of both the strains showed

significant increase in CFU than that of the respective unirradiated counterparts. However, in

contrast to the findings in case of tolerance of Cd and Zn by these two strains of fungi where

gamma irradiation resulted in tolerance of the metals in higher concentration and manifested

upsurge in CFU in media supplemented with metals in concentration where the unirradaited

groups could not grow at all, in case of Pb, gamma irradiated fungi could not tolerate

concentration of Pb in the media higher than the MIC of the control (unirradiated) group. As

such while the MIC value of A.niger against Pb was 3100ppm, the gamma exposed A.niger

could not tolerate Pb above 3000ppm, though the exposed groups showed higher CFU in

terms of higher number of colonies with respect to their non irradiated counterparts against

all the tested concentration of Pb. Results illustrated that in both cases i.e against 2000ppm

& 3000ppm of Pb , a significant increase in CFU was noticed in gamma exposed groups

showing maximum increase in CFU when exposed at an absorbed dose of 100Gy (Fig 5.13) .

The radiation dose dependent increase in CFU of A. niger was more noticeable in case of

2000ppm of Zn in the growth medium.

Fig 5.13 Colony Forming Unit of A.niger with or without exposure to gamma irradiation grown in Pb supplemented media (Error bars represent standard deviation for n = 6. p≤0.05 was considered significant).

20Gy

40Gy

60Gy

80Gy

100Gy

0 200 400 600 800 1000

Change (%)in CFU/ml in comparison with unirradiated conterparts

Dos

e

2000ppm Pb 3000ppm Pb

Results

82 | P a g e

Significant role of gamma irradiation in modulating uptake and removal of Pb by A.

niger was evident from analysis of AAS data. Mycelia of the gamma irradaited A.niger

showed significant increase in Pb uptake and a rise in Pb removal potential of the fungi from

different Pb supplemented liquid media was observed as compared to the non irradiated but

same Pb stressed condition. A 100 Gy exposed A.niger showed 1.5 times more Pb uptake in

the mycelia and 10.23% more removal potential from 2000ppm Pb supplemented media,

whereas from 3000 ppm Pb supplemented media its uptake and removal potential were 2

times and 7% more respectively , with respect to the non-irradiated counterparts (Fig-5.14).

A B C D

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

A-2000ppmB-2000ppm+100GyC-3000ppmD-3000ppm+100Gy

Uptake(mg/g of tissue) Removal (%)

Dose

Myc

elia

l upt

ake(

mg/

g tis

sue)

64

66

68

70

72

74

76

78

80

82

Rem

oval (%)

Fig-5.14 Uptake and removal of Pb by Aspergillus niger exposed to different absorbed doses of gamma irradiation (20-100 Gy) and grown in media supplemented with different concentrations of Pb (Error bars represent standard deviation for n = 3. p≤0.05 was considered significant).

FTIR spectral analysis for A.niger (Fig 5.15) biomass grown in media containing

2000 ppm of Pb revealed shifting of peaks at 3383 Cm-1, 2856 Cm-1 , 1647 Cm-1, 1552 Cm-1,

1149 Cm-1,1080 Cm-1 regions. Similar changes in peak spectra were observed in gamma

exposed A.niger grown in same concentration of Pb in the media. However, changes in peak

positions were more prominent in gamma irradiated groups than that in unirradaited group of

the fungi both grown in media supplemented with same concentration of Pb. The peak at

2856 Cm-1 region was not observed in unirradiated fungi treated with only Pb.

Results

83 | P a g e

Fig 5.15 FTIR spectra of A.niger biomass

(a) Control biomass (b) 2000ppm Pb loaded biomass (c) 100Gy gamma exposed biomass

grown in 2000ppm Pb enriched media

Unlike A.niger, gamma exposed P.cyclopium grown in Pb supplemented media

showed maximum CFU when exposed at an absorbed dose of 80Gy of gamma. A 80Gy

exposed P.cyclopium displayed 1.9 and 1.6 fold increase in colonies when grown in media

having 2000ppm and 3000ppm of Pb respectively as compared to the respective un-irradiated

Pb stressed counterparts. The groups irradiated at 100Gy absorbed dose of gamma although

showed a decrease in CFU as compared to that irradiated at 80Gy both grown in media

supplemented with Pb, the number of colonies were significantly higher than that of un

irradiated groups of the fungi grown in media having same concentration of Pb (Fig-5.16).

The 80Gy gamma exposed P.cyclopium grown in different concentrations of Pb (2000ppm

and 3000ppm) demonstrated almost two times more Pb accumulation in addition to 10%-11%

higher efficacy for Pb removal from growth media than their unirradiated but same

concentrations of lead exposed counterparts (Table-5.12) .

Results

84 | P a g e

Fig 5.16 Colony Forming Unit of P.cyclopium with or without exposure to gamma irradiation grown in Pb supplemented media (Error bars represent standard deviation for n = 6. p≤0.05 was considered significant). Table5.12. Uptake and removal of Pb by P.cyclopium with or without exposure to

gamma irradiation (Mean ±SD for n=3)

Cultured condition

2000ppm of Pb

Uptake Potential

(mg/g of tissue)

Removal (%)

Unirradiated P. Cyclopium 14.086±1.25 80±4.5

80Gy gamma exposed P.cyclopium 25.32±2.0 90±4.5

3000ppm of Pb

Unirradiated P. Cyclopium 8.972±0.5 75±5.5

80Gy gamma exposed P.cyclopium 14.254±0.5 89±4.5

20Gy 40Gy 60Gy 80Gy 100Gy

0

20

40

60

80

100

120

140

160

180

200

2000ppm Pb 3000ppm Pb

Dose (Gy)

Cha

nge

in C

FU(%

) with

resp

ect t

o un

irrad

iate

d on

e

-20

0

20

40

60

80

100

120

140

160

180

Change in C

FU(%

) with respect to

unirradiated one

Results

85 | P a g e

Fig 5.17 FTIR spectra of P.cyclopium biomass

(a) Control biomass (b) 2000ppm Pb loaded biomass (c) 80Gy gamma exposed biomass

grown in 2000ppm Pb enriched media

FTIR analysis illustrated the involvement of amide group (-NH), hydroxyl group (-

OH), carboxylate group (-COO), carbonyl group (-CO) in Pb adsorption of P.cyclopium.

Significant changes observed in 3394 cm-1, 1548 cm-1 and 1240 cm-1 regions in 2000ppm Pb

treated biomasses (in both gamma exposed as well as unexposed one). Changes in spectral

characteristics were more prominenet in gamma irradiated group than unirradiated ones when

both treated with same Pb concentrations. 3394 cm-1 region in control biomass was shifted to

3376 region in Pb treated group while in the 100Gy gamma exposed group grown in Pb

enriched media, it was noted at 3366 cm-1 region. Spectra at 1548 cm-1 and 1230 cm-1 regions

in control biomass was shifted to 1543-1542 cm-1 and 1240 cm-1 regions respectively in

other two experimental groups (Fig 5.17).

Although both A.niger and P.cyclopium showed involvement of same functional

groups against 2000ppm Pb stress, the FTIR spectral characters compared between the two

strains showed some distinct differences. In P. cyclopium the peak observed at 2856 cm-1

Results

86 | P a g e

region in the spectrum of the control group, was not noted in cases of the group exposed to

2000ppm of Pb and the group exposed to gamma irradiation and then grown in same

concentration of Pb (Fig 5.17) . In contrast the FTIR spectra of all the groups of A.niger

showed peak at 2856 cm-1 region (Fig 5.15). Second, instead of the peak observed at 1240

cm-1 region in A. niger (all groups), in case of all the groups of P.cyclopium peak was noted

in 1230 cm-1 region.

The FTIR spectra of the three selected fungi exposed to different doses of gamma rays

is shown in Fig 5.18 (A-C).

Fig 5.18A FTIR analysis of A.niger biomass exposed to different gamma absorbed doses

(a) Control biomass (b) 60Gy treated biomass (c) 100Gy treated biomass

Results

87 | P a g e

Fig 5.18B FTIR analysis of P.cyclopium biomass exposed to different gamma absorbed

doses (a) Control biomass (b) 60Gy treated biomass (c) 100Gy treated biomass

Results

88 | P a g e

Fig 5.18C FTIR analysis of A.terreus biomass exposed to different gamma absorbed

doses (a) Control biomass (b) 80Gy treated biomass (c) 100Gy treated biomass

5.9 pH and temperature optimisation for removal of metals by gamma exposed and unexposed fungal strains

pH and temperature are two factors that are essential for growth of fungi and also play

important roles in modulating uptake and removal potential of fungi. Table 5.13- 5.18

represent comparative tabulation of the effects of different temperature and pH as maintained

for growth of the selected fungal strains to determine their respective metal removal

efficiencies against the different pH and temperature. Results showed maximum metal

removal efficacy of all the three selected strains of fungi to be maximum in groups

maintained in growth media having at neutral pH and at temperature 29-300C. This also

complied with the data of maximum mycelia growth of the fungi at the same pH and

temperature range. Optimum pH and temperature for maximum metal removal were noted to

be same for both gamma irradiated and unirradiated fungal groups.

4000 3500 3000 2500 2000 1500 1000 500

2030405060708090

1004000 3500 3000 2500 2000 1500 1000 5000

1020304050604000 3500 3000 2500 2000 1500 1000 5001020304050607080

Wavenumbers (Cm-1)

(a)

Tra

nsm

ittan

ce (%

)

(b)

C

1030.12

1078

.51149

.813

75.6

1

1456

.415

49.3

916

47.4

7

1745.542854.98

2225.253348.68

639.

87769.

73906.

84

1029.26

1078.27

1149

.412

47.2

313

72.1

315

48.7

61644.61746.03

2854.09

2924.73359.9

1027.74

1079.68

1151

.2912

47.2

313

72.8

714

59.1

515

44.7

6

1645.86

1746.46

2853.25

2925.723380.15

(c)

Results

89 | P a g e

Table: 5.13 Effect of pH on removal of Zn (100ppm) by Gamma irradiated and

unirradiated fungal strains

Strain Removal (%)

pH 5 pH7 pH 9

Unirradiated A.niger 70 80 75

Gamma exposed A.niger 75 87 81

Unirradiated A.terreus 60 70 66

Gamma exposed A.terreus 65 76 70

Table: 5.14 Effect of temperature on removal of Zn (100ppm) by Gamma irradiated

and unirradiated fungal strains

Strain Removal (%)

Temp.250C Temp.29-300C Temp.350C

Unirradiated A.niger 72 80 71

Gamma exposed A.niger 76 87 76

Unirradiated A.terreus 61 70 65

Gamma exposed A.terreus 65 76 70

Table: 5.15 Effect of pH on removal of Cd by Gamma irradiated and unirradiated

fungal strains

Strain Removal (%)

pH 5 pH7 pH 9

Unirradiated A.terreus grown in 90ppm Cd 60 65 61 Gamma exposed A.terreus grown in 90ppm Cd 66 73 70.23 Unirradiated P.cyclopium grown in 300ppm Cd 79 82 80 Gamma exposed P.cyclopium grown in 300ppm Cd 88 95 90.1

Results

90 | P a g e

Table: 5.16 Effect of temperature on removal of Cd by Gamma irradiated and


Strain Removal (%)


Unirradiated A.terreus grown in

90ppm Cd

59 65 62

Gamma exposed A.terreus grown

in 90ppm Cd

67.5 73 66.7

Unirradiated P.cyclopium grown

in 300ppm Cd

76 82 78

Gamma exposed P.cyclopium

grown in 300ppm Cd

85 95 87.9

Table: 5.17 Effect of pH on removal of Pb (2000ppm) by Gamma irradiated and


Strain Removal (%)

pH 5 pH7 pH 9

Unirradiated A.niger 65 70 66 Gamma exposed A.niger 76.5 81 78 Unirradiated P.cyclopium 75 80 77 Gamma exposed

P.cyclopium 84.9 90 86.5

Table: 5.18 Effect of temperature on removal of Pb (2000ppm) by Gamma irradiated

and unirradiated fungal strains

Strain Removal (%)


Unirradiated A.niger 62 70 65 Gamma exposed A.niger 72.9 81 77.2 Unirradiated P.cyclopium 72 80 75 GammaexposedP.cyclopium 82.5 90 86.2

Results

91 | P a g e

Section III Alteration in metabolic / lignocellulosic enzyme activity and ultrastructure of fungi after

gamma exposure

Results showed that gamma exposure has the potential in stimulating activities of the

metabolic/ lignocellulosic enzymes (CMCase and α- amylase ) in fungi grown under metal

stress. While only metal stress caused decrease in those enzyme activities.The fungi exposed

to both gamma radiation and metals had higher activities of both the enzymes. However, the

changes in intensity of activity of the enzymes are fungal strain specific and metal specific i.e

the changes were different in case of the different fungal strains and also varied from metal to

metal. Scanning electron microscopic study showed metal stress induced structural

deformities in fungi, while gamma exposed fungi when grown in metal enriched growth

media showed much lesser or no such deformity.

5.10 Effect of gamma on metabolic/lignocellulosic enzyme activities and ultrastructure

of Aspergillus terreus stressed with Cd and Zn

A.terreus grown in Cd supplemented medium showed decrease in activities of the

both the enzymes. Interestingly, prior exposure of the fungi to different absorbed doses of

gamma (20Gy-100Gy), followed by their culture in Cd supplemented medium manifested

enhancement of activity of both the enzymes when compared to their unirradiated but Cd

exposed counterparts. All the groups of A. terreus exposed to gamma absorbed doses and

grown with 90ppm Cd in the medium showed significant enhancement (p≤0.05) of CMCase

activity with respect to that of unirradiated group grown in same concentration of Cd (Fig

5.19a). Results showed an overall increase of 18-25% activity of CMCase in the 20Gy and

40 Gy gamma exposed A. terreus when compared to the activity of enzyme in the

unirradiated but Cd stressed group. Though not much difference in stimulation of activity

was noted between the groups exposed to 60Gy and 80 Gy absorbed dose of gamma, the

increase in activity of the enzyme was 39-40% when compared to that of the unirradiated

but Cd stressed group. Maximum increase (59%) in CMCase activity was observed in the

group exposed to 100 Gy absorbed dose of gamma Contrary to CMCase activity, no

significant enhancement of α- amylase activity in A. terreus was noted till 40 Gy (absorbed

dose) of gamma exposure. A. terreus exposed to 60Gy manifested 35% increase in α-amylase

activity with respect to that of the unirradiated Cd stressed control. Results showed a steep

increase in the activity of this enzyme in groups exposed to 80 Gy (1.06 fold), representing

the maximum enhancement of α-amylase activity and at an exposure to 100 Gy (1.09fold)

Results

92 | P a g e

absorbed dose of gamma.Further increase in metal concentration in the media i.e. from

90ppm to 150ppm Cd, also reflected increasing trend in activities of both the enzymes

starting from the lowest dose of gamma irradiation showing maximum stimulation in

activities in groups exposed at 80Gy absorbed dose of gamma (Fig-5.19b). In groups

irradiated at 100Gy absorbed dose of gamma and grown in 150ppm Cd, a slight decline in

both enzyme activities were noticed as compared to that of the group irradiated at 80Gy with

same Cd concentration. As without gamma exposure A.terreus could not tolerate 150ppm of

Cd so it could not be compared with the non-irradiated counterparts.

Results

93 | P a g e

Fig-5.19 α-Amylase and CMCase activity of Aspergillus terreus

(a)A. terreus exposed to different absorbed doses of gamma rays and then grown in media containing 90ppm Cd (b) A. terreus exposed to different absorbed doses of gamma rays and then grown in media containing 150 ppm Cd (Error bars represent standard deviation for n = 6. p≤0.05 was considered significant.)

Cont

90 ppm Cd

90 ppm Cd +20 Gy

90 ppm Cd + 40 Gy

90 ppm Cd +60 Gy

90 ppm Cd + 80 Gy

90 ppm Cd + 100 Gy

2

4

6

(a)

- Amylase activity(IU/ml) CMCase activity(IU/ml)

-Am

ylas

e ac

tivity

(IU/m

l)

32

34

36

38

40

42

44

46

48

50

52

54

56

58

CMCase activity(IU/m

l)

Cont

150 ppm Cd +20 Gy

150 ppm Cd + 40 Gy

150 ppm Cd + 60 Gy

150 ppm Cd + 80 Gy

150 ppm Cd + 100 Gy

2

4

6

(b)

-Amylase activity(IU/ml) CMCase activity(IU/ml)

-Am

ylas

e ac

tivity

(IU/m

l)

32

34

36

38

40

42

44

46

48

50

52

54

56

CM

Case activity(IU

/ml)

Results

94 | P a g e

Zn stress caused decrease in activities of both CMCase and α- amylase in A.terreus.

However effect of Zn stress was found to be more on α- amylase activity than on CMCase ;

Table 5.19 represents changes in activity of CMCase and α- amylase in A.terreus under

different conditions of metal and radiation stress. A.terreus when grown in media containing

7000-9000ppm , a 43%- 78% decrease in CMCase activity and 37%- 82% decrease in α-

amylase activities were noticed respectively when compared with the control groups ( neither

stressed with metal nor with gamma) . Interestingly gamma exposed A.terreus (at the

effective dose showing maximum CFU) when grown in same Zn enriched growth media

(7000ppm, 8000ppm and 9000ppm) showed significant enhanced enzyme activities as

compared to its unirradiated but Zn stressed counterparts. Effect of gamma on enhancing

enzyme activity against Zn stress was more for CMCase than α- amylase. A 80Gy exposed

A.terreus manifested 74%, 62% and 53% enhancement in CMCase activity when grown in

7000, 8000 and 9000ppm of Zn respectively with respect to the unirradiated but metal

stressed (same conc.) counterparts. Whereas 80Gy exposed A.terreus manifested only 33%-

40% α- amylase activity under 7000-9000ppm Zn stressed when compared with their

unirradiated but Zn stressed counterparts.

Cd and Zn caused deformities in normal hyphal morphology of A.terreus. After

exposed to metals the normal shaped cylindrical hyphae changed into narrower with

flocculations and cages and pores although no such significant changes found in conidial

shape. However,the same fungal strain when exposed to gamma a priori and then grown in

metal enriched growth media, showed lesser structural deformities. Morphological changes in

A.terreus due to metal and gamma exposure are described in Table 5.20 and Plates I and Plate

II showed the SEM photographs.

Results

95 | P a g e

Table 5.19 CMCase (IU/ml) and α amylase activity (IU/ml) of A.terreus with or without exposure to gamma under Zn stress (Mean±SD for n=6)

Dose

CMCase activity(IU/ml)

α amylase activity(IU/ml)

A.terreus grown in control condition

38.29±1.5

6.59±0.15

A.terreus grown in 7000ppm Zn

21.54±1.21

2.50±0.25

80Gy exposed A.terreus grown in 7000ppm Zn

37.56±2.1

3.43±0.21


16.02±0.5

2.08±0.05


25.96±0.6

2.77±0.12


8.28±0.25

1.24±0.05


12.7±0.5

1.74±0.04

Table 5.20 Comparative analysis of ultratructural changes in A.terreus with or without exposure to Cd, Zn and gamma

Treatment Changes

Control Regular hyphae with bulging conidia without any flocculation

90ppm Cd treated Hyphal surfaces with irregular cages, Flocculated, folded, narrow hyphae No changes in conidia

100Gy exposed fungi grown in 90ppm of Cd

Lesser deformation with no flocculation with respect to unirradiated one

Well shaped hyphae present with some narrow one 150ppm Cd treated Death of Fungi

80Gy exposed fungi grown in 150ppm Cd

Hyphal wall with cages, corrugation and pores which signifies metal accumulation

Only gamma(100Gy) exposed

Hyphal wall is wavy/undulated in nature Conidia is embedded

7000ppm Zn treated Hyphal surfaces with irregular cages, Flocculated, folded, narrow hyphae Hyphal shrinkage


Some deformed hyphae tried to reform its structures

Results

96 | P a g e

5.11 Effect of gamma on metabolic/lignocellulosic enzyme activities and ultrastructure

of Aspergillus niger stressed with Cd ,Zn and Pb

Significant decrease in CMCase and α- amylase activities of A.niger was observed

after treating the fungi with metals (Cd,Zn and Pb), when compared to that of normal group,

i.e. without any metal stress. Changes in activity of the enzymes was found to be metal

specific Zn was found to have more drastic effect on α- amylase activity of A.niger than on

its CMCase activity. The depletion of activity of CMCase is observed to be directly

proportional to the metal concentrations. CMCase activity of A,niger is found to be decreased

by only 24%-52% while α- amylase activity of A.niger is found to be decreased 61%-64%

when grown in Zn supplemented media (250ppm, 500ppm &1000ppm respectively) (Fig-

5.20a). Effect of Pb on the activity of the two enzymes of A.niger is shown in Fig-5.21 (a).

Change in CMCase activity of A.niger was noted to be slightly varying with Pb

concentration. 2000ppm & 3000ppm of Pb showed a decrease of 68% and 71% respectively

in CMCase activity. However a 74% decrease in α- amylase activity was observed against

both the concentration of Pb. In comparison to Zn and Pb, Cd was observed to have more

drastic changes in α- amylase activity of A. niger. Effect of Cd on CMCase activity of A.

niger was intermediary to that observed in case of Zn and Pb. Cd induced depletion of

CMCase activity was higher than that noted in case of Zn but lower than that observed in

case of Pb stress. 70ppm of Cd salt in the medium produced 63% decrease in CMCase

activity and 56% decrease in α- amylase activity, 85ppm of Cd salt showed 68% less activity

of CMCase and 65% less in α- amylase activity (Fig-5.22a) with respect to the

corresponding control fungi not exposed to any metal stress.

Interestingly, however, while A.niger grown under metal stress resulted in depletion

of enzymes activity, prior exposure of the fungi to different absorbed doses (20Gy-100Gy) of

gamma and then to metals in the growth media, shows enhancement of enzyme activity when

compared to that of the metal stressed ones (Fig-5.20 b-d,5.21 b-c ,5.22 b-c). Results showed

a dose-dependent relation between enhancement of CMCase and α- amylase activities and the

absorbed dose of gamma exposure to A.niger under metal stress. Additionally, the activity of

both the enzymes is also observed to be metal specific and manifests differential trend

depending upon concentration of a particular metal. In the group exposed to 100Gy absorbed

dose of gamma a priori and then grown in media containing 250ppm and 500ppm Zn, a 93%

and 1.78 fold increase respectively in CMCase activity is observed with respect to the groups

only exposed to 250ppm and 500ppmZn (Fig-5.20b and 5.20c). Similar to the observation

Results

97 | P a g e

found in case of Zn, gamma irradiation at 100 Gy absorbed dose resulted in 1fold increase in

CMCase actitvity in the group of A. niger having 2000ppm Pb in the medium when compared

to the group exposed to Pb stress of same concentration but unirradiated (Fig-5.21b). Further

increase in concentration of these metals ( i.e Zn, from 250ppm to 1000ppm and Pb, from

2000ppm to 3000ppm), the maximum CMCase activity in both the cases is found in the

groups exposed to 80Gy absorbed dose (Fig 5.20d and 5.21c). Similarly, while A.niger under

70ppm Cd stress exposed to 60Gy absorbed dose showed maximum enzyme (CMCase)

activity (89.76%) (Fig-5.22b), further increase in Cd concentration to 85ppm, reflected

maximum activity (64%) of the enzyme in A. niger irradiated at 40Gy (Fig-5.22c) all

compared to the metal (Cd) stressed counterparts.

100Gy exposed A.niger showed 3.23 fold and 2.88 fold more α- amylase activity

when grown in 250ppm and 500ppm Zn respectively as compared to their un-irradiated but

Zn stressed counterparts (Fig 5.20b and 5.20c). A 100 Gy exposed A.niger under 2000ppm

Pb stress caused 55% more α -amylase activity with respect to non-irradiated counterparts

(Fig 5.21b). Further increase in metal concentration i.e from 250ppm to 1000ppm of Zn and

from 2000ppm to 3000ppm of Pb maximum α -amylase activities were found to be at 80Gy.

A 80Gy gamma exposed A.niger showed 1.88 fold and 1 fold more α- amylase activity when

grown in 1000ppm Zn and 3000ppm Pb respectively (Fig 5.20d and 5.21c). Likewise 60Gy

and 40Gy gamma exposed groups of A.niger showed 1 fold and 1.16 fold more α -amylase

activity under 70ppm and 85ppm of Cd respectively than that of unirradiated but Cd stressed

counterparts (Fig 5.22b and 5.22c). Maximum increase in CMCase and α- amylase activities

were noted in groups exposed at 80Gy - 100Gy of absorbed dose of gamma rays in case of

Pb and Zn stress while for Cd stress the dose range producing maximum CMCase and α-

amylase activities were in the range of 40Gy-60Gy. Comparing these three heavy metal

stress, A.niger shows higher stimulation of CMCase and α- amylase activities were under Zn

stress when exposed to same dose (absorbed) of gamma as compared to the group under Pb

stress. On the other hand, exposure to much lower dose of gamma irradiation produced

significant stimulation in enzyme activities when under Cd stress.

Results

98 | P a g e

Cont.

250 ppm Zn

500 ppm Zn

1000ppmZn0

5

10

15

20

25

30

35

40 CMCase activity

Enz

yme

Activ

ity (I

U/m

l)

Cont.

250 ppm Zn

500 ppm Zn

1000 ppm Zn0

2

4

6

8

10

12

14

(a)- Amylase activity

Cont.

250ppm Zn

250 ppm Zn + 20 Gy

250 ppm Zn + 40 Gy

250 ppm Zn + 60 Gy

250 ppm Zn + 80 Gy

250 ppm Zn + 100 Gy26283032343638404244464850525456586062

CMCase activity - Amylase activity

CM

Case

act

ivity

(IU/

ml)

4

6

8

10

12

14

16

18

20

22(b)

- Amylase activity (IU/m

l)

Results

99 | P a g e

Fig 5.20 CMCase and α-Amylase activity of Aspergillus niger

(a) A. niger grown in medium supplemented with 250-1000ppm Zn (b) A. niger exposed to different absorbed doses of gamma rays and then grown in media containing 250ppm Zn (c) A. niger exposed to different absorbed doses of gamma rays and then grown in media containing 500ppm Zn (d) A. niger exposed to different absorbed doses of gamma rays and then grown in media containing 1000ppm Zn(Error bars represent standard deviation for n = 6. p≤0.05 was considered significant.)

Cont.

500 ppm Zn

500 ppm Zn +20 Gy

500 ppm Zn + 40 Gy

500 ppm Zn + 60 Gy

500 ppm Zn + 80 Gy

500 ppm Zn + 100Gy

20

25

30

35

40

45

50

55

60

65 (c)

CMCase activity -Amylase activity

CM

Case

act

ivity

(IU/

ml)

4

6

8

10

12

14

16

18

20

22

-Amylase activity (IU/m

l)

Cont.

1000 ppm Zn

1000 ppm Zn +20 G

y

1000 ppm Zn +40 Gy

1000 ppm Zn +60 Gy

1000 ppm Zn +80 Gy

1000 ppm Zn +100 Gy15

20

25

30

35

40

45

50

55

60


CM

Cas

e ac

tivity

(IU/

ml)

4

6

8

10

12

14(d)


l)

Results

100 | P a g e

Fig 5.21 CMCase and α-Amylase activity of Aspergillus niger (a) A. niger grown in medium supplemented with 2000-3000 ppm Pb (b) A. niger exposed to different absorbed doses of gamma rays and then grown in media containing 2000ppm Pb (c) A. niger exposed to different absorbed doses of gamma rays and then grown in media containing 3000ppm Pb (Error bars represent standard deviation for n = 6. p≤0.05 was considered significant.)

Cont. 2000ppmPb 3000ppmPb05

1015202530354045 - Amylase activityCMCase activity

Enzy

me

activ

ity (I

U/m

l)

Cont. 2000ppmPb 3000ppmPb0

2

4

6

8

10

12

14(a)

Cont.

2000 ppm Pb

2000 ppm Pb + 20 Gy

2000 ppm Pb + 40 Gy

2000 ppm Pb +60 Gy

2000 ppm Pb + 80 Gy

2000 ppm Pb + 100 Gy5

10

15

20

25

30

35

40(b)


CMCa

se a

ctiv

ity (I

u/m

l)

2

4

6

8

10

12

14

-Am

ylas

e ac

tivity

(IU/

ml)

Cont.

3000 ppm Pb

3000 ppm Pb + 20 Gy

3000 ppm Pb + 40 Gy

3000 ppm Pb + 60 Gy

3000 ppm Pb + 80 Gy

3000 ppm Pb + 100 Gy

10

15

20

25

30

35

40 (c)

CMCa

se a

ctiv

ity (I

u/m

l)

2

4

6

8

10

12

14



l)

Results

101 | P a g e

Fig 5.22 CMCase and α-Amylase activity of Aspergillus niger (a) A. niger grown in medium supplemented with 70 -85 ppm Cd (b) A. niger exposed to different absorbed doses of gamma rays and then grown in media containing 70ppm Cd (c) A. niger exposed to different absorbed doses of gamma rays and then grown in media containing 85ppm Cd (Error bars represent standard deviation for n = 6. p≤0.05 was considered significant.)

Cont. 70ppm Cd 85ppmCd05

1015202530354045 - Amylase activity CMCase activity

Enzy

me

activ

ity (I

U/m

l)

Cont. 70ppm Cd 85ppmCd0

2

4

6

8

10

12

14(a)

Cont.

70 ppm Cd

70 ppm Cd + 20 Gy

70 ppm Cd +40 Gy

70 ppm Cd + 60 Gy

70 ppm Cd + 80 Gy

70 ppm Cd + 100 Gy05

10152025303540

(b) CMCase activity - Amylase activity

CMCa

se a

ctiv

ity (I

U/m

l)

0

2

4

6

8

10

12

14

- A

myl

ase

activ

ity (I

U/m

l)

Cont.

85 ppm Cd

85 ppm Cd + 20 Gy

85 ppm Cd + 40 Gy

85ppm Cd +60 Gy

85ppm Cd +80 Gy

85ppm Cd + 100 Gy

10

15

20

25

30

35

40(c)

CMCa

se a

ctiv

ity (I

U/m

l)

0

24

6

810

12

14 CMCase activity - Amylase activity


l)

Results

102 | P a g e

Exposure to Zn and Pb results in distortion of normal shape of A.niger hyphae as

revealed through SEM study. Formation of many irregular cages and flocculations were

observed in metal stressed A.niger. Table 5.21 showed the comparison between the effects of

the two different metals (Zn and Pb) on hyphal surface ultrastructure of A. niger. The normal

cylindrical hyphae became narrow and shrinked due to metal accumulation. Increase in

concentration of Zn from 250ppm to 500ppm resulted in more shrinkage. Similar to A.terreus

gamma exposed A. niger when grown in metal enriched growth medium showed lesser

deformities. Their microscopic pictures are listed in Plate III and Plate IV.

Table: 5.21 Comparative analysis of ultrastructural changes in A.niger with or without exposure to Pb, Zn and gamma

Treatment Changes

Control Regular hyphae with bulging conidia without any flocculation

250ppm Zn treated A.niger Hyphal surfaces with irregular cages, Flocculated, folded, narrow hyphae No changes in conidia

100Gy exposed A.niger grown in 250ppm of Zn

Less deformation , less flocculation with respect to unirradiated one

Well shaped hyphae present with some narrow one 500ppm Zn treated A.niger More hyphal shrinkage and deformities

60Gy exposed A.niger grown in 500ppm of Zn

Hyphal wall with cages, corrugation and pores which signifies metal accumulation

2000ppm Pb treated A.niger Hyphal surfaces with irregular cages, Flocculated, folded, narrow hyphae Hyphal shrinkage

80Gy exposed A.niger grown in 2000ppm of Pb

Less deformities with some narrow one

Results

103 | P a g e

5.12 Effect of gamma on metabolic/ lignocellulosic enzyme activities and ultrastructure

of P.cyclopium grown in Cd and Pb riched media

Similar trend of observations were obtained when P.cyclopium was subjected to Cd

and Pb stress. P.cyclopium grown in 300ppm Cd showed 57% decrease in CMCase activity

and 54% decrease in α -amylase activity. Further increase in Cd concentration i.e from

300ppm to 650ppm enzyme activities were found to be more declined (86% CMCase and

80% α -amylase) with respect to its control counterparts (Table 5.22).80Gy exposed

P.cyclopium manifested 95% more CMCase and 1.15 fold more a α -amylase activity when

grown in 300ppm of Cd with respect to its unirradiated but Cd stressed counterparts. Parallel

to this against 650ppm of Cd in growth medium, 60Gy exposed P.cyclopium was able to

exhibit 3.56fold CMCase and 1.14fold more α -amylase activity when compared to their

unirradiated but Cd stressed (650ppm) counterparts (Table 5.22).

Alike Cd stress, 2000ppm Pb caused 52% decrease in CMCase and 80% α -amylase

activities in P.cyclopium than control counterparts. Further increase in Pb concentration i.e

from 2000ppm to 3000ppm Pb, enzyme activities were showed to be declined more, 69%

CMCase and 86% α -amylase activities. While P.cyclopium prior exposed to gamma and

then grown in Pb riched media interestingly enhanced its both enzyme activities.80Gy

exposed P.cyclopium when grown in 2000-3000ppm Pb enriched media showed 45%- 96%

more CMCase activity and 85%-98% more α-amylase activity respectively when compared

with their un - irradiated but Pb stressed (2000-3000ppm) counterparts (Table 5.22).

Cd and Pb caused deformities in normal hyphal morphology of P.cyclopium. After

exposed to metals the normal shaped cylindrical hyphae changed into narrower with

flocculations and cages and pores although no such significant changes found in conidial

shape. However, the same fungal strain when exposed to gamma a priori and then grown in

metal enriched growth media, showed lesser structural deformities. Morphological changes in

P.cyclopium due to metal and gamma exposure are described in Table 5.23 and Plates V

showed the SEM photographs.

Results

104 | P a g e

Table: 5.22 CMCase (IU/ml) and α amylase activity (IU/ml) of P.cyclopium with or

without exposure to gamma under Cd and Pb stress (Mean±SD for n=6)

Dose

Metal : Cd

CMCase activity (IU/ml)

α amylase activity (IU/ml)

P. cyclopium grown in control condition

45.84±2.1

3.19±0.25

P. cyclopium grown in 300ppm Cd

26.68±1.54

1.46±0.02

80Gy exposed P. cyclopium grown in 300ppm Cd

38.66±2.25

3.60±0.15

P. cyclopium grown in 650ppm Cd

6.3±0.5

0.64±0.1

60Gy exposed P. cyclopium grown in 650ppm Cd

32.36±1.75

2.29±0.1

Metal : Pb CMCase activity(IU/ml) α amylase activity(IU/ml)

P. cyclopium grown in 2000ppm Pb

22.09±0.5

0.63±0.05

80Gy exposed P. cyclopium grown in 2000ppm Pb

70.53±1.5

1.24±0.06

P. cyclopium grown in 3000ppm Pb

33.91±1.65

0.45±0.005

80Gy exposed P. cyclopium grown in 3000ppm Pb

61.14±2.12

0.84±0.01

Table: 5.23 Comparative analysis of ultrastructural changes in P.cyclopium with

or without exposure to Cd ,Pb and gamma

Treatment Changes Control P.cyclopium Normal healthy hyphae with branches and spores

300ppm Cd treated P.cyclopium

Total hyphal structures are deformed with pores and cages; No uniform structures are observed as hyphae became narrower

80Gy exposed P.cyclopium grown in 300ppm Cd

Some portions of hyphal body become healthy and uniform although in some portions still structural deformities are there

2000ppm Pb treated P.cyclopium

Hyphal surfaces with irregular cages Flocculated, folded, narrow hyphae Hyphal shrinkage

80Gy exposed P.cyclopium grown in 2000ppm Pb

Less deformities

Results

105 | P a g e

5.13 Effect of gamma irradiation on metabolic/lignocellulosic enzyme activities and

ultrastructure of A.terreus, A.niger and P.cyclopium.

Fig 5.23 (a,b,c) depicted significant enhancement (p≤0.05) of activity of CMCase and

α- amylase in gamma irradiated A. Terreus, A.niger and P.cyclopium as a function of increase

in absorbed dose of gamma (20-100Gy).Gamma induced effect on α -amylase activity was

noted to be more than that noted in case of CMCase in case of A.terreus and P.cyclopium. For

A.niger the trend is just opposite i.e gamma caused more potential enhancement of CMCase

activity than α -amylase. Gamma exposed fungal groups showed dose dependant

enhancement of both the enzyme activities against every tested fungal strains. In every cases

maximum escalation was obtained when the fungal strains were exposed at 100Gy of gamma

absorbed dose. While a 100Gy exposed group of A.terreus showed 1.13 fold increase in

activity of α-amylase, the same dose showed 48% increase in activity of CMCase, when both

compared to the un-irradiated normal control counterparts (Fig 5.23a)

Similarly a 100Gy exposed A.niger showed 83% increase in α-amylase activity, while

the same group showed 1.08 fold increase in CMCase activity when both are compared with

their non-irradiated counterparts (Fig 5.23b). Parallel to this observations 100Gy exposed

P.cyclopium showed 40% more CMCase activity but 2.03fold α-amylase activity with respect

to their unirradiated normal control counterparts ( Fig 5.23c).

Only gamma exposure causes no noticeable structural changes in A.niger and

P.cyclopium (Plate VI). But in case of A.terreus slight changes in conidial shape and hyphal

structures ( mentioned in Table 5.20).Gamma exposed A.terreus showed some undulated

shape of conidial heads with respect to their unirradiated counterparts (Plate VI).

Results

106 | P a g e

Fig 5.23 CMCase and α-Amylase activity of different fungi exposed to different doses of gamma rays (20-100Gy) (a) Aspergillus terreus (b) A.niger (c) P.cyclopium (Error bars represent standard deviation for n = 6. p≤0.05 was considered significant.)

Cont 20Gy 40Gy 60Gy 80Gy 100Gy

36384042444648505254565860 (a)


CM

Cas

e ac

tivity

(IU

/ml)

6

8

10

12

14

-Amylase activity (IU

/ml)

Cont. 20Gy 40Gy 60Gy 80Gy 100Gy30

40

50

60

70

80 (b)


CMCa

se a

ctiv

ity (I

U/m

l)

12

14

16

18

20

22

24

26


l)

Cont. 20Gy 40Gy 60Gy 80Gy 100Gy4244464850525456586062646668

(c)


CMCa

se a

ctiv

ity (I

U/m

l)

2

4

6

8

10


l)

Results

107 | P a g e

Section IV

Possible mechanism for metallo-resistance in fungi in response to gamma exposure

Results revealed that administration of the three heavy metals i.e. Zn, Cd and Pb,

significantly boosted the antioxidative status of the selected fungal strains exposed to these

metals. Stress induced by these metals stimulated activities of the antioxidative enzymes,

SOD and CAT, increased total content of reduced glutathione (GSH) and enhanced metal

responsive protein Metallothionein (MT) in fungi grown in metal enriched media with respect

to their normal control counterparts. Evaluation of these antioxidative marker enzymes and

proteins of the fungi grown under metal enriched growth media but exposed a priori to

gamma irradiation at an absorbed dose that resulted in highest CFU and maximum metal

uptake and removal, showed more remarkable increase in response of SOD, CAT, GSH and

MT. Increase in activities of the antioxidative stress marker enzymes was observed to be

different in the different experimental groups reflecting strain specific and metal specific

response of the enzymes.

5.14 Antioxidative enzymes (SOD ,CAT) and antioxidant protein(GSH) response in

gamma exposed Aspergillus niger and A.terreus grown in Zn enriched growth media

Fig 5.24(a) represents Zn induced effects on antioxidant enzymes and proteins in A.

niger as compared to the respective control groups. Administration of 250ppm of Zn in

growth media showed nearly 2-fold enhancement in SOD and CAT activity and glutathione

level in A.niger. Further increase in Zn concentration i.e from 250ppm to 1000ppm Zn in

growth media exhibited 3-fold increase in SOD activity but 6-fold more CAT activity than

that in the control group. The glutathione level in this group (having 1000ppm Zn in the

medium) however, was only two times higher than that of the control. Exposure to gamma

irradiation showed interesting influence on antioxidant response of the fungi grown under

metal stress. A 5-6 fold increase in activities of SOD and CAT (Fig 5.24b) and 1.3 fold

increase in glutathione level (Fig 5.24c) were noted in the group of A.niger grown in 250ppm

of Zn enriched media but exposed 100Gy absorbed dose of gamma rays a priori to culture

with Zn, all compared to their unirradiated but metal stressed (250ppm Zn) counterparts . In

contrast to 250ppm Zn treated and gamma irradiated A.niger showing highest induction of

antioxidative response when exposed to 100 Gy absorbed dose of gamma, the same strain of

fungi grown in 1000ppm of Zn showed maximum escalation of both the enzymes and

glutathione level when exposed to 60Gy absorbed dose of gamma. The 60Gy exposed

Results

108 | P a g e

1000ppm Zn treated A.niger expressed 5fold and 2fold increase in SOD and CAT activity

respectively (Fig 5.24b) in addition to more than 1fold higher glutathione level (Fig 5.24c)

than its un-irradiated counterparts. Gamma showed more potential in inducing CAT activity

than SOD activity when A.niger was treated higher concentration (1000ppm).

Response of Zn induced effect on SOD activity of A. terreus treated with 8000ppmof

Zn in the medium was observed to be very similar to that of A. niger treated with 250 ppm Zn

Similar to that observed in A.niger , higher concentration of Zn (8000ppm) manifested more

induction in CAT activity in A. terreus also. In contrast to a 2 fold increase in SOD activity,

A.terreus treated with 8000ppm of Zn showed a 4-fold enhancement in CAT activity as

compared to the respective control counterparts (Fig 5.25a). GSH content was nearly 2-fold

more than the glutathione level in the control group (Fig 5.25a). Similar to the observation

noted in case of A. niger, prior exposure of A. terreus to gamma irradiation induced much

strong stimulation in activities of SOD and CAT and GSH level as compared to the group

treated with Zn but not exposed to irradiation (Fig 5.25b and 5.25c). A.terreus when grown in

Zn treated (8000ppm) media following exposure to 80Gy absorbed dose of gamma showed

more than 2fold SOD activity, 1.75times more CAT activity (Fig 5.25b) and 1.4 times more

glutathione level (Fig 5.25c) than its un-irradiated but metal stressed (Zn) counterparts.

Further increase in Zn concentration ( from 8000ppm to 9000ppm ) also exhibited enhanced

level of both enzyme activities and glutathione level, while gamma exposed group when

grown in 9000ppm Zn enriched media showed significantly higher level of enzyme activities

and glutathione level than that of its un-irradiated counterparts (Fig 5.25b and Fig

5.25c).Antioxidative response revealed through the results clearly stated that gamma showed

its better potential in escalating antioxidative system in A.niger than A.terreus when both

were treated with Zn.

Results

109 | P a g e

Fig 5.24 Antioxidative response of Aspergillus niger

(a) A.niger grown in medium supplemented with 250-1000 ppm Zn (b) SOD and CAT response in A.niger exposed to different absorbed doses of gamma rays and then grown in media containing 250-1000 ppm Zn (c) GSH content in A.niger exposed to different absorbed doses of gamma rays and then grown in media containing 250-1000 ppm Zn(Error bars represent standard deviation for n = 6. p≤0.05 was considered significant)

Control

250ppmZn

1000ppm Zn0.00

0.02

0.04

0.06

0.08

0.10(a)

Antio

xida

tive

resp

onse

Control

250ppmZn

1000ppm Zn0

20406080

100120140160

GSH content(nMoles/mg of protein)

CAT activity(moles/sec/mg of protein)

SOD activity (U/mg of protein)

Control

250ppmZn

1000ppm Zn0

5

10

15

20

25

Control250ppm Zn

250 ppm Zn +100Gy1000 ppm Zn

1000 ppm Zn +60Gy

0.0

0.1

0.2

0.3

0.4

SOD activity CAT activity

SOD

act

ivity

(U/m

g of

pro

tein

)

0

50

100

150

200

250

300

350(b)

CAT activity

( mole/sec/m

g of protein)

Control250 ppm Zn

250 ppm Zn +100Gy1000ppm Zn

1000 ppm Zn +60Gy0

5

10

15

20

25

30 (c)

GSH

con

tent

(nM

oles

/mg

of p

rote

in)

Results

110 | P a g e

Fig 5.25 Antioxidative response of Aspergillus terreus

(a) A.terreus grown in medium supplemented with 8000-9000 ppm Zn (b) SOD and CAT response in A.terreus exposed to different absorbed doses of gamma rays and then grown in media containing 8000-9000 ppm Zn (c) GSH content in A.terreus exposed to different absorbed doses of gamma rays and then grown in media containing 8000-9000 ppm Zn(Error bars represent standard deviation for n = 6. p≤0.05 was considered significant.)

Control

8000 ppm Zn

9000 ppm Zn0.00

0.02

0.04

0.06

0.08

0.10

Antio

xida

tive

resp

onse

Control

8000 ppm Zn

9000 ppm Zn0

50100150200250300350400(a)

Control

8000 ppm Zn

9000ppm Zn0

5

10

15

20

25GSH content(nMoles/mg of protein)



Control8000ppm Zn

8000 ppm Zn+80Gy9000ppm Zn

9000 ppm Zn +80Gy

0.05

0.10

0.15

0.20

0.25

0.30(b)


SOD

activ

ity

(U/m

g of

pro

tein

)

0

100

200

300

400

500

600

CAT activity ( m

ole/sec/mg of protein)

Control8000ppm Zn

8000 ppm Zn +80Gy9000ppm Zn

9000 ppm Zn +80Gy0

5

10

15

20

25

30

35

(c)

GSH

con

tent

(n

Mol

es/m

g of

pro

tein

)

Results

111 | P a g e

5.15 Antioxidative enzymes (SOD ,CAT) and antioxidant protein (GSH) response in gamma exposed Aspergillus terreus A .niger and Penicillium cyclopium grown in Cd enriched growth media

Cd induced antioxidative response of the selected fungal strains was observed to be

very much similar to that noted in case of Zn treatment. Role of gamma irradiation in

boosting up the response in terms of activity of SOD , CAT and GSH content in Cd treated

tested fungal strains was also similar to that as observed in Zn treated groups. A.terreus

having 90ppm of Cd in growth media manifested 1.8times more SOD activity , 2times more

CAT activity and 1.5times more glutathione level than that of its normal counterparts

(unexposed to Cd) (Fig 5.26a). A.niger when grown in 70ppm Cd enriched media

demonstrated 3.88 times more SOD, 1.5 times more CAT activity and 1.4 times more

glutathione level than its normal counterparts (Fig 5.27a). Thus Cd induced alterations in

CAT activity and GSH content was very much comparable in both the strains, when A.terreus

had 90ppm of Cd and A.niger had 70ppm of Cd in growth media. SOD activity however was

observed to be significantly different reflecting strain specific response of the enzyme.

Further increase in Cd concentration i.e from 70ppm to 85ppm in growth media of A. niger

resulted in a remarkable boost up in activity of SOD manifesting 5-fold increase than that of

its control (unexposed to Cd) counterparts .Increase in Cd concentration to 85ppm in the

medium however did not produce much change in CAT activity or GSH level than that

observed in groups having 70ppm Cd.

Fig. 5.28(a) shows Cd induced changes in antioxidative status of P. cyclopium in

terms of activities of SOD,CAT and GSH level. 300ppm of Cd in growth media of

P.cyclopium resulted in two times increase in SOD and CAT activity and 1.8times more

glutathione level as compared to control group of the fungi without having any metal

treatment. A higher concentration of Cd in growth media (650ppm) manifested further

increase in all the three parameters showing 3-fold increase in SOD activity, more than 2-fold

higher CAT activity and reduced glutathione content as compared to their control

counterparts. Stimulatory role of gamma irradiation in enhancing antioxidant response in Cd

treated fungi was observed in all the three fungal strains with respect to their un-irradiated but

metal exposed counterparts. A 100 Gy exposed A.terreus when grown in 90ppm Cd

supplemented growth media demonstrated 1.7 times more SOD, 3 times more CAT activity

and 1.6 times more glutathione level than that of its un-irradiated counterparts (Fig 5.26b and

5.26c), whereas a 80Gy exposed P.cyclopium grown in 300ppm Cd supplemented growth

media manifested 2fold increase in activity of both SOD and CAT (Fig5.28b). Response of

Results

112 | P a g e

this group of P.cyclopium in terms of its GSH content was found to be similar to that

observed in case of 100 Gy exposed A. terreus grown in media containing 90ppm Cd. A.niger

grown in 70ppm Cd enriched media following an exposure to an absorbed dose of 60Gy of

gamma rays showed more than 1 fold enhancement in activity of the antioxidant marker

enzymes in addition to 1.7 times more glutathione level as compared to their un-irradiated but

Cd stressed (70ppm) counterparts. In contrast to the 60Gy exposed Cd (70ppm) treated

A.niger (Fig 5.27b-c), a 60Gy exposed P.cyclopium grown in 650ppm of Cd showed higher

stimulation in activities of the two marker enzymes manifesting more than 2 fold increase

in SOD activity, 3 fold increase in CAT activity as compared to that of its unirradiated

counterparts when grown under same metal stress (Fig 5.28b). Alteration in glutathione level

was almost similar in the two groups of fungi. Although similar stimulation (2 fold) in SOD

activity was observed in 40Gy exposed A.niger , increase in CAT activity and GSH content

was only 1.2 to 1.5times more respectively than that observed in the un-irradiated control

counterparts(Fig 5.27b and 5.27c).

Results obtained so far clearly stated the potential of gamma in manifesting anti

oxidative response in A.niger , A.terreus and P.cyclopium exposed to Zn and Cd.

Results

113 | P a g e

Fig 5.26 Antioxidative response of Aspergillus terreus (a) SOD and CAT response in A.terreus exposed to effective absorbed dose of gamma rays and then grown in media containing 90ppm Cd (b) GSH content in A.terreus exposed to effective absorbed dose of gamma rays and then grown in media containing 90ppm Cd(Error bars represent standard deviation for n = 6. p≤0.05 was considered significant).

Control 90ppm Cd 90ppm Cd +100Gy0.04

0.06

0.08

0.10

0.12

0.14

0.16

SOD activityCAT activity

SOD

activ

ity (U

/mg

of p

rote

in)

50

100

150

200

250

300

350(a) CAT activity (moles/sec/m

g of protein)

Control 90ppm Cd 90ppm Cd +100Gy0

5

10

15

20

25 (b)

GSH

con

tent

(nM

oles

/mg

of p

rote

in)

Results

114 | P a g e

Fig 5.27Antioxidative response of Aspergillus niger

(a) A.niger grown in medium supplemented with 70-85ppm Cd (b) SOD and CAT response in A.niger exposed to different absorbed doses of gamma rays and then grown in media containing 70-85ppm Cd (c) GSH content in A.niger exposed to different absorbed doses of gamma rays and then grown in media containing 70-85ppm Cd(Error bars represent standard deviation for n = 6. p≤0.05 was considered significant.

Control

70 ppm Cd

85ppm Cd0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

Antio

xida

tive

resp

onse

Control

70 ppm Cd

85 ppm Cd0

10

20

30

40

50CAT activity(moles/sec/mg of protein)


Control

70 ppm Cd

85ppm Cd0

5

10

15

20

25(a)


Control70ppm Cd

70ppm Cd +60Gy 85ppm Cd

85ppm Cd +40Gy

0.00

0.05

0.10

0.15

0.20

0.25

0.30(b)


SOD

act

ivity

(U

/mg

of p

rote

in)

202530354045505560

CAT activity

( mole/sec/m

g of protein)

Control70ppm Cd

70ppm Cd +60Gy 85ppm Cd

85ppm Cd +40Gy0

5

10

15

20

25

30

35(c)

GSH

con

tent

(n

Mol

es/m

g of

pro

tein

)

Results

115 | P a g e

Fig 5.28 Antioxidative response of Penicillium cyclopium

(a) P.cyclopium grown in medium supplemented with 300-650 ppm Cd (b) SOD and CAT response in P.cyclopium exposed to different absorbed doses of gamma rays and then grown in media containing 300-650 ppm Cd (c) GSH content in P.cyclopium exposed to different absorbed doses of gamma rays and then grown in media containing 300-650 ppm Cd(Error bars represent standard deviation for n = 6. p≤0.05 was considered significant.)

Control

300 ppm Cd

650 ppm Cd0.00

0.04

0.08

0.12

0.16

0.20

Antio

xida

tive

resp

onse

Control

300 ppm Cd

650 ppm Cd0

20

40

60

80

100

120

(a) CAT activity(moles/sec/mg of protein)


Control

300 ppm Cd

650 ppm Cd0

4

8

12

16

20GSH content(nMoles/mg of protein)

Control300ppm Cd

300ppm Cd +80Gy650ppm Cd

650ppm Cd +60Gy

0.1

0.2

0.3

0.4


SOD

activ

ity

(U/m

g of

pro

tein

)

050100150200250300350(b)

CAT activity (m

oles/sec/mg of protein)

Control300ppm Cd

300ppm Cd +80Gy650ppm Cd

650ppm Cd +60Gy0

5

10

15

20

25

30

35(c)

GSH

con

tent

(nMol

es/m

g of

pro

tein

)

Results

116 | P a g e

5.16 Antioxidative enzymes (SOD , CAT) and antioxidant protein (GSH) response in

gamma exposed A .niger and P. cyclopium grown in Pb enriched growth media

Fig 5.29 and Fig5.30 represent Pb induced effects on antioxidant enzymes and

proteins in A. niger and P.cyclopium with or without exposure to gamma irradiation as

compared to the respective control groups. Pb was observed to have more effect on SOD and

CAT activity of P. cyclopium than that of A.niger . While P. cyclopium manifested more

than 4 fold increase in SOD activity when grown in 2000ppm Pb in the growth media, there

was less than 3 fold increase in SOD activity in A. niger grown under the same conditions

(Fig 5.29a and Fig 5.30a). The increase in CAT activity of P. cyclopium was also much

higher (more than 3 fold ) than that noted in A. niger (less than 2 fold ) both grown in

2000ppm Pb supplemented growth media and compared to their respective control

counterparts (Fig 5.29a and Fig 5.30a). Interestingly however further increase in Pb

concentration i.e from 2000ppm to 3000ppm in the growth medium resulted significantly

more stimulation of SOD activity in A. niger, while enhancement of CAT activity and

increase in total content of GSH was much more high in P.cyclopium. There was more than 6

fold increase in SOD , activity in A. niger in contrast to 4 fold increase in activity of the same

enzyme in P.cyclopium both grown under same conditions of 3000 ppm of Pb in the growth

medium compared to their control counterparts. CAT and GSH level were observed to be

2.08 times and 1.36 times more respectively in A.niger whereas in P.cyclopium there was a

4fold increase in CAT activity and more than 2 fold increase in GSH level when both grown

in media supplemented with 3000ppm of Pb and compared to their respective control groups

(Fig 5.29a and Fig 5.30a).

A 100Gy exposed A.niger manifested 1.9 times more SOD ,1.3 times more CAT

activity and 1.18 times more GSH level with respect to its un-irradiated counterparts when

grown in 2000ppm Pb riched media (Fig 5.29b and Fig 5.29c), whereas a 80Gy exposed

P.cyclopium when grown in 2000ppm Pb showed 2.06 times more SOD, 1.7 times more CAT

activity and 2.4 times more GSH level than that of its un-irradiated counterparts (Fig 5.30b

and Fig 5.30c).2000ppm Pb caused enhanced SOD activity in A.niger than P.cyclopium

whereas for CAT activity the results obtained was just opposite i.e more enhanced CAT

activity was found for P.cyclopium than A.niger. 100Gy exposed A.niger showed 1.55 times

more SOD, 1.2 times more CAT and 1.2 times more GSH level than that of its un-irradiated

counterparts where as 80Gy exposed P.cyclopium showed 1.98 times more SOD activity,

Results

117 | P a g e

1.25 times more CAT activity and 1.23 times more GSH level with respect to its un-irradiated

counterparts when both A.niger and P.cyclopium were grown in 3000ppm of Pb enriched

growth media. Against 3000ppm of Pb, gamma caused more enhanced SOD level in

P.cyclopium than A.niger.

5.17 Antioxidative enzymes (SOD,CAT) and antioxidant protein (GSH)response in

gamma exposed A.niger A.terreus and P.cyclopium

Effect of gamma irradiation on antioxidative response of all the tested fungal strains

showed increase in SOD, CAT activity and GSH content as compared to the unirradaited

control groups of the respective fungi (Fig 5.31 a-c). The upsurge of antioxidant status of the

fungi was observed to be radiation dose dependent and with increase in absorbed doses of

gamma irradiation both the enzymes and antioxidant protein (GSH) expressed higher

stimulation. The groups of fungi exposed to the absorbed dose of 100Gy showed maximum

enhancement in antioxidative response showing more than 4fold increase in SOD activity and

2 fold higher GSH content as compared to that of the respective normal control counterparts.

Change in CAT activity was observed to be differential manifesting strain specific

modulation of activity by gamma radaiation. While an exposure to an absorbed dose of

100Gy showed 4.6 fold stimulation of CAT activity in A.niger the change in activity of the

enzyme in P.cyclopium and A.terreus was noted to be only 3.22 fold and 2.51fold more

respectively , all compared to the respective unirradiated control groups.

Results

118 | P a g e

Fig 5.29 Antioxidative response of Aspergillus niger (a) A.niger grown in medium supplemented with 2000-3000ppm Pb (b) SOD and CAT response in A.nigrt exposed to different absorbed doses of gamma rays and then grown in media containing 2000-3000ppm Pb (c) GSH content in A.niger exposed to different absorbed doses of gamma rays and then grown in media containing 2000-3000 ppm Pb(Error bars represent standard deviation for n = 6. p≤0.05 was considered significant).

Control

2000 ppm Pb

3000 ppm Pb0.00

0.05

0.10

0.15

Antio

xida

tive

resp

onse

Control

2000 ppm Pb

3000 ppm Pb0

10

20

30

40

50

60(a) GSH content(nMoles/mg of protein)



Control

2000 ppm Pb

3000 ppm Pb0

5

10

15

20

25

30

35

Control2000ppm Pb

2000 ppm Pb +100Gy3000ppm Pb

3000 ppm Pb +100Gy

0.00

0.05

0.10

0.15

0.20

0.25

0.30

SOD activityCAT activity

SOD

activ

ity

(U/m

g of

prot

ein)

2025303540455055606570

(b)

CAT activity (m

oles/sec/mg of prootein)

Control2000ppm Pb

2000 ppm Pb +100Gy3000ppmPb

3000 ppm Pb +100Gy05

10152025303540 (c)

GSH

con

tent

(n

Mol

es/m

g of

pro

tein

)

Results

119 | P a g e

Fig 5.30 Antioxidative response of Penicillium cyclopium (a) P.cyclopium grown in medium supplemented with 2000-3000ppm Pb (b) SOD and CAT response in P.cyclopium exposed to different absorbed doses of gamma rays and then grown in media containing 2000-3000ppm Pb (c) GSH content in P.cyclopium exposed to different absorbed doses of gamma rays and then grown in media containing 2000-3000 ppm Pb(Error bars represent standard deviation for n = 6. p≤0.05 was considered significant).

Control

2000 ppm Pb

3000 ppm Pb0.00

0.05

0.10

0.15

0.20

0.25

(a)

Antio

xida

tive

resp

onse

Control

2000 ppm Pb

3000 ppm Pb0

20406080

100120140160180200

Control

2000 ppm Pb

3000 ppm Pb0

5

10

15

20

25 GSH content(nMoles/mg of protein)



Control2000ppm Pb


3000 ppm Pb +80Gy0.0

0.1

0.2

0.3

0.4

0.5 (b)


SOD

act

ivity

(U

/mg

of rp

otei

n)

50

100

150

200

250

300

CAT activity

(moles/sec/m

g of protein)

Control2000ppm Pb


3000 ppm Pb +80Gy0

5

10

15

20

25

30

35(c)

GSH

con

tent

(m

oles

/mg

of p

rote

in)

Results

120 | P a g e

Fig 5.31 Antioxidative response in fungi exposed to different absorbed doses of gamma

(a)Aspergillus niger (b) Aspergillus terreus (c) Penicillium cyclopium (Error bars represent standard deviation for n = 6. p≤0.05 was considered significant).

Control 40Gy 60Gy 100Gy0.00

0.02

0.04

0.06

0.08

0.10

0.12 SOD activity (U/mg of protein)

Antio

xida

tive

resp

onse

Control 40Gy 60Gy 100Gy0

20

40

60

80

100

120

Control 40Gy 60Gy 100Gy0

5

10

15

20

25

30

(a) GSH content(nMoles/mg of protein)


Control 80Gy 100Gy0.00

0.05

0.10

0.15

0.20

0.25

(b)

Antio

xida

tive

resp

onse

Control 80Gy 100Gy0

20

40

60

80

100

120

140

Control 80Gy 100Gy0

5

10

15

20

25

30 GSH content(nMoles/mg of protein)



Control60Gy 80Gy 100Gy0.00

0.05

0.10

0.15

0.20

0.25

Antio

xida

tive

resp

onse

Control60Gy 80Gy 100Gy0

20406080

100120140160



Control60Gy 80Gy 100Gy02468

101214161820

(c) SOD activity (U/mg of protein)

Results

121 | P a g e

5.18 Gamma radiation induced modulation of Metallothionein in A.terreus, A.niger and P.cyclopium with or without metal stress Spectrophotometric values of metallothionein (MT) protein depicted that 250ppm of

Zn enhanced 68% more metallothionein content in Aspergillus niger when compared to their

normal control counterparts; 100 Gy exposed A.niger when grown in 250ppm Zn showed

18% more MT with respect to their un-irradiated but 250ppm Zn exposed counterparts (Table

5.24). Flow-cytometric values of mean fluorescence intensity of MT protein expression also

are in tune with the quantified data of MT protein revealed through spectrophotometric

observations. Mean fluorescence intensity was found to be increased 1.67 times after addition

of 250ppm of Zn in growth media, while A.niger prior exposed to 100Gy of gamma and then

grown in 250ppm of Zn supplemented growth media showed 1.08 times more fluorescence

intensity with respect to their un-irradiated but Zn (250ppm) exposed counterparts (Fig

5.32a). Further increase in Zn concentration from 250ppm to 1000ppm 1.2 fold increase in

MT content was observed, while 60Gy exposed A.niger grown in 1000ppm of Zn exhibited

10% more MT when compared to their un-irradiated counterparts. Flow cytometric data also

revealed same trend of expression when Zn concentration was further increased from 250ppm

to 1000ppm (Table 5.24).70ppm Cd enhanced 61% more metallothioneincontent in A.niger

when compared with normal control counterparts ; 60Gy exposed A.niger when treated with

70ppm of Cd showed 8% more metallothionein with respect to their un-irradiated but 70ppm

Cd exposed counterparts. Flow cytometric data of mean fluorescene intensity of MT

expression also are in tune with the quantified data of MT protein obtained through

spectrophotometric observations (Table 5.24 and Fig 5.32a).

A.terreus when grown in 8000ppm of Zn supplemented media MT content was

supposed to be increased 1.01 fold while Mean fluorescence intensity was found to be

upregulated 1.06times (Table 5.25 and Fig 5.32b). 80Gy exposed A.terreus when grown in

8000ppm of Zn enriched media showed 17% more MT content and its fluorescence intensity

was found to be increased 1.09 times more with respect to their un-irradiated but 8000Zn

exposed counterparts. Further increase in Zn concentration from 8000ppm to 9000ppm

enhanced MT content 1.4 fold and fluorescence intensity was found to be increased 1.3 times

more when compared to their control ( without exposed to Zn) counterparts. Interestingly

when A.terreus prior exposed to 80Gy of absorbed doses of gamma and then grown in Zn

supplemented media (9000ppm) manifested 14% more MT content and mean fluorescence

intensity was found to be increased 1.09times more with respect to their un0irradiated but Zn

Results

122 | P a g e

exposed counterparts (Table 5.25). Similar to the effect of Zn, A.terreus when grown in

90ppm of Cd riched growth media showed 1.32fold increase in MT content and 1.16 times

more fluorescence intensity, while 100Gy exposed A.terreus when grown in same Cd

supplemented growth media showed 22% more MT content with respect to their un-irradiated

counterparts which supposed to be coincide with 1.12 times more mean fluorescence

intensity (Fig 5.32b).

Effect of Cd on MT expression was similar in Penicillium cyclopium as found in

A.terreus.MT content was found to be 48% more in P.cyclopium when grown in 300ppm Cd

supplemented media, while 80Gy exposed P.cyclopium when grown in same Cd enriched

media (300ppm) it showed 23% more MT content and 1.05 times more mean fluorescence

intensity when compared to their un-irradiated but Cd exposed counterparts (Table 5.26 and

Fig 5.26c).

Table- 5.24 Metallothionein (µmole/mg of protein) in A.niger

Sample Total Metallothionein (µmole/mg

of protein)

Control A.niger 12.05 x 10-5

250ppm Zn exposed A.niger 20.32 x 10-5

100Gy gamma exposed A.niger grown in 250ppm Zn 24.04 x 10-5

1000ppm Zn exposed A.niger 26.63 x 10-5

60Gy gamma exposed A.niger grown in 1000ppm Zn 29.35 x 10-5

70ppm Cd exposed A.niger 19.45 x 10-5

60Gy exposed A.niger grown in 70ppm Cd 21.34 x 10-5

100Gy gamma exposed A.niger 16.33 x 10-5

60Gy gamma exposed A.niger 18.2 x 10-5

Results

123 | P a g e

Table- 5.25 Metallothionein (µmole/mg of protein) in A.terreus

Sample Total Metallothionein (µmole/mg of

protein)

Control A.terres 11.06 x 10-5

8000ppm Zn exposed A.terres 22.32 x 10-5

80Gy exposed A.terres grown in 8000ppmZn 26.04 x 10-5

9000ppm Zn exposed A.terres 26.63 x 10-5

80Gy exposed A.terres grown in 9000ppmZn 30.35 x 10-5

90ppm Cd exposed A.terreus 25.62 x 10-5

100 Gy exposed A.terreus grown in 90ppm Cd 31.25 x 10-5

80Gy exposed A.terreus 17.56 x 10-5

100Gy exposed A.terreus 15.25 x 10-5

Table- 5.26 Metallothionein (µmole/mg of protein) in P.cyclopium

Sample Total Metallothionein (µmole/mg of

protein)

Control P.cyclopium 10.35 x 10-5

300ppm Cd exposed P.cyclopium 15.36 x 10-5

80Gy exposed P.cyclopium grown in 300ppmCd 18.85 x 10-5

80Gy exposed P.cyclopium 13.65 x 10-5

Interestingly all the tested fungal strains when exposed to only gamma absorbed doses

showed upregulation of MT content revealed both the data of spectrophotometer and Flow-

cytometer (Fig 5.32). Effect of gamma on MT expression was found to me more on A.niger

than A.terreus when both the strain were exposed to 100Gy of gamma absorbed dose. While

effect of gamma was more on A.terreus than P.cyclopium when both the strains were exposed

to 80Gy absorbed doses of gamma. Upregulation of MT expression was found higher at

lower doses of gamma, i.e 60Gy exposed A.niger showed more upregulation of

metallothionein than 100Gy exposed one, similarly A.terreus showed more enhancement in

MT content when exposed to 80Gy of gamma absorbed dose than 100Gy absorbed dose.

Results

124 | P a g e

Fig 5.32 Metallothionein (MT) expression (Mean Fluorescence Intensity) of A.niger,

A.terreus and P.cyclopium in Flow cytometer (a) A.niger exposed to Zn, Cd and gamma

absorbed doses (b) A.terreus exposed to Zn, Cd and gamma absorbed doses (c)

P.cyclopium exposed to Cd and gamma absorbed dose.

Cont.

250ppm Zn

250ppm Zn+100Gy

1000ppm Zn

1000ppm Zn+60Gy0

500

1000

1500

2000

Mea

n Fl

uore

scen

ce

Inte

nsity

Cont.

70ppm Cd

70ppm Cd+60Gy0

200400600800

10001200140016001800

(a)

Cont. 60Gy 100Gy0

200400600800

10001200140016001800

Cont.

8000ppm Zn

8000ppm Zn+80Gy

9000pm Zn

9000ppm Zn+80Gy0

200

400

600

800

1000

1200

1400

Mea

n Fl

uore

scen

ce

Inte

nsity

Cont.90ppm Cd

90ppm Cd+100 Gy0

200

400

600

800

1000

1200

Cont. 80Gy 100Gy0

200

400

600

800

(b)

Cont. 300ppm Cd 300ppm Cd+80 Gy 80Gy0

200

400

600

800

1000

(c)

Mea

n flu

ores

cenc

e In

tens

ity

6. General Discussion

Dhapa, located in the eastern fringe of Kolkata, a megapoly in eastern part of India, is

the prime garbage dump site of Kolkata municipal area. Since the last couple of years a major

portion of this area is being converted to cultivable land thus offering provision for use of the

waste land for production of edible vegetables. Huge production of edible crops and

vegetables in this garbage dumping site is complemented by the results of the present study

of physico-chemical parameters of the soil, collected from three sites of Dhapa. Data

pertaining to the organic carbon content in the range of 2- 3.5% validate the soil of Dhapa as

a good agricultural soil type as endorsed earlier by Choudhury et al., (2011) who

demonstrated similar good quality soil in northern region of India having organic content 1-

3.5% in most of the areas. Electrical Conductivity (EC), defined by the level of ability of the

soil water to carry electrical current, is a good indicator of the amount of nutrients available

for crops to absorb. Although EC does not provide a direct measurement of specific ions or

salt compounds, it can be correlated with the concentrations of nitrates, potassium, sodium,

chloride, sulfate, and ammonia. Ionic conductivity of soil collected from Dhapa having a

value between 400-600 µS/cm and pH value reflecting the alkaline nature of soil together

with data of available nitrogen, phosphorus, potassium content, confirms good health of the

soil. Concentrations of heavy metals detected in the soil sample reveal higher concentration

of Zn, followed by Cd and very negligible amount of Pb. Although concentration of Zn is

comparatively higher than the other two metals, all are within the permissible limit set by

World Health Organisation (WHO). The high concentration of zinc ion (Zn2+) might be

because Zn readily hydrates and combines with other metals to form its ores.

Metals are very crucial for growth and development of living organisms. They are

directly or indirectly involved in various biochemical pathways and have important roles in

different functional processes of life. Although metals are highly essential for life systems

and metals like K, Na, Mg, Ca, Mn, Fe, Cu, Zn, Co, Ni etc, have been documented to have

specific functions in metabolic processes of living organisms, many metals (e.g. Rb, Cs, Al,

Cd, Ag, Au, Hg, Pb) have no apparent essentiality. In the present study the three fungal

strains selected from the ten different fungal isolates are observed to have differential

response towards the heavy metals Cd, Pb and Zn in relation to their growth, colony forming

ability and some other metabolic parameters. Such differential response is dependent on

sensitivity and tolerance towards metals as seen in cases of yeasts and other saprophytic fungi

(Ortiz et al., 1992, 1995; Gadd, 1993). Although metals have critical role in biological

General Discussion

126 | P a g e

functioning of the organisms, the concentration of the metals determine their precise role or

effect within the body. As such, even essential metals, if present in concentrations above

threshold, might pose serious deleterious effects on the concerned organism. Hence the titer

or bioavailable concentration of the metal within the cells is an important determinant of

normal functioning of any organism. In this regard essential metals when available in higher

concentration could be considered as toxic. Fungal survival in presence of any toxic metals

mainly depends on intrinsic biochemical and structural properties, physiological and/or

genetic adaptation. Speciation of metal and environmental modification of speciation, is

another important factor which determine toxicity and intensity of toxicity of the metal.

Arsenic (As) and its compounds are ubiquitous in nature and exhibit both metallic and

nonmetallic properties. The trivalent and pentavalent forms are the most common oxidation

states. Trivalent arsenic is more potential in toxicity than pentavalent arsenic (Wan et al,

1982; Jacobson-Kram and Montailbano, 1985; Kochhar et al, 1996; Moore et al, 1997). Hg+ ion is not stable under environmental conditions since it dismutates into Hg0 and Hg2+. A

second potential route for the conversion of mercury in the soil is methylation to methyl or

dimethyl mercury by anaerobic bacteria (Rodriguez et al., 2005) Mercury is a persistent

environmental pollutant with bioaccumulation ability in fish, animals, and human beings

(Chang et al.,2009). In the present study the selected fungal strains showed specific range of

tolerance towards Cd, Zn and Pb, where beyond certain level, i.e the threshold level, visible

growth of the fungal strains were restricted. Various group of researchers reported that MIC

values are fungal strain specific and metal specific i.e it varies from one species to other

against a particular metal. Iram et al., (2009) showed the range scale of different metal

tolerance in Aspergillus niger, Mucor and Fusarium strain. Ahmed et al., (2005) isolated

twenty different fungal strains from local soil samples of Aligarh polluted with industrial

effluents, municipal wastes. Their group also studied MIC values of Cu, Cr and Cd for

Aspergillus, Penicillium, Alternaria, Fusariu, Rhizopus, Trichophytes and Mycelia species. In

the present study A.niger is observed to be more sensitive towards Cd while P .cyclopium is

more tolerant towards Cd. It has been reported that a number of fungi from all taxonomic

groups may be found in metal polluted soils and they possessed the ability to survive in that

environment by detoxification of the metal (Ross, 1975; Gadd and White, 1989a; Baldi et al.,

1990, Turnau, 1991). Gadd, (1978) reported that all these elements interact with fungal cells

and can be accumulated by physico-chemical mechanisms and transport systems of varying

specificity.

General Discussion

127 | P a g e

Similar to their response to metals, response of fungi towards antibiotic is also strain-

specific. the objective of in vitro assessment of susceptibility of fungal isolates towards a

regime of antibiotics has been an important factor for fundamental research in addition to

clinical point of view. Result of the present investigation showed that the three selected

fungal strains are differentially resistant to different antibiotics. Antibiotic sensitivity or

susceptibility test showed fungus to be resistant to certain antibiotics and not to some others;

degree of resistance is also found to be different in case of different fungi. This is due to

differences in their genetic makeup (Davies and Davies, 2010). This supports the present

finding showing nearly 1.5 fold difference in response of one fungi than another towards the

same antibiotic. Hemambika et al., (2011) studied the anti-fungal susceptibility of

Aspergillus, Penicillium, Cephalosporium fungi towards nystatin and intraconazole.

pH and temperature are two important factors responsible for fungal growth and

colony formation. The neutral pH supporting maximum mycelial growth observed in the

present study is perfectly in tune with the observation of Lewis et al., (2012). This group also

reported that temperature, growth medium, carbon starvation, low oxygen concentration and

a wide range of chemicals including N-acetylglucosamine, proline, and alcohols are the other

critical factors playing important role for fungal growth. A group of researchers reported

earlier that the extracellular pH is one of the important environmental factors that modified

the physiology of the cell (Cornett et al., 2005). Maximum growth of all tested fungal species

were at pH 7 and temperature 29 0 –30 0 C. Much lesser growth observed at higher

temperature i.e. higher than 300C. This is possibly due to the effect of higher temperature on

metabolic enzyme activity, transport carriers and cell membrane integrity since higher

temperature denatures functional proteins and consequently disrupts vital functions. The

present observation and subsequent postulation gets support from the report of Prescott et al.,

(2002) who have noted decrease in growth due to high temperature induced effect on enzyme

activity, membrane integrity in microbial system. Effect of temperature and pH on growth of

yeast cells were studied by Nadeem et al., (2013).They observed maximum growth of

Candida albicans at pH 7.4 and 340 C. Effect of incubation temperature and media pH on

growth of different fungi were reported by several research groups since decades (Mcclary,

1952; Buffo et al., 1984; Sudbery et al., 2004; Kabli, 2006).

Data of the present study establish the potential of gamma irradiation in modulating

metal tolerance in fungi. Effect of gamma irradiation on metal tolerance by the selected fungi

indicates strain-specific sensitivity and response of the fungi which is dependent on the

absorbed dose of gamma exposure and also on concentration of the metal in the medium.

General Discussion

128 | P a g e

Such strain specific behavior of microbes towards metals have been reported by a host of

researchers (Ahmed et al., 2005; Iram et al., 2009) .Influence of absorbed dose of gamma

irradiation on various properties of fungi including growth, colony forming ability and other

metabolic parameters have been reported earlier by Hassanein, (1987) and Dadachova et al.,

(2007).

The present study reflecting the change in pattern of sensitivity of fungi towards

metals after exposure to radiation further demonstrates potential of gamma in modulating

intrinsic characters of fungi. This is validated by the findings showing gamma irradiated

A.niger, originally sensitive for Zn became more Zn resistant than A.terreus, which when

isolated was a more Zn tolerant strain. Comparison of effects on Cd tolerance by A.terreus

and P cyclopium before and after exposure to gamma, also corroborates the above.

Concomitant to enhancing metal tolerance, gamma induced increase in accumulation and

removal potential of irradiated strains with respect to their non-irradiated counterparts reflect

the role of ionizing radiation on interaction and response of fungi towards heavy metals.

While some groups have worked on use of UV radiation to induce metal resistance in

bacteria (Ling et al., 2007; Dib et al., 2008), , no reports are available showing gamma rays in

regulating metal tolerance in fungi. Some groups reported about developing metal resistant

fungi by chemical mutagens (Levine and Marzluf, 1989; Pali et al., 2007). As such, the

present study showing gamma-induced enhancement of metal tolerance in three different

fungi in addition to their increased uptake and removal potential, is the first of its kind and

presents an important contribution in the relevant field conveying possibility of employing

ionizing radiation in modulating heavy metal tolerance and uptake by fungi.

Results of the present study showed that the respective absorbed doses of gamma

irradiation which were most effective in inducing metal tolerance and maximum potential of

removal and uptake of the metals from specific metal supplemented liquid media by the

respective fungi, also induced maximum growth of the concerned fungi in terms of its colony

forming ability. Colony-forming ability is considered to be an important parameter denoting

growth of fungi where colony forming unit (CFU) provides an estimate of the viable fungal

numbers. CFU has been used as a standard method of expressing growth/survival response of

microbes (fungi and bacteria) in normal condition as well as exposed to any physical and

chemical stress. Several earlier researchers have used CFU to determine tolerance of

microbes towards different heavy metals and chemical stress effectors (Baath et al., 2005;

Peciulyte and Volodkiene, 2009; Liu et al., 2010; Iram et al., 2011). Increase in colony

growth of Aspergillus flavus when exposed to lower doses of gamma irradiation and

General Discussion

129 | P a g e

complete inhibition at higher doses has been reported earlier by Hassanein, (1987). Parallel to

the data of present investigation, effect of radiation on CFU of fungi has been demonstrated

by Dadachova et al., (2007) who reported higher CFUs with increased biomass of some

fungal strains isolated from areas having higher radiation level. Increase in dry mycelial

weight of Paecilomyces violacea upon exposure to gamma irradiation has been reported

earlier by Ibrahim (1986). Ziombra (1994) reported 100Gy and 200Gy of absorbed doses of

gamma radiation accelerate spore germination of Pleurotus sp. However, contrary to our

report showing maximum stimulation of CFU in Aspergillus terreus at 80-100 Gy absorbed

dose of gamma, Geweely and Nawar (2006) found maximum stimulation of germination and

growth at a dose of 250Gy in Aspergillus tenuissima and Stemphylium boryosum. Many fungi

especially melanized ones are very radioresistant. Photochemical properties of melanin make

it an excellent photoprotectant. Electronic complexity of melanins allows them to scatter

/trap photons and electrons which supports the hypothesis of Dadachova et al., (2007). He

proposed that melanin harness metabolic energy from ionizing radiation, and melanised cells

might consume more energy when exposed to radiation. As postulated by Robertson et

al.,(2012) melanin upon exposure to ionizing radiation plays dual role- 1) upregulation of

different transporter genes which subsequently help melanised cells to take more carbon and

amino acids as nutrition and expel toxic metabolites generated by radiation stress 2)

upregulation of the gene expression involved in ribosomal biogenesis. These properties of

melanin support the enhanced growth and survivability of radiation exposed fungi than

unirradiated counterparts and indicate efficient protective role of melanin against different

stresses viz. UV or gamma radiation, high temperature, chemical stress including heavy

metals and oxidizing agents and other stress effectors that produce reactive oxygen species,

which induce cell damage or other biochemical threats. As A. niger is a melanised fungal

strain, increased growth of A.niger simultaneous with induction of other metabolic properties

including higher tolerance towards heavy metals observed in the present study might be due

to involvement of melanin.

Uptake of heavy metal ions by microorganisms from the ambient habitats has been an

effective way for remediation of metal burden. As such, microbes by virtue of their ability to

uptake heavy metals have offered a way of bioremediation. Fungi are known to detoxify

metals by several mechanisms including ion exchange, chelation, adsorption (cell wall

binding), crystallization, valence transformation, extra and intracellular precipitation and

active uptake (Ashida, 1965; Gadd, 1993). Endurance of fungi in presence of heavy metals

depends on a biphasic process which may be metabolism independent or dependent. In the

General Discussion

130 | P a g e

former condition initial cell surface binding can occur either in living or inactivated

organisms, while in metabolic dependent condition, intracellular accumulation takes place

only in living cells. Metals initially bind to the cell surface followed by intracellular

accumulation (Green & Clausen, 2003).

Although metal removal capacity of fungi has been studied by host of earlier

researchers, role of ionizing radiation on metal removal efficiency have not been worked out

yet. It is well established that micro organisms are able to grow in heavy metal enriched

environment with a significant metal uptake capacity (Srivastava and Thakur, 2006). Reports

regarding potential of A. niger for removing heavy metals including Zn has been cited by host

of earlier researchers. Akthar and Mohan (1995) reported that killed mycelium of A. niger

could remove copper and zinc from contaminated lake waters. Use of A. niger to remove

copper, zinc and other metals from incinerated municipal fly ash by organic acid leaching

was also reported by Bosshard et al.,(1996). Price et al., (2001) has shown potential of A.

niger to remove 91% of Cu and 70% of Zn from a treated swine effluent. The potential of

living Aspergillus niger to remove cadmium and zinc from aqueous solution, under the

optimal conditions, showed the maximum uptake capacities of Cd and Zn ions were 15.50

mg/g and 23.70 mg/g at initial concentrations of 75 mg/L and 150 mg/L, respectively (Liu et

al., 2006). In contrast to these reports results of the present study revealed that irradiated

A.niger (60Gy) gained its potential to remove 45% and 40% of zinc from 1500ppm and

2000ppm of zinc treated media respectively while without gamma irradiation it could not

tolerate that high zinc concentration. Potential of Aspergillus sp. for removing heavy metals

including Cd has been cited by Kumar et al., (2011) who showed acclimated biomass of

A.niger could remove 51.05% Cd from growth media. Earlier Massaccesi et al., (2002)

observed 60% removal of Cd by A.terreus in shaking condition . Hemambika et al., (2011)

reported the potential of Penicillium sp to remove 97.21% of cadmium after immobilization.

Dugal et al., (2012) reported the Penicillium isolate could remove 95% of cadmium from the

medium after 96 h of incubation in the same context. The potential use of the fungus

Penicillium purpurogenum to remove cadmium, lead, mercury, and arsenic ions from

aqueous solutions was evaluated by Ridvan et al., (2003). In contrast to these reports,

however data of the present investigation reflected much higher removal potential of gamma

irradiated A. terreus as nearly 66% -75% removal of Cd was observed by the irradiated

groups exposed to 100Gy and 80Gy absorbed dose respectively. The study also clearly

indicates that effect of gamma irradiation on metal removal potential is strain specific as

reflected by much higher potential of Penicillium cyclopium in removing the same metal Cd

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131 | P a g e

in comparison to Aspergillus terreus and A.niger. Heavy metal removal efficacy of

Penicillium has been reported by earlier researchers (Niu et al., 1993, Fan et al.,2008) . The

present study also revealed P.cyclopium to be more efficient for Pb removal than A.niger.

From 2000ppm and 3000ppm Pb enriched media A.niger was able to remove 65-70% of Pb

while for P.cyclopuim removal potential from same Pb supplemented media was reflected as

75-80% respectively. Pb removal efficacies of A.niger, P.austurianum, Saccharomyces

cerevisiae, Mucor arcindloides, Trichoderma reesi were reported earlier by Awofulo et al.,

(2006) for dead and live biomass respectively. According to their reports living biomass are

most effective in metal removal than dead biomass. Iskandar et al., (2011) also highlighted

the Pb and Cu uptake efficacies of Aspergillus niger, A. fumigatus, Trichoderma asperellum,

Penicillium simplicissimum and P. janthinellum, where the maximum removal of Cu(II) and

Pb(II) was performed by A. niger.

Though use of gamma radiation for modulating metal tolerance have not been

investigated earlier, but gamma radiation induced salt tolerance in Trichoderma harzianum

has been reported by Mohammad and Hagagg, (2005) while Sridhar et al.,(2002) showed UV

radiation could impart thermo-tolerance or osmo-tolerance in Trichoderma sp.

Metal binding in fungi involves functional biomolecules. Initial cell surface binding

of metals is considered to occur with proteins, lipids and different polysaccharides like

glucans, mannan, chitin and chitisan present on the cell wall (Korn and Northcote, 1960;

Ruiz-Herrera, 1992). For the binding of metals during the biosorption, the multilaminate,

microfibrillar cell wall structures containing a large number of functional groups as carbonyl,

hydroxyls, amides are known to be responsible (Tobin et al., 1990 ; Akar et al.,2005). Role of

different functional groups as well as cell wall components in total intake of metal ions by

fungal biomass has been reported earlier by a host of researchers (Zhou and Banks, 1993;

Baik et al., 2002). FTIR spectral analysis suggested that OH, -NH,-C=O (broad and sharp

peak) are the key binding sites for metals on the surface layer of tested fungal strains. Some

other functional groups viz. CH,CH2, C-N, COO are also reflected to be involved in metal

adsorption as indicated by the observed peak shifting in the regions denoting these functional

groups in strains treated with metals. Similar shifting in peaks in fungal biomass treated with

heavy metals have been reported earlier by a host of researchers (Das and Guha, 2009; Xu et

al., 2012; Damodaran et al., 2013) .First two research groups studied the peak shifts in

filamentous fungi treated with Cr, while Damodaran et al., (2013) observed the effects of Cd,

Pb, Zn, Cu and Cr treated groups of fungi. Use of FTIR to detect the presence of both

primary and secondary stress factors have been cited earlier (Qian and Krimm, 1994; Yang et

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132 | P a g e

al., 1999; and Shi et al., 2002). Modulatory role of gamma in inducing metal tolerance in the

selected fungi is corroborated by FTIR spectral data of samples exposed to gamma and

treated with metal, where more drastic peak shifting were noted in regions of the involved

functional groups. In contrast, the only gamma exposed groups showed slight changes in

FTIR spectra with respect to their control counterparts. Thus, it may be postulated that

gamma irradiation being an external stress factor, caused some metabolic changes involving

the functional groups in the exposed fungal strains., However significant changes in the same

functional groups in the gamma exposed fungi additionally treated with metals, confirms

potential of gamma irradiation in regulating response of the concerned fungi towards heavy

metals involving important functional groups.

Results portrayed that temperature 29-30°C and pH 7 were most favorable condition

for removal as well as uptake of metals by the selected groups of fungi. Interestingly the

gamma exposed groups of those fungi also showed same favorable conditions for removal of

metal. Actually the dependence of metal uptake on pH is related to both surface functional

groups present on fungal biomass and metal solution. At low pH, the cell surface sites are

closely linked to the H+ ions, thereby making these sites unavailable for metal cations.

However, with increase in the pH, there is an increase in ligand with negative charges which

favored electrochemical attraction and increased binding of cations resulting in more

adsorption of metal. This view supports the findings of the present study where at pH 7

removal of metals form media was much higher than that in pH 5 . The less bioaccumulation

capacity at lower pH is reported due to the competition of hydrogen ions with metal ions on

the sorption sites (Congeevaram et al., 2007). Similar observation was also reported earlier by

Lovely (1995) who observed sorption of heavy metals by the fungi is strongly pH dependent

and biosorption rate increases with increase in pH. Nasseri et al., (2002) showed that at pH

value above 7, metals exist as hydroxide colloids and precipitate at alkaline pH due to

osmotic changes and hydrolyzing effect, thus resulting reduced uptake rate; This validates the

findings of the present study where at higher pH (pH 9) uptake of metals was observed to be

much lesser than that at pH 7 in case of all the three selected fungal strains. At low pH most

of the carboxylic groups is not dissociated and cannot bind the metal ions to fungal cell wall

(Choudhary and Sar, 2009) and at high pH value metals get precipitate which causes low

biosorption of metal ions at high pH(Pinoa etal., 2006).

Role of temperature in the biosorption of metal ions has been reported earlier where it

has been proposed that higher temperature damages metabolic functioning via damaging

enzymes transport carriers, integrity of cell membrane (Prescott et al., 2002), and thus might

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133 | P a g e

hinder compart- mentalization of metal ions leading to reduced metal uptake (Faryal et al.,

2007). Srivastava and Thakur (2006) highlighted about the physical damage to the biosorbent

and exothermic nature of some adsorption processes which might reduce the biosorption

capacity of the biomass in high temperature. These might be the explanation of lower

uptake/removal of metals observed in the present study in case of all the three strains of fungi

at higher temperature. However in contrary to the present data Shivakumar et al.,(2014)

observed maximum accumulation of heavy metals at 25°C temperature and at pH 5.

Distortion of normal cylindrical shape and structure of the hyphae in fungi exposed to

the heavy metals as observed under SEM is perfectly in tune with reports from earlier

researchers (Srivastava and Thakur, 2006; Xu et al.,2012). Results, showing maintenance of

near normal hyphal morphology in samples exposed to gamma irradiation before metal

treatment, indicate possible potential of gamma in providing protection against metal-induced

morphological alterations. It might also be presumed that gamma induced metal tolerance had

influenced the fungal metabolism in a way where the concerned fungi could efficiently utilize

the metal without having any perturbations in the morphological characteristics. The present

postulation is complemented by the observed data reflecting significant enhanced activity of

CMCase and α- amylase, the two enzymes involved in growth and reproduction of fungi in

metal treated samples pre-exposed to gamma irradiation Potential of gamma irradiation in

enhancing activities of these enzymes have been documented earlier by a host of workers.

Abostate et al.,(2010) reported higher production of cellulases (CMCase, Avicelase) by

gamma irradiated (0.5 K Gy) Aspergillus than that of the parental non-irradiated counterparts

. Fawzi and Hamdy (2011) observed different doses of gamma irradiation could induce

enhanced production of CMCase in Chaetomium cellulolyticum . It is well established that

stress in any form - physical (viz. ionising radiation etc.) or chemical (viz. heavy metals like

Cd, Pb, Zn etc.), causes perturbation in the dynamic equilibrium within the living system. In

such condition the cells must be able to adjust their physiological processes to reach a

homeostasis. Data of the present study show metal (Cd, Pb and Zn) induced depletion in

activities of CMCase and α-amylase of all fungal strains, the two enzymes involved in

nutrient assimilation and subsequent growth of the fungi. This ensures role of metal in

distortion of enzyme functioning in the concerned fungi which is possibly due to association

of metal with transcriptional as well as translational pathways as postulated by Baldrian et al

(2003). Heavy metals are known to disrupt normal growth and metabolic activity of the

exposed organisms (Baldrian et al., 2005). Usually heavy metal induces uncontrolled

efflux/influx of electrolytes or other vital ions resulting in disruption of the ionic homeostasis

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134 | P a g e

and subsequent deregulation of activities of many enzymes crucial for basic cell metabolism

.In parallel to the present observation Huang et al.,(2006) found inhibition of CMCase

activity and cellulose degradation capacity of Phanerochaete chrysosporium under Pb stress.

Earlier, Baldrian et al.,(2000) also reported decrease of ligninolytic activities in fungi under

metal stress. Several studies have shown that certain metals like Fe, Hg Ag , Cd, Pb

inhibited the laccase activity ( Robles et al.,2002 , Hatyani and Mecs, 2003).

In the present study, boosting up of activity of CMCase and α-amylase, either to near

normal values or even higher, as a result of exposure to gamma irradiation preceding metal

exposure, might possibly be an attempt of the fungi to cope up with the stress through

stimulation of the enzymes that are directly linked with growth and reproduction of fungi

(Tuckwell et al.,2006). Remarkable increase in CFU observed in these samples validates the

role of gamma irradiation in conferring protection against metal induced toxicity through

enhancement in CMCase and α-amylase activity. This might be attributed to the gamma

induced enhancement of fungal metabolism to maintain energy in response to exposure to

metal stress as some earlier researchers have talked about ability to harness radiation for

metabolic energy by some fungi (Dadachova et al., 2006).

Thus, it may be conjectured that gamma irradiation being an effective mutagenic

agent for fungi, has imparted mutation which induces up regulation of CMCase and α-

amylase resulting in better survival of the fungi with higher tolerance towards metal.

Recently Huma et al., (2012) developed gamma induced mutants of Phialocephala humicola

having much higher amylase production. Up regulation of proteins in response to various

stressed environments to favor growth and survival of microbes has been well cited by

several groups of researcher (Len et al., 2004). Weber et al., (2006) observed secondary

adaptive mechanisms including up regulation of metabolic enzymes in E coli under severe

stressed condition of high osmolarity. Runic et al., (2009) reported, in addition to antioxidant

proteins, up regulation of proteins involved in nitrogen assimilation or amino acid transport in

Pseudomonas putida grown under the condition with limited nitrogen.

In short, our data documenting higher CMCase and α-amylase activity in gamma

irradiated heavy metal stressed fungi put a strong support for the recent proposed hypothesis

of need-based responsiveness of microbes or any living system per se, mediated via

regulation of proteins. This could play an adaptive role in maintenance of functions that are

compromised by natural genetic, environmental or any perturbation (Deluna et al., 2010).

Precise mechanism for such adaptive strategy needs detailed investigation with

molecular approach. It is well established that oxidant –antioxidant status of a cell or the

General Discussion

135 | P a g e

system concerned is an important marker of stress induced effects. Response of antioxidant

proteins and enzymes towards stress not only portrays immediate sensitivity or tolerance of

the exposed organism but also might give an indication towards strategic development of

adaptive response.

Although under normal conditions of growth of an organism reactive oxygen species

(ROS) are formed as a by-product of various metabolic pathways, there exists a balance

between ROS generation and its scavenging. However the equilibrium is disturbed when the

body is under stress condition. Both heavy metals and gamma irradiation are two external

stress effectors which stimulate excess generation of reactive oxygen species which

subsequently imparts deleterious effects on normal functional processes and produce

pathobiological conditions or toxicity (Macfarlane and Burchett,2001; Poli et al., 2004). So

there is a need to combat or defend excess ROS generation in order to detoxify the internal

environment of the concerned organism. ROS is a known initiator of antioxidative defense

system (ADS) (Andric et al.,2003; Ozmen et al.,2007). To combat the damaging effects of

ROS, living organisms, including fungi, are known to have evolved both non-enzymatic and

enzymatic antioxidant defense mechanisms. As a part of the enzymatic defense system,

superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione

peroxidase (GPx,) and glutathione reductase (GR), take the key role, while non-enzymatic

defense systems comprise of glutathione (GSH), melatonin, vitamins C, E and ß-carotene, all

involved in protecting the cells from oxidative injury in aerobic organisms. Under such

circumstances SOD plays its role as cell’s first line of defense against ROS catalyzing the

dis-proportionation of superoxide radicals to hydrogen peroxide and oxygen (Hassan and

Scandalios, 1998). CAT and peroxidases reduce H2O2 to O2 as well as H2O (Scandalios,

2005). GSH is also established to play crucial role in the defense mechanism of this category

especially against heavy metals in a variety of organisms (Wysocki and Tamás 2010;

Yadav,2010; Hossain et al.,2012). Data of the present study showing enhancement of anti-

oxidative enzymes as well as non-enzymatic antioxidants in the tested fungal strains treated

with heavy metals reestablish the toxic effect of heavy metals in producing cellular

perturbations through production of ROS as has been cited by host of earlier researchers

(Jacob et al., 2001; Ott et al.,2002). The escalation of SOD, CAT and GSH level revealed

through this study depicted their role in protecting fungi from the toxicity of heavy metals.

Participation of SODs in heavy metals toxicity has been reported by a group of researchers in

plants, animals and microorganisms (Yoo et al.,1999;Vido et al.,2001) but reports about

filamentous fungi is limited .The present findings showing Cd induced 3.88-1.5 times more

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136 | P a g e

increase in antioxidative enzyme in three different fungi are in tune with the pronouncement

of the Ott et al. (2002) who reported antioxidative enzyme fluctuations (SOD,CAT,GR etc.)

in Cd administered fungal culture Paxillus involutus. Jacob et al. (2001) demonstrated

increase of SOD activity in response to Cd in Paxillus involutus. Effects of cadmium on

cellular glutathione levels has been worked by Li et al. (1993). Their group reported a dose-

dependent increase in both cellular glutathione levels and cytoskeletal protein sulfhydryl

when cultured cells were exposed to cadmium. Yildirim and Asma (2010) reported the

enhancement of SOD, CAT and GSH in Cd treated Phanerochaete chrysosporium. Krumova

et al.,(2011) demonstrated the induction of SOD in Humicola lutea after administration of

copper. The observed data showing 3 fold increase in SOD activity in Zn treated A.niger is

similar to the findings of Vallino et al., (2009) ,who reported 2.5 fold more SOD activity in

ericoid mycorrhizal fungus Oidiodendron maius under Zn stress Interestingly interaction and

response of microbes to prior exposure to metals are shown to ameliorate toxic effect of that

metal. Saccharomyces cerevisiae cells exposed to sublethal levels of mercury induces

protection against toxic levels of mercury (Westwater et al.,2002).Although some detailed

research on SOD mediated response of fungi towards metal exposure and oxidative stress has

been carried out (Azevedo et al.,2007) and Frealle et al. (2005) nicely reviewed SODs of

filamentous fungi. Reports pertaining to heavy metal stress effect on CAT activity in fungi is

scanty. Abhrasev et al.,(2005) observed enhanced level of SOD and CAT activities against

thermal stress in Aspergillus niger. It is reported that in Candida intermedia, concentration of

superoxide, hydrogen peroxide radicals, GSH content as well as catalase activity was

increased in presence of Cu and tolerance to metal ions depended on the rate ROS generation

and antioxidative defense system effectiveness (Fujs et al.,2005).

Significant additional acceleration in activity of anti oxidative enzymes and non-

enzymic components in gamma exposed fungal groups grown in metal stressed condition as

compared to their un-irradiated but metal stressed counterparts, reconfirms the role of

ionizing radiation in generation of reactive oxygen species. The up-regulation of CTT1 and

SOD2 genes, in radiation exposed W. dermatitidis cells reported by Robertson et al. (2012),

verified the response of the microbial cells towards oxidative stress in order to eliminate

ROS. In short, this validates ionizing radiation activates DNA repair mechanism and anti

oxidative defence systems. This group also observed enhanced growth and extend lifespan of

W. dermatitidis upon exposure to low doses of ionizing radiation. In tune with their

observation, the present data showing more colony forming ability of all the three selected

fungal strains exposed to gamma irradiation and then grown in metal enriched media might

General Discussion

137 | P a g e

be mediated through enhancement of antioxidative defense mechanisms as opined by

Balaban et al., (2005) who suggested that induction of oxidative stress responses might

promote longevity by protecting against reactive oxygen damages. Paecilomyces lilacinus

isolated from Chernobyl Nuclear Power Plant having 3 to 5 orders higher radioactivity than

the background showed activation of antioxidant enzymes (Belozerskaya et al, 2010).

Metallothionein (MTs), a cysteine -rich protein, having serine, lysine and aromatic

amino acids, has high affinity for metal ions and bind metals via thiole groups. Some

metallothionein genes are known to be positively regulated by metals and involved in heavy

metal detoxification and homeostasis (Cobbett and Goldsbrough, 2002). Thus MTs play a

very important role in intracellular sequestrations of heavy metals. MTs are also known to

provide protection from oxidative damage (Tamai et al.,1993). Along with metal ion transfer

in fungi and yeast, metallothioneins sequester Cu, Zn, Cd, Hg and silver ions (Peterson et al.,

1996; Freisinger,2013). This protein shows high affinity towards zinc and possesses high

antioxidant activity. Significant increase in metallothionein in Zn and Cd induced fungal

strains observed in the present investigation, indicates up-regulation of metallothionein in

metal treated fungi. Results reflect role of MT in inducing heavy metal tolerance of the fungi

allowing the fungi to survive in metal rich growth media as MTs have been shown to be

associated in metal homeostasis and oxidative stress along with other numerous cellular

functions (Gadd, 1993; Cobett and Goldsbrough, 2002). The present results are in tune with

the findings of Pal et al., (2008), who also reported upregulation of mtallothionein in Cd

exposed Aspergillus niger with respect to wild type . Relation of GintMT1 with Cu stress has

been reported by Gonzalez et al.,(2007) in Glomus intraradic. Earlier Kameo et al.,(2000)

accounted induction of metallothioneins in Beauveria bassiana treated with Cd and Cu.

Association of the protein metallothionein in fungi has primarily been studied in response

to metal toxicity and in stress response (Tucker et al.,2004). Although involvement of genes

responsible for various important functions like repair (RAD50, RAD51), recombination

(HRP1),chromosome stability (CHL1, CTF4), endocytosis (VID21) have been reported in

microbes (Sachharomyces cerevisiae)in response to ionizing radiation (Bennett et al.,2001),

role of metallothionein in radiation exposed fungi or precise effect of radiation on

metallothionein has not been worked out. However, some earlier research groups

documented induction of metallothionein as a significant factor in the mechanism of

protection against radiation in mice (Matsubara et al.,1987;Ono et al.,1998), while Ding et

al.,(2005) reported similar upregulation in gamma induced human fibroblast cells .Saha et

al.,(2013) reported gamma radiation induced upregulation of metallothionein in Plantago

General Discussion

138 | P a g e

ovata forsk. Modulation of photolyase/ cryptochrome family genes in filamentous fungi

exposed to radiation has been reported (Berrocal et al., 2000).

The findings of the present study offer a two-fold contribution to the scientific field of

relevance; first, potential of gamma rays in modulating tolerance of fungi towards heavy

metals is revealed through this study which reflects role of ionizing radiation in imparting

metallo-resistance through regulation of important functional biomolecules. Upregulation of

stress responsive enzymes viz.SOD, CAT, antioxidant protein GSH and the metal responsive

ROS scavenger protein metallothionein in gamma exposed fungal groups grown under metal

stress, suggest role of gamma in conferring better ability of the fungi to combat metal stress

thereby helping them to manifest better growth. This is complemented by the observation of

ultrastructural analyses of the fungi. This part of the findings present a comprehensive view

of gamma induced changes in some selected fungal strains with respect to metal tolerance.

Secondly, results of the study offer a distinct implication to the field of environmental

application with respect to management of heavy metal pollution. The higher efficiency of

the radiation exposed fungi to uptake and remove heavy metals from the media, in addition to

signals of gamma induced metabolic changes in functioning of enzymes which have positive

role in degradation of wastes, highlights the possibility of employing ionising radiation to

confer potential to the fungi to be used in bioremediation of heavy metals.

7. Conclusion and Major Highlights

Microbial metal bioremediation is an eco-friendly, cost-effective, high efficiency

strategy for combating heavy metal pollution. Advances made in understanding metal

microbe interaction has created a thrust for employing technology for developing more

efficient microbes having higher potential for metal accumulation and /or detoxification to

minimize bioavailability and biotoxicity of heavy metals. Thus bioremediation has been

taken up as a unique management procedure for controlling heavy metal pollution , one of the

serious impending global issue in the present era of industrial boom.

Observations and findings of the present study aimed at evaluating the potential of gamma

radiation on modulating heavy metal tolerance in fungi elucidate the following highlights

Prospective role of fungi in metal bioremediation can be further boosted by

employing gamma irradiation which imparts significantly higher metal tolerance in

fungi.

Exposure to gamma radiation has potential modulatory role on the fungal extracellular

enzymes that are responsible for lignocellulosic waste and plastic polymer

degradation.

Distinct difference in intensity of metal tolerance in case of different fungi against

different metals indicates strain-specific sensitivity and response of the fungi.

Regulation of metal tolerance in fungi exposed to gamma irradiation show a dose-

dependent characteristic with respect to absorbed dose of gamma.

Higher tolerance of the fungi towards heavy metals after being exposed to gamma

irradiation is evident from significant increase in their colony forming ability as

expressed by increased number of Colony Forming Unit (CFU) than that of its un-

irradiated counterparts under metal stressed condition. The effective dose showing

maximum CFU depends upon metal concentration.

Gamma irradiation also stimulates higher efficiency of uptake and removal of metals

in the exposed fungi. The most effective absorbed dose of gamma irradiation that

resulted in maximum metal tolerance reflecting maximum number of colonies (CFU),

is observed to be the same which imparted the fungi the maximum potential of uptake

and removal of metals from specific metal supplemented liquid media.

Involvement of specific functional groups like OH, -NH,-C=O are observed to be

most crucial for higher metal adsorption by the gamma irradiated fungi. Some other

Conclusion and Major Highlights

140 | P a g e

functional groups like CH, CH2, C-N, COO are also found to be involved in metal

adsorption.

Metal stress causes decrease in metabolic enzymes of the fungi CMCase and ά

amylase activity, and decrease in activity of these enzymes is directly proportional to

metal concentration.

Metal stress causes stimulation of antioxidative response of the fungi in terms of

activity of SOD and CAT and total GSH content.

SEM photographs revealed that metal stress causes structural deformities; Gamma has

antagonistic effect on hyphal deformities of tested fungal strains caused by metal

exposure

Gamma radiation induces significant enhancement of activity of metabolic enzymes

of the fungi grown under metal stress. Gamma radiation also has stimulatory effect on

antioxidant marker enzymes and proteins of irradiated fungi grown in metal

supplemented media. Role of gamma rays in modulating enzyme activity is metal

specific.

Significant enhancement of the growth regulatory metabolic enzymes CMCase and ά

amylase activity in the fungi induced by gamma irradiation might be directly

responsible for better survival of the fungi with higher tolerance towards Cd , Zn, Pb.

The mechanism of increase in metal tolerance might also be mediated via stimulation

of activities of the stress marker enzymes (SOD, CAT) and protein (GSH).

Involvement of metallothionein, the metal responsive protein, in response to metal

stress and adaptation of the gamma irradiated fungi conferring more metal tolerance is

noteworthy.

Upregulation of SOD, CAT and GSH in fungi were obtained when they treated to

higher doses of gamma (at 100Gy) whereas upregulation of MT obtained when the

fungi were treated with lower doses of gamma (e.g MT was higher at 60Gy than that

of 100Gy in A.niger).

Utilisation of low doses of gamma irradiation stands out to be an option for

developing microbes with higher metal tolerance to formulate better strategy for

combating heavy metal pollution using fungi in bioremediation.

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RESEARCH ARTICLE

Ionising Radiation in Modulating Zinc Tolerance Potentialof Aspergillus niger

Dipanwita Das • A. Chakraborty • S. C. Santra

Received: 25 January 2014 / Revised: 5 June 2014 / Accepted: 16 July 2014

� The National Academy of Sciences, India 2014

Abstract The present study describes the potential of

gamma rays in enhancing zinc tolerance of Aspergillus

niger. Gamma exposed group of A. niger showed 1.82

times more zinc tolerance than the unirradiated ones.

Gamma exposed A. niger grown in different zinc enriched

media, showed increase in growth (expressed in terms of

colony forming unit), higher efficiency in zinc uptake and

removal, when compared to that of their unirradiated

counterparts. The present investigation throws light

towards utilizing gamma radiation for formulating more

metal-tolerant fungi for bioremediation.

Keywords Gamma � Aspergillus niger �Colony forming unit (CFU) � Zinc uptake and removal �Bioremediation

Introduction

Environmental contamination through discharge of haz-

ardous effluents from various industries has become a

serious global issue. Heavy metals constitute a major

component of such unwanted industrial effluents. Pollution

caused by these heavy metals has created an alarming sit-

uation in recent years. Zinc (Zn) occupies an important

position in the series of heavy metal environmental pollu-

tants that gets its source from various factories and

industries like those involved in strip mines, coal burning

power plant and smelters.

Zinc is an essential divalent metal ion in all life forms

where it serves catalytic and structural roles and as such

this micronutrient element is required for various cellular

metabolic processes in all living organisms including

microbes. However if present in high concentration, it may

become toxic. Its elevated level has been widely reported

to be a soil contaminant [1, 2]. In aquatic systems too,

elevated level of Zn has been shown to pose severe threats

[1, 3]. Industrial and smelter complexes that emit metal

oxides, including those of Zn, have been reported to cause

a decrease in growth, development, metabolic activity and

an induction of oxidative damage in various plant species

growing in and around such industrial complexes [4].

Higher concentrations of Zn is known to cause reductions

in the number of soil bacteria, including actinomycetes,

fungi, and lichens. So, Zn is considered as an important

contender for remediation of soil or environment.

For proper abatement of heavy metal pollution, strate-

gies are being planned and various methods are being

implemented by Government and other public sectors.

However, those are mostly chemical and physical treatment

procedures and are not only costly but rather less effective.

As such, bioremediation is now attaining more focus and

considered as an effective tool for abatement of metal

pollution for its low cost and high efficacy. For controlling

metal pollution through bioremediation, different micro-

organisms like bacteria, fungi, algae and yeasts are usually

utilized as they have the potential to internally accumulate

different heavy metals. Among these microbes, fungi are

considered to have better prospective to be used in biore-

mediation as they can accumulate more metal due to their

high surface to volume ratio than other microbes. Potential

of filamentous fungi in bioremediation of heavy metals

D. Das (&) � S. C. SantraDepartment of Environmental Science, University of Kalyani,

Nadia, Kalyani 741235, WB, India

e-mail: [email protected]

A. Chakraborty

UGC-DAE, Consortium for Scientific Research, Kolkata

Centre,3/LB-8, Saltlake, Kolkata 700098, WB, India

123

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

DOI 10.1007/s40011-014-0397-5

Author's personal copy

containing industrial effluents and waste waters has been

increasingly reported from different parts of the world [5,

6]. Aspergillus niger is considered as one of the top metal

biosorbents consistently by a host of researchers [5, 7, 8].

Evidence for internal absorption and the mechanism used

by A. niger to detoxify environmental Cu and Zn has been

observed by earlier researchers [9].

Now a days genetic engineering and biotechnology

research is becoming more important for extracting more

usability and to gain better economic output in all fields. It

is being established that microbial strain improvement can

possibly be done through inducing mutation using physical

stress like exposure to ionizing radiation. Cordeiro et al.

[10] reported that exposure to gamma rays showed maxi-

mum potential to induce mutation in fungi (Metarhizium

anisopliae) than that of UV or other chemical mutagens. As

such the authors tried the possibility to utilize gamma

induced mutation in fungi for its prospect to be used in

bioremediation.

Earlier reports from authors’ lab showed that high doses

of gamma radiation cause dose-dependent inhibitory

effects in fungi [11] while some other group of workers

[12] reported low doses of gamma radiation to produce

stimulatory effects. Gamma irradiation caused increases in

total protein in Alternaria tenuissima, Botrytis cinerea,

Penicillium expansum and Stemphylium botryosum [12]. It

is suggested that protein might play an important part in

protection against the harmful effect of radiation. Signifi-

cant improvement in production of a- and b-galactosidasesenzymes by A. niger has been propounded by Awan et al.

[13] as the result of gamma-ray induced mutagenesis.

Considering several modulatory effects of gamma radiation

on fungal growth and other metabolic activities, the present

investigation has been designed to evaluate the potential, if

any, of low doses of gamma irradiation in inducing Zn

tolerance in A. niger. The main objective of the present

work is to make A. niger more tolerant to Zn so as to be

used as potential candidate for remediation of Zn con-

tamination in the environment.

Material and Methods

Isolation, Identification and Culture of Fungi

Aspergilus niger was isolated from soil of garbage dump

site of Dhapa, Kolkata by standard plating methods [14] in

Potato dextrose media. The species was purified by

streaking repeatedly on the same medium and the fungus

was identified microscopically, followed by standard fun-

gal identification keys [15, 16].Isolated strain was cultured

in potato dextrose broth with the following composition:

Peeled potato (400 gm), Dextrose (25 gm) and dissolved in

one liter of distilled water. The final pH was around seven.

The medium was autoclaved at 121 �C for 20 min. Cul-

tures were maintained in agar slants (potato dextrose broth

plus 20 g/l agar).

Assessment of Metal Tolerance

Determination of minimum inhibitory concentration (MIC)

of Zn against growth of A.niger was assessed in two steps,

initially after isolating the fungi from soil near garbage

dump site of Dhapa, Kolkata. Different concentrations of

Zn solutions were added separately to PDA medium after

being plated. The plates were inoculated with 100 ll ofspore suspension. Three replicates of each concentration

and controls without metal were used. The inoculated

plates were incubated at 29 �C for at least 7 days. The MIC

is defined as the lowest concentration of metal that inhibit

visible growth of the isolate. ZnS04, 7H20 (Merck) salt was

used for running all experiments.

Finally the metal tolerance with different gamma

absorbed doses was studied considering the number of

colonies (in terms of colony forming unit (CFU) with

respect to their unexposed counterparts.

For these two steps preparation of spore suspension and

gamma exposure to the spore suspensions were necessary,

which are briefly stated below.

Preparation of Spore Suspension

Eight days old culture (grown in potato dextrose broth

media) was agitated fully and then hyphal mat was

removed and the liquid was filtered through a Whatmann

filter paper No-1. Filtrate was centrifuged in 10,000 rpm

for 15 min, supernatant was discarded and the pellet was

suspended in Tween 20 (0.02 %v/v) and NaCl (0.85 %w/v)

solution [10].

Gamma Radiation Exposure for Spore Suspension

Prepared spore suspensions (5 9 105 spores/ml) were

equally divided in six parts, one part taken as control (i.e.

without irradiation) and rests for gamma irradiations.

Spore suspensions were taken in small centrifuge tubes

and then exposed to 20, 40, 60, 80 and 100 Gray of

absorbed doses of gamma radiation from Co60 as gamma

source (GC 1,200, BRIT). The dose range was selected on

the basis of previous references [12, 17]. These irradiated

suspensions were serially diluted (10-3) and being inocu-

lated in agar plates (containing different Zn concentration)

with 300 ll inoculation and allowed for incubation. After

14 days of incubation colony numbers were considered and

finally expressed in CFU. In each case six replicates were

done.

D. Das et al.

123


Estimation of Metal Uptake and Removal Potential

from Respective Liquid Media

After 14 days of incubation 8 mm disk [18] of A.niger

colony (randomly selected) was transferred from petri

plates to same Zn concentrated liquid medium with a

sterilized cork borer and allowed for growth in an orbital

shaker at 100 rpm for 14 days in incubation at 29 �C.Estimation of metal uptake and removal from respective

liquid media were carried out following the method of

Srivastava and Thakur [19] using Atomic Absorption

Spectrophotometer (FI-HG-AAS Perkin Elmer Analyst

400).

Results and Discussion

Results of the present study revealed that gamma irradia-

tion has the potential to modulate Zn tolerance of A. niger.

Data analyzed in terms of CFU of the selected fungal strain

showed that the group of A. niger exposed to gamma

irradiation (Dose range 20–100 Gy; from Co60 source) has

tolerated Zn nearly 1.82 times more than the unirradiated

ones (Fig. 1a).

Minimum inhibitory concentration of Zn for A.niger as

estimated after isolation from Dhapa soil is observed to be

around 1,200 ppm, while maximum tolerable concentration

is around 1,100 ppm. The soil from which the strain was

isolated contained 225 ppm Zn. In contrast to the tolerance

limit of A.niger against Zn assessed after isolation from the

waste dump, a significant increase in tolerance is noted in

case of the gamma irradiated groups of the same strain

(Fig. 2a–e) in terms of their CFU. Dose dependant rela-

tionship between gamma absorbed dose and CFU (except

250 ? 20 Gy and 500 ? 20 Gy; Fig. 2a and 2b respec-

tively) has been observed up to a certain dose range which

varied with the concentration of Zn. In case of 250 ppm of

Zn in the medium, maximum CFU is noted with an

absorbed dose of 100 Gy (Fig. 2a), in all other cases (i.e.

with increase in Zn conc. from 500 to 2000 ppm) maxi-

mum number of colonies were obtained when exposed to

an absorbed dose of 60 Gy of gamma irradiation (Fig. 2b–

e). Similar to the present result, Dadachova et al. [20]

reported higher CFUs with increased biomass of some

fungal strains isolated from areas having higher radiation

level. Increase in dry mycelial weight of fungi upon

exposure to gamma irradiation has been reported by a

group of researchers [21, 22]. Geweely and Nawar [12]

described significant increase in radial growth and dry

weight in A. tenuissima and S. botryosum induced by lower

doses of gamma irradiation. However, no reports are

available in the context of increase in Zn tolerance in

addition to CFU of A. niger upon exposure to gamma

irradiation. Morphological changes have been noticed in

unirradiated groups with increase in Zn concentration

above 300 ppm in the medium. Zn above 300 ppm resulted

in color change of the colony from blackish to brownish

yellow, further the colony shape also changed from round

to oval (Fig. 1b).

Zinc treatment caused much spreaded growth of the

colony with, more dense hyphal growth at the periphery.

Similar changes in morphology have been reported by

Lanfranco et al. [23] in ericoid mycorrhiza-forming asco-

mycete treated with milimolar concentrations of Zn. This

group observed apical swellings and increased branching in

the sub-apical parts as well as a significant increase in the

amount of chitin in metal-treated hyphae as a consequence

of Zn treatment. Recently it has been established that the

fungal color and morphology both are affected by high Zn

concentrations [24]. No significant change in morphology

is observed in A. niger after irradiation.

Atomic absorption spectroscopic (AAS) analysis for Zn

uptake and removal of Zn from the respective media by the

gamma irradiated groups of A. niger showed significant

(p B 0.05) increase in Zn uptake as compared to that of the

unirradiated groups (except 250 ? 20 Gy and 500 ? 20 Gy;

Fig. 3a and 3b).

With respect to the unirradiated group of A. niger, the

group exposed to 100 Gy absorbed dose of gamma rays,

could uptake 1.5 times more and could remove 10 %

(a)

(b)

Fig. 1 a Zn tolerance efficiency of gamma exposed and unexposed

A. niger; A: Before gamma irradiation and B: After gamma

irradiation, b change in color and morphology of A. niger exposed

to higher concentrations of Zn (at 325 ppm immediately after

315 ppm)

Modulation of Zinc Tolerance in Gamma Exposed Aspergillus niger

123


more Zn from the medium containing 250 ppm of Zn (F

of Fig. 3a). Though in terms of Zn tolerance as mea-

sured by CFU, no difference is observed between the

groups exposed to 80 Gy and 100 Gy of absorbed doses

(Fig. 2a), the latter showed better prospect in terms of

modulating Zn uptake efficiency simultaneously with the

Zn removal potential of the respective group of the

fungi(F of Fig. 3a).

For the other groups of the selected fungi (A.niger) exposed

to higher concentration of Zn (500–2,000 ppm) in the media,

exposure to 60 Gy of absorbed dose of gamma rays showed

maximum potential of modulating Zn uptake and removal

efficiency of A.niger. This complies with the present data of

Zn tolerance of the fungi in terms of its CFU as detailed above

(Fig. 2b–e) showing 60 Gy as the most effectively absorbed

dose of gamma. Precisely an exposure to 60 Gy of absorbed

dose of gamma stands out to be the most effective against Zn

(a)

(b)

(c)

(d)

(e)

Fig. 2 Colony Forming Unit of irradiated A. niger with different

doses of gamma rays (20–100 Gy) and grown in medium enriched

with different concentrations of Zn; 250 ppm Zn (a), 500 ppm Zn (b),1,000 ppm Zn (c), 1,500 ppm Zn (d), and 2,000 ppm Zn (e). [n = 6].

Significant increase (p\=0.05) in CFU was noted in gamma exposed

groups of A.niger with respect to their unirradiated counterparts when

grown in different Zn treated media (except 250 ppm ? 20 Gy and

500 ppm ? 20 Gy). 100 Gy exposed A.niger showed maximum CFU

when grown in 250 ppm of Zn in growth media while against

500–2,000 ppm of Zn in growth media the effective dose showing

maximum CFU was at 60 Gy

D. Das et al.

123


concentrations from 500 to 2,000 ppm for not only inducing

higher tolerance towards Zn but also in modulating the metal

uptake and removal potential of A.niger.

An interesting observation is noted between groups

having different concentration of Zn in medium but

exposed to the same dose i.e. 60 Gy of absorbed dose of

gamma irradiation. While A.niger treated with 500 and

1,000 ppm of Zn and prior exposed to 60 Gy of gamma

absorbed dose showed more Zn uptake (D of Fig. 3b and D

of 3c), by means of 20 % more removal (in both cases

stated earlier) as compared with their unirradiated

counterparts.

(a)

(b)

(c)

(d)

(e)

Fig. 3 Uptake of Zn by A. niger [plotted against Y1 axis] and

removal of Zn by A. niger [plotted against Y2 axis] exposed to

different absorbed doses of gamma irradiation (20–100 Gy) and

grown in media supplemented with different concentrations of Zn;

250 ppm Zn(a), 500 ppm Zn(b), 1,000 ppm Zn (c), 1,500 ppm Zn(d),and 2,000 ppm Zn (e). [n = 3]. Atomic absorption spectroscopic

analysis for Zn uptake and removal of Zn from the respective media

by the gamma irradiated groups of A. niger showed significant

(p\= 0.05) increase in Zn uptake and subsequent removal efficacies

as compared to that of the unirradiated groups (except 250 ? 20 Gy

and 500 ? 20 Gy; a and b respectively).The effective dose showing

maximum CFU (Fig. 2) at each concentration also coincided with the

effective dose reflecting maximum uptake and removal potential in

every cases


123


The maximum tolerable concentration of Zn for gamma

exposed A.niger is 2,000 ppm in contrast to only

1,100 ppm as maximum tolerable limit for unirradiated

A.niger. Hence the two groups exposed to 1,500 and

2,000 ppm of Zn in the media could not be compared with

their un-irradiated counterparts but both the groups showed

significant uptake of Zn upon exposure to 60 Gy of

absorbed dose of gamma (group C of both Fig. 3d and 3e).

Detailed analysis of Zn uptake and removal potential of

A.niger irradiated upon exposure to different doses of

gamma and treated with different concentration of Zn in

media showed the group exposed to 100 Gy of gamma,

treated with 250 ppm of Zn in the media has the maximum

uptake potential (F of Fig. 3a), while the same group has

the maximum removal potential (F of Fig. 3a).

Reports regarding potential of A. niger for removing

heavy metals including Zn has been cited by many of

earlier researchers. Akthar and Mohan [8] reported that

killed mycelia of A. niger could remove Cu and Zn from

contaminated lake waters. Price et al. [9] has shown

potential of A. niger to remove 91 % of Cu and 70 % of Zn

from a treated swine effluent. The potential of living A.

niger to remove Cd and Zn from aqueous solution, under

the optimal conditions, showed that the maximum uptake

capacities of Cd and Zn ions were 15.50 and 23.70 mg/g at

initial concentrations of 75 and 150 mg/L, respectively

[25]. In contrast to these reports the present results (C of

both Fig. 3d and 3e) revealed that irradiated A.niger

(60 Gy) gained its potential to remove 45 and 40 % of Zn

from 1,500 and 2,000 ppm of Zn treated media respec-

tively while without gamma irradiation it could not tolerate

the high Zn concentration. Recently Kumar et al. [26]

documented that acclimated A. niger isolated from soil and

sludge, has an efficacy for removing 58 % Zn.

Mutation of fungi for making them metal resistant is

usually done by chemical applications [27]. Some groups

have worked on the use of UV radiation to induce metal

resistance in bacteria [28, 29]. Earlier the authors have

reported that the same fungal strain (Aspergillus sp.) prior

exposed to gamma absorbed doses, showed better CFU,

metal uptake and removal potential than that of their un-

irradiated counterparts when grown in Cd enriched media

[30]. In contrast to these reports the present study shows

the use of gamma irradiation to induce Zn tolerance and

also to impart potential for removal of in A. niger, one Zn

in of the most common air-borne fungi.

Conclusion

The study was initiated to evaluate the potential of gamma

radiation in modulating Zn potential in A. niger. The

present results depicted that gamma exposed A. niger could

uptake more Zn as compared to the unirradiated counter-

parts and subsequent removal potential of the irradiated

group was also higher. This result thus reflects higher tol-

erance of the gamma irradiated group of A. niger towards

Zn. So it can be hypothesized that gamma irradiation has

the potential in inducing Zn tolerance of A. niger. As metal

uptake and subsequent removal by the microbes may be an

important bioremediation strategy, so it is plausible to

advocate the use of these gamma-induced metal tolerant

fungal strain in Zn stressed environment to mitigate the Zn

pollution to some extent. Utilisation of gamma irradiation

for developing microbes with higher metal tolerance might

possibly lead to formulate better strategy for combating

heavy metal pollution.

Acknowledgments Authors are thankful to UGC-DAE-consortium

for Scientific Research, Kolkata centre for giving gamma radiation

facility as well as financial support to carry on this research and

Department of Environmental Science, Kalyani University for using

AAS.

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11. Maity JP, Kar S, Banerjee S, Chakraborty A, Santra SC (2009)

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12. Geweely SIN, Nawar LS (2006) Sensitivity to gamma irradiation

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IOSR Journal Of Environmental Science, Toxicology And Food Technology (IOSR-JESTFT)

e-ISSN: 2319-2402,p- ISSN: 2319-2399. Volume 3, Issue 6 (May. - Jun. 2013), PP 51-55 www.Iosrjournals.Org

www.iosrjournals.org 51 | Page

Gamma irradiation in modulating cadmium bioremediation

potential of Aspergillus sp.

Dipanwita Das¹,², A. Chakraborty², S.Bhar1,M.Sudarshan

2, S.C.Santra¹

¹ Department of Environmental Science, University of Kalyani, Kalyani,Nadia 741235,W.B, India

²UGC-DAE Consortium for Scientific Research, Kolkata Centre,3/LB-8,Saltlake,Kolkata 700098,India

Abstract: The present paper describes the potential of gamma irradiation in modulating Cd tolerance of

Aspergillus sp. After being exposed to different gamma absorbed doses (20-100Gy), Aspergillus sp. grown in

different Cd supplemented media, showed increase in growth (expressed in terms of colony forming unit), higher

efficiency in Cd accumulation and removal, when compared to that of their unirradiated counterparts. Our

results throw light towards a new step of Cd bioremediation. Key words: Aspergillus sp, Bioremediation, Cadmium, Gamma irradiation

I. INTRODUCTION In recent years increase in pollution load from industrial effluents has become a serious global issue.

Heavy metals constitute a major component of such unwanted industrial effluents. Pollution caused by these

heavy metals has created an alarming situation as they persist for very long time in the abiotic components of

the ecosystem and are discharged into thereby creating severe hazards to the various strata of living world. In

most of the countries Cd, Hg, Pb are included among the „priority pollutants‟ because of their high toxicity.

For controlling such heavy metal pollution different strategies are being planned and implemented, but

most of these processes are not only costly but also less effective. As such abatement of heavy metal pollution

through bioremediation has gained a major focus and is being regarded as an cost-effective alternative solution.

Metal accumulation potential of different microorganisms like bacteria, fungi, algae has placed them to be considered as effective agents for bioremediation of heavy metals. Among these microbes, fungi have better

prospective to be used in bioremediation as they can accumulate more metal due to their high surface to volume

ratio than the other microbes. Potential of filamentous fungi in bioremediation of heavy metals containing

industrial effluents and waste waters has been reported from different parts of the world [1,2,3] .Among

different filamentous fungi Aspergillus niger has been consistently listed among the top metal biosorbents [1,4-

7]. Actually research in mycology has taken a different term due to the natural inherent potential of various

fungi in having productive values which are highly beneficial for human welfare. While some fungi produce

enzymes that are required for industrial purposes [8], while some are effective in waste detoxification [9-10]. To

have more such economic output, recently research and developmental activities in terms of genetic engineering

is being used for microbial strain improvement. Studies are also being carried out to improve the strains so as to

make them more efficient accumulators of metals. Among different strain improving agent physical mutagen like ionizing radiation treatment deserves mention. Geweely and Nawar [11] reported that low doses of gamma

causes stimulatory effects in fungi like enhancement in spore germination percentage, mycelial growth ; while

higher doses causes killing of those fungi. Aspergillus niger, Rhizopus microsporus and Penicillium

atrovenetum showed enhance production of industrially important enzyme lipase when these were exposed to

various low doses of gamma irradiation (20, 40, 60, 80, 100, 120, 140 and 160 Gy)[12].

The present study is designed to observe the potential of gamma in modulating Cd tolerance of

Aspergillus sp grown in Cd stress.

II. MATERIALS AND METHODS 2.1.Isolation , Identification and Culture of fungi :

Aspergillus sp. (black conidial heads) was isolated from soil of garbage dump site of Dhapa, Kolkata

by standard plating methods [13] in Potato dextrose media. The species was purified by streaking repeatedly on

the same medium and the fungus was identified by high resolution microscopy.

Fungal strain was cultured in potato dextrose broth with the following composition: Peeled potato (400gm),

Dextrose (25 gm) , dissolved in one liter of distilled water. Final pH was around 7. The medium was autoclaved

at 121ºC for 20 minutes. Cultures were maintained in agar slants (potato dextrose broth plus 20 g/l agar).

Gamma irradiation in modulating cadmium bioremediation potential of Aspergillus sp.


2.2 Assessment of metal (Cd) tolerance of isolated Apergillus sp.: Assessment of metal (Cd) tolerance was done in two steps. Initially, determination of Minimum

Inhibitory Concentration (MIC) of Aspergillus sp.(isolated from soil near garbage dump site of Dhapa, Kolkata )

against Cd was done. Different concentrations of metal solutions were added separately to PDA medium and

being inoculated with 100μl of spore suspension to determine MIC (minimum inhibitory concentration).

CdCl2,H20 (Merck) salt was used for running all experiments. Secondly, further evaluation of Cd tolerance after

being exposed to different gamma absorbed doses were studied considering the changes in number of colonies

(in terms of colony forming unit; CFU) between gamma exposure and non-exposure groups. For these,

preparation of spore suspension and gamma exposure to that spore suspensions were necessary , The steps are

briefly stated below.

2.2.1 Preparation of Spore suspension: 8days old culture (grown in potato dextrose broth media) was agitated fully and then hyphal mat was

removed and the liquid was filtered through a Whatmann filter paper No-1.Filtrate was centrifuged in10,000

rpm for 15mins, supernatant was discarded and the pellet was suspended in Tween 20 (0.02%v/v) and NaCl

(0.85%w/v) solution [14-15].

2.2.2 Gamma radiation Exposure to spore suspension : Spore suspensions (5 x 105 spores/ml) were taken in small centrifuge tubes and then exposed to 20, 40,

60, 80 and 100 Gray of gamma absorbed doses from a Co60 as gamma source ( GC 1200, BRIT). The dose

range was selected on the basis of previous references [11-12,16].Then these irradiated suspensions were being

serially diluted (10-.3 ) and 300μl of diluted suspensions were inoculated in agar plates containing different Cd

concentrations . After 3rd day to 14days of irradiation colony numbers were counted. In each case six replicates

were done.

2.3 Estimation of metal accumulation and removal potential (from respective liquid media) of

Apergillus sp.: After 14days of irradiation one 8 mm disk of Aspergillus sp. colony was transferred from petri plates to

same Cd concentrated liquid medium with a sterilized cork borer and allowed for growth in an orbital shaker at

100 rpm for 14days of incubation at 29°. Estimation of total Cd accumulation in fungal body and removal

percentage from respective culture media were carried out following the method of Srivastava and Thakur [17]

using Atomic Absorption Spectrophotometer (FI-HG-AAS Perkin Elmer Analyst 400).

III. RESULTS AND DISCUSSION

Our results depict that gamma irradiation induced increase in Cd tolerance in Aspergillus sp.(grown

under Cd stress) as reflected in increase in colony forming units (CFU) in gamma exposed groups in comparison

to that of the unirradiated counterparts. Exposure to gamma (20-100Gy absorbed dose) could also effectively

increase Cd accumulation and removal efficiencies of the fungi. Analysis of Cd tolerance in case of unirradiated

Aspergillus sp isolated from the waste dumping site showed that 90 ppm of Cd completely inhibited growth of the fungi in lab condition, i.e. MIC of Cd is 90ppm. Soil of the site contained 3.6ppm of Cd.

Fig-1 reflects change in number of colony forming units (CFU) of Aspergillus sp in response to

different absorbed doses of gamma when grown under different concentrations of Cd in the medium. The

pattern of change in CFU is different in case of different concentration of Cd supplemented in the medium.

While gamma exposed Aspergillus sp. grown in 70ppm Cd in the medium showed gradual increase in CFU upto

60Gy of gamma irradiation followed by decline in number of CFU (not below that of unirradiated but 70ppm

Cd stressed group). 85ppm of Cd in the medium manifest a sharp increase in CFU from 20-40Gy (40Gy being

maximum).In contrast 100ppm Cd supplementation which was not at all tolerated by Aspergillus sp in

unirradiated condition ( MIC-90ppm) showed maximum number of CFU in group irradiated at 20Gy of gamma

followed by gradual decline in CFU in the groups exposed to other absorbed doses of gamma(Fig-1C] (as

without gamma exposure Aspergillus sp could not tolerate 100ppm of Cd so it is not comparable with that of unirradiated counterparts).

While irradiation at 60Gy of absorbed dose showed a 3 fold increase in CFU in Aspergillus sp grown

in 70ppm Cd supplemented medium (Fig1A), a five-fold increase in CFU was observed in case of the group

irradiated at 40Gy absorbed dose of gamma and grown in 85ppm of Cd in the medium (Fig-1B), compared to

that of the unirradiated but Cd exposed counterparts .

Similar to our results Dadachova et al.,[18] reported higher CFUs with increased biomass of some

fungal strains isolated from areas having higher radiation level . Reports from some earlier researchers



demonstrate stimulation of spore germination and growth of fungi exposed to low doses of gamma rays [19-

20].

Atomic Absorption Spectrophotometric (AAS) analyses reveal increase in Cd concentration in the

fungal body together with decrease in Cd level in the growth media of the gamma irradiated groups of

Aspergillus sp. This reflects that gamma exposed Aspergillus sp showed better Cd accumulation and removal

potential than their unirradiated counterparts (Fig-2) .The most effective dose which reflected maximum CFU in

all the three sets of fungal groups grown with three different concentrations of Cd in the medium following prior irradiation was considered for studying further accumulation and removal efficacies. A 60Gy exposed

Aspergillus sp could accumulate 1.73 times more Cd and could remove 11% more cadmium from 70ppm Cd

supplemented media with respect to its unirradiated counterpart . From 85 ppm Cd supplimented media 40Gy

exposed Aspergillus sp showed 1.65 times more accumulation and 10.5 % more removal with respect to

unirradiated counterparts .Without being exposed to gamma irradiation Aspergillus sp could not tolererate

100ppm Cd but after being irradiated, a 20 Gy exposed Aspergillus sp could remove 45% Cd from the 100 ppm

Cd supplimented media . Removal of Cd by Aspergillus niger was reported by Kumar et al. [21], who showed

acclimated biomass of A.niger could remove 51.05% Cd from liquid Cd supplemented media. A 43% Cd

biosorption efficiency of A.niger from single metal (Cd) liquid system has also been described earlier [22].

However. our study entailed an increase in removal efficiency of gamma irradiated Aspergillus sp. While

unirradaited Aspergillus sp could show around 45% removal of Cd from the medium there was nearly 56% Cd removal by the gamma irradiated groups. Thus it is evident that exposure to gamma triggers the metal tolerance

of Aspergillus sp and simultaneously induces augmentation in the uptake or accumulation potential and removal

efficiency of the fungi. It may be postulated that gamma being a strong mutagenic agent, the concerned fungi

subjected to gamma irradiation might have experienced mutation induction so as to become more tolerant

towards Cd. No such reports are available so far where gamma has been used for enhancing metal tolerance in

fungi and as such this is an effective report where gamma irradiation is being employed to enhance tolerance of

fungi towards Cd. Mutation of fungi for making them metal resistant are usually done by chemical applications

[23-24]. Some groups have worked on use of UV radiation to induce metal resistance in bacteria [25-26].It is

already reported that exposure to gamma showed maximum potential to induce mutation in fungi (Metarhizium

anisopliae) than that of UV or other chemical mutagens [14].Gamma exposed fungal strain improvement for

different industrial as well as ecological purposes are also reported earliar [27-28]. Thus our study showed that

possibility of utilising gamma irradiation for increasing Cd removal efficiency of fungi.

IV. CONCLUSION This study is an effective report that might lead a new step forward towards Cd bioremediation.

Utilisation of low doses of gamma irradiation for developing microbes with higher metal tolerance might

possibly show the way us to formulate better approach for abatement of heavy metal pollution.

ACKNOWLEDGEMENT Authors are very much thankful to UGC-DAE Consortium for Scientific Research, ,Kolkata centre ,for

giving gamma radiation facility as well as financial support to carry on this study and Department of

Environmental Science , Kalyani University, for giving AAS facility.

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FIGURES

70ppm 70+20Gy 70+40Gy 70+60Gy 70+80Gy 70+100Gy

0

20

40

60

80

100

(A)

CF

U/m

l

85ppm 85+20Gy 85+40Gy 85+60Gy 85+80Gy 85+100Gy

0

10

20

30

40

50

60

70

80

(B)

CF

U/m

l

http://www.ncbi.nlm.nih.gov/pubmed?term=Tugay%20T%5BAuthor%5D&cauthor=true&cauthor_uid=17139845

http://www.ncbi.nlm.nih.gov/pubmed?term=Zhdanova%20NN%5BAuthor%5D&cauthor=true&cauthor_uid=17139845

http://www.ncbi.nlm.nih.gov/pubmed?term=Zheltonozhsky%20V%5BAuthor%5D&cauthor=true&cauthor_uid=17139845

http://www.ncbi.nlm.nih.gov/pubmed?term=Sadovnikov%20L%5BAuthor%5D&cauthor=true&cauthor_uid=17139845

http://www.ncbi.nlm.nih.gov/pubmed?term=Dighton%20J%5BAuthor%5D&cauthor=true&cauthor_uid=17139845

http://www.ncbi.nlm.nih.gov/pubmed/



Fig-1 Colony forming unit (CFU) of irradiated Aspergillus sp with different doses of gamma

irradiation (20-100Gy) and grown in medium supplimented with different concentrations of Cd;

70ppm Cd(A), 85ppm Cd(B) ,100ppm Cd (C)[n=6]

Fig-2 Bioaccumulation of Cd in Aspergillus sp [plotted against Y1 axis] and removal of Cd by

Aspergillus sp [plotted against Y2 axis] exposed to different absorbed doses of gamma

irradiation (20-100 Gy) and grown in media supplemented with different concentrations of Cd;

[n=3]

100ppm+20Gy 100ppm+40Gy 100ppm+60Gy 100ppm+80Gy 100ppm+100Gy

0

10

20

30

40

50

60

(C)

CF

U/m

l

70ppm 70ppm+60Gy 85ppm 85ppm+40Gy 100ppm+20Gy

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

Accumulation

Removal

Accu

mu

lati

on

(m

g/g

of

tissu

e)

[Y1]

40

42

44

46

48

50

52

54

56

58

Rem

oval(%

)

[Y2]

LIST OF PAPERS PRESENTED IN CONFERENCES

Presentated a poster on ‘Zinc bioremediation potential of Aspergillus niger and A.terreus’-

D.Das, A. Chakraborty, S.C.Santra ,in the Thematic Orientation Workshop on Trace Element

Analysis and Radiological Sciences,organized by Manipur University , Imphal &UGC-DAE-

CSR, Kolkata centre,12-14th March,2013.

Oral Presentation on ‘Potential of ionising radiation for modulating amylase and

cellulase activity of Aspergillus niger under lead and zinc stress’- D.Das, A. Chakraborty,

S.C.Santra. International conference on “Environment and human health”, 28-29th

November, 2012,National Environmental Science Academy (NESA), NewDelhi

Presented a poster and awarded for best presentation on ‘Modulation of cadmium

tolerance of Aspergillus terreus by Ionising radiation’- D.Das, A.Chakraborty, S.C.Santra.

3rd World Congress on Biotechnology. 13-15th September, 2012. Omics groups, Hyderabad,

India.

Presented a poster entitled ‘Ionising Radiation and Its Potential to Induce Mutation in

Fungi- An Overview’ - D.Das, A. Chakraborty, S.C.Santra, In 15th All India Congress of

Cytology and Genetics and Fogarty International Workshop,Organised by Dept. of Botany,

Magadh University,Bodhgaya, In collaboration with IICB, Kolkata and Fogarty Programme

at University of California,Berkeley,USA, November 21-23,2011

Presented a Poster entitled ‘Metallo-Resistance in fungi exposed to Ionising Radiation’-

D.Das, S.Majumdar,S.S.Ram,A. Chakraborty, S.C.Santra In one day Symposium ‘Trends in

Electron Microscopy in Frontier Science’ Organised by WBUT.

Chapter 1 ----------------------------------

INTRODUCTION

Chapter 2 ----------------------------------

REVIEW OF LITERATURE

Chapter 3 ----------------------------------

AIMS &

OBJECTIVES

Chapter 4 ----------------------------------

MATERIALS &

METHODS

Chapter 5 ----------------------------------

RESULTS

Chapter 6 ----------------------------------

GENERAL DISCUSSION

Chapter 7 ----------------------------------

CONCLUSION &

MAJOR HIGHLIGHTS

Chapter 8 ----------------------------------

BIBLIOGRAPHY

Plate I Ultrastructure (SEM) of A.terreus (1500X)

{ Green circles represent structural deformities in hyphae due to Cd exposure in B and in E it represents hyphal modification due to gamma exposure; Red circles represent less deformities in gamma exposed group grown Cd stress}

[ A : Control A.terreus B: 90ppm Cd treated A.terreus C: 100Gy exposed A.terreus grown in 90ppm Cd D :80Gy exposed A.terreus grown in 150ppm Cd E : 100Gy exposed A.terreus ]

C

A

B

E D

C

Plate II Ultrastructure (SEM) of A.terreus (1500X)

{ Green circles represent structural deformities in hyphae due to Zn exposure; Red circles represent less deformities in gamma exposed group grown Zn stress}

[A : Control A.terreus B: 8000ppm Zn treated A.terreus C: 80Gy exposed A.terreus grown in 8000ppm Zn]

A

B

C

Plate III Ultrastructure (SEM) of A.niger (1000-3000X)

{ Green circles represent structural deformities in hyphae due to Zn exposure; Red circles represent less deformities in gamma exposed group grown Zn stress}

[A :Control A.niger, B: 250ppm Zn treated A.niger, C: 100Gy exposed A.niger grown in 250ppm Zn, D : 500ppm Zn treated A.niger, E : 60Gy exposed A.niger grown in 500ppm Zn]

A

C

D E

B

Plate IV Ultrastructure (SEM) of A.niger (1000-1500X)

{ Green circles represent structural deformities in hyphae due to Pb exposure; Red circles represent less deformities in gamma exposed group grown Pb stress}

[ A : Contol A.niger ,B : 2000ppm Pb treated A.niger, C : 100Gy exposed A.niger grown in 2000ppm Pb]

C

A

B

Plate V Ultrastructure (SEM) of P.cyclopium (1500-2000X)

{ Green circles represent structural deformities in hyphae due to Cd & Pb exposure; Red circles represent less deformities in gamma exposed group grown Cd & Pb stress}

[ A : Control P.cyclopium, B : 300ppm Cd treated P.cyclopium, C : 80Gy exposed P.cyclopium grown in 300ppm Cd, D : 2000ppm Pb treated P.cyclopium, E : 80Gy exposed P.cyclopium grown in 2000ppm Pb]

A

C B

D E

Plate VI Ultrastructure (SEM) of A.terreus, A.niger and P.cyclopium exposed to gamma absorbed dose (1000-3000X)

[A : Control A.terreus, B : 100Gy exposed A.terreus, C : Control A.niger, D : 100Gy exposed A.niger, E : Control P.cyclopium, F : 80Gy exposed P.cyclopium]

F

A B

C D

E

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