Syed Agha Hassan - Higher Education...

KINETIC STUDY OF BIODEGRADATION OF TEXTILE

DYESTUFFS BY WHITE ROT FUNGI

By

Syed Agha Hassan

A thesis submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

IN

CHEMISTRY

DEPARTMENT OF CHEMISTRY,

UNIVERSITY OF SARGODHA,

SARGODHA, PAKISTAN

2009

Certificate

It is certified that the work contained in this thesis has been carried out under my

supervision and is approved for submission in fulfillment of the requirements for

the degree of Doctor of Philosophy in Chemistry.

………………………………….. Prof. Dr. Muhammad Ali

Dedicated

To

MY LATE MOTHER

Who was very anxious for my higher education, Though she is invisible to our bodily eyes,

her soul must definitely be contented, satisfied and happy

&

MY LOVING FATHER

Whose prayers enabled me to accomplish my goals of excellence

in academics

CONTENTS

Page # List of Tables ................................................................ i List of Figures .............................................................. v List of Abbreviations ................................................... ix Acknowledgement ....................................................... x Abstract ....................................................................... xi

CHAPTER 1 Introduction ....................................................... 1 CHAPTER 2 Review Of Literature ......................................... 18 Chapter 3 Experimental ...................................................... 64

3.1 Materials required .............................................................................64 3.1.1 Textile dyestuffs.....................................................................64 3.1.2 Chemicals ...............................................................................65 3.1.3 Facilities used ........................................................................66 3.1.4 Microbial strains and their maintenance ...............................66

3.2 Inoculum preparation .........................................................................67 3.3 Basal nutrient media ..........................................................................68

3.3.1 Medium I ................................................................................68 3.3.2 Medium II ..............................................................................68 3.3.3 Medium III .............................................................................69 3.3.4 Medium IV .............................................................................69

3.4 Decolorization protocol ....................................................................69 3.4.1 Dyestuff analysis/determination of percent decolorization .. 3.4.2 Screening of WRF cultures on different dyestuffs ................69

3.5 Screening on reactive dyes.................................................................69 3.5.1 Selection of basal nutrient medium ........................................70 3.5.2 Initial medium pH ..................................................................71 3.5.3 Effect of incubation temperature ..........................................71 3.5.4 Addition of carbon sources ....................................................71 3.5.5 Effect of varying concentration of glucose ............................72 3.5.6 Effect of additional nitrogen sources .....................................73 3.5.7 Effect of varying concentration of CSL .................................74 3.5.8 Effect of low molecular mass mediators ................................74 3.5.9 Effect of metal ions ................................................................75 3.5.10 Effect of different dye concentration .....................................75 3.5.11 Dye absorption on fungal mycelia .........................................76

3.6 Disperse dyes .....................................................................................76 3.6.1 Declorization protocol ..........................................................76 3.6.2 Dyestuff analysis/determination of percent decoloriz.76 ...... 3.6.3 Screening of WRF on disperse dyes ......................................77 3.6.4 Optimization process for decolorization of Foron Turquise SBLN

200 by Ganoderma lucidum ..................................................77

3.6.5 Effect of media composition 3.6.6 Effect of additional carbon sources ........................................78 3.6.7 Effect of varying concentrations of wheat bran .....................78 3.6.8 Effect of addition of different nitrogen sources .....................79 3.6.9 Effect of varying concentration of MGM 60% ......................79 3.6.10 Effect of low molecular mass mediators ................................80 3.6.11 Effect of different metal ions .................................................80 3.6.12 Effect of varying concentration of dye ..................................81 3.6.13 Study of dye adsorption on fungal mycelia ..........................81

3.7 Direct dyes .........................................................................................82 3.7.1 Declorization protocol ...........................................................82

3.8 Dyestuff analysis/determination of percent decolorization ..............82 3.8.1 Screening of WRF on direct dyes ..........................................83

3.9 Optimization of process for declorization of solar golden yellow R .............................................................................................83 3.9.1 Selection of basal salt medium ..............................................83 3.9.2 Addition of carbon source ......................................................84 3.9.3 Effect of varying concentration of wheat bran ......................84 3.9.4 Effect of nitrogen sources ......................................................84 3.9.5 Effect of varying concentrations of nitrogen source87 ..........85 3.9.6 Effect of low molecular mass mediators ................................85 3.9.7 Effect of metal ions ................................................................86 3.9.8 Effect of different dye concentration .....................................86 3.9.9 Effect of different dye adsorption

3.10 Vat dyes .............................................................................................87 3.10.1 Declorization protocol ..........................................................87 3.10.2 Dyestuff analysis/determination of percent 88

declorization ..........................................................................88 3.10.3 Development of method for Vat dyes solution

preparation ............................................................................88 3.10.4 Screening for selection of best fungus and dye

combination ...........................................................................88 3.11 Optimization for decolorization of Cibaron blue GFJ-MD

by Coriolus versicolor .......................................................................89 3.11.1 Media optimization ................................................................89 3.11.2 Addition of carbon source ......................................................89 3.11.3 Effect of varying concentrations of glycerol ........................90 3.11.4 Addition of nitrogen source ...................................................90 3.11.5 Effect of mediators ................................................................90 3.11.6 Effect of metal ions ................................................................90 3.11.7 Effect of different dye concentration .....................................91 3.11.8 Dye adsorption on fungal mycelia ........................................92 3.11.9 Enzyme activity assays .........................................................92

3.12 Statistical analysis .............................................................................93 CHAPTER 4 RESULTS AND DISCUSSION ............................... 94

4.1 Decolorization of reactive dyes .........................................................94

4.1.1 Screening of white rot fungi on reactive dyes .......................94 4.1.2 Optimization of ramzole brilliant yellow 3-GL

decolorization by Coriolus versicolor ..................................98 4.2 Decolorization of disperse dyes .........................................................136

4.2.1 Screening of white rot fungi on disperse dyes .......................137 4.2.2 Optimization of Foron turquoise SBLN-200

decolorization by G. lucidum .................................................166 4.3 Decolorization of direct dyes .............................................................167

4.3.1 Screening of white rot fungi on direct dyes ...........................169 4.3.2 Optimization of solar golden yellow R decolorization

by Pleurotus ostreatus ..........................................................169 4.4 Decolorization of vat dyes……………………………………….198

4.4.1 Screening of white rot fungi on vat dyes decolorization .........................................................................198

4.4.2 Process optimization ..............................................................198 Conclusion ........................................................ 225 Recommendations .............................................. 226 Literature Cited ................................................. 227 Appendices ........................................................ 262 s

i

List of Tables

Table # Title Page #3.1a Composition of sporulation medium for white rot fungi 67 3.1b Composition of trace elements solution 67 3.2 Composition of inocculum media for white rot fungi 68 3.1.2 Composition of growth media for decolorization of Remazole brilliant yellow

3-GLby C.versicolor with varying pH 71

3.1.3 Composition of growth media for decolorization of Remazole brilliant yellow 3-gl by C.versicolor with varying incubation temperatures

71

3.14 Composition of growth media with different carbon sources 72 3.1.6 Composition of growth media with varying concentrations of Glucose 72 3.1.7 Composition of growth media with different Nitrogen sources 73 3.1.7’ Composition of growth media with varying concentrations of CSL 73 3.1.8 Composition of growth media with different Mediators 74 3.1.9 Composition of growth media with different metal ions 75 3.1.10 Composition of growth media with varying concentrations of Remazole

brilliant yellow 3GL 75

3.1.11 Composition of adsorption flasks 76 3.2.1 Composition of growth media with different carbon sources for

decolorization of Foron Turquise SBLN200 by G.lucidum 78

3.2.2 Composition of growth media with varying concentrations of wheat bran 79 3.2.4 Composition of growth media with different nitrogen sources 79 3.2.5 Composition of growth media with varying concentrations of MGM 60%. 80 3.2.6 Composition of growth media with Low Molecular mass Mediators 80 3.2.7 Composition of growth media with different metal ions 81 3.2.8 Composition of growth media with varying concetrations of the dye 81 3.2.9 Composition of adsorption flasks 82 3.3.1 Composition of growth media with different carbon sources for

decolorization of Solar Golden Yellow R by P.ostreatus 84

3.3.2 Composition of growth media with different varying concentrations of wheat bran

84

3.3.3 Composition of growth media with different Nitrogen sources 85 3.3.4 Composition of growth media with varying concentrations of nitrogen source 85 3.3.5 Composition of growth media with different mediators 86 3.3.6 Composition of growth media with different metal ions. 86 3.3.7 Composition of growth media with different dye concentration 87 3.3.8 Composition of adsorption flasks 87 3.4.1 Composition of growth media with different carbon sources for Cibanon Blue

GFJ-MD 89

3.4.2 Composition of growth media with varying concentration of Glycerol 90 3.4.3 Composition of growth media with different nitrogen sources. 90 3.4.4 Composition of growth media with different Mediators 91 3.4.5 Composition of growth media with different Metal ions 91 3.4.6 Composition of growth media with different dye concentration 92 3.4.7 Composition of adsorption flasks 92

ii

Table # Title Page #4.1a Decolorization of reactive dyes by different white rot fungi in time course

study 95

4.1b Summary of decolorization of reactive dyes by white rot fungi 97 4.2 Decolorizatio of Remazole Brilliant Yellow 3-GLBY C.versicolor using

different basal nutrient media and its lignolytic enzyme profile 100

4.3 Decolorization of Remazole Brilliant Yellow 3-GL by C. versicolor and its lignolytic enzyme profile with varying pH levels

103

4.4 Decolorization of Remazole Brilliant Yellow 3-GL by C. versicolor and its lignolytic enzymes at different Temperatures under optimum conditions

107

4.5 Decolorization of Remazole Brilliant Yellow 3-GL by C. versicolor and its lignolytic enzyme profile at with different carbon sources under optimimum conditions

110

4.6 Decolorization of Remazole Brilliant Yellow 3-GL by C. versicolor and its lignolytic enzyme profile with varying concentrations of glucose under optimum conditions

113

4.7 Decolorization of Remazole Brilliant Yellow-3GL by C. versicolor and its lignolytic enzyme profile using different nitrogen sources under optimimum conditions

117

4.8 Decolorization of Remazole Brilliant Yellow-3GL by C.versicolor and its Lignolytic enzyme profile using varying concentrations of CSL under optimimum conditions

120

4.9 Decolorization of Remazole Brilliant Yellow 3-GL by C.versicolor using low molecular mass mediators and its lignolytic enzymes profile under optimimum conditions

123

4.10 Decolorization of Remazole Brillaint Yellow 3-GL by C.versicolor and its lignolytic enzyme profile using different metal ions under optimimum conditions

127

4.11 Extent of decolorization of varying concentrations of Remazole Brilliant Yellow 3-GL C. versicolor and its lignolytic enzyme profile under optimimum conditions

132

4.12 Adsorption of Remazole Brilliant Yellow 3-GL on fungal mycelia under optimimum conditions

135

4.13a Decolorization of Disperse dyestuffs by different white rot fungi 138 4.14 Decolorization of Foron Turquoise SBLN-200 by Ganoderma lucidum and

its lignolytic enzyme using different basal nutrient media 140

4.15 Decolorization of Foron Turquoise SBLN- 200 BY Ganoderma lucidum and its lignolytic enzyme profile using different carbon sources under optimum conditions

143

4.16 Decolorization of Foron Turquoise SBLN- 200 by Ganoderma lucidum and its lignolytic enzyme profile using varying concentrations of wheat bran under optimum conditions

146

4.17 Decolorization of Foron Turquise SBLN- 200 by Ganoderma lucidum and its lignolytic enzyme profile using different nitrogen sources under optimimum conditions

149

4.18 Decolorization of Foron Turquiose SBLN- 200 by Ganoderma lucidum and its lignolytic enzyme profile using varying concentrations of Maize Glutein Meal 60% under optimum conditions

152

4.19 Decolorization of Foron Turquise SBLN- 200 by Ganoderma lucidum and its 156

iii

Table # Title Page #Lignolytic enzyme profile using low molecular weight mediators under optimimum conditions.

4.20 Decolorization of Foron Turquoise SBLN- 200 by Ganoderma lucidum and its lignolytic enzyme profile using different metal ions under optimimum conditions

159

4.21 Decolorization of Foron Turquoise SBLN- 200 by Ganoderma lucidum and its lignolytic enzyme profile using varying concentrations of the dye under optimimum conditions

163

4.22 Adsorption of Foron Turquoise SBLN-200 on fungal mycelia at optimum pH and temperature

166

4.23a Decolorization of direct dyes by different white rot fungi in time course study 168 4.24 Decolorization of Solar Golden Yellow R by Pleurotus ostreatus and its

lignolytic enzyme profile using different basal nutrient media 170

4.25 Decolorization of Solar Golden Yellow R by Pleurotus ostreatus and its lignolytic enzymes profile using different carbon sources under optimum conditions

174

4.26 Decolorization of Solar Golden Yellow RSN by Pleurotus ostreatus and its lignolytic enzyme profile with varying concentrations of wheat bran optimum conditions

177

4.27 Decolorization of Solar Golden Yellow R by Pleurotus ostreatus and its lignolytic enzymes profile using different nitrogen sources optimum conditions

180

4.28 Decolorization of Solar Golden Yellow R by P.ostratus and lignolytic enzymes profile with varying Concentrations of Maize Glutein Meal 60% under optimum conditions

184

4.29 Decolorization of Solar Golden Yellow R by Pleurotus ostreatus and its lignolytic enzymes profile using low molecular mass mediators under optimum conditions

187

4.30 Decolorization of Solar Golden Yellow R by Pleurotus ostreatus and its lignolytic enzyme profile using different metal ions under optimum conditions

191

4.31 Decolorization of Solar Golden Yellow RSN by Pleurotus ostreatus and its lignolytic enzyme profile using varying concentrations of the dye under optimum conditions

194

4.32 Adsorption of Solar Golden Yellow R by fungal mycelia at optimum temperature and pH

196

4.33 Decolorization of Vat dyestuffs by different white rot fungi in time course study

199

4.34 Decolorization of Cibanon blue GFJ-MD by C. versicolor and its lignolytic enzyme profile using different basal media

202

4.35 Decolorization of Cibanon blue GFJ-MD by C.versicolor and its lignolytic enzyme profile carbon sources carbon sources under optimum conditions

205

4.36 Decolorization of Cibanon blue GFJ-MD by C. versicolor and its lignolytic enzyme profile using varying varying concentrations of glycerol under optimimum conditions

208

4.37 Decolorization of Cibanon blue GFJ-MD by C. versicolor and its lignolytic enzyme profile using different nitrogen sources under optimisation conditions

211

4.38 Decolorization of Cibanon blue GFJ-MD by C.versicolor and its lignolytic 216

iv

Table # Title Page #enzyme profile using using low molecular mass mediators under optimum conditions

4.39 Decolorization of Cibanon blue GFJ-MD by C. versicolor and its lignolytic enzyme profile under optimum conditions

219

4.40 Decolorization of Cibanon blue GFJ-MD by C.versicolor and its lignolytic enzyme profile under optimum conditions

222

4.41 Adsorption of Dye on fungal mycelia 225

v

List of Figures

Figure #

Title Page #

4.2a Effect of varying media compositions on decolorization of Remazole Brilliant Yellow 3-GL by C.versicolor and its lignolytic enzyme production in 7 days

101

4.2b Relationship between dye decolorization and enzyme activities with different media compositions

101

4.3a Effect of varying pH on decolorization of Remazole Brilliant Yellow 3-GL by C. versicolor and its lignolytic enzyme activities production in 6 days

104

4.3b Relationship between dye decolorization and enzyme activities with varying pH

104

4.4a Effect of varying temperatures on decolorization of Remazole Brilliant Yellow 3-GL by C.versicolor and lignolytic enzyme production in 5 days

108

4.4b Relationship between dye decolorization and enzyme activities with varying temperatures

108

4.5a Effect of different carbon sources on decolorization of Remazole Brilliant Yellow 3-GL by C. versicolorand its lignolytic enzyme production

111

4.5b Relationship between dye decolorization and enzyme activities with different carbon sources

111

4.6a Effect of varying concentrations of glucose on decolorization of Remazole Brilliant Yellow 3-GL by C.versicolor and its lignolytic enzyme production in 4 days

114

4.6b Relationship between dye decolorization and enzyme activities with varying concentrations of glucose

114

4.7a Effect of different nitrogen sources on decolorization of Remazole Brilliant Yellow 3-GL by C. versicolor and its lignolytic enzyme production in 3 days

118

4.7b Relationship between dye decolorization and enzyme activities with different nitrogen sources.

118

4.8a Effect of varying concentrations of CSL on decolorization of Remazole Brilliant Yellow 3-GL by C. versicolor and lignolytic enzyme production in 3 days

121

4.8b Relationship between dye decolorization and enzyme activities with varying concentrations of CSL

121

4.9a Effect of low molecular mass mediators on decolorization of Remazole Brilliant Yellow 3-GL by C. versicolor and lignolytic enzyme production

124

4.9b Relationship between dye decolorization and enzyme activities with low molecular mass mediators in 28 hours

124

4.10a Effect of different metal ions on decolorization of Remazole Brilliant Yellow 3- GL and lignolytic enzymes production by C.versicolor 24 hours

128

4.10b Relationship between dye decolorization and enzyme activities with different metal ions

128

4.11a Effect of varying concentrations of Remazole Brilliant yellow 3-GL decolorization by Coriolus versicolor and lignolyitic enzymes production in 24 hours

133

4.11b Relationship between dye decolorization and enzyme activities with varying concentrations of the dye

133

vi

Figure #

Title Page #

4.12a Effect of varying dye concentrations on adsorption by fungal mycelia 135 4.14a Effect of varying media compositions on decolorization of Foron Turquoise

SBLN-200 by Ganoderma lucidum and its lignolytic enzyme production in 7 days

141

4.14b Relationship between dye decolorization and enzyme activities with varying media compositions

141

4.15a Effect of different carbon sources on decolorization of Foron Turquoise SBLN-200 by Ganoderma lucidum and lignolyitic enzymes production

144


144

4.16a Effect of varying concentrations of wheat bran on decolorization of Foron Turquoise SBLN-200 by Ganoderma lucidum and lignolyitic enzymes production in 4 days.

147

4.16b Relationship between dye decolorization and enzyme activity with varying concentrations of wheat bran

147

4.17a Effect of different nitrogen sources on decolorization of Foron Turquoise SBLN-200 by Ganoderma lucidum and lignolyitic enzymes production in 3 days.

150


150

4.18a Effect of varying concentrations of MGM60% on decolorization of Foron Turquoise SBLN-200 by Ganoderma lucidum and lignolyitic enzymes production in 3 days

153

4.18b Relationship between dye decolorization and enzyme activities with varying concentrations of MGM 60%.

153

4.19a Effect of low molecular mass mediators on decolorization of Foron Turquoise SBLN-200 by Ganoderma lucidum and production of lignolyitic enzymes

157

4.19b Relationship between dye decolorization and enzyme activity with low molecular mass mediators

157

4.20a Effect of metal ions on decolorization of Foron Turquoise SBLN-200 by Ganoderma lucidum and lignolyitic enzymes production

160


160

4.21a Effect of varying concentrations of dye on decolorization of Foron Turquoise SBLN-200 by Ganoderma lucidum and its lignolyitic enzymes production in 24 hours

164

4.21b Relationship between decolorization and enzyme activity with varying concentrations of the dye in 24 hours

164

4.22a Dye adsorption on fungal mycelia of Foron Turquoise SBLN200 with varying concentrations of the dye

166

4.24a Effect of varying media compositions on decolorization of Solar Golden Yellow R by Pleurotus ostreatus and its ezyme production in 7 days

171

4.24b Relationship between dye decolorization and enzyme activities with varying media compositions

171

4.25a Effect of different carbon sources on decolorization of Solar Golden Yellow R by Pleurotus ostreatus and its lignolyitic enzymes aproduction in 5days

175

4.25b Relationship between dye decolorization and enzyme activities with different 175

vii

Figure #

Title Page #

carbon sources 4.26a Effect of varying concentrations of wheat bran on decolorization of Solar

Golden Yellow R by Pleurotus ostreatus and lignolytic enzymes production in 5 days

178

4.26b Relationship between dye decolorization and enzyme activities with varying concentrations of wheat bran

178

4.27a Effect of different nitrogen sources on decolorization of Solar Golden Yellow R by Pleurotus otreatus and its lignolyitic enzymes aproduction in 4 days

181


181

4.28a Effect of varying concentrations of MGM 60% on decolorization of Solar Golden Yellow R by Pleurotus ostreatus and lignolytic enzymes production in 3 days

185

4.28b Relationship between dye decolorization and enzyme activities with varying concentrations of MGM 60%

185

4.29a Effect of low molecular mass mediators on decolorization of Solar Golden Yellow R by Pleurotus otreatus and lignolytic enzymes production in 2 days

188

4.29b Relationship between dye decolorization and enzyme activities with low molecular mass mediators

188

4.30a Effect of different metal ions on decolorization of Soar Golden Yellow R byPleurotus otreatus and lignolytic enzymes production in 24 hours

192


192

4.31a Effect of varying concentrations of the dye on decolorization of Solar Golden Yellow R by P.ostreatus and lignolytic enzymes production in 24 hours

195

4.31b Relationship between decolorization and enzyme activities at varying concentrations of Solar Golden Yellow R

195

4.32a Dye adsorption on fungal mycelia of Solar Golden Yellow R with varying concentrations of the dye

196

4.34a Effect of varying medium compositions on decolorization of Cibanon Blue GFJ-MD by Coriolus versicolor and lignolytic enyme production in 6 days

203

4.34b Relationship between dye decolorization and enzyme activities with different media

203

4.35a Effect of different carbon sources on decolorization of Cibanon Blue GFJ-MD and its lignolytic enyme production by Coriolus versicolor in 3 days

206


206

4.36a Effect of different carbon sources on decolorization of Cibanon Blue GFJ-MD by Coriolus versicolor and its lignolytic enyme production in 3 days

209

4.36b Relationship between dye decolorization and enzyme activities with varying concentrations of glycerol

209

4.37a Effect of different nitrogen sources on decolorization of Cibanon Blue GFJ-MD by Coriolus versicolor and its lignolytic enyme production in 3 days

212


212

4.38a Effect of low molecular mass mediators on decolorization of Cibanon Blue GFJ-MD by Coriolus versicolor and its lignolytic enyme production in 2days

217

viii

Figure #

Title Page #

4.38b Relationship between dye decolorization and enzyme activities with different nitrogen sources

217

4.39a Effect of metal ions on decolorization of Cibanon Blue GFJ-MD by Coriolus versicolor and its lignolytic enyme production in 2 days

220

4.39b Relationship between decolorization and enzyme activities with different metal ions

220

4.40a Effect of varying concentrations of dye on the decolorization of Cibanon Blue GFJ-MD and lignolytic enzymes production by C.versicolor

23

4.40b Relationship between dye decolorization and enzyme activities with varying concentrations of the dye

223

4.41a Adsorption of Cibanon Blue GFJ-MD on C.versicolor mycelia 225

ix

List of Abbreviations

ABTS 2,2’-Azinobis (3-ethylbeuzothiazoline-6-sulfonic acid)

SSF Solid state fermentation

VA Veratyl alcohol

PVA Polyvinyl alcohol

LME Lignin modifying enzymes

RBBR Remazle Brilliant Red 3

RBY Remzole Brilliant Yellow

Lip Ligni Peroxidase

MnP Manganese peroxidase

Lac Laccase

IBL Industrial Biotechnology Lab.

EDTA Ethyl diamine Acetate

BPEs Bleach Plant Effluents

TEMED N-N-N0-N0-tetramethylenediammine

WRF White Rot Fungi

DMSO Dimethyl Sulfomide

OMWW Olive Mills Waste Water

MWW Molasses Waste Water

WAD Waste water from alcohol distillery

MBR Membrane Bioreactors

x

Acknowledgment

I feel exalted while expressing my sincere and heartfelt gratitude to my research supervisor, Prof. Dr. Muhammad Ali, Chairman Department of Chemistry, University of Sargodha, Sargodha, Pakistan. I would like to express my myriad and deepest appreciations to my second supervisor Prof. Dr. Muhammad Asghar, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad for his dedication and conscientious guidance. His generosity and encouragement will stay with me in life. This thesis is, in fact, outcome of his fraternal attitude, pragmatic approach and constructive criticism without which it would have been impossible to accomplish such a research project in due time. May God help him to achieve the apogee of fortunes. Special thanks are for Prof. Dr. Munir A. Sheikh, Chairman, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad who fully cooperated in providing all facilities involved in my research work. I would like to thank Dr. Haq Nawaz Bhatti, Associate Professor, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad who provided his laboratory in the beginning of my research. I have been specially fortunate to have the encouragement of Dr. Sajjad-ur-Rehman, Associate Professor in Department of Veternary Microbiology, University of Agriculture, Faisalabad. who never said ‘NO’’ for spore counting even in his soft tone. I cannot forget the assistance of Prof. Dr. Rukhsana Bajwa, Chairperson Department of MPPL, the University of Punjab, Lahore and Miss. Zahida PCSIR Laboratories, Lahore in fugal cultures identification. Dr. Muhammad Ilyas Tariq, Associate professor, Department of Chemistry, University of Sargodha, Sargodha guided me amicably during short span of interaction with him in absence of Prof. Dr. Muhammad Ali.

Heartiest thanks are for research fellows like Sheikh Kashif, Hafiz Nasir, Mu- hammad Rmzan, Muhammad Irshad, Saadia Javaid Naqvi, Shaheera Batool, Razia Naureen and Shaista Javaid for their timely help and cooperation in working with research group are unforgettable for me.

I owe immense feeling of love and respect to my real benefactor during my whole academic career, Syed Jawad Hussain, the eldest brother, whose inspiration and motivation lead me to this level. Prayers of my brothers Syed Alamdar Hussain and Syed Abrar Hussain proved an asset for me during this research work.

At the end, I will never for get my wife who always proved to be a source of encouragement for me during the Ph.D. study. In addition, My kids (Tauhaw, Tanzeel and Ali Hassan) smiles and warm welcome always removed my tiredness when I saw them at home on the eve of holidays.

SYED AGHA HASSAN

xi

Abstract

Potentials of five locally isolated white rot fungi Pleurotus ostreatsus IBL-02,

Phanerochaete chrysosporium IBL-03, Coriolus versicolor IBL-04, Ganoderma lucidum

IBL-05, and Schizophyllum commune IBL-06, for biodegradation of textile dyes

commonly used in Fasalabd texile dyeing process units, were investigated. Dyes

(Reactive, Disperse, Direct and Vat) dyes were provided free of cost from Clarient (pvt)

limited, Ciba (pvt) limited and Dyestar (pvt) limited. Reactive dyes included Drimarine

Blue K2RL, Cibacron blue FG3A, Drimarine Orange KGL, Drimarine Brilliant Red

K4BL, Prucion Blue PX5R, and Remazol Brilliant Yellow 3G. The dyes of disperse class

were Foron Turquize SBLN-200, Foron Blue RDGLN, Foron Red RDRBLS and Foron

Yellow SE4G. Direct dyes group comprised of Solar Golden Yellow R, Solar Brilliant

Red BA, Solar Orange RSN and Solar Blue A and Vat dyes included were Cibanon Blue

BFMD, Cibanon Golden Yellow RK-MD, Indanthrene Direct Black RBS.

White Rot fungi cultures were applied on reactive dyes and a combination of best

fungus and best decolorized dye was selected. Reactive Remazole Brilliant Yello3-GL

was maximally decolorized by coriolus versicolor in 7 days of incubation and it was

processed for further process optimization. Activities of LiP, MnP and laccase were124,

254 and 354 IU/mL were respectively. The C. versicolor strain decolorized 0.01% dye up

to 99.6% in 24 hours in Kirk’s medium, I; pH, 4, temperature, 30±20C with the addition

of 1% Glucose, 0.1% CSL, 1mM ABTS and CuSO4. Activities of MnP and laccase were

389 and 795 IU/Ml. Adsorption on fungal mycelia was negligible in 0.01 % dye solution

but it increased with higher dye the concentration.

Disperse dyes were also subjected to decolorization by WRF cultures and

maximum decolorization (91.87%) of Foron Turquoise SBLN-200 was caused by

Ganoderma lucidumon on 8th day. After optimization of varying media ompositions the

dye was effectively decolorized in medium IV which was nitrogen rich and its lignolytic

enzyme profile was 282, 115 and 116 IU/mL for LiP, MnP and laccase respectively on

7th day of incubation. Further process of optimization revealed that 99.20% dye was

degraded in solution receiving 0.01% dye concentration with production of LiP, MnP

nand laccase (636, 531and 382 IU/mL respectiely) under optimum conditions

xii

(MediumIV; pH 4.5, temperature, 35±20C, in the presence of 1.5%wheat bran, 0.1% of

MGM60%, 1mM Veratryl Alcohol and 1mM MnSO4).

Direct dyes like reactive and disperse were subjected to decolorization by WRF and

screening experiment proved that Pleurotus ostreatus was efficient in decolorizing Solar

Golden Yellow R up to 93.10% in 7 days. After optimization of media composition the

dye color removal reached at 98.86% in MediumIII and activities of Lip,MnP, AND

laccase were 61,163 and 140 IU/ML respectively. Uner Complete optimum conditions (

at pH 3.5 and temperature 30±20C in presene of 1.0% Wheat Bran, 0.05% of MGM

(60%), 1Mm of H2O2 and FeSO4 maximum dye decolorization was achieved in 24 hours

of incubation. Activities of major enzymes MnP and Lac were 687 and 376 IU/mL

respectively. Dye adsorption was negligible.

Vat dyes usually were not soluble in water and dye solution were prepared in

dilute NaOH. sluccinic Acid was used to adjust optimum pH. Of all WRF cultures

applied on vat dyes, C.versicolor showed maximum decolorization (92.32%) of Cibanon

Blue GFJ-MD in 6 days of incubation in Kirk’s basal medium. After media compositions

optimization the dye was degraded up to 97.12% in medium II and enzyme activies were

54, 35 and 185 IU/m. After the completion of process optimization medium II ; pH, 4 ;

temperature, 30±20C; Glucose, 1%; 1m M ABTS and CuSO4. 0.01% Cibanon Blue GFJ-

MD was degraded up to 99.12% in 24 hours. Major enzyme involved in dye degradation

was laccase and its was 595 IU/mL. Dye adsorption was 0.06% after 24 hours of

incubation which declined with the passage of time, due to degradation of dyes by the

enzymes. Addition of nitrogen showed inhibitory effect on fungal enzyme activities and

dye removal.

1

Chapter 1

Introduction

Environmental pollution is rapidly augmenting with increase in industrial sector

development (Gulnaz et al., 2006). Water is contaminated by an ample variety of

contaminants which is a great threat to the environment owing to their toxicity for human

beings. A large volume of water is being used by textile industry during wet processing

and consequently produces a bulk quantity of contaminated water having dyestuffs and

other products like emulsifiers, dispersing agents, leveling agents and heavy metals

(Noroozi et al., 2007). The entire dying process causes production of intense color that

needs to be removed readily before its discharge into the water bodies (Martina et al.,

2006; Vanhulle et al., 2008).

According to some recent researchers (Kapdan et al., 2000; Levin et al., 2005;

Romero et al., 2006) different kinds of pigments and dyes are widely applied in a number

of industrial fields like paper, textile, pharmaceutical, food and cosmetic industries due to

their easy use, low cost in synthesis, variety of color and high level of satability

compared with natural dyes. Dyestuffs classification can be based on their physical

properties or chemical nature, source, or properties related to their nature of application

for a particular process. Classification of dyestuffs can be based on origin, chemical and

or physical properties and characteristics related to the nature of their application for a

particular process. Just a division into native and synthetic dyes is insufficient, since now

a days the synthesis of many natural substances possible. Another reasonable method of

classification for textile dyes is based upon the method of applications of the modern

dyeing technologies involving Reactive, Direct, ink, Vat dyes or pigments, and Disperse

dyes. Thers is another wel recognized method of classification based on structure is the

color Index (C.I). This system has also useful as for investigating the biodegradibilty of

dyes concerned.

At present, there are more than 10, 000 dyes existing in the market (Noroozi et

al.,2007). The annual world wide production of dye is 7x105 tonnes ( D; Souza etal.,

Chapter 1 Introduction

2

2006; Noroozi et al., 2007). Due to inefficiencies of the industrial dyeing operations

highly coloured substances are discharged into water (Vidya and Datye, 1982; Boer et al.,

2004). According to an estimate 280000 tons of textile dyes arereleased in such industrial

effluents every year world wide. worldwide (Mass & Chaudhari, 2005). According to the

confirmed reports that direct release of thes effluents might cause formation of poisnous

aromatic amines under anerobic conditions in receiving media. Structural formulas of

some representative colorants are given as under.

(Wesenberg et al., 2003).

According to Poots et al., (1976) and Macky, (1979), textile dyes applied commercially

are found to stand firm vanishing on contact with light, water, sweat, chemicals like

oxidizing agents and microbial attack. As a result these make them more stable and less

acquiescent to biodegradation (Fewson, 1988; Seshadri et al., 1994; Fewson, 1988;


3

Seshadri et al., 1994). During processing, up to 10-15% of the dye stuffs are released into

the process water during dying process (Vaidya and Datye, 1982; Robinson et al., 2001;

Selvam et al., 2002; Wesenbrg et al., 2003). According to Chung and Stevens (1993)

concern over synthetic dyes potential fo causing pollution has primarily been provoked

owing to their possible carcinogenicity and toxicity. In addition to their visual effect and

their adverse impact in terms of chemical oxygen demand (COD), many synthetic dyes

arecarcinogenic, toxic, and mutagenic. A serious attention towards Stringent legislation

concerning environmental protection has compelled the researchers and owners to pay

serious attention and find out asuitable treatment technology on priority basis (O’Neill,

2000; Libra et al., 2003). De Angelis and Rodriguesm (1987) said becaus many textile

were rural based and municipal treatment costs were rising both industries and scientists

were compelled to search for inventive novel technologies and treatments directed chiefly

towards the decolorization of dyes effluents.

Churechly et al, (1994)and Yeslida et al. (1991) reported , the prevailing state of

the art for the treatment of waste water containing dyes are physiochemical techniques,

such as precipitation, adsorption, photo degradation, chemical oxidation, or membrane

filtration. Qther available technologies like electrochemical destruction (Jia et al., 1999),

sorption (Vandevivere et al., 1994) though effective as tertiary treatment but is not

economically feasible (Nillesh et al., 2006). Banat et al. (1996) and McMullan et al.

(2001) said that biological methods being inexpensive and easy to use have been the

main focal point of current studies on decolorization and dye degradation. Being a low

cost, ecofriendly and widely acceptable treatment technology Bioremediation of textile

effluents seems to be an effective process. This seems to be the most practicable for

effluent treatment or decolorization and the most practical in terms of man power

necessities and operational expenses to take on and develop.

Synthetic nature and complex structure of dyes make the degradation of dyes,

particularly azo dyes which consist of about 70% of all dyes used, is difficult (Swamy &

Ramsay 1999; Maas & Chaudhari, 2005). Synthetic dyestuffs are opposed to biological

decolorization and color removal by bioprocesses is also difficult (Shaul et al., 1991;

Willmott et al., 1998). Chung and Stevens (1993) and Chander et al.(2004) gane their


4

findings that dye decolorization generally occurred by adsorption of dyestuffs on

bacteria, instead of oxidation in aerobic systems. There are some some bacteria that are

capable to biodegrade dyestuffs by their azo reductase activity. However, the effluent at

the end of biotransformation of dyestuffs could be toxic. These troubles hinder pilot scale

application of bacterial decolorization.

Xueheng and Hardin (2007) have said tht at present there is no cost-effective and

dependable process to detoxify and decolorize textile waste water. Modern research

points towards the potential of fungal waste water treatment methods for textile

industries. Pointing (2001) studies reflectd that WRF were physiological group

comprising fungi that were able to biodegrade lignin and the name white rot fungi was

given from the manifestation of white color from the wood attacked by WRF, where

delignification gave bleached appearance. Many researchers (Fu & Virarhagavan, 2001;

Zilly et al., 2002; Libra et al., 2003; Christian et al., 2005) have said, that taxanomically,

WRF, with the exception of a few ascomycetes, belong to phylum basidiomycota that

decay wood powerfully (Eaton and Hale, 1993). The presence of extracellular non-

specific enzymes of WRF make them better dye-decolorizers as compared to prokaryotes.

This property is associated with extracellular non-specific lignin mineralizing enzymes

(LME) including Lignin peroxidase (LiP), Manganese peroxidase (MnP), and Laccase

(Lac), Versatile peroxidase (VP) and some H2O2 producing enzymes system capable of

degrading a wide range of xenobiotics including textile dyes.

These LMEs with the synergetic effect of low-molecular mass mediators enhance

the bioavailability of contaminants to WRF (Pointing, 2001). WRF possess a property to

tolerate a wide range of pH further enhances their pollutant degradation capabilities

(Verma and Madamwar, 2002). Several researchers (Kirk and Farrell, 1987; Cullen and

Kersten, 1992; Gold and Alic, 1993; Barr and Aust, 1994; Hatakka, 1994, 2001; Reddy,

1995; Pointing, 2001; Hofrichter, 2002; Martı´nez, 2002; Shah and Nerud, 2002;

Wesenberg et al., 2003) have investigated a variety of aspects of WRF and their enzyme

systems.


5

WRF have been effectively used for dye decolorization and most of the earlier

studies were based mainly on Phanerochaete chrysosporium and Trametes versicolor

(Toh et al., 2003). However, locally isolated WRF like Schizophyllum commune and

Ganoderma lucidum have been found best competitor of Phanerochaete chrysosporium.

Nagai et al. (2002), Boer et al. (2004) and Kamitsuji etal.(2005) have said, WRF

offer major advantages over bacteria. Their LMEs system including lignin peroxidase

(Lip). Mn dependent peroxidase (MnP), Mn independent laccase and versatile

peroxidases (VP) being non specific in naure can attack a wide range of complex

aromatic dyestuffs. Fungal decolorization by fungi involving an oxidative mechanism

produces less toxic by- products than original dye (Heinfling et al., 1997; Martin et al.,

2006). Knapp et al.(1997) elucidated that because the enzymes were extracellular, the

substrate diffusion restraint into the cell, usually observed in bacteria, was not

encountered. WRF do not necessitate preconditioning to meticulous pollutants, because

enzyme secretion depends on nitrogen or carbon, nutrient limitation, rather than presence

of pollutant. In addition to this this, extracellular enzyme system also enables white rot

fungi to tolerate high concerntrations of pollutants. Physiological differences among

WRF may justify differences in their pollutant degradation capabilities (Yesilada et al.,

2003; Chander et al., 2004). Verma & Mdamwar (2002) sreported in that the dyes differ

in structures also so the best way is to opt for coculturing of different fungi. For instance,

azo acid Reactive biodegradation is carried out effectively by P. chrysosporium and P.

ostreatus with the help of all three types of LMEs (Lip, MnP and Laccase).

There are more than a few reasons for the attractiveness of white rot fungi in the

cleansing of pollutant sites (Bumpus, 1993a). Verma and Madamwar (2002) have said

WRF have potential for mineralizing a large range of toxic xenobiotics because of non

specific nature of LMEs. They occur far and wide in the natural habitat and have the

capibilityl to oxidize substrates which have low solubility because the major enzymes

involved in the oxidation of pollutants are extracellular. The constitutive nature of the

LMEs urges the need (in most cases) for these organisms to be adapted to the chemical

going to be degraded. The favored substrates for the growth of white-rot fungi, such as

forest wastes and agroindustrial have low-cost. The extracellular enzyme system of most

WRF are active under nutrient-limited conditions, which are available in many soils. And


6

finally, since filamentous fungi grow by hyphal extension and get longer through the soil

with growth, they can approach pollutants in the soil in behavior that bacteria cannot

(Reddy, 1995).

These lygnolytic enzymes (LMEs) have also applications in the pulp and paper

industry, in biopulping and bioleaching and pollutants and degradation of xenobiotics

(Feijoo et al., 1995). Collins and Dobson (1996), say that biopulping has the potential to

be financially and ecologically viable alternate to existing costly and polluting chemical

methods and it also improves quality of the pulp. These enzymes have broad range of

substrate specifcity, particularly for phenolic compounds and potentially valuable to

clean up pollutants such as dyes, chlorinated phenol derivates, polycyclic aromatics and

making them potential biocatalysts in the field of environmental biotechnology.

Some researchers (Kirk and Farrell, 1987; Buswell, 1987; Boominathan and

Reddy, 1992; Cullen and Kersten, 1992; Reddy, 1993; Hatakka, 1994) have said that the

main LMEs include two types of peroxidases (LiP and MnP) and a phenoloxidase, Lac.

These LMEs are used in lignin and xenobiotic degradation by WRF with different low

molecular mass mediators. WRF synthesize different isoforms of these extracellular

oxidases. Previous research by Verma and Madamwar (2002) and Silva et al.( 2005)

showed that ligninolytic system of WRF is in a straight line involved in the degradation

of different dyes and xenobiotic compounds. Different levels of these lignin mineralizing

enzymes (LMEs) are produced by different species of white-rot fungi.

LiPs are extracellular glycosylated heme proteins that catalyze H2O2-dependent

one-electron oxidation of a wide range of of lignin type aromatic compounds, resulting in

the synthesis of aryl cation radicals which go through various non-enzymatic reactions

giving out a multiplicity of end-products (Kirk and Farrell, 1987; Higuchi, 1993) to the

lignin substructures. The H2O2 dependent LiP catalysed reaction is found to be neither

substrate specific or stereoselective (Kalyanaraman, 1995). Veratryl alcohol (VA) -- a

natural fungal secondary metabolites and 2-chloro-1,4-dimethoxybenzene play the role of

low molecular mass redox mediators to enhance the LiP catalyzed oxidation of a wide

range of recalcitrant substrates (Teunissen and Field, 1998; Christian et al., 2005) and


7

facilitating in the turnover of the enzyme, and shielding the enzyme from H2O2

inactivation (Buswell and Odier, 1987).

Lignin peroxidases (LiPs) are capable to mineralize a wide of recalcitrant

aromatic compounds (Shrivastava et al., 2005). The molecular mass of LiPs obtained

from different WRF strains varies from 37 to 50 kDa (Asgher et al. 2006; Hirai et al.

2005). Temperature and pH profiles of LiPs from different sources vary considerably

with maximum activities shown between pH 2–5 and 350C–550C, respectively (Yang et

al., 2004; Asgher et al., 2007). Optimum pH and temperature as well as thermostability

and catalytic properties can be enhanced by using immobilization of LiP (Asgher et al.,

2007).

Kinetic studies revealed that cationic radical of VA increases the catalytic cycle of

LiP possibly by converting LiP (II) and/or LiP(III) to LiP (Lan et al., 2006). Cationic

surfactant cetyltrimethylammonium bromide (CTAB) acts as an inhibitor for LiP; it

reduces the Vmax and causes Km of H2O2 to decrease spectacularly and that of VA to

increase probably by reshaping the enzyme conformation (Liu et al., 2003). Chang and

Bumpus (2001) in his finding said that Phanerochaete chrysosporium LiP was also

inhibited by ethylenediamine tetraacetic acid (EDTA) and N-N-N0-N0-

tetramethylenediamine (TEMED). However, in the presence of excess of ions like Zn2+

and other metal ions the TEMED and EDTA and mediated noncompetitive inhibition of

LiP catalyzed veratryl alcohol oxidation can be reversed. According to Yu et al. (2006)

and Lan et al. (2006) opinion it was observed that the oxidation caused by LiP depends

on the optimum molar ratio of H2O2 to pollutant. At lower concentrations, H2O2 is an

activator of Phanerochaete chrysosporium LiP while at higher concentrations it is an

inhibitor and swiftly deactivates the enzyme.

MnPs are extracellular glycoproteins (Nie et al., 1999) containing an iron

protoporphyrin IX (heme) prosthetic group (Glenn and Gold, 1985), with molecular mass

ranging between 32 and 62.5 kDa (Hofrichter, 2002; Ürek and Pazarlioglu, 2004b;

Baborova´ et al., 2006) and are produced in various isoforms in nitrogen and carbon


8

deficient media supplemented with Mn2+ and VA (2004; Hakala et al. 2005; Cheng et al.

2007).

Proof about expression of different isoforms are obvious from the noticeable

differences in characteristics and structure (pI 3.4 & 3.9; molecular masses 47 & 52 kDa)

and differential regulation of two different MnP encoding genes MnPA and MnPB in

Physiisporinus rivulosus T24li and their inconsistent properties (Hakala et al. 2005,

2006).

Sundramoorthy et al. ( 2005) research shows that Mn2+ plays the role of mediator

for MnP. Ma¨kela¨ et al. (2005) have said that high resolution crystal structure has

exposed that MnP catalyzes the peroxide dependent oxidation of Mn2+ to Mn3+ and Mn3+

is released from the enzyme in complex with oxalate or with other chelators along with

pyrophosphate, the various Mn2+ chelators that increases the activity are oxalate, L-

tartrate, malonate, Lmalate, oxaloacetate and methylmalonate. Chelated Mn3+ acts as a

highly reactive low molecular weight, diffusible redox-mediator (Warrishi et al., 1988

.This complex free Mn diffuses out from the enzyme’s active site (Mester and Field

1998; Kamitsuji etal., 2005) and oxidizes secondary substrates, lignin model compounds

such as phenolic compounds, (Tuor et al. 1992; Wariishi et al. 1989), high molecular

weight chlorolignin (Lackner et al. 1991), chlorophenols (Grabski et al., 1998),

nitroaminotoluenes (Schiebner et al., 1997; Van Aken et al., 1999a) and textile dyes

(Heinfling et al. 1998a; Compos et al., 2001a; Boer et al. 2004)

Urek and Pazarlioglu (2005) said that MnP could also utilize both H2O2 and a

range of organic hydroperoxides, such as, m-chloroperoxybenzoic acid, peracetic acid,

and p-nitroperoxybenzoic acid, as its source of oxidizing equivalents. Hofrichter et al.

(2002) and Urek and Pazarlioglu (2005) research showed that MnP activity was inhibited

by NaN3, b-mercaptoethanol, ascorbic acid and dithreitol, whereas the activity could be

enhanced in the presence of cooxidants such as unsaturated fatty acids, glutathione and

Tween 80. The stability of MnP could be enhanced by immobilization with sodium

alginate, chitosan or gelatin as carriers and glutaraldehyde as crosslinking driving force

(Cheng et al., 2007).


9

Laccases (EC 1.10.3.2) are N-glycosylated extracellular (Wells et al. 2006) are

blue multicopper oxidoreductases that are ubiquitious enzymes synthesized by

basidiomycetes (Brandi et al., 2006). These were first reported in detail by FYoshida

(1983).These are widespread and found in several , bacteria, plants and fungi (Gianfreda

et al., 1999). Laccases have a wide range of industrial applications, including wine

clarification, dye decolorization, drug analysis, bioremediation, ethanol production and

denim washing and paper pulp bleaching industry (Mayer and Staples, 2002),

Nanobiotechnology, soilbioremediation, synthetic chemistry and Cosmetics, Biosensors

development (Couto et al., 2006). Laccases are capable to oxidize many recalcitrant

substances, such as aromatic dyes (Rodríguez et al., 1999; Abadulla et al. 2000; Balan

and Monteiro, 2001; Nagai et al. 2002). Laccase Mediated System (LMS) catalyses the

oxidation of ortho and paradiphenols, polyphenols, aminophenols polyamines, aryl

diamines and lignins as well as some inorganic ions together twith the reduction of

molecular oxygen to water. (Yaropolov et al. 1994; Solomon et al. 1996, Couto et al.,

2006). Wesenberg etal., (2003) proposed catalytic cycle is given as :

Fig. 2. The catalytic cycle of laccases.

Laccases can be grouped into low or high redox potential oxidoreductases (Xu et al.

1996) having molecular masses varying from 58 to 90 kDa (Murugesan et al. 2006;

Salony et al. 2006; Zouari-Mechichi et al. 2006; Quaratino et al. 2007). Purified laccases

from Cyathus bulleri and Panus tigrinus CBS577.79 have shown 16% and 6.9% (high


10

mannose type) glycation, respectively (Salony et al. 2006; Quaratino et al. 2007). The

temperature and pH optima of laccases from different WRF vary from 2 to 10 and 40 to

65°C, respectively (Lu et al. 2005; Ullrich et al. 2005; Murugesan et al. 2006; Zouari-

Mechichi et al. 2006; D’Souza et al. 2006; Quaratino et al. 2007). Two laccase isozymes

LacI and LasI have been investigated in Physisporinus rivulosus T241i (pI 3.1 &

3.3;MW66 & 68 kDa), Cerrena unicolor 137 (pI 3.6 & 3.7; MW 64 & 57 kDa; optimum

pH 7 & 10; optimum temperature around 60°C), Trametes trogii (pI 4.3 & 4.5; MW

around 62 kDa; optimum pH 2.0 & 2.5; optimum temperature around 500C), and Panus

tigrinus (identical optimum pH 7.0 and 60–650C temperature) (Cadimaliev et al. 2005;

Lorenzo et al. 2006; Michniewicz et al. 2006; Ma¨kela¨ et al. 2006; Zouari-Mechichi et

al. 2006). It has been reported that there are two different laccases encoding genes Prlac1

and Prlac 2 in Phlebia radiata 79 which are differentially expressed and regulated

(Makela et al., 2006). According to some researchers (Geng et al., 2004; Cho et al., 2006;

Minussi et al., 2007; Lu et al. 2007; Quaratino et al., 2007) the Prlac 2 gene has a very

high (66%) sequence resemblance with the laccase of Trametes versicolor.There are

different types of fungal metabolites such as3-hydroxyanthranilate, N-hydroxyacetanilide

(NHA), N-(4-cyanophenyl)acetohydroxamic acid (NCPA), ABTS (2,20-azino-bis(3-

ethylbenzthiazoline-6-sulphonate), syringaldehyde, 2,6-dimethoxyphenol (DMP), 1-

hydroxybenzotriazole (HBT), violuric acid, 2,2,6,6-tetramethylpipperidin-N-oxide

radical, acetovanillone (AV), acetohydroxamic acid and acetosyringone which perform

the role of mediators for laccase synthesis and enhance pollutant degradation. However,

for denim washing Trametes versicolor laccase was found more effective without a

mediator as compared to a commercial laccase (Pazarlioglu et al. 2005). According to

Ullrich et al.(2005) and Murugesan et al. (2006) findings it was noted that addition of

metal ions affected fungal physiology and influenced catalytic role of laccases. Some

metals are prerequisite but to a variable extent depending on nature of strain and

substrates going to be degraded. Cu, Mn Cd, Mo and Ni addition at lower concentration

make active the WRF laccases, in a good number cases, whereas Zn, Ag, Hg, Pb, sodium

azide, H2O2 and sodium chloride leave toxic effect and hamper its activity. However,

laccase from Trametes trogii was not affected by Al, Cd, Li and Ca (Zouari-Mechichi et

al., 2006), whereas the activity of Pleurotus ostreatus laccase is increased by Cu and Zn


11

(Baldrian et al. 2005). Increase in activity and stability as well as dye degradation can be

obtained by immobilization of laccase on alginate-chitosan microcapsules which is useful

for its repeated use (Lu et al., 2007).

Palmieri et al. (2003) said that laccases were syntheis in multiple isoforms depending on

the environmental conditions and fungal species. This biochemical variety of laccase was

due to multiplicity of laccase gene.

Versatile peroxidase (VP) is a heme containing dual nature enzyme because it has

hybrid structure between MnPs and LiPs, since they can oxidize not only Mn2+ but can

also oxidize veratryl alcohol, phenolic and nonphenolic and high molecular weight

aromatic compounds including dyes in manganese-independent reactions (Kamitsuji et

al., 2004; Pogni et al., 2005; Shrivastava et al., 2005). VPs have been detected in

Pleurotus and Bjerkandera (Mester and Field, 1998; Kamitsuji et al., 2005 a, b; Pogni et

al., 2005; Honda et al., 2006; Rodakiewicz-Nowak et al., 2006). The decreased sequence

of MnP2 isolated from P. ostreatus and VP (Vaxpdgvnta) from a novel strain of

Bjerkandera sp. (MW 45 kDa) is more than 95% indistinguishable to that of a typical VP

(VP-PS1) from P. eryngii (Moreira et al., 2006). A homologous enzyme, MnP-GY, from

recombinant strains P. ostreatus ATCC66376 (Kamitsuji et al., 2004) and MnP2 from P.

ostreatus TM2-10 (Tsukihara et al.2006) could directly oxidize VA and high molecular

weight compounds, such as Poly R-478 and RNaseA, although much less capably than it

oxidized Mn2+ in absence of mediators indicating that enzyme is has properties of both

LiP and MnP. Pogni et al. (2007) and Tinoco et al. (2007) have said that the structure of

VP on molecular level explained that Bjerkandera adusta and P.eryngii included an

exposed neutral tryptophan 172 (Trp 172) radical close to the heme prosthetic group,

which was accountable for aromatic substrate oxidation and had Mn2+ oxidation site.

Crystallographic and Biodegradation side chain orientation studies` have cclearly shown

that shown that Trp164 is the site used in wide-range electron transfer for aromatic

substrate oxidation (Pogni et al. 2006). The crystal structures (solved up to 1.3 A°) of

Pleurotus eryngii VP, before and after exposure to Mn2+ showed a variable orientation of

the Glu36 and Glu40 side chains that, together with Asp175, helps in Mn2+ coordination

(Ruiz-Duen˜as et al., 2007). Verdı´n et al., (2006) said, Bjerkandera adusta VP needs

Ca2+ for long range electron transfer during oxidation of pollutants. Poisoning of VP by


12

Ca2+ depletion at optimum pH 4.5 because of the differences in the Fe3+ spin states

suggested that Ca2+ deficient VP is able to form the active intermediate compound I but it

is unable to work for long range electron transfer. Effect of medium polarity by the

addition of organic solvents like dimethylsulfoxide (DMSO), acetonitrile, ethanol, and n-

propanol leads to restriction of Bjerkandera fumosa VP (Rodakiewicz-Nowak et al.,

2006). Oxidizing mediators like veratryl alcohol (VA), cetosyringone and 2,2,6,6-

tetramethyl-1- piperidinyloxy (TEMPO) increase the VP catalyzed decolorization of a

few textile dyes by Bjerkandera adusta (Tinoco et al. 2007).

BIOREMEDIATION OF INDUSTRIAL POLLUTANTS BY WRF

Textil dyes dyes and dye based effluents are posing severe danger to the

environment on global level, in general, and Pakistan, in particular.Several different

types of industries like textile, cosmetics, paper, plastic, pharmaceutical and food

industries make an widespread use of a variety of dyes and pigments (Levin et al., 2005).

Absolute degradation of dyestuffs can be achieved by biological or chemical oxidation

methods (Kapdan and Kargi, 2002; Supaka et al., 2004). In the natural environment, the

dyes can be changed or degraded by a number of of microorganisms, including fungi,

aerobic / anaerobic bacteria and mixed microbial consortia (Chung and Stevens, 1993;

Banat et al., 1996; Asgher et al., 2007). Bioremediation because of its low cost, eco-

friendly impact, and publicly favoured treatment technology, for textile dyes effluents

using WRF seems to be an impressive choice (Banat et al., 1996; McMullan et al. 2001)

Attention of researchers diverted towards Biodegradation due to serious concerns

over possible carcinogenicity and toxicity of synthetic dyes (Maas and Chaudhari 2005;

Salony et al., 2006; Revankar and Lele, 2007). WRF are found better dye-degraders than

prokaryotes due to their extracellular non-specific LME system capable of degrading a

wide range of dyes (Christian et al., 2005). In previous research work by Toh et al. (

2003) showed, “dye decolorization studies were carried out mainly on Phanerochaete

chrysosporium and Trametes versicolor (Toh et al., 2003). However, other WRF

including Pleurotus sajor-caju, Phellinus gilvus, Pycnoporus sanguineus (Balan and

Monteiro, 2001), Daedalea flavida, Irpex flavus, Dichomitus squalens, Polyporus


13

sanguineus (Chander et al., 2004; Eichlerova ´ et al. 2006; Chander and Arora, 2007),

Funalia trogii ATCC200800 (Ozsoy et al., 2005), Ischnoderma resinosum (Eichlerova´

et al. 2006) and Ganoderma sp. WR-1 (Revankar and Lele, 2007) have been

demonstrated to have higher dye decolorization rates than Trametes versicolor and P.

chrysosporium. Mechanisms of biodegradation and profiles of biodegradation products

of different dyes have also been explained (Zhao et al. 2005, 2006; Gavril and Hodson,

2007; Zhao and Hardin, 2007). Kinetic studies of purified enzymes as well as

immobilized oneshave been reported in the literature. Decolorization of Direct Blue 15

by Phanerochaete chrysosporium immobilized on ZrOCl2-activated pumice follows the

first-order kinetics with respect to initial dye concentration and MnP plays the principle

role in decolorization with insignificant adsorption on mycelia (Pazarlioglu et al. 2005).

Asgher et al, (2008) rerported decolorization of Solar golden yellow R by Schyzophyllum

commune indicating the involvement of MnP as a major enzyme while LiP and Laccase

activities profile was very low.

Lu et al., (2005 & 2007), research has mentioned earlier that LME’s work in the

presences of some low molecular mass mediators. Low molecular mass redox mediators

like ABTS are necessary for laccase-catalyzed decolorization of most of the dyes..

However, some WRF like P. pulmonarius and L. edodes SR-1 produced only

extracellular laccase to decolorize dyes of different spectra in absence of mediators

(Nagai et al., 2002). MnP mediated decolorization of azo dyes, Direct Green 6, Direct

Blue 15 and Congo red by Phanerochaete chrysosporium can be enhanced by the

addition of Tween-80 (U¨ rek and Pazarlioglu 2005) and copper (Tychanowicz et al.,

2006).Whereas, LiP produced by Trametes versicolor can decolorize Remazol Brilliant

Blue R (RBBR) in the presence as well as in the absence of Veratryl Alcohol (Christian

et al., 2005). The decolorization ability of WRF can be considerably increased by

cautiously optimizing the operational conditions such as initial dye concentration, age of

fungus, nutrient content of the media and carbon and nitrogen sources (Ozsoy et al.,

2005; Nilsson et al., 2006; Sanghi et al., 2006). Asgher et al.( 2008) have said that

Carbon source like glucose enhanced decolorization to a remarkable extent. Solar golden

yellow R IBL-06 decolorization was faster after the addition of glucose as carbon source,

whereas additional nitrogen sources inhibited MnP fsynthesis and dye decolorization.


14

D’Souza et al.(2006) says that pulp and paper industry is one of the major

sources of pollutants. Bleach plant effluents (BPEs) having toxic chloroorganics and

colored compounds are difficult to decolorize and are source of environmental problems.

LMEs from WRF have the potential to degrade highly toxic phenolic compounds from

BPEs (Minussi et al., 2007). BPE-decolorizing and dechlorination/ detoxification

activities of Trametes versicolor, Phanerochaete chrysosporium, Fomes lividus and

Thelephora sp. and the enzyme systems have been illustrated (Selvam et al., 2002, 2006).

It has been further reported that only laccase play the major role in decolorization and

detoxification of effluents from pulp and paper industry by WRF (D’Souza et al., 2006;

Font et al., 2006). However, some studies point out that both laccase and MnP are active

enzymes involved in BPE decolorization by Coriolus versicolor. This decolorization

process is directly proportional to the initial color intensities and glucose addition which

stimulates color removal (Driessel and Dhristov, 2001). Font et al.,( 2006) have said that

immobilization of Trametes versicolor in polyurethane foam cubes and nylon made

possible only 36% color removal, 54% reduction in aromatic compounds and up to 5.7

fold decline in toxicity as compared to Trametes versicolor pellets causing 84.8% color

removal and 70.2% aromatic compounds reduction with laccase as the major enzyme. It

suggested that immobilization of the fungus caused a significant reduction in laccase

activity. Acetohydroxamic acid is very efficient mediator for Trametes versicolor laccase

catalyzed bioremediation of BPEs reducing 70% and 73% of the total phenol and organic

carbon, respectively as compared to 23% phenol diminution in the presence of HBT or in

the absence of any mediator (Minussi et al., 2007).

Thereare other two types of industrialwastes namely Olive mill wastewater

(OMWW) and dry residues of olives (ADOR) containing acidic pH, high organic load,

and contain recalcitrant and toxic substances such as lipidic and phenolic compounds that

is a source of serious concern of the olive industry (Ahmadi et al., 2006; Aranda et al.,

2006). This (OMWW) as high chemical oxygen demand (COD) of up to 200 g l-1 and

organic fraction includes sugars, polyphenols, tannins, polyalcohols, lipids and pectins.

Traditional biological wastewater treatments are ineffective for OMWW treatment since


15

phenolics possess antimicrobial activity (Ahmadi et al., 2005). At present, most of the

studies have been focused on bioremediation as a means of reducing the polluting effect

of OMWW and its bio-transformation into valuable products (Ramos-Cormenzana et al.,

1995). There are reports (Sanjust et al. 1991; Zervakis et al., 1996; Kalmis and Sargin,

2004) on the cultivation of Pleurotus spp., Pleurotus sajor-caju and P. cornucopiaevar.

citrinopileatus using wheat straw moistened with mixtures containing 25% and 50%

OMWW. from OMWWrelative to the total organic load consumed indicates the highest

capability for free as well as loofahmmobilized Phanerochaete chrysosporium (Garcia et

al., 2004; Ahmadi et al., 2005). Dhouib et a., (2006) findings showed, “dexification of

OMWW can be possible through removal of organic matter, decreasing COD/BOD ratio

by Phanerochaete chrysosporiumor, Trametes versicolor in the presence of complex

microbial consortia in combined aerobic/anaerobic systems for its reuse and biogas

productionon industrial scale.” Pycnoporus coccineus, Pleurotus sajor-caju, Coriolopsis

polyzona and Lentinus tigrinus, are also very active in colorand COD removal of

OMWW at 50 and 75 g l-1COD (Jaouani et al., 2003). At 100 g l-1 COD only

Pycnoporus coccineus and Pleurotus sajor-caju are effective, Panus trigrinus

CBS577.79 gives better COD reduction (60.9%), dephenolization (97.2%) and

decolorization (75%) of OMWW in bubbled column bioreactor (BCB) as compared to

stirred tank reactors (STR) (D0Annibale et al. 2006).

Tsioulpas et al.(2002) have said that laccase have a detrimental effect on OMWW

decolorization when incorporated at high concentration level. There is an intimate

relatioinship between laccase level and phenol contents., high laccase activity produced

by Pleurotus spp. in the growth medium reduce in phenol content. Pycnoporus

cinnabarinus and Coriolopsis rigida also produce high laccase activities responsible for

73% phenol reduction of ADOR in 15 days (Aranda et al., 2006).


16

Vahabzadeh et al.(2004) have said that sugar cane industries using fermentation

technology are source of toxic molasses wastewater (MWW) that contains considerable

quantities of organic compounds. A large amount of the organic matter of MWW is

removed by means of trivial biodegradation treatments but the removal of dark color due

to the presence of melanoidin-type high molecular weight compounds is only

insignificant. White Rot Fungi are however, best alternative for catalyzing degradation of

numerous recalcitrant organic compounds often present in MWW (Fu and Viraraghavan,

2001; Lacina et al., 2003). The decolorizaing ability of Phanerochaete chrysosporium is

linked with the activity of ligninolytic enzymes like LiP and MnP (Vahabzadeh et al.,

2004). Better expression of laccase genes in Trametes sp. I-62 (lcc1 & lcc2) and

basidiomycetous fungus NIOCC # 2a upon contact with MWW accompanied by

improved color removal, proved the involvement of laccase in the melanoidins

metabolism (D’Souza et al., 2006; Gonza´lez et al., 2007). Molasses spent wash (MSW)

or digested Gonza´lez et al., 2007). Molasses Spent Wash (MSW) or digested spent wash

(DSW) or alcohol distillery wastewater (WAD) is another wastewater from molasses-

based alcohol distilleries (Raghukumar, 2002; Chopra et al., 2004). Quite a lot of species

of WRF have been reported to remove about 70–80% of the color present in MSW based

effluents (Raghukumar 2002; D’Souza et al., 2006).

Bredberg et al.( 2002) have said that rubber waste mainly cames out in the form

of tyres and huge amount of waste rubber material produced as a byproduct was an

environmental problem of great concern. Natural rubber waste serum (NRWS) is

obtained as a by-product during coagulation of latex and is a major pollutant from the

rubber industry (Lau and Subramaniam 1991). Recycling of spent rubber material is

tricky due to the vulcanisation, which generates strong sulfur bonds between the rubber

molecules (Liu et al. 2000). Different methods for desulfurization of rubber material and

to facilitate the reuse of waste rubber have been developed, including biotechnological

processes (Christiansson et al., 1998; Bredberg et al., 2001). Microbial devulcanisation is

a promising way to increase the recycling of rubber materials. However, a number of


17

microorganisms tested for devulcanisation are sensitive to rubber additives (Christiansson

et al. 2000). According to Bredberg et al. (2002) study WRF have the potential to

bioremediate (NRW). Most of the common rubber additives are aromatic compounds and

can be effectively removed by LMEs of WRF. Resinicium bicolor is the most effective

fungus for detoxification of rubber material, especially the ground waste tire rubber.

Atagana et al. (1999) said that treatment of rubber waste with Resinicium bicolor

enhanced the growth of Thiobacillus ferrooxidans bacterium as well as desulfurization

compared to the untreated rubber. The biodegradation of rubber waste using co-cultures

of WRF with desulfurizing bacteria can thus be an attractive option for developing a

potentially feasible process.

Faisalabad is one of the major textile cities of Pakistan. The textile dyes of

different selected in this study are extensively used intextile dying plants. The major

objective of this study was to investigate the potential of some local white rot fungi

cultures for decolorization but widely used textile dyestuffs with the following

objectives:

1. Screening and evaluating the potential of local fungal cultures as industrial

microorganism for decolorization of textile dyes.

2. Optimization of decolorization process for selected dyes using classical method.

3. To study the patterns of ligninolytic enzymes of different fungi secreted during

dye decolorization.

18

eChapter 2

Review of Literature

White rot fungi (WRF) have extensive potential for degradation of toxic

pollutants including dyes and dye based effluents. WRF have been used to develop

biotechnological processes. Highly non-specific nature of their Lignin degrading

enzymes suggested WRF may be the best choice available for the bioremdeation of

textile dyes and textile industry effluents.

Cripps et al. (1990) have demonstrated biodegradation of three dyes namely

Azure B, Orange II, Congo Red, and Tropaeolin O and using cultures of the white rot

fungus, Phanerochaete chrysosporium. The degree of decolorization was calculated by

watching the decline in absorbance at or near the highest wavelength for each dye. The

metabolite synthesis was also monitored. Decolorization of these dyes was most

widespread in ligninolytic cultures, but substantial decolorization also occurred in non

ligninolytic cultures. Incubation with crude lignin peroxidase resulted in decolorization of

Azure B, Orange II and Tropaeolin O but not Congo red, indicating that lignin peroxidase

is not required in the initial step of Congo red degradation.

Spadro et al; (1992) investigated that under nitrogen deficient conditions, the

white rot basidiomycete Phanerochaete chrysosporium widely mineralized the

exclusively 14C-ring-labeled azo dyes Disperse Yellow 3 [2-(4'-acetamidophenylazo)-4-

methylphenol], 4-phenylazo-2-methoxyphenol, 4-phenylazoaniline, 4-phenylazophenol, ,

Solvent Yellow 14 (1-phenylazo-2-naphthol Disperse Orange 3 [4-(4'-nitrophenylazo)-

aniline]) and N,N-dimethyl-4-phenylazoaniline. According to them, the dyes were

mineralized from 23.1 to 48.1% twelve days after the addition to cultures. Aromatic rings

with substituents such as hydroxyl, amino, acetamido or nitro, functions were mineralized

to a greater extent than unsubstituted rings. The majority of the dyes were degraded

extensively only under nitrogen-limited and ligninolytic conditions. However, 4-

Chapter 2 Review of Literature

19

phenylazo-[U-14C]2-methoxyphenol and 4-phenylazo-[U-14C] phenol were degraded to

a less significant extent under nitrogen-sufficient, non ligninolytic conditions as well.

These results proved that P. chrysosporium had potential applications for the clean up of t

dye basd effluents and for the bioremediation contaminated soil.

Young and Jian (1997) reported that the majority of the textile dyes in industrial

waste waters were opposed to to degradation in minor biological treatment process. Their

study included decolorization of eight synthetic dyes including anthraquinone, azo,

indigo and metal complex by using cultures of white-rot fungi and their peroxidase-

catalysed oxidation. They said that the dyes were not decolorized by Mn dependent

peroxidase whereas more than 80% colo rremoval was by ligninase-catalysed oxidation.

The rate of dye decolorization improved directlyy with ligninase concentrations. As

compared with fungal cultures in which ligninase enzyme was detected, partly purified

ligninase showed a steady and higher level of dye decolorization with other indispensable

components being provided such as hydrogen peroxide, veratryl alcohol, and acidic pH

(3.5–5). Veratryl alcohol had a critical concentration level above which no further effect

on dye decolorization was observed. Depending on the influence of H2O2 on dye

decolorization, the eight dyes can be divided into two groups; one had an optimum H2O2

concentration and the other showed increased decolorization with high H2O2 doses. Dye

concentration had a negative effect on decolorization rate in general. The dye

concentration above which the negative effect was observed varied from 10 to 125 mg/L,

depending on individual dye structure.

Cloete and Celliers (1999) found that addition of solid MnO addition improved

the production of manganese(II)-dependant peroxidase and H2O2. It increased the rate of

biodegradation of Aroclor 1254 in a nitrogen-limited condition by the white rot fungus

Coriolus versicolor.They noted MnP activity 48 h after the addition of MnO2 to the

cultures and was absent in cultures that did not receive MnO2. According to them, the

rate of Aroclor 1254 removal by C. versicolor was affected by the concentration of

MnO2. 34.5 mM of MnO only increased the H2O2 production. Removal of Aroclor 1254

in the absence of MnO2 still took place which showed the presence of (LiP) or

nonspecific absorption. The cultures having 57.5 mM MnO2 removed ca. 84% of the


20

initial 750 mg l-1 Aroclor in 6 days of incubation. Cultures with out MnO2 and 34.5 mM

removed 79 and 76%, respectively. Cultures recieving MnP or LiP as the dominant

enzyme species removed penta- and hexachlorobiphenyls at a slower rate than tri- and

tetrachlorobiphenyl.

Swamy and Ramsay (1999) investigated potential of five cultures of white rot

fungi for their ability to decolorize Amaranth, Remazol Black B, and Tropaeolin O in

agar plates. According to their findings, Bjerkandera sp. BOS55, Phanerochaeta

chrysosporium and Trametes versicolor showed the greatest extent of decolorization. In

static aqueous culture, the three cultures formed fungal mats, which did not decolorize

any dye beyond some mycelial sorption. When agitated at 200 rpm, the biomass grew as

mycelial pellets. Bjerkandaera sp. BOS55 pellets decolorized only Amaranth, Remazol

Black B and Remazol Orange, P. chrysosporium and T. versicolor pellets were capable of

decolorizing most dyes with decoloration by T. versicolor being several times more rapid.

Batch cultures of Bjerkandaera sp. BOS55 and P. chrysosporium had a limited ability to

decolorize repeated dye additions; however, T. versicolor rapidly decolorized repeated

additions of the different dyes and dye mixtures without any visual sorption of any dye to

the pellets. The choice of the buffer left a deeper effect on pH stability upon dye addition

and as a result, decoloration. The choice of 2, 2΄ dimethyl succinic acid proved excellent

for pH maintenance and gave better result as for as decolorization ability was concerned.

Wong and Yu (1999) worked on some textile dyes which were not uniformly

degraded by microbial attack in conventional aerobic treatment because of their unique

and stable chemical structures. Both the reachers used three synthetic dyes

anthraquinone, azo and indigo with typical chromophores.These were decolorized by a

white-rot fungus Trametes versicolor. Laccase was the responsible enzyme for dye

degradation. It was an extracellular oxidase synthesised by the fungus under the

conditions of slow increase or in its stil condition. The mechanism of laccase-catalyzed

dye decomposition, however, it was different depending on dye structures.

Anthraquinone dye acted as an enzyme substratewhich was directly oxidized by laccase

while decolorization of rest of the two dyes utilised some smaller molecules (<8 kDa)


21

metabolites. It was established that azo and indigo dyes dide not play the role of`

substrates for laccase rather metabolites of smaller molecules made the possibilty of

interaction between the dyes and laccase. The decolorization rate of the nonsubstrate dyes

was actually limited by the concentration of mediating compounds rather than laccase

activity in the solutions. Some syn-thetic compounds such as 2,2'-azino-bis(3-

ethylthiazoline-6-sulfonate) or ABTS and anthraquinone dye could also mediate the

decolorization of azo and indigo dyes. The mediating function of ABTS and

anthraquinone dye was quantitatively compared in the decomposition of two nonsubstrate

dyes. This fact implies that the laccase-substrate dyes in an industrial e.uent can promote

the decolorization of those nonsubstrate dyes. E.uent decolorization, therefore, may not

be limited by the small molecule metabolites which are not produced in large amount by

fungus in most industrial events. A laccase-catalyzed and mediator-involved dye

degradation mechanism is proposed for further kinetic studies.

Zhang et al. (1999) studied the decolorization of cotton bleaching effluent by a

wood rotting fungus . According to them, fungus No.7 could removed more than 70% of

the original colour (initial A400=2.0–2.4) from the textile effluent within 96 hours in the

presence of agitated conditions. They reused the fungal mycelia for a prolonged time and

the decolourisation potential of mycelial pellets was fairly stable during a long period of

cool storage A lot of factors touching the decolorization process were investigated,

including: concentration of effluent, glucose, NH4+ and Mn(II); temperature; initial pH;

The activity of manganese peroxidase appeared to link fine with the decolourization rate.

After doing the fungal treatment, better treatment of the effluent was observed by using

other microorganisms.

Abdullah et al. (2000) used Trametes hirsuta and its purified laccase to degrade

triarylmethane, indigoid, azo, and anthraquinonic dyes. According to their research

findings, initial decolorization velocities depended on the substituents on the phenolic

rings of the dyes. Thermal stabilities of the enzyme were enhanced by the immobilization

of the T. hirsuta laccase on alumina and its tolerance against some enzyme inhibitors,

such as copper chelators, halides, and dyeing additives. At 50 mM NaCl the laccase lost


22

50% of its activity while the 50% inhibitory concentration (IC50) of the immobilized

enzyme was 85 mM. Treatment of dyes with the Immobilized laccase reduced thE

toxicity of dyes (based on the oxygen consumption rate of Pseudomonas putida) up to

80% (anthraquinonic dyes). Textile effluents were decolorized with T. hirsuta or the

laccase were used for dyeing. Enzyme protein or metabolites strongly interacted with the

dyeing process indicated by lower staining levels (K/S) values than obtained with a blank

using water. However, when immobilized laccase decolorized the effluents they could be

used for dyeing and acceptable color differences (ΔE*) below 1.1 were measured for

most dyes.

Cuoto et al. (2000) studied the effect of some low molecular mass mediators

forligninases enzyme synthesis, veratryl alcohol, manganese (IV) oxide, and Tween 80 to

semi-solid-state cultures of Phanerochaete chrysosporium BKM-F-1767 (ATCC

24725).They ustilised cubes of a polymer (polyurethane foam. According to them,

supplementing the cultures with Tween 80, a maximum manganese-dependent peroxidase

(MnP) activity of 350 U/l was achieved. The value obtained was about 3-times higher

than that noted in the control cultures (without Tween 80). On the other hand when not

only Tween 80 but also veratryl alcohol or manganese (IV) oxide were added into the

culture medium, the end product was greater and activities noted were 600 and 1100 U/l

for MnP with oxide or alcohol, respectively. These activities profile was remarkably

higher than those obtained in the control cultures (about 3-fold and 7-fold,

respectively).The decolorization of the dye Poly R-478 by the afore-mentioned cultures

was investigated. Theextent of fungal decolorization reached at the end of all the

cultivations was higher than 95%.

Kirby et al. (2000) used Phlebia tremellosa to decolorize eight synthetic dyes.

After invstigating the fungus potential for decolorization they observed that Phlebia

tremellosa decolorized eight synthetic textile dyes (200 mg l−1) by greater than 96%

within 14 days under still incubation conditions. HPLC analysis of the culture

supernatants showed that Remazol Black B was degraded by the fungu. However,

outright mineralization did not happen due to accumulation of colourless organic


23

breakdown. Activity of laccase was measurable in culture supernatants after 120 hours.

The fungus was grown in the presence of an artificial textile effluent,where the activity

was approaching a maximum of 15 U l−1 on day 14.

Nigam et al. (2000) tested three agricultural residues, wheat straw, wood chips

and corn cob shreds. Their objective was to investigate the individual dyes and dye

mixture adsorption potential in solutions up to 70-75 %. They noted that color removal

was achieved from 550 ppm dye solution at room temperature using corn-cob shreds and

wheat straw. Increase in temperature had little effect on the adsorption capability of the

residues. The dye-absorbed residues left at the end were found to be suitable substrates

for solid-state fermentation (SSF) by two white-rot fungi, Phanerocheate chrysosporium

and Coriolus versicolor. The fungal strains grew unconstrained and synthesised highest

protein content of 16, 25 and 35 g and 19, 23 and 50 g dry weight wood chips and wheat

straw corn-cob shreds respectively, sin presence of ammonical nitrogen to give a C: N

ratio of 20:1. Infact,this approach provided resulted for the remediation of textile

effluents and the exchange of agricultural residues into soil conditioners.

Sumathi and Manju (2000) used culture of Aspergillus foetidus which was

efficient to decolorize media containing azo reactive Drimarene dyes.Their result

showed, the color removal was greater than 95% within 2 days of growth of the fungus.

The dye was found to be strongly adsorbed to the rapidly settling fungal pallets of

biomass instead of undergoing significant biotransformation. Their investigations

revealed that the process of decolorization was associated with the exponential growth

phase of the Aspergillus foetidus and had required for a biodegradable substrate such as

glucose. The fungal strain was also able to decolorize media receiving mixture of dyes to

an extent of 85% within 3 days of growth. Kinetic analyses of fungal decolorization

indicated that the process was time dependent and followed first order kinetics with

respect to initial dye concentration. Color uptake rates (k values) decreased to a

significantlevel with increasing initial concentrations of dye. Their selected strain was

able to grow and decolorize media in the presence of 5 ppm of chromium and 1% sodium

chloride. An alternating and economical carbon source like starch enhanced the growth


24

and accelerated the decolorization process. These results suggested that dye uptake

process mediated by A. foetidus had a potential for large-scale treatment of textile mill

discharges.

Balan and Regina (2001) discussed that indigo dye was rapidly used by textile

industries and was considered a recalcitrant substance, which caused a threat to the

envirnment. According to their research report, Chemical products used during textile

processing left enormous effect on environment while treating effluents through their

color variation. Commonly used dyes for cellulosic fibre are vat and indigo. The

decolorization of these dyes in liquid medium was investigated with white rot

basidiomycete fungi obtained from Brazil. It was found that decolorization commenced

in short period and after 4 days the removal of dye by Phellinus gilvus culture was in

100%, by Pycnoporus sanguineus 91% and by Phanerochaete chrysosporium 75%. No

color reduction wasnoted in a sterile control. On applying TLC of fungi culture extracts

revealed only one unknown metabolite of Rf=0.60, as a consequence of dye degradation.

Chagas and Durrant (2001) studied decolorization of azo dyes by Phanerochaete

chrysosporium and Pleurotus sajor-caju. A lot of synthetic dyes used in industrial

wastewater were opposed to to degradation bytivial methods for treatments. They

examined decolorization of four synthetic dyes using two white rot fungal cultures

medium. P.chrysosporium mainly decolorized all the dyes under investigation, whereas

Pleurotus sajor-caju entirely decolorized New Coccine, Orange G, Amaranth and 60%

tartrazine. Neither fungus showed lignin peroxidase or veratryl alcohol oxides activities,

indicating that those enzymes might not be involved in decolorization. β-glucosidase and

Manganese-peroxidase maight be concerned in decolorization of the dyes by P.

chrysosporium, whereas in Pleurotus sajor-caju a laccase was active towards O-

dianisidine, and glucose 1-oxidase might participate in the whole process.

Driessel and Christov (2001) studied decolorizationof dye based effluents in a

rotating biological contactor (RBC) reactor using Coriolus versicolor and Rhizomucor

pusillus strain RM7, a mucoralean fungus. The results showed that decolorization by the

selected two fungi was in a straight line proportional to original color intensities.


25

Moreover, it was noted that the level of decolorization was not badly affected by

intensity of color, except at the minimum level examined. In addition to this 53% to 73%

decolorization might be obtained by making use of a hydraulic retention time (HRT) of

almost one day. 55% of AOX were removed with R. pusillus, and 40% by C. versicolor.

Teatment with these fungal cultures R. pusillus and C. versicolor left the the effluent in

essence non-hazardous. Glucose incorporation into decolorization media stimulated

color reduction by C. versicolorandremained unaffected with R. pusillus. Ligninases

enzymes (laccase and manganese peroxidase) were only found in effluent treated by C.

versicolor. It also shows that decolorizing mechanisms are certainly between the white-

rot fungi. WRF showed adsorption + biodegradation while the mucoralean fungus

showed adsorption only. This feature is needed to be to be examined in more detail to

confirm the manners responsible for the decolorization process.

Fu and Viraraghavan (2001) worked on decolorization of waste water by white rot

fungi. During the last few decades there had been much focus on on fungal decolorization

of dye wastewater. Bioremediation by WRF is becoming a promising alternative to

replace or supplement present treatment processes. Their research work included

investigation of several fungi, alive or lifeless cells. They found that these had a

potential for decolorizing textile dyes effluents and explained different processes. They

reported a number of elution as wel as regeneration methods for fungal biomass. Their

study also summarized the current before treatment methods for growing the biosorption

potential of fungal biomass. They also discussed the effect of several factors on

decolorization ability of both types of fungal cultures.

Martins et al. (2001) have found that azo dyes are important chemical pollutants

of industrial origin. Synthesis of textile azo dyes with bioaccessible groups for lignin

degrading fungi, such as 2-methoxyphenol (guaiacol) and 2, 6-dimethoxyphenol

(syringol), were possible using different aminobenzoic and aminosulphonic acids as

diazo components. The inoculum of the most suitable biodegradation assays were

obtained from a pre-growth medium (PAM), containing one of the synthesized dyes.

They evaluated the dye biodegradation results after every 7 days, by the decline in


26

absorbance at the highest wavelength of each dye, by the dropping of the sucrose level in

the culture medium and by the increase in the biomass during the 28 days. It was noted

that the dye degradation level depended mainly on the concentration sucrose, structure

of the dye degraded and the dye added into the PAM medium.

Tekere et al. (2001) collected white rot fungi from Chimanimani hardwood

forests Chirinda and in Zimbabwe and studied the best temperature for growth

temperature and dye decolorization. Range of optimum temmperature was found to vary

between 25–37°C amongst the isolated fungal cultures. The fungal cultures were

screened for their ability to degrade the triphenylmethane dyes; (Cresol Red, Crystal

Violet and Bromophenol Blue) and polymeric dyes (Blue Dextran and Poly R478). The

activities of hydrolytic enzyme exhibited by the White Rot Fungi using the API ZYM

system. They also determined the activities of lignin peroxidase (LiP), manganese

peroxidase (MnP) and laccase. Activityies of LiP waere not detectectable in any of the

isolates but manganese peroxidase and laccase activities among all isolates only. Relation

ship between time , decolorization% and optimum pH level was determined for Poly

R478 degradation were studied in liquid cultures. The most significant rates of the dye

decolorization in liquid cultures were found with the following isolates:, Trametes pocas,

Trametes versicolo, Trametes cingulata, DSPM95 (a species to be identified),

Pycnoporus sanguineus and Datronia concentrica.

Gill et al. (2002) investigated the potential of nine WRF cultures by screening

nine white-rot fungal strains for biodecolorization of brilliant green, cresol red, crystal

violet, congo red and orange II. Dichomitus squalens, Phlebia fascicularia and P.

floridensis, possesed color removal of all the dyes on solid agar medium and showed

better dye decolorization ability than Phanerochaete chrysosporium under nitrogen-

limited broth medium.

Hatvani and Me´cs (2002) investigated the the synthesis of laccase and

manganese peroxidase (MnP) by Lentinus edode by studying on a solid medium in

relation with the decolorisation of the dyes Poly R-478, Remazol Brilliant Blue and

Orange II. They noted that L. edodes mycelium was able to decolorize all three dyes.


27

They focused their study on role of Nitrogen (N) concentration-dependence using three

sources peptone, ammonium chloride and malt extract revealed that the range 1–3 mM N

was optimum for both enzyme production and dye degradation, regardless of the N

source or dye used. Result showed, MnP production and the decolorisation of Poly R-478

and Orange II were repressed entirely more than 8 mM N. The enzymatic processes also

reflected dependence on Mn concentration. It was found that 20 M Mn provided

maximum dye decolorization. The incorporation of supplements like oak sawdust and

wheat straw significantly improved MnP production. Oak sawdust had a encouraging

effect on the decolorization of each of the dyes investigated. A medium containing 10 g/l

starch, 3.5 g/l malt extract and 20 g/l oak sawdust proved optimum for the enzymatic

processes investigated.

Kapdan and Kargi (2002) have studied biological decolorization of textile

dyestuffs including, a Phthalocyanine type reactive dyestuff and Everzol Turquoise Blue

G by white-rot fungus Coriolus versicolor MUCL in a rotating biological contactor. They

studied effects of multiple parameters such as, rotational speed (10-40 rpm), disc type,

glucose (5-10 g/L) and dyestuffs concentration (50-500 mg/liter) on the decolorization

potential of WRF. The whole system worked efficiently in repeated-batch mode for 2

days hydraulic retention time at temperature of 28˚C and pH = 4.5-5.0. Glucose, TOC,

and dyestuff concentrations were noted during the experiments. After having used three

different disc types; metal mesh covered plastic discs, metal mesh discs and plastic, it

was found that plastic disc was the best one. According to them, the maximum

decolorization efficiency (80%) was achieved with a speed of 30 rpm. Minimum glucose

concentration (5 g/L) resulted in 77% dye decolorization. Efficiency was around 80% for

50-200 mg/liter initial dyestuff concentrations and declined to 33% for 500 mg

dyestuff/liter.

Koroleva et al. (2002) comparatively studied the LME production by co-

cultivated C. hirsutus and Cerrena maxim in defined medium under static and shaken

conditions, in media with birch sawdust and sulfonate lignin and under SSF of birch

sawdust. It was found that the structure of residual lignin before and after fungal


28

treatment indicated that residual lignin was enriched in carboxylic acid groups and the

oxidation of phenolic component of lignin occurred. Three laccase isoenzymes were

produced by Cerrena maxima growing in defined media. The isoenzyme with pI 3.5 was

largely present and its metal content, molecular mass, isoelectric point and carbohydrate

composition were calculated. The laccase was exhibited broad substrate specificity. It had

high redox potential laccases (750 mV vs. normal hydrogen electrode) and might be

useful in pilot scale.

Leung and Stephen (2002) have studied the correlation between effect of different

nitrogen and sources on Poly R decolorization by white-rot fungi. Synthesis of LMEs by

10 white-rot fungi during decolorization of Poly R 478 dye, showed variable trends in

response to different carbon and nitrogen sources. The result indicated, best

decolorization % was achieved with wood polysaccharide like glucose, xylose as carbon

source, despite the fact that cellulose was the only carbon source to make possible

decolorization by allthe fungal cultures under investigation. Enzyme synthesis by most

isolates was strongly dependant on nitrogen concentration. Under high nitrogen

conditions generally enzyme production was suppressed. An unidentified isolate

(HKUCC 4062) showed that nitrogen deregulated enzyme synthesis. A cellulose with

nitrogen deficient growth medium is recommended for agar-based screening measures of

LMEs production by WRF.

Maximo et al. (2003) selected Geotrichum sp. from quite a lot of fungi because of

its ability to transformation of Reactive Red 158, Reactive Yellow 27 and Reactive

Black 5 used in industry. According to them, none of the white rot fungi tested (,

Rigidoporus sp. Irpex lacteus, Bjerkandera adusta, Phanerochaete magnoliae, and

Trametes versicolor) could transform the red dyes and yellow, although Remazol

Brilliant Blue R was rapidly degraded. When Geotrichum sp. was cultivated together

with each dye the fungus transformed the black dye rapidly while the other two dyes took

twice as long to be degraded. When 20-day old cultures were supplemented with

consecutive amounts (200 ppm) of respecteive dyes, the time required for total

transformation was reduced to about 5 days for all the three dyes. It is possiblethat the


29

LMEs (Mn peroxidase, Mn-independent peroxidase and laccase) were involved in the

black dye transformation They suggest that additional enzymes or factors might be

involved for the yellow and red dyes. The unrelenting ability of Geotrichum sp. to

transform large amounts of dyes (800 ppm after consecutive additions) suggested that it

might have potential application in the decolorizationtion of textile wastewater.

Salvam et al. (2003) used a white rot fungus Thelephora sp.in order to check its

potential for decolorization of azo dyes like amido black 10B, congo red, Orange G and.

According to reported results, decolorization caused by the fungus was 33.3%, 97.1% and

98.8% for orange G, congo red and amido black 10B, respectively. An enzymatic dye

decolorization study showed that lignin peroxidase (LiP) and manganese dependent

peroxidase (MnP) at the same concentration decolorized 13.5% and 10.8%, orange G, ra

maximum of 19% orange G was decoloorized by laccase at 15 U/ml. Laccase caused

maximum decolorization of 12.0% and 15.0% for congo red and amido black 10B,

respectively. In an other experiment a dye industry effluent was treated by the fungus in

continuous and batch modes. A maximum decolorization of 61% was noted on the third

day in the batch mode and a maximum decolourization of 50% was experienced on

seventh day in the continuous mode. Their findings suggested that the batch process for

treatment using Thelephora sp. Might be more effective than the continuous for color

removal from dye based industrial effluents.

Tuomela et al. (2002) investigated the degradation of synthetic lignin by different

white-rot fungi in soil. According to their experimental strategy, soil sample contained

wheat straw – a lignocellulose substrate and the sterilized mixture was autoclaved t

before the inoculation. They used fungal cultures used were Dichomitus squalens,

Abortiporus biennis, Phanerochaete chrysosporium, Bjerkandera adusta, Phanerochaete

sordida, Phlebia radiata, Pleurotus ostreatus, Trametes versicolor, and Trametes hirsuta.

They incubated fungal cultures for 56 days in presence of a straw-to-soil ratio of 1:5, and

T. hirsuta was also cultured on straw in absence of soil. In addition, B. adusta and T.

hirsuta were studied with three other straw-to-soil ratios (1:2.5; 1:10; and 1:25). The

researchers collected 14CO2 during incubation, and incubated cultures were


30

consecutively extracted with dioxane, water, the residue and alkali, was combusted.

Mineralization by fungal inocula in soil was 4% with P. sordida while 23% with T.

versicolor. Without soil T. hirsuta mineralized 30% of 14C-DHP, whereas only 0.2%.

mineralization in autoclaved controls was observed. With the decrease in amount of

straw, fungi growth declined, and T. hirsuta transformed lesser quantityies of 14C-DHP

to 14CO2. However, the transformation by B. adusta did not change appreciably until the

straw-to-soil ratio was brought to 1:25. The unmodified fraction dioxane decreased

significantly during incubation of fungi with all straw treatments. By day 0, 62–64% of 14C-DHP was previously confined to humic substances without fungal activity, but the

mass balance of radiolabel in controls remained unchanged in spite of the incubation.

WRF were, in fact, were able to degrade the lignin, because the amount of radiolabel in

humic fractions decreased during incubation. However, the binding increased as the

amount of straw decreased.m The most of 14C-DHP remained bound to humic substances,

Verma and Madamwar (2002) used Neem hull waste for lignin peroxidase

production by Phanerochaete chrysosporum under SSF conditions. According to them,

maximum dye decolorization achieved partly by purified LiP was 80% for Porocion

while Brilliant Blue HGR, 83 for Ranocid Fast Blue, 70 for Acid Red 119 and 61 for

Navidol Fast Black MSRL. The different concentrations of veratryl alcohol, hydrogen

peroxide, dye and enzyme affecting the efficiency of decolorization were investigated.

Highest decolorization potential was observed pH 5.0 , at 0.2 and 0.4 mmol/L hydrogen

peroxide, and 2.5 mmol/L veratryl alcohols after a 1-h reaction. It involved 50 ppm of

dyes and 9.96 mkat/L of enzyme.

Wu and Xia (2002) studied both laccase synthesis by the white-rot fungus

Coriolus versicolor and decolorization of dyestuff and dying wastewater with crude

solution of laccase. Their research findings showed that laccase production met the

definition of secondary metabolism. For laccase synthesis the optimum initial pH was

4.5. Addition of veratryl alcohol or elevated trace metals both enhanced the laccase

activity, while Tween-80 showed some inhibition. The immobilization of C. versicolor

mycelia in polyurethane foam exhibited less laccase synthesis ability than mycelial


31

pellets. An economical way for laccase synthesis was the use of repeated batch

cultivation process. The same fungal pellets could be used repeatedly up to 14 times and

average laccase activity of each batch could reach up to 6.72 IU/mL. This method

reduced the enzyme synthesis route, medium utilization and the chance of contamination,

providing highly efficient and great economic benefit. Experiment of decolorization with

crude solution of laccase even gave good results. With 3.3 IU/mL initial laccase activity,

color removal of Acid Orange reached 98.5% after with in 1 day reaction. Also with 2.6

IU/mL initial laccase activity, color removal of dyes effluents reached 93% after 24 h

reaction.

Yesilada et al. (2002) investigated the effects of different culture conditions like

initial pH, dye concentrations, amount of pellet, temperature and agitation on

decolourizing activity of Funalia trogii. According to them, “these, except initial pH,

were all found to be important for dye decolourizing activity of F. trogii. The

decolourization of the dye involved adsorption of the dye compound by fungal pellets at

the initial stage, followed by the decolourization through microbial metabolism. Heat-

killed pellets were also tested for their ability to decolourize Astrazon Red dye. These

pellets adsorbed the dye and 55% decolourization was obtained in 24 h. But at the

second cycle there was only 24% decolourization. Our observation showed that Astrazon

Red dye decolourization by heat-killed pellets was mainly due to biosorption. The

longevity of the decolourization activity of F. trogii pellets was also investigated in

repeated batch mode. Variations in the amount of pellet increased % decolourization and

stability of pellets.”

Adosinda et al. (2003) performed a screened g several fungal strains like

Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor and

Aureobasidium pullulans on the degradation of syringol derivatives of azo dyes

possessing either carboxylic or sulphonic groups, under optimized conditions previously

established by us. Result showed, “T. versicolor showed the best biodegradation

performance and its potential was confirmed by the degradation of differently substituted

fungal bioaccessible dyes. Enzymatic assays (lignin peroxidase, manganese peroxidase,


32

laccase, proteases and glyoxal oxidase) and GC-MS analysis were performed upon the

assay obtained using the maximally degraded dye. The identification of hydroxylated

metabolites permitted us suggest a possible metabolic route. Quantificatification of

biodegradable dyes were performed to assess the possibility of wastewater treatment by

fungal cultures for textile industries.

Baldrian (2003) investigated the heavy metal interactions with WRF and their

effect on enzymes which played crucial role in dye degradation. According to him,

White-rot fungi required minute quantities of indispensable heavy metals such as Cd, Mn

or Zn for their growth, but their excess is sources of toxicity when available in excess.

Toxicity of these heavy metals can retard the growth, cause physiological and

morphological changes and consequently affect the reproduction of Basidiomycota.

Fungal strains and species differ in their sensitivity towards metals and in the defence

mechanisms involved. Some heavy metals such as Hg, Cu or Ni has been used for the

development of antifungal wood preservatives. Extracellular ligninolytic and cellulolytic

enzymes are regulated by heavy metals on the level o their action as wel as during during

transcription. During the degradation of lignocellulose and xenobiotics by white-rot fungi

or isolated enzymes from these fungi heavy metals obstruct with both the activity of

extracellular enzymes involved in the fungal colonization and process. The adsorption nd

accumulation of ability of metals by white-rot fungi together with the excellent

mechanical properties of fungal mycelial pellets provide an a prospect for application of

fungal mycelia in selective sorption of individual heavy metal ions from contaminated

water.

Cing et al. (2003) evaluated the potential use of fungal pellets for decolorization

of the textile dyeing wastewater. Result showed the live pellets of the fungus

Phanerochaete chrysosporium were found to remove more than 95% of the color of this

wastewater within 24 hours. The dye-removal capacity was a dependent on time and was

proportional to the agitation rate. The optimum temperature was 30 degrees C.. The

decolorization performance of live pellets remained high and stable for 5 days and they

showed twice to thrice higher decolorization capacity than dead pellets.


33

Hai et al. (2003) investigated the performance of a bench-scale submerged

microfiltration bioreactor using the Coriolus versicolor (NBRC 9791) for treatment of

textile dye wastewater following the confirmation of the decoloration capacity of the

fungus strain in agar-plate and aqueous batch studies. According to their protocol, the

temperature and pH of the reactor was controlled at 291 C and 4.52, respectively. The

bioreactor was operated with an average flux of 0.05 m/d (HRT=15hrs) for a month.

Extensive growth of fungi and their attachment to the membrane led to its fouling and

associated increase of transmembrane pressure requiring periodic withdrawal of sludge

and membrane cleaning. However, stable decoloration activity (approx. 98%) and TOC

removal (>95%) was achieved using the entire system (fungi+membrane), while the

contribution of the fungi culture alone to color and TOC removal, as indicated by the

quality of the reactor supernatant, was 35-50% and 70%, respectively. Comparison of

UV-visible spectra of the influent and permeate revealed subsequent biodegradation of

the aromatic group following the breakdown in the chromophoric group of the dye.

Rayan et al. (2003) reported that S. rolfsii secretesd two laccases (SRL1 and

SRL2) having molecular weights of 55 and 86 kDa, respectively. According to them,

laccase production was found to be triggered by the addition of 2, 5-xylidine to the

cultural media. Two different laccases were isolated from the sclerotia treatment with a

combination of chitinase and 1,3-glucanas depending on the stage of sclerotia

development. The more prominent laccase, SRL1, was purified and found to decolorize

the industrially important wool azo dye Diamond Black PV200 without the addition of

redox mediators. The acidic pH (2..4 ) mdae the enzyme (PI5.2) more active indicating

more activity with ABTS as substrate.The optimal temperature for activity was

determined to be 62 •C. The study of enzyme stability studies revealed that SRL1 was

remarkably stable at 18 °C and pH 4.5, retaining full activity after seven days. Tyrosine

Oxidation was not mesurable under the reaction conditions but the enzyme did oxidize a

variety of the usual laccase substrates. SRL1 was strongly repressed by sodium azideand

fluoride. Dye solutions decolorized with the Laccase immobilisation were successfully

used for redyeing.


34

Martins(2003) worked on screening of several fungi including Phanerochaete

chrysosporium, Trametes versicolor Pleurotus ostreatus, and Aureobasidium pullulans

performed on the degradation of syringol derivatives of azo dyes possessing either

carboxylic or sulphonic groups, under optimized conditions previously established by us.

T. versicolor showed the best biodegradation performance and its potential was

confirmed by the degradation of differently substituted fungal bioaccessible dyes.

Enzymatic assays forignin peroxidase, manganese eroxidase, laccase, proteases and

glyoxal oxidase and GC-MS analysis were performed upon the assay obtained using the

most degraded dye. The identification of hydroxylated metabolites allowed us to propose

a possible metabolic pathway. Biodegradation assays using mixtures of these

bioaccessible dyes were performed to evaluate the possibility of a fungal wastewater

treatment for textile industries.

Wesenberg et al. (2003) summarized the state of the art in the research and

prospective use of WRF and their enzymes for the treatment of dye based industrial

effluents. According to their report, White rot fungi producd a variety of extracellular

oxidases i.e lacasse, Mn peroxidase and lignin peroxidase which are utilised in the

degradation of various dyes and xenobiotic compounds. The textile industry by far the

most avid user of synthetic dyes is in need of coefficient solutions for its colored

effluents. The detoxification and degradation and potential of WRF can be harnessed

using up and coming knowledge knowledge of the physiology of these organisms as well

as of the biocatalysts and stability characteristics of their enzymes. This knowledge is

needed to be transformed into forceful and reliable waste treatment processes.

Alessandra et al. (2004) screened filaments fungi for their ability to decolorize a

commercial reactive dye. They reported ability of 19 isolates of 13 different fungal

cultures to decolorize the reactive dye. In accordance with their result, the isolates of

Schizophyllum commune and other obtained from textile effluents were evaluated in

minimum liquid medium. Different isolates presented variable trend (the fastest and

slowest growth) and decolorized the dye actively.


35

Bllanquez et al. (2004) reported biotranformation mechanism of textile metal dye

biotransformaby Trametes versicolor. They studied biodegradation of Grey Lanaset G,

which consists of a mixture of metal complexed dye. Their research protocol, revealed

that experiments were carried out in a bioreactor with fixed pellets of the fungus

Trametes versicolor that was functional under conditions of laccase synthesis. Although

decolorization was highly efficient (90%), yet no direct relationship to extracellular

enzyme was understandable. Moreover, the extracellular enzyme was found to be unable

to degrade the dye in vitro and the whole process involved several steps. The process

involves several steps. Thus, the initial adsorption of the dye and its transfer into cells is

followed by breaking of the metal complex bond in the cells release of the components.

The Co and Cr contents of the biomass and treated solutions, and their closer relationship

to intracellular enzyme and degradation of the dye, confirmed the initial hypothesis.

Chander et al. (2004) worked on screening of eight white-rot fungal strains for

biodecolorization of eight dyes commercially used in various industries. Decolorisation

of Poly R 478 was used as a standard todetermine the dye-decolorisation potential of

various fungi. Result showed , ‘all the fungi tested significantly decolourized Poly R 478

on solid agar medium. When tested in a nitrogen-limited broth medium, Dichomitus

squalens, Irpex flavus, Phlebia spp. and Polyporus sanguineus were better industrial dye

decolourisers than Phanerochaete chrysosporium.’’

Fang et al. (2004) investigated the biodegradation mechanisms and kinetics of azo

dye 4BS by a microbial consortium consisting of a white-rot fungus 8-4 and a

Pseudomonas 1-10 was isolated from wastewater treatment facilities of a local dyeing

house by enrichment.They used azo dye Direct Fast Scarlet as the sole source of carbon

and energy, which had a high capacity for rapid decolorization of 4BS. To explain the

decolorization process in detail, decolorization of 4BS was compared between individual

strains and the microbial consortium under diverse treatment processes. The microbial

consortium showed a significant dye decolorization rates under either static or shaking

culture, which could be be due to the synergetic reaction of single strains. From the UV–

visible spectra of 4BS dye solutions before and after decolorization and the curve of


36

UV vlues cultivation with the microbial consortium. It was found that 4BS could be

mineralized totally, and the results had been used for comprehending the degrading

pathway of 4BS. This study also examined the kinetics of 4BS decolorization by

immobilized microbial consortium. The results demonstrated that the optimal

decolorization activity was observed in pH range between four and 9, temperature range

between 20 and 40°C and the maximal specific decolorization rate occurred at 1000

mgl/L of 4BS. The distribution and proliferation of microbial consortium were also

microscopically observed, which further con-.rmed the decolorization mechanisms of

4BS.

Ge et al (2004) focused their research on biological decolorization of textile

dyestuff Basic Blue 22, a phthalocyanine type reactive dyestuff, by the white-rot fungus

Phanerochaete sordida ATCC90872 in a rotating biological contactor. The method

adopted for their study showed effect of effects of different operating parameters

including disc type, rotational speed, glucose and dyestuff concentration on the

decolorization performance of white-rot fungi were investigated. The system was

operated in repeated-batch mode with 48 h hydraulic retention time. Three different disc

types; plastic, metal mesh covered plastic discs and metal mesh discs were used and the

plastic disc was found to be most suitable. Result showed, the highest decolorization

efficiency was obtained with a rotational speed of 40 rpm. Minimum glucose

concentration for 78% decolorization efficiency was 5 g/l. TOC removal efficiency was

around 80% for 50–200 mg/l initial dyestuff concentrations and decreased to 52% for 400

mg dyestuff/l.

Kandelbauer et al. (2004) studied the kinetics of WRF laccase-catalyzed

transformation of the azo-dye Diamond Black PV 200 and synthesized derivatives were

analyzed for dependence on pH and substrate structure. GC-MS analysisis results

showed, upon laccase oxidation, reactive chinoid fragments of lower molecular weight

were formed. These may further oligomerize as indicated by the appearance of a number

of compounds with increased molecular weight. The pH for the decolorization was pH 5

for Diamond Black PV 200 which did not change significantly when the substitution


37

pattern of its basic structure was varied. Biodegradability, however, was strongly

dependent on the structure of the dyes.

Kim etal. (2004)used a membrane bioreactor using white-rot fungi for the

decolorization of dye solutions. They elucidated that decolorization of dye solutions by

Trametes versicolor KCTC 16781 and membrane filtration were combined, and the

applicability of this process was investigated using reactive dye solutions. The

practicability of MBR using fungal biodegradation was studied with nano filtration and

reverse osmosis membranes to improve permeate flux and separation efficiencies. The

effects of dye types on fungal biodegradation and membrane filtration (rejection and

permeate flux) were also investigated.

Novotny et al. (2004) have explained the ole of lignolytic enzymes in

remediation of pollutants in water and soil. They found that xtracellular peroxidases and

laccases have been shown to oxidize recalcitrant compounds in vitro but the likely

significance of individual enzyme levels in vivo remained unclear. This study

documented the quantities and activities of Mn-dependent peroxidase (MnP), lignin

peroxidase and laccase (LAC) in various species of ligninolytic fungi grown in liquid

medium and soil and their effect on degradation of polycyclic aromatic hydrocarbons

(anthracene and pyrene), a polychlorinated biphenyl mixture (Delor 106) and a number of

synthetic dyes. Stationary cultures of a highly degradative strain Irpex lacteus exhibited

380-fold and 2-fold increase in production of MnP and LAC, respectively, compared to

submerged cultures. Addition of Tween-80 to the submerged culture increased MnP

levels 260-fold. High levels of MnP correlated with efficient decolorization of Reactive

Orange 16e but not of Remazol Brilliant Blue R dye. Degradation of anthracene and

pyrene in spiked soil by straw-grown explorative mycelium of Phanerochaete

chrysosporium, Pleurotus ostreatus and Trametes versicolor showed the importance of

LAC and MnP levels secreted into the soil. The importance of high fungal enzyme levels

for efficient degradation of recalcitrant compounds was better demonstrated in liquid

media compared to the same strains growing in soil.

Pazarlioglu and Urek (2004) study showed, the effect of some activators on Mn


38

dependent peroxidase production, Mn, Tween 80, phenyl methyl sulphonide fluoride

(PMSF) oxygen, temperature, pH, glycerol and nitrogen in solid state cultures of

Phanerochaete chrysosporium BKM - F 1767 (ATCC24725). Polystyrene foam beads

were employed as support material. Supplementing the cultures with Tween 80(0.05%

v\v) and Mn (174uM) a maximum MnP activity of 421U/L was achieved. This activity

was just about 2 times higher than that obtained in the controlled cultures (without Tween

80). Decolorization of some azo dyes was also achieved with MnP produced in solid

state.

Ramsay and Chris (2004) have worked on decoloration of a carpet dye effluent

using Trametes versicolor. Their result showed, although a non-sterile, undiluted carpet

dye effluent (containing two anthraquinone dyes) restricted the growth of Trametes

versicolor, the pre-grown fungus removed 95% of its color in shake-flasks after 10 h of

incubation. After decolorisation, the COD of the cell-free supernatant increased and the

toxicity was unchanged as calculated by the Microtox assay using Vibrio fischeri.

Decoloration rates decreased when either glucose alone or Mn2+ and glucose were

added. T. versicolor, immobilized on jute twine in a rotating biological contacting

reactor, also decolorized four successive batches of the effluent. There was no

decolorization in any of the uninoculated, non-sterile controls culture of P.

chrysosporium.

Shin (2004) reported that the textile industry wastewater is decolorized efficiently

by the white rot fungus, Irpex lacteus, without any chemical. The degree of the

decolorization of the dye effluent by shaking or stationary cultures is 59 and 93%,

respectively, on the 8th day.. Laccase activities were equal in both cultures and its level

was not affected considerably by the culture duration. Neither lignin peroxidase LiP nor

RBBR ox. could be investigated in both cultures. The absorbance of the dye effluent was

significantly decreased by the stationary culture filtrate of 7 days in the absence of Mn

(II) and veratryl alcohol. In the sttil culture filtrate, three or more additional peroxidase

bands were detected by the zymogram analysis.

Christian et al. (2005) explained the role of mediator like veratryl alcohol in the


39

lignin peroxidase-catalyzed oxidative decolorization of Remazol brilliant blue R. Result

showed, lignin peroxidase produced by Trametes versicolor decolorizes Remazol brilliant

blue R (RBBR) in the presence as well as in the absence of veratryl alcohol (VA). VA

increases and stabilizes the RBBR-decolorization rates by lignin peroxidase. RBBR has

better substrate reactivity than VA for LiP. RBBR is also decolorized directly by LiP and

competitively inhibits VA oxidation by LiP. In the presence of higher concentrations of

RBBR (i) RBBR ecolorization rates improve, (ii) veratryl aldehyde appears after a lag

and (iii) VA oxidation rates decrease. The lag is due to consumption of VA cation radical

(VA?) generated upon LiP-catalyzed VA oxidation, during RBBR oxidation.That might

result in the formation of compound III in the absence of VA+ and contributes to the

inhibitory influence of RBBR on LiP activity.

Eichlerova et al. (2005) screened thirty different white rot strains for Orange G

and Remazol Brilliant Blue R (RBBR) decolorization on agar plates. Three promising

strains, Dichomitus squalens, Ischnoderma resinosum and Pleurotus calyptratus, selected

on the basis of this screening, were used for decolorization study in liquid media. All

three strains efficiently decolorized both Orange G and RBBR, but they differed in

decolorization capacity depending on cultivation conditions and ligninolytic enzyme

production. Two different decolorization patterns were found in these strains: Orange G

decolorization in I. resinosum and P. calyptratus was caused mainly by laccase, while

RBBR decolorization was effected by manganese peroxidase (MnP); in D. squalens

laccase and MnP cooperated in the decolorization processes.

Georgiou et al. (2005) treated textile wastewater, on-site, by means of a two-

stage fixed-bed-reactor pilot plant and immobilized strain on special porous carriers made

from reticulated sintered glass. According to their research report, acetic acid solution,

enriched with nutrients and trace elements, was utilized, both as a pH-regulator and as an

external substrate for the growth of methanogenic bacteria. Ther esearch objective was

decolorization of the wastewater and transformation of the non-biodegradable azo dyes

(reactive in nature) to the degradable metabolites, under aerobic biological conditions,

like aromatic amines. Complete decolorization , by microbial technology, of the textile


40

wastewater, at very low hydraulic residence times (less than 4 h), can be achieved

utilizing this technique, while biogas, rich in methane,was also produced. Furthermore,

the resulting effluent presents high biodegradation ability under aerobic biological

conditions, as proved by means of a special microbial sensor. Thus, the utilization of the

above-described anaerobic technique with activated-sludge treatment as a second stage,

seemed to be a very attractive method for processing textile wastewater since it is cost-

effective and environment-friendly.

Harazono and Kazunori (2005) tried to decolorize mixtures of four reactive textile

dyes, including azo and anthraquinone dyes, by a white-rot basidiomycete Phanerochaete

sordida. According to them, P. sordida decolorized dye mixtures (200 mgl/1 each) by

90% within 2 days in presence of nitrogen-limited glucose–ammonium media.

Decolorization of dye mixtures needed Mn2+ and Tween 80 in the media. Manganese

peroxidase (MnP) played a major role in dye decolorization by P. sordida. Decolorization

of dye mixtures by P. sordida was partly inhibited by polyvinyl alcohol (PVA) which

was normally found in textile waste water. This was caused by an inhibitory effect of

PVA on the decolorization of Reactive Red 120 (RR120) with MnP reaction system.

Second addition of Tween 80 to the reaction mixtures in the presence of PVA improved

the decolorization of RR120. These results suggest that PVA could interfere with lipid

peroxidation or subsequent attack the dye.

Echlerova´et al. (2005) carried out screening of thirty different white rot strains

for Orange G and Remazol Brilliant Blue R decolorization on agar plates. They used

three best strains, Dichomitus squalens, Ischnoderma resinosum and Pleurotus

calyptratus for decolorization study in liquid media. Result showed, all three strains

efficiently decolorized both Orange G and RBBR, but they di.ered in decolorization

capacity depending on cultivation conditions and ligninolytic enzyme production. Two

different decolorization patterns were found in these strains: Orange G decolorization in

I. resinosum and P. calyptratus was caused mainly by laccase, while RBBR

decolorization was effected by manganese peroxidase (MnP); in D. squalens laccase and

MnP synergatic effect in the decolorization processes.


41

Mazmanci and Unyayar (2005) investigated the the decolorisation of Reactive

Black 5 by immobilised Funalia trogii was investigated. Reported results shwed, cultures

of F. trogii immobilised on Luffa cylindrica sponge could effectively decolourise the dye.

The effect of mycelial age was also studied, and decolourisation rate of a 3-day-old age

culture was higher (8.22 mg dye/g dmw day) than those of 0- and 6-day-old cultures

(6.86 and 7.80 mg dye/g dmw day). Macroscopic and microscopic examinations showed

that dye was not biosorbed on the fungal mycelium. The growth of F. trogii was inhibited

by all tested dye concentrations with compared to controls but this effect was minimised

when the fungus was completely immobilised on the sponge. Using optimal mycelial age,

cultures of L. cylindrica sponge were tested for their ability for dye decolorization at

different initial concentration. The kinetic parameters of decoloziation were calculated

according to Lineweaver–Burk plots (Km of 106.04 mg dye/l and Vmax of 117.64 mg

dye/l day).

Palmieri et al. (2005) investigated decoloriztion of the recalcitrant dye Remazol

Brilliant Blue R by the fungus basidiomycete Pleurotus ostreatus. According to them,

P. ostreatus is able to decolorize RBBR on agar plate. When grow in liquid media

supplemented with veratryl alcohol, the fungus completely decoloizes RBBR in 3 days.

In these conditions, P. ostreatus produces among other enzymes, laccases, vreratryl

alcohol oxidase and dye-decolorizing peroxidase but only laccases seemed to be

responsible of RBBR transformation. Two purified laccases (POXC and POXA3) were

found able to degrade RBBR in vitro, in the absence of any redox mediators. These

laccases differ significantly in their efficiency of decolorization of the tested dye, as

suggested by comparison of their catalytic efficiency (kcat/Km values) towards RBBR.

Furthermore, using a mixture of both POXC and POXA3 a remarkable improvement in

the reaction rate and in the final level of dye decolorization was observed. The extent of

RBBR decoloriztion by laccase mixture also depended on incubation temperature and

enzyme concentration. The dye was decolorized by laccase isoenzymes most efficiently

under acidic conditions. Treatment of RBBR with the laccase mixture reduced its toxicity

by 95%.


42

Pazarlioglu et al. (2005) studied in vitro and in vivo biodecolorization of nine

different direct azo dyes by Phanerochaete chrysosporium immobilized on ZrOCl2-

activated pumice in stationary cultures. After investigation it was found that no lignin

peroxidase activity was detected in the extracellular medium of P. chrysosporium. In

order to support dye degradation, ligninolytic culture filtrate from fungus, containing

mainly manganese peroxidase, was treated with dye. Direct Blue 15 (DB15, 120 mg/l)

was determined as the best decolourized dye and its decolorization by immobilized P.

chrysosporium was studied in a small-scale packed-bed reactor (PBR). The colour

removal efficiency in repeated batches was found as 95–100%. Kinetic analysis of

enzymatic decolourization of DB15 indicate that the process is time dependent and

follows first-order kinetics with respect to initial concentrations of dye. The rates of

colour removal (k values) decrease to a significant extent with increasing

initialconcentrations of dye. Moreover, this decolorization process it was observed that

MnP played an important role while there was no obvious role for LiP and adsorption

was determined as a minor mechanism in decolourizing DB15.

Radha et al. (2005) worked on decolorization of synthetic dyes using

Phanerochaete chrysosporium. It was a commonly used white-rot fungus able to degrade

several synthetic dyes of varying structures, namely Azo, Anthraquinone, Thiazine and

Vat dyes. The decolorization potential of P. chrysosporium for seven dyes namely, Acid

Orange, Congo Red, Methyl Violet, Vat Magenta, Acid Red 114, Methylene Blue and

Acid Green was studied. Studies were carried out using free cells and fungal cell

entrapped Calcium alginate beads of different sizes. The kinetics parameters K dye and V

dye max for the decolorization process for all the seven dyes were estimated..

Shrivastava et al. (2005) carried out his research on enzymatic decolorization of

sulfonphthalein dyes. Tthe white rot fungus (WRF) Pleurotus ostreatus produced

manganese peroxidase (MnP) and manganese-independent peroxidase (MIP) activities

during solid state fermentation of wheat straw, a natural lignocellulosic substrate. Most

of the sulfonphthalein (SP) dyes were decolorized by MnP at pH 4.0. The higher Km for

meta-cresol purple (40_M) and lower Km for ortho-cresol red (26_M) for MnP activities


43

illustrated the preference for the position of methyl group at ortho than at meta

chromophore. Bromophenol blue decolorizing activity was higher at pH 3.5 and

decreased as the concentration of MnII was increased. SP-decolorizing activity was

associated not only withnP but also with MIP. Additional bromine group along with the

methyl group on SP chromophores decreases the rate of decolorization.Bromination of

sulfonphthalein chromophore makes them the poorer substrate for MnP. This is evident

from the higher Km for bromocresoleen (117 _M) when compared to bromocresol purple

(36 _M) and bromophenol blue (78 _M). The order of preference for the SP dyes as

substrate for the MnP-catalyzed decolorizing activity is phenol red > ortho-cresol red >

meta-cresol purple > bromophenol red > bromocresolpurple > bromophenol blue >

bromocresol green and the order of preference for the SP dyes as substrate for the MIP-

catalyzed decolorizing activity is bromocresol green > bromophenol blue > bromocresol

purple > bromophenol red > meta-cresol purple > ortho-cresol red > phenolred.

.Restricted decoloriztion of PR by NaN3 proved that decolorizing activity was an

oxidative process.

Asgher et al. (2006) ued four white rot fungi, Phanerochaeta chrysosporium,

Coriolus versicolor, Gonoderma lucidium and Pleurotus ostreatus for decolorization of

Drimarene Orange K-GL, Remazol Brilliant Yellow 3GL, Procion BluePX-5R and

Cibacron Blue P-2RGR for 10 days in shake flasks. Samples were removed every day,

centrifuged and the absorbances of the supernatants were read to determine percentage

decolorization. It was observed that P. chrysosporium and C. versicolor could effectively

decolorize Remazol Brilliant Yellow 3GL, Procion BluePX-5R and Cibacron Blue P-

2RGR. Drimarene Orange K-G2 was completely decolorized (0.2 g/l after 8 days) only

by P. chrysosporium, followed by P. ostreatus (0.17 g/l after 10 days). P. ostreatus also

showed good decolorization efficiencies (0.19-0.2 g/l) on all dyes except Remazol

Brilliant Yellow (0.07 g/l after 10 days). G. lucidium did not decolorize any of the

dyestuffs to an appreciable extent except Remazol Brilliant Yellow (0.2 g/l after 10

days).

Chairattanamanokorn et al. (2006) detected synthesis of lignin peroxidase,


44

manganese peroxidase MnP, and laccase, decolorization, and removal of total phenol and

chemical oxygen demand to select a thermotolerant fungal strain for decolorization of

wastewaters.They found that thirty-eight fungal strains were used for enzyme production

at 35 and 43 degrees C on modified Kirk agar medium including 2,2'-azino-bis (3-

ethylbenzothiazoline-6-sulfonic acid) (ABTS) and MnCl2. According to them, “thirteen

strains grew on manganese-containing agar and provided green color on ABTS-

containing agar plates under culture at 43 degrees C. Decolorization of wastewater from

alcohol distillery (WAD) by these strains was compared under static culture at 43

degrees C, and Pycnoporus coccineus FPF 97091303 showed the highest potential.

Thereafter, immobilized mycelia were compared with free mycelia for WAD

decolorization under culture conditions of 43 degrees 0C and 100 rpm. The immobilized

mycelia on polyurethane foam enhanced the ligninolytic enzyme production as well as

total phenol and color removal. At about the same COD removal, MnP and laccase

produced by immobilized mycelia were 2 and 19 times higher than by free mycelia; the

simultaneous total phenol and color removal were 3.1 and 1.5 times higher than the

latter. Moreover, decolorization of synthesis dye wastewater was carried out at 43

degrees C and 100 rpm. More than 80% of 300 mg/L of reactive blue-5 was decolorized

by the immobilized mycelia within 1 to 2 d for four cycles”.

Cho et al. (2006) investigated the role of laccase from white-rot fungi, Cerrena

unicolor and Trametes versicolor, with low molecular weight mediators, acetovanillone

(AV) and acetosyringone (AS), in decolorizing of the textile dye, Chicago sky blue 6B

(Direct Blue 1, DB1). Result showed, “the tested fungi were shown the ability to remove

colour form DB1. The decolorizing activity of the dye was closely related to the laccase

from fungi. In the presence of AV or AS as co-substrate, decolorization was more

extensive than that of the control. Moreover, at the presence of AS, the decolorization

process was more effective than AV. The highly purified laccase of C. unicolor and T.

versicolor also decolorized the dye. The addition of AV or AS enhanced this process.”

D’Souza et al. (2006) reported the decolorization of textile and dye-making

industries and alcohol distilleries effluents by a marine fungal isolate, NIOCC # 2a


45

cultured from decaying mangrove wood. According their result, “the fungus also

decolorized several synthetic dyes. Laccase was the most dominant lignin-degrading

enzyme produced by this fungus with very low activities of manganese-dependent

peroxidase and no lignin peroxidase activity. The growth and production of laccase was

best in a medium prepared with seawater having salinity in the range of 25-30 ppt. The

pH optimum for the laccase activity was 3.0 and 6.0 and the temperature optimum was

60oC. Laccase production was increased in the presence of phenolic and non-phenolic

inducers. A several fold enhancement in laccase production was found during treatment

of colored effluents form textile, paper and pulp mill and distillery waste. Industrial

effluents and synthetic dyes added to the growing culture of this fungus were decolorized

to a great extent. The culture supernatant without the fungal biomass was also effective

in decolorization of these effluents to various degrees within 6 h of incubation.

Extracellular polymeric substances (EPS) produced by this fungus were also useful in

decolorization of these effluents. Thus, efficiency of this fungus in decolorization of

various effluents with laccase that is active at pH 3.0 and 3.0 and 60oC in the presence of

seawater has great potential in bioremediation of industrial effluents. Enhanced laccase

production in the presence of industrial effluents in this fungus is an added advantage

during bioremediation of effluents.”

Eichlerova et al. (2006) tested eight different Pleurotus species for the Orange G

and Remazol Brilliant Blue R decolorization capacity and their ligninolytic properties.

According to them, “all the species produced laccase and manganese peroxidase (MnP)

and in five species we found aryl-alcohol oxidase (AAO) activity. Strain CCBAS 461 of a

very little studied species Pleurotus calyptratus was chosen for a more detailed study.

This strain produced a relatively high amount of Lac, MnP and also AAO. Within 14 days

the strain decolorized up to 91% of Orange G and 85% of RBBR in liquid culture and

more than 50% of these dyes on agar plates. P. calyptratus is able to decolorize

efficiently also other azo and phthalocyanine dyes, but only a limited decolorization

capacity was found in the case of polyaromatic and triphenylmethane dyes.Lac and MnP

production was strongly influenced by the kind of cultivation media and by the dye

present.”


46

Frijters et al. (2006) have worked on decolorization and detoxification of textile

wastewater, containing both soluble and insoluble dyes, in a full seale combined

anaerobic/aerobic system. Their result showed, “the colour was largely removed in the

pre-acidification basin and the anaerobic reactor. Colour deriving from both reactive as

well as disperse, was anaerobically removed, indicating that these types of dyes were

reduced to colorless products. Apparently the anaerobic system is capable of effectively

removing the colour of both soluble and insoluble dyes.”

Gao et al. (2006) used white rot fungus Phanerochaete chrysosporium to degrade

reactive brilliant red K-2BP dye under non-sterile conditions. The result showed, “ there

was no decolorizing effect under non-sterile condition if white rot fungus was incubated

under non-sterile condition, and the decolorization was always near to 0% during

decolorizing test for 3 d; in the meantime, a lot of yeast funguses were found in liquid

medium when white rot fungus was incubated under non-sterile conditions; however, if

white rot fungus was incubated under sterile condition firstly, its decolorization was

above 90% under non-sterile condition, which was similar to that of sterile condition.”

Kamei et al. (2006) carried out degradation experiment of model polychlorinated

biphenyl compound 4,4’-dichlorobiphenyl (4,4’-DCB) and its metabolites by

Phanerochaete chrysosporium and newly isolated 4,4’-DCB-degrading white-rot fungus

strain MZ142. According to them, “although P. chrysosporium showed higher

degradation of 4,4’-DCB in low-nitrogen (LN) medium than that in potato dextrose broth

(PDB) medium, Phanerochaete sp. MZ142 showed higher degradation of 4,4’-DCB

under PDB medium condition than that in LN medium. The metabolic pathway of 4,4’-

DCB was elucidated by the identification of metabolites upon addition of 4,4’-DCB and

its metabolic intermediates. 4,4’-DCB was initially metabolized to 2-hydroxy-4,4’-DCB

and 3-hydroxy-4,4’-DCB by Phanerochaete sp. MZ142. On the other hand, P.

chrysosporium transformed 4,4’-DCB to 3-hydroxy-4,4’-DCB and 4-hydroxy-3,4’-DCB

produced via a National Institutes of Health shift of 4-chlorine. 3-Hydroxy-4,4’-DCB was

transformed to 3-methoxy-4,4’-DCB; 4-chororbenzoic acid; 4-chlorobenzaldehyde; and

4-chlorobenzyl alcohol in the culture with Phanerochaete sp. MZ142 or P.


47

chrysosporium. LN medium condition was needed to form 4-chlorobenzoic acid, 4-

chlorobenzaldehyde, and 4-chlorobenzyl alcohol form 3-hydrozy-4,4’-DCB, indicating

the involvement of secondary metabolism. 2-Hydroxy-4,4’-DCB was not methylated. In

this paper, we proved for the first time by characterization of intermediate that

hydroxlylation of PCB was a key step in the PCB degradation process by white-rot

fungi”.

Koray et al. (2006) evaluated the potential of a recently isolated wood-degrading

fungus, Trichophyton rubrum LSK-27, for efficient decolorization of textile azo dyes was

evaluated. According to Korey and his fellows, “within two days of dye addition, the

fungus was able to decolorize 83% of Remazol Tiefschwarz, 86% of Remazol Blue RR

and 80% of Supranol Turquoise GGL in liquid cultures. The reactive dyes, Remazol

Tiefschwarz and Remazol Blue, were removed by fungal biodegradation, while

decolorization of the acid dye, Supranol Turquoise GGL, was accomplished mainly by

bio adsorption. Therefore the fungus proved to be E. ciently capable of both

biodegradation and biosorption as the major dye removal mechanisms. The extent of

biodegradation was associated with the levels of the extracellular ligninolytic enzymes

such as manganese peroxidase and laccase.”

Kumar et al. (2006) have dicscussed in his present study the decolorisation,

biodegradation and detoxification of Direct Black-38, a benzidine based azo dye, by a

mixed microbial culture isolated from an aerobic bioreactor treating textile wastewater.

According to their research study, “a biotransformation of Direct Black-38 into benzidine

and 4-aminobiphenyl followed by complete decolorisation and biodegradation of these

toxicintermediates. From cytotoxicity tudies, it was concluded that detoxication of the dye

took place after degradation of the toxic intermediates by the culture.”

Lorenzo et al. (2006) have studied the effect of heavy metals on the production of

several laccase isoenzymes by Trametes versicolor and on their ability to decolourise

dyes. They said, “the white-rot fungus Trametes versicolor growing in submerged culture

on a basal medium, with barley bran as a carbon source, produced two laccase

isoenzymes LacI and LacII. The addition of metal ions to the culture medium was


48

performed to improve the total laccase activity and to determine the e.ect on the

production of laccase isoenzymes.From all the tested metals, only Cu2+ increased

laccase activity (up to 12-fold with respect to control cultures) and T. versicolor in

presence of all metals produced the two isoenzymes in di.erent proportion with ratios of

activity (LacI/LacII) varying between 0.11 and 0.51. This factor played an important role

in the decolourisation of the textile dye Indigo Carmine.”

Mohorcici et al. (2006) have concentrated their research on fungal and enzymatic

decolorisation of artificial textile dye baths. Dcolorizing ability of 25 strain was

investigated on a textile dye Reactive Black 5Result showed, “the most promising strains

were tested in a medium containing species constituents of a dye bath in order to

approach real application conditions. It was shown that the concentrations of the

constituents had to be reduced to allow fungal growth. Decolourisation started in

cultures of Geotrichum candidum but was not complete. Only Bjerkandera adusta was

able to decolourise the black-blue colour through violet and red to pale yellow. After 17

days spectral absorption coefficients, a, at three wavelengths, 620, 525 and 436 nm

almost reached the permitted values. A partly purified manganese peroxidase prepared

from B. adusta was tested for decolourisation of several artificial dye baths. The

constituents seemed not to be inhibitory to the enzyme and no dilution was necessary.

Evaluation of decolourisation gave different results, depending on the method used. The

most efficient decolourisation on a percentage basis was observed in the dye bath of the

anthraquinone dye Reactive Blue 19, followed by the diazo dye Reactive Black 5.

However, based on absorbance units, the largest reduction was achieved with the

Reactive Black 5 and Acid Orange 7 dye baths. Comparing the a values after 120 h

fungal and enzymatic treatments of Reactive Black 5 dye bath the enzyme showed about

1.5 times greater colour reduction than the fungus. Given the tolerance to the

constituents and concentration of dye baths, the enzyme proved to be a promising tool for

their treatment”.

López et al. (2006) applied ligninolytic microorganisms isolated from composting

environment. The decolorization of Remazol Brilliant Blue R, Poly R-478 and Poly S-


49

119 by fungi isolated from composting piles and the relationship of this ability to other

ligninolytic activity tests was analyzed. According to them, “ Poly S-119 decolorizing

capability was the most widespread among all tested microorganisms, followed by poly

R-478, with RBBR being the most difficult to decolorize. Decolorizing ability was

Fstrongly correlated to ligninase production according to Sundman test. However, no

relationship was found between this capability and laccase, tyrosinase, oxidase,

polyphenoloxidase or peroxidase activities. Three mesophile fungi, one thermophile

fungus, and one bacterium were able to significantly decolorize all the dyes. The

mesophile fungi had the higher decolorization efficiency with more than 95% for Poly R-

478 and Poly S-119 and more than 50% for RBBR. The thermophile fungus decolorized

12, 14 and 40% of Poly R-478, RBBR and Poly S-119, respectively, and the bacterium

decolorized 24, 45 and 96% of Poly R-478, RBBR and Poly S-119, respectively. This is

the first report of non-filamentous aerobic bacteria showing dye decolorization in axenic

culture. These microorganisms can be potential candidates for use in biodecolorization

processes”.

Murugesan et al. (2006) have isolated and purified that an extracellular laccase

from Pleurotus sajor-caju grown in submerged culture in a bioreactor, and used to

investigate its ability to decolorize three azo dyes. Their research findings showed, “the

extracellular laccase production was enhanced up to 2.5-fold in the medium amended

with xylidine (1 mM). Purification was carried out using ammonium sulfate (70% w/v),

DEAE-cellulose, and Sephadex G-100 column chromatography. The enzyme was purified

up to 10.3-fold from the initial protein preparation with an overall yield of 53%. The

purified laccase was monomeric with an apparent molecular mass of 61.0 kDa. The

purified enzyme exerted its optimal activity with 2,2-azino-bis(3-ethylbenzothiazoline-6-

sulfonate (ABTS) and oxidized various lignin-related phenols. The catalytic efficiencies k

cat/K m determined for ABTS and syringaldazine were 9.2×105 and 8.7×105,

respectively. The optimum pH and temperature for the purified enzyme was 5.0 and

40°C, respectively. Sodium azide completely inhibited the laccase activity. The

absorption spectrum revealed type 1 and type 3 copper signals. The purified enzyme

decolorized azo dyes such as Acid Red 18, Acid Black 1, and direct blue 71 up to 90, 87,


50

and 72%, respectively. Decolorization ability of P. sajor-caju laccase suggests that this

enzyme could be used for decolorization of industrial effluents”.

Renganathan et al. (2006) studied the role of growing Schizophyllum commune in

Accumulation of Acid Orange 7, Acid Red 18 and Reactive Black 5.They studied the

effect on growth and decolorizing properties of Schizophyllum commune using Acid

Orange 7, Acid Red 18 and Reactive Black 5 with respect to the initial pH varying from

1 to 6 and initial dye concentration (10–100 mg/L). The optimum pH valuewas found to

be 2 for both growth and color removal of these azo dyes. Increasing the concentration of

azo dyes inhibited the growth of S. commune. According to them, “ it was observed that

S. commune was capable of removing Acid Orange 7, Acid Red 18 and Reactive Black 5

with a maximum species uptake capacity of 44.23, 127.53 and 180.17 (mg/g) respectively

for an initial concentration of 100 mg/L of the dye. Higher decolorization was observed

at lower concentrations for all the dyes. Finally it was found that the percentage

decolorization was more in the case of Reactive Black 5 dye compared to the other two

dyes used in the present investigation”.

Romero et al. (2006) have used Trametes versicolor pellets were in a pulsed

fluidised bioreactor with laccase production media to decolourise the textile dye Grey

Lanaset G. According to them, “the effect of the enzyme on dye degradation was

analysed. In a batch process, degrading the dye with the fungus results in a

decolorization percentage higher than90%(initial dye concentration 150 mg/L) while the

results were lower than35%using enzymatic degradation. Although most of the

decolorization was initially due to an adsorption process, later on in both phases, the

biomass and the culture broth became colourless. In order to check the possibility of

improving the degradation capacity in the batch mode operation, after decolourising the

initial dye solution, different pulse dye adding strategies were tested. Adding a large

pulse resulted in fast enzymatic deactivation while adding a small pulse caused the

system to operate below its optimum degradation capacity. There was a close correlation

between the amount of laccase produced and the amount of dye degraded. The system

worked in a bioreactor for one month without any operational problems. Finally, in the


51

continuous mode the dye degradation has been demonstrated because it is possible to

maintain the continuous production of the laccase enzyme.”

Sanghi et al. (2006) have found that the white rot fungus Coriolus versicolor

could decolorize reactive dye Remazol Brilliant Violet to almost 90%. According to their

result, “ fungal mycelia removed color as well as COD up to 95% and 75%, respectively,

in a batch reactor. Decolorizing activity was observed during the repeated reuse of the

fungus. It was possible to substantially increase the dye decolorising activity of the

fungus by carefully selecting the operational conditions such as media composition, age

of fungus and nitrogen source. The fungal pellets could be used for eight cycles during

the long term operation, where medium and dye was replenished at the end of each cycle

and the fungus was recycled. Presence of a nitrogen source and nutrient content of media

played an important role in sustaining the decolorisation activity of the fungus. The form

of nitrogen source (e.g. peptone vs. urea) was also important to maintain the decolorising

activity with peptone showing better decolorization.”

Yesiladalý et al. (2006) have discussed the Bioremediation of textile azo dyes by

Trichophyton rubrum LSK-27.According to them, “the potential of a recently isolated

wood-degrading fungus, Trichophyton rubrum LSK-27, for effective decolorization of

textile azo dyes was evaluated. Within two days of dye addition, the fungus was able to

decolorize 83% of Remazol Tiefschwarz, 86% of Remazol Blue RR and 80% of Supranol

Turquoise GGL in liquid cultures. The reactive dyes, Remazol Tiefschwarz and Remazol

Blue, were removed by fungal biodegradation, while decolorization of the acid dye,

Supranol Turquoise GGL, was accomplished mainly by bioadsorption. Therefore the

fungus proved to be efficiently capable of both biodegradation and biosorption as the

major dye removal mechanisms. The extent of biodegradation was associated with the

levels of the extracellular ligninolytic enzymes such as manganese peroxidase and

laccase.”

Yu et al. (2006) carried out the production of the ligninolytic enzymes at C/N

rations of 28/44 mM and 56/2.2 mM in nonimmersed liquid culture of P.chrysosporium. I

According to them, “decolorization of one industrial azo dye, Reactive brilliant red K-


52

2BP, by crude lignin peroxidase (LiP) and manganese peroxidase (MnP) obtained under

carbon and nitrogen limitation respectively, was examined invitro conditions and

compared for their degradation characteristics. Both decolorization by LiP and MnP

were sensitive to pH, peaking around pH 3.0, and improved at higher enzyme activities.

Decolorization by LiP can be enhanced to the greatest degree (83%) with higher addition

of H2O2 and veratryl alcohol, whereas decolorization by MnP was optimized only with a

suitable dose of H2O2 (0.1 mM) and decreased by the addition of Mn2+. Decolorization

declined at high dye concentration; LiP was able to decolorize a dye concentration of 60

mg/l and below to no less than 85%, and MnP of 10mg/l to a maximum of 71%.

Decolorization by LiP and MnP together was somewhat lower than that by LiP alone. It

is suggested that the optimization of the H2O2 supply was mainly responsible for a higher

efficiency in continuous dye degradation by crude LiP and MnP.”

Zhao et al. (2006)have investigated the biodegradation of a model azo disperse

dye by the white rot fungus Pleurotus ostreatus Disperse Orange 3, 4-(4-

nitrophenylazo)aniline, by the white-rot fungus Pleurotus ostreatus grown in submerged

culture under controlled conditions. According to them, “degradation was investigated

using a commercial preparation of Disperse Orange 3 that contained 20% dye plus

dispersing agents, and an high-performance liquid chromatography purified preparation

of the dye. The metabolites generated by Pleurotus ostreatus were identified as 4-

nitroaniline, 4-nitrobenzene, 4-nitrophenol, and 4-nitroanisole. Veratryl alcohol, a redox

mediator for lignin peroxidase of white-rot fungi, and its oxidant veratraldehyde were

also detected in cultures grown in the presence of Disperse Orange 3. 4-Nitroanisole was

the major metabolite when 4-nitrophenol was incubated with Pleurotus ostreatus. Kinetic

profiles of these degradation products were determined and a partial degradation

pathway is proposed”.

Aksu et al .(2007) have studied the inhibitory effects of chromium(VI) and

Remazol Black B on chromium (VI) and dyestuff removals by Trametes versicolor many

dye-bearing wastewaters also contain heavy metal ions. According to them, “ although

the decolorization of single reactive dye or the uptake of single heavy metal ions by

various growing cells has been extensively studied, very little attention has been given to


53

the simultaneous bioremoval of reactivedye-metal ion systems. In this study, the single

and combined effects of chromium(VI) and Remazol Black B reactive dye on the

chromium(VI) anddye removal properties of adapted Trametes versicolor, a white-rot

fungus, was investigated in a batch system at different levels of chromium(VI)and dye.

Removal studies were performed at an initial pH of 4.0. Chromium(VI) uptake studies

were carried out in two different growth media;mainly containing glucose and reduced

quantity of glucose and whey. As the maximum microbial chromium(VI) uptake was

accomplished inglucose + whey medium, single dye and binary dye-hromium(VI)

bioremoval studies were also performed in this culture medium. Although thesingle

removal of chromium(VI) and dye was enhanced with increasing initial concentration of

each component up to 30 mg l-1 for chromium(VI)and up to 400 mg l-1 for dye, in

general the presence of increasing concentrations of chromium(VI) ions much more

severely inhibited the dyebioremoval by T. versicolor. While single chromium(VI) uptake

efficiency was 32.2% in 30 mg l-1 chromium(VI) containing growth medium andsingle

dye removal percent was 77.0% in 400 mg l-1 dye-bearing growth medium, the fungus

was only capable of 10.8 and 13.3% removals of chromium(VI) and Remazol Black B

dye, respectively, in the growth medium containing the binary mixture of these

components at the above concentrations.”

Chander and Arora (2007) have tested white-rot fungi, Dichomitus squalens,

Daedalea flavida, Irpex flavus and Polyporus sanguineus for their potential to decolorise

various chromophoric groups of eight dyes, employed in different industries. According

to them, “the fungal-based biocleaning systems have been suffering from drawback of

adsorption, thus, in order to overcome this limitation, the cell free enzyme extracts

obtained from fungal cultures have been used. D. squalens and I. flavus were found to be

competitive industrial dye decolourisers in comparison to much studied white-rot fungus

P. chrysosporium.”

Eichlerova et al. (2007) have investigated the decolorization of high

concentrations of synthetic dyes by the white rot fungus Bjerkandera adusta strain

CCBAS 232. Result showed, “among thirty different basidiomycetes screened,

Bjerkandera adusta strain CCBAS 232, Phanerochaete chrysosporium strain CCBAS


54

571and Pleurotus ostreatus strain CCBAS473 under these conditions. Unlike P.

chrysosporium and P. ostreatus, B. adusta was able to efficiently decolorize all tested

dyes. These properties and also a good ligninolyticenzyme production predetermine B.

adusta strain CCBAS 232 for biotechnological applications.”

Erkurt et al (2007) studied the decolorization of synthetic dyes involving laccase

enzymes in the process. According to them, “decolorization of Remazol Brilliant Blue

Royal and Drimaren Blue CL-BR was investigated using three white rot fungi named as

P. ostreatu,C. versicolo, an F.trogii. Decolorization studies were continued for 48 h

under static conditions at 30º C and pH 5.0. They anlysed the degree of pH, dry mycelium

weight (DMW), dye concentration, laccase activity and protein content and the enzyme

responsible for decolorization was detected for both dyes. Maximum and minimum

decolorizations were obtained by F. trogii and P. ostreatus, respectively. Result showed,

“ both dyes at all concentrations were found to be toxic for P. ostreatus growth, whereas

only DB above 60 mg/L was found to be toxic for C. versicolor growth. Maximum and

minimum laccase activities were detected in decolorization media of F. trogii and P.

ostreatus, respectively. Results of activity staining following SDS-PAGE showed that

laccase is the only enzyme that isresponsible for decolorization of DB and RBBR.”

Hamedaani et al. (2007) carried out the decolorization of 12 different azo, diazo

and anthraquinone dyes using a new isolated white rot fungus, strain L-25. According to

them, “a decolorization efficiency of 84.9-99.6% was achieved by cultivation in 14 days

using an initial dye concentration of 40 mg L-1. The strain L-25 produces manganese

peroxidase (MnP) as its a major ligninolytic enzyme. The adsorption of dye by cells was

observed during the decolorization at the beginning of the process. However, this color

disappeared when MnP was released by the strain L-25. The activity of MnP in the

cultures was over 1.0 U mL-1 at the end of cultivation. Meanwhile, MnP produced by

strain L-25 was used for the enzymatic decolorization of the dyes thus confirming the

capability of the enzyme for this purpose.”

Kokol et al. (2007)investigated that Ischnoderma resinosum produced

extracellular ligninolytic enzymes laccase and MnP. Accoriding to them, “the activity of


55

laccase achieved the maximum on day 10 (29.4 U L−1), the MnP on day 14 (34.5 U L−1).

Laccase and Mn-peroxidase were purified from the culture liquid using gel permeation

and ion-exchange chromatographies. Purified Mn-peroxidase performed decolorization

of all textile dyes tested (Reactive Black 5, Reactive Blue 19, Reactive Red 22 and

Reactive Yellow 15). Laccase was inactive with Reactive Black 5 and Reactive Red 22,

while all dyes were decolorized after addition of the redox mediators violuric acid (VA)

and hydroxybenzotriazole (HBT). The culture liquid from I. resinosum cultures was also

able to decolorize all dyes as well as the synthetic dyebaths in the presence of VA and

HBT. The highest decolorization rates were detected in acidic pH (3–4)”.

Murugesan et al. (2007) applied response surface methodology (RSM) for the

decolourization of the azo dye reactive black 5 (RB-5) using purified laccase from a

white rot fungus Pleurotus sajor-caju. According to their observation, “that the presence

of 1- hydroxybenzotriazole (HBT) is essential for decolourization of RB-5 by purified

laccase from P. sajor-caju. BoxeBehnken design using RSM with four variables namely

dye (25e100 mg l_1), enzyme (0.5e 2.5 U ml_1), redox mediator (0.5e1.5 mM)

concentrations and incubation time (24e48 h) was employed in this study to optimize

significant correlation between the effects of these variables on the decolourization of

RB-5. The optimum concentrations of dye, enzyme, HBT, and time were found to be 62.5

mg/L, 2.5 U/mL, 1.5 mM and 36 h, respectively, for maximum decolourization of RB-5

(84.4%). A quadratic model was obtained for dye decolourization through this design.

The experimental values were in good agreement with predicted values and the model

was highly significant, the correlation coefficient being 0.999. Increased decolourization

was observed with increase in enzyme concentration at lower dye concentration.

Interaction between HBT and dye concentrations was negligible. The optimization of

HBT WAS independent of dye concentration.”

Parshetti et al.(2007) studied the biodegradation of Reactive blue-25 by

Aspergillus ochraceus NCIM-1146.The present study dealt with the decolorization and

degradation of textile dye Reactive blue-25 (0.1g/L) by mycelium of Aspergillus

ochraceus NCIM-1146. According to their findings, “spectrophotometric and visual


56

examinations showed that the decolorization was through fungal adsorption, followed by

degradation. Shaking condition was found to be better for complete and faster adsorption

(7 h) and decolorization (20 days) of dye Reactive blue-25 (100 mg/l) as compared to

static condition. Presence of glucose in medium showed faster adsorption (4 h) and

decolorization of dye from bound (7 days) mycelium. FTIR and GCMS analysis study

revealed biodegradation of Reactive blue-25 into two metabolites phthalimide and di-

isobutyl phthalateose.”

Revankar and Lele (2007) investigated decolorization of dyes by a white rot

fungus isolated from bark of dead tree, WR-1 identified as Ganoderma sp. The

fermentation medium was optimized using a combination of one factor at a time and

orthogonal array method. Result showed, “maximum decolorization (96%) of 100 ppm

amaranth was achieved in 8 h with optimized medium containing 2% starch and 0.125%

yeast extract. Rate of dye decolorization by the indigenous isolate Ganoderma sp. WR-1

was very high compared to the most widely used strains of Trametes versicolor and

Phanerochaete chrysosporium. The broad-spectrum decolorization efficiency of the

isolate was assessed using chemically different dyes. The isolate was further evaluated

for the decolorization of industrial effluent. Complete decolorization was achieved in 12

days.”

Šušla et al. (2007) have focused their tudy on the production of ligninolytic

enzymes and dye degradation capacity of Dichomitus squalens immobilized on

polyurethane foam or pine wood PW in a fixed bed reactor at a laboratory scale (working

volume of 0.6 l). According to them, “immobilization of fungal cultures on pine wood

improved eminently laccase production in comparison to the liquid cultures. Immobilized

D. squalens was able to decolorize an anthraquinone dye Remazol Brilliant Blue R and

an azo dye Reactive Orange 16, However, only a limited decolorization of

Copper(II)phthalocyanine dye was observed in both types of reactor cultures. The

involvement of a laccase activity in dye decolorization was suggested. Further, two

different chromatographical forms of laccases, Lc1 and Lc2, were isolated from PW

cultures of D. squalens using a fast, two step FPLC method. Enzymes revealed identical


57

molecular masses of 68 kDa (estimated by SDS-PAGE) and similar pI’s, however, they

differed in their catalytic properties such as pH dependence of the activity and ABTS

oxidation rates. In this study, we demonstrated different dye decolorization capacities of

Lc1 and Lc2 as well.”

Trovaslet et al;(2007) applied the frame of the development of a bioprocess using

the competences of White Rot Fungi for decolorization and detoxification of dye

contaminated effluents, laccases were produced by the strain Pycnoporus sanguineus

MUCL 41582 in a malt extract medium. According to the result of their study, “two

isoenzymes were detected among which LAC-1 was concentrated. A survey of the

composition of industrial effluents showed that waste waters from textile industry usually

contain high concentrations of Na2SO4 or NaCl. Regarding the activity profile of the

biocatalyst against pH, salts, temperature and target substrates, LAC-1 appears to be a

good candidate for application in acid dye bath treatments. Studying the model

anthraquinonic dye ABu62 decolourisation, we proved that this dye was a good substrate

for LAC-1. Furthermore, unusual kinetic behaviour was observed suggesting that LAC-1

was activated in presence of ABu62. On the contrary, a classical Michaelis-Menten

behaviour was observed for the oxidation of ABTS and LAC-1 showed a high affinity for

this substrate as compared to data available for other laccases. To our knowledge, this is

the first report showing this atypical behaviour of a laccase in the presence of dyes.”

Zhao and Hardin (2007) employed a white rot fungus Pleruotus ostreatus for

degradation of two commercially used disperse azo dyes, Disperse Orange 3 and

Disperse Yellow 3. UV-visible spectrophotometric method and high performance liquid

chromatography (HPLC) for decolorization and products from fungal degradation of

these azo dyes in liquid medium were determined Their decolorization studies showed,

“that both azo dyes were removed by more than 50% in 5 days and HPLC analysis

determined several degradation products. These results suggest that P. ostreatus has

potential in color removal from textile wastewater containing disperse.”

Asgher et al. (2008) explained optimization of medium for decolorization of Solar

golden yellow R direct textile dye by Schizophyllum commune IBL-06 he ability of two


58

new white rot fungi Schizophyllum commune IBL-06 and Ganoderma lucidum IBL-05 to

decolorize direct dye Solar golden yellow R was investigated using Kirk's basal salts

medium. according to them in the maximum decolorization (73%) of Solar golden yellow

R was caused by S. commune IBL-06 after 6 days of incubation at pH 4.5 and 35 °C.

Different parameters like incubation time, pH, temperature, and additional carbon and

nitrogen sources were optimized to achieve maximum decolorization of Solar golden

yellow R by S. commune IBL-06 in minimum possible time period. Supplementation of the

medium with additional carbon sources enhanced dye decolorization to a variable extent.

Addition of glucose (1%) gave the best results and caused a dramatic increase in

decolorization; complete decolorization (100%) of the dye was achieved after only 2 days

of incubation under optimum conditions. All the additional nitrogen sources showed an

inhibitory effect on enzyme induction and dye decolorization. Manganese peroxidase

(MnP) was found to be the major enzyme (764 U/ml) secreted by S. commune IBL-06

followed by laccase and only a minor activity of lignin peroxidase (LiP). The results

suggest an excellent potential of S. commune IBL-06 for dye decolorization that can be

enhanced by careful optimization of process parameters.”

Asghar et al. (2008) have worked on screening of five indigenous white rot fungi

Pleurotus ostreatus IBL-02 Phanerochaete chrysosporium IBL-03, Coriolus versicolor

IBL-04, Ganoderma lucidum IBL-05 and Schyzophyllum commune IBL-06 for

decolorization of four Vat dyes, Cibanon red 2B-MD, Cibanon golden-yellow PK-MD,

Cibanon blue GFJ-MD and Indanthrene direct black RBS. Result showed that the

screening experiment was run for 10 days with 0.01% dye solutions prepared in alkaline

Kirk’s basal nutrient medium in triplicate (250ml flasks). Every 48 hrs samples were read

on their respective wavelengths (λmax) to determine the percent decolorization. It was

observed that C. versicolor IBL-04 could effectively decolorized all the four Vat dyes at

varying incubation times but best results were shown on Cibanon blue GFJ-MD (90.7%)

after 7 days, followed by golden yellow (88%), Indathrene direct black (79.7%) and

Cibanon red (74%) respectively. Phanaerochaete chrysosporium also showed good

decolorization potential on Cibanon blue (87%), followed by Cibanon golden yellow

(74.8%), Red (71%), and Indathrene direct black (54.6%). However, rest of the strains


59

showed poor decolorization potential on four Vat dyes. C. versicolor showing maximum

decolorization of Cibanon blue GFJ-MD was therefore, selected for process

optimization. The effect of varying pH, temperature, initial dye concentration and

addition of carbon and nitrogen sources was investigated. Maximum decolorization

(98.5%) of 0.01% Cibanon Blue GFJ-MD could be achieved after 3 days at pH 5 and

30C temperature in the nutrient medium supplemented with 1% starch as additional

carbon source. All the supplementary nitrogen sources were found inhibitory to laccase

activity and dye decolorization. It was also noted that there was negligible adsorption of

the dye on fungal mycelia and laccase catalyzed biodegradation was the major

decolorization route”..

Blla´nquez et al.(2008) have illustrated the scale-up of an air pulsed bioreactor for

the continuous treatment of textile wastewater by of the white rot Trametes versicolor

has been carried out, based on the geometric similitude with lab-scale bioreactors (0.5

and 1.5 L). According to them, “decolourisation experiments of150 mg/ L Grey Lanaset

G dye solution carried out in the pilot-scale bioreactor showed that in both discontinuous

and continuous treatment withan HRT of 48 h, the decolourisation levels were higher

than 90%. Some operational changes were carried out in the continuous

decolourisationtreatment of the dye solution in order to adapt the process to industrial

conditions such as, non-sterilization of the dye solution, use of tap water instead of

distilled water plus macronutrients and micronutrients and the use of industrial quality

co-substrate instead of reagent grade. The pilotsystem was working continuously during

3 months and over 70 days without sterilization of the dye feeding solution, achieving

gooddecolourisation levels (78% average during the treatment). Continuous treatment of

real industrial textile wastewater under non-sterile conditionswas carried out during 15

days in the pilot-scale bioreactor, with colour reduction levels between 40 and 60%.

These dye concentrations areregarded as environmentally acceptable to be discharged

into a municipal wastewater treatment plant if necessary according to the local

regulation.”.

Ciullini et al (2008) have explained the combined action of fungal laccase,

cellobiose dehydrogenase, and chemical mediators during decolorization of different


60

classes of textile dyes. According the report presente by them, “ dyes belonging to the

mono-, di-, tri- and poly-azo as well as anthraquinonic and mono-azo Cr-complexed

classes, chosen among the most utilized in textile applications, were employed for a

comparative enzymatic decolorization study using the extracellular crude culture extracts

from the white rot fungus Funalia (Trametes) trogii grown on different culture media and

activators able to trigger different levels of expression of oxidizing enzymes: laccase and

cellobiose dehydrogenase. Laccase containing extracts were capable to decolorize some

dyes from all the different classes analyzed, whereas the recalcitrant dyes were subjected

to the combined action of laccase and the chemical mediator HBT, or laccase plus

cellobiose dehydrogenase. Correlations among the decolorization degree of the various

dyes and their electronic and structural diversities were rationalized and discussed. The

utilization of cellobiose dehydrogenase in support to the activity of laccase for the

decolorization of azo textile dyes resulted in substantial increases in decolorization for

all the refractory dyes proving to be a valid alternative to more expensive and less

environmentally friendly chemical treatments of textile dyes wastes”.

Gao et al. (2008) concluded that WRF were capable of oxidizing many persistent

organic pollutants including dyes. The fungi has limited application under non-sterile

conditions for waste water treatment due to contamination by bacteria and other

microorganism. Their findings showed , “they developed a treatment approach by using

immobilized white rot fungi Phanerochaete chrysosporium to degrade reactive dye K-

2BP under nonsterile condition. Four different inert carriers were tested for

immobilization of the white rot fungi with orthogonal experiments in comparison with

suspension culture. The activity of manganese peroxidase (MnP) was used for the

evaluation of oxidization performance in order to understand whether contamination of

bacteria and other micro-organisms was suppressed. Under non-sterile conditions, the

immobilized fungal cultures successfully restrained the growth of microzymes, coccies,

and bacillus but suspension culture was highly contaminated with poor MnP activity.

Under non-sterile conditions, higher MnP enzymatic activity (690 U/L vs. 125 U/L),

higher decolorization efficiency (93.5% vs. 15%) and shorter reaction period (3 days vs.

6 days) were achieved in immobilized cultures in comparison with suspension culture.


61

With the immobilized fungal cultures, no difference was observed under non-sterile and

sterile conditions for the degradation of reactive dye K-2BP.”

Jnghanns et al. (2008) did a comparative study of aquatic fungi and white rot

fungi for decoulorizing synthetic azo dyes and anthraquinone dyes. Research result

showed, “a total of 37 strains of aquatic hyphomycetes and 95 fungal isolates derived

from diverse freshwater environments were screened on agar plates for the

decolourisation of the disazo dye Reactive Black 5 and the anthraquinone dye Reactive

Blue 19. The decolourisation of 9 azo and 3 anthraquinone dyes by 9 selected aquatic

fungi was subsequently assessed in a liquid test system. The fungi were representatives of

mitosporic anamorphs, and 6 strains had proven ascomycete a.liations. For comparison,

5 white rot basidiomycetes were included. The majority of dyes were decolourised by

several mitosporic aquatic isolates at rates essentially comparable to those observed with

the most e.cient white rot fungus. Under certain conditions, particular aquatic strains

decolorised dyes even more e.ciently than the best performing white rot basidiomycete.

Upon fungal treatment of several dyes, new absorbance peaks appeared, ndicating

biotransformation metabolites. All together, these results point to the potential of fungi

occurring in freshwater environments for the treatment of dye-containing effluents.”

Kaushik and Anusheree (2008) reviewed perspectives and prospect of dyes

released by the textile industries pose a threat to the environmental safety. According

their review, “ dye decolorization through biological means has gained momentum as

these are cheap and can be applied to wide range of dyes. This review paper focuses on

the decolourization of dye wastewaters through fungi via two processes (biosorption and

bioaccumulation) and discusses the effect of various process parameters like pH,

temperature, dye concentration etc. on the dye removing efficiency of different fungi.

Various enzymes involved in the degradation of the dyes and the metabolites thus formed

have been compiled. Genetic manipulations of microorganisms for production of more

efficient biological agents, various bioreactor configurations and the application of

purified enzymes for decolourization, which constitute some of the recent advances in this

field, have also been reviewed. The studies discussed in this paper indicate fungal

decolourization has a great potential to be developed further as a decentralized


62

wastewater treatment technology for small textile or dyeing units. However, further

research work is required to study the toxicity of the metabolites of dye degradation and

the possible fate of the utilized biomass in order to ensure the development of an eco-

friendly technology.”

Lucas et al. (2008) have developed a microtitre plate-based for a fast screening of

numerous fungal strains for their ability to decolourise textile dyes. According to their

findings, “ this method allowed to estimate significant fungal decolourisation capability

by measuring the absorbance decrease on up to ten dyes. Morethan 325 white-rot fungi

(WRF) strains belonging to 76 fungal genera were compared with regards to their

capability to decolourise five azo and two anthraquinone dyes as well as the dyes

mixture. The most recalcitrant dyes belonged to the azo group. Several new species

unstudied in the bioremediation field were found to be able to efficiently decolourise all

the dyes tested.”

Nozaki et al. (2008) have worked on the screening and investigation of dye

decolorization activities of basidiomycetes. The decolorization trend of industrial dyes

was investigated using enzymes produced by 21 basidiomycetes, mainly edible

mushrooms. Result showed, “among the 27 dyes used in this study, nine were

decolorized by over 40%. Most fungi decolorized Acid Orange 20, but they showed

different specificities in the case of the other dyes. Determination of activity staining by

native polyacrylamide gel electrophoresis revealed that all the decolorization activities

corresponded to ABTS.”

Vanhulle et al. (2008) have revealed in his work that coupling reactions occurred

before breakdown during biotransformation of Acid Blue 62 by white rot fungi. There is

a few data existed on the metabolites produced during the biotransformation of

anthraquinonic dyes by (WRF).According to them, “during the biotransformation of an

anthraquinonic dye Acid Blue 62 (ABu62) using Pycnoporus sanguineus MUCL 41582

strain, it was previously demonstrated that the blue colour of the medium turned to red

before complete dye decolourisation. To better understand the phenomenon, this study

carried out ABu62 biotransformation with .ve di.erent WRF strains (Coriolopsis


63

polyzona MUCL38443, Perenniporia ochroleuca MUCL 41114, Perenniporia

tephropora MUCL 41562, P. sanguineus MUCL 38531 and Trametes versicolor MUCL

38412) and compared with P. sanguineus MUCL 41582 previously described. A multi-

methodological approach (using capillary electrophoresis, mass spectrometry, HPLC,

UV, NMR and IR spectroscopies) was developed to characterise the metabolitesinvolved

and monitor their apparition. Seven metabolites were commonly found with all strains,

suggesting a common metabolic pathwayfor Acid Blue 62 biotransformation. During the

.rst days, dimer and oligomers of the initial ABu62 molecule were observed: the main

oneabsorbed in the 500 nm region, explaining the red colour appearance of the medium.

This main metabolite was made up of two moleculesof ABu62 linked by an azo bond,

minus a cyclohexyl moiety. After a longer incubation time, breakdown products were

observed. Basedon these products characterizations, a bioconversion pathway was

proposed.”

64

Chapter 3

Experimental

3.1 MATERIALS REQUIRED

3.1.1 Textile Dyestuffs

The dyes were selected on the basis of their applications in industry and their

market demand. Following four main groups of dyes were selected for this research

project.Following dyestuffs of different groups being extensively used in local industry

were selected for decolourization studies

Reactive Dyes Company λmax (nm)

i. Drimarine Blue K2RL Clariant 582

ii. Cibacron blue FG3A Ciba 465

iii. Drimarine Orange KGL Clariant 430

iv. Drimarine Brilliant Red K4BL Clariant 541

v. Prucion Blue PX5R DyeStar 670

vi. Remazol Brilliant Yellow 3GL DyeStar 430

Disperse Dyes

i. Foron Turquize SBLN-200 Clariant 380

ii. Foron Blue RDGLN Clariant 549

iii. Foron Red RDRBLS Clariant 495

iv. Foron Yellow SE4G Clariant 489

Direct Dyes

i. Solar Golden Yellow R Clariant 398

ii. Solar Brilliant Red BA Clariant 528

iii. Solar Orange RSN Clariant 425

iv. Solar Blue A Clariant 560

Vat Dyes

i. Cibanon Red 2B-MD Ciba 522

ii. Cibanon Blue BFMD Ciba 665

Chapter 3 Experimental

65

iii. Cibanon Golden Yellow RK-MD Ciba 467

iv. Indanthrene Direct Black RBS DyeStar 580

3.1.2 Chemicals

Chemicals required for preparation of different types Buffers, media, and enzyme

assays were purchased from Sigma (Germany), Fluka(Germany), Oxide(U.K), and

Panareac().

1. Agar (Oxide Hampshire, England)

2. ABTS: (2.2/- azino – di (3-ethy-1-benzo thiazoline -sulphonate)

3. Copper Sulphate

4. Ammonium tartarate

5. Calcium Chloride

6. Chloromphenicol

7. Potassium Dihydrogen phosphate

8. Glucose

9. Maganese sulphate

10. Ferric sulphate

11. Dipotassium hydrogen phosphate

12. Hydrochloric acid

13. Veratryl alcohol

14. Veratryl aldehyde

15. Malonic acid

16. Succinic Acid

17. Sodium malonate

18. Sodium molybdate

19. Sodium succinate

20. Sodium acetate

21. Sodium hydroxide

22. Thiamine

23. Tween-80

24. Urea

25. Yeast extract


66

26. *Maize gluten meal (30% &60%)

27. *Corn steep liquor

These were obtained from Rafhan Mills (pvt) Ltd. Faisalabad.

3.1.3 Facilities Used:

1. T60 U.V.Spectrophotometre (PG Instruments (Pvt.) Ltd. England.)

2. Autoclave (Sanyo, MLS-3020U, Japan)

3. Laminr Air Flow (9au-300BN Dalton, Japan).

4. Centrifuge ((TGL-16, Changzhou, Guohua, China)

5. Fermentor (Eyela) Japan

6. Orbital Shaker(Gallen Kamp)

7. Distillary(Eyela SA-2000 ET Japan)

8. Refrigerator Dawlance

9. Oven (WTC-Binder,Germany)

10. pH meter (Inolab WTW Series, A070601185, Germany)

11. Electronic balance (Shamidizo MODEL:D432612180 Japan)

12. Incuabtor (Sanyo-GallenKemp PLC, London, Japan)

3.1.4 Microbial strains and their maintenance

Five indigenous white rot fungi were obtained from Industrial Biotechnology

Laboratory, Department of Chemistry, University of Agriculture, Faisalabad. These

cultures were labeled as: Pleurotus ostreatsus IBL-02, Phanerochaete chrysosporium

IBL-03, Coiolus versicolo IBL-04, Ganoderma lucidum IBL-05, and Schizophyllum

commune IBL-06.

Pure cultures of WRF were raised on potato dextrose agar (PDA) slants 30 ±20C.

The PDA media (table3.1a) were prepared in one litre flaks and adjusted to pH 4.0 using

M NaOH / M HCl solutions. The pH was checked with the help of pH meter (WTW-

Inolab. 720). The media were sterilized (121oC) in autoclave (Sanyo, MLS-3020U,

Japan) for 15 minutes. After cooling at room temperature, the contents were poured into

the sterilized cotton plug test tubes. The test tubes were left undisturbed in slanting

position at room temperature for solidification of slants. Five groups of 20 slants each

were labelled with the names of fungi and spores of respective fungi were transferred

onto the slants with help of sterilized inoculation loops aseptically in laminar air flow


67

(Dalton, Japan). The inoculated slants were incubated at 30 ±20C for 3 days for getting

fungal growth on PDA media. The slants with fungal growth were stored in refrigerator

at 4 oC.

Table 3.1a Composition of sporulation medium *for white rot fungi

Ingredients Quality(g/L)

PoataoDextrose agar(PDA) 250

Glucose 20

Agar 15

Ammoniumtartarate 0.22

K2HPO4 0.21

MgSO4 0.05

CaCl2 0.01

Thiamine 0.001

10% Tween80 10 ml

*Trace elements Solution (table3.1b) 10 ml

100mM Veratryl alcohol 10 ml

Chlorophenicol 1c.c (to inhibit bacterial contamination)

Table3.1 b Composition of trace elements solution

Trace elements Quality (g/L)

CuSO4 0.08

H2Mo O4 0.05

MnSO4.7H2O 0.07

ZnSO4.7H2O 0.043

Fe2(SO4)3.7H2O 0.05

3.2 INOCULUM PREPARATION

For preparation of inocula, the 100 mL inoculum media (table3.2) were prepared

in labelled flasks for individual fungi. The media were adjusted to pH 4.0 using M NaOH

/ M HCl solutions and sterilized (121oC) in autoclave for 15 minutes. After cooling at

room temperature, spores of individual fungi from PDA slants were aseptically


68

transferred into the respective flaks. The inoculum flasks were incubated at 300 ± 2 C in a

shaking incubator (120rpm) for 72 hours to get homogenopus spore suspensions

containing 1x108 spores/ml(Kay-Shoemake and Watwood, 1996).The spore counting was

performed in Department of Microbiology, Universirty of Agriculture, Faisalabd by the

method of Kolmer using hemoctometre. Fresh inocula were prepared for each experiment

Table 3.2 Composition of inocculum media for white rot fungi

Ingredients Quality(g/L)

Glucose 20

Ammoniumtartarate 0.22

K2HPO4 0.21

MgSO4 0.05

CaCl2 0.01

Thiamine 0.001

10% Tween80 10 ml

*Trace elements Solution(Table2.1B) 10 ml

100 mM Veratryl alcohol 10 ml

Chloromphenicol 1c.c

3.3 BASAL NUTRIENT MEDIA

Following four dfferent basal nutrient media (Kirk’s basal medium and its three

modifications) were used for selecting the best basal medium one for each group of dyes.

3.3.1 Medium 1

Kirk’s basal salts medium (Tien and Kirk, 1988) with the following composition

(g/l)was used: ammonium tartarate, 0.22; KH2PO4, 0.2; MgSO4.7H2O, 0.005; CaCl2,

0.01; thiamine, 1 mg/l; 10 ml/l of 10% (w/v) Tween-80 solution; 100 mM veratryl

alcohol and 10 ml/l trace elements solution was added. Trace elements solution(3.1b)

3.3.2 Medium II

Same composition as Medium I excluding veratry laicohol and Tween-80.


69

3.3.3 Medium III (g/l)

Urea 0.03; KH2PO4 0.2; MgSO4, 7H2O 0.01; CaC12 0.01.

3.3.4 Medium IV (g/l)

Urea 0.04; KH2 PO4 0.1; K2HPO4; MgSO4.7H2O 0.5; Cac12 0.05.

3.4 DECOLORIZATION PROTOCOL

Triplicate set of flasks (500 ml) were prepared for studying decolorization process

and each contained 100 ml of 0.01% solution of the Reactive Dye prepared in basal

nutrient medium I. The pH of media was adjusted with M NaOH/Methyl Succinic Acid

to 4.5 (except the pH optimization experiment) and the flaks were sterilized (121oC) in

autoclave (Sanyo, MLS-3020U, Japan) for 15 minutes. The triplicates were inoculated

with 5 ml (5 % v/v) of spore suspension in laminar air flow (Dalton,) and the flasks were

incubated at 30oC for 7 days at 120 rpm in shaking incubator (Sanyo-GallenKemp PLC,

London, Japan). The incubation period reduced gradually with the optimization of

different conditions. Control flasks were provided only dyestuff and nutrients, but with

out inoculum. Triplicate Samples were removed from triplicates after every 24 hours and

centrifuged at 48,000x g for 10 min. in a high speed centrifuge (TGL-16, Changzhou,

Guohua, China). The supernatants from the triplicates were analyzed for residual dyestuff

concentrations and lignolytic enzymes.

3.4.1 Dyestuff Analysis/Determination of Percent Decolourization

The culture supernatants recovered after filtration and centrifugation of the

fermented samples collected after every 24 hours were subjected to residual dyestuff

analysis. Absorbance measurements were done by using a UV-Visible spectrophotometer

(T-60, PG instruments, UK). Wavelength resulting in maximum absorbance (λmax) for

each individual dye was used for absorbance measurements. The absorbance values for

respective supernatants at each time period were corrected by subtracting the values for

respective blanks (containing only the nutrient medium). The corrected absorbance

values were used to calculate percentage decolourization using the absorbance values for

original dye solutions as standards.

3.4.2 Screening of WRF cultures on different dyestuffs

The five WRF cultures were used in screening for decolorization of different

groups of dyes.All the five WRF cultures were used on all groups of dyes in triplicate


70

flasks in a 10 days time course study to select the best cultures on different dyestuffs on

the basis of maximum decolourization of individual dyes.

3.5 SCREENING ON REACTIVE DYES

Four reactve dyes (Drimarene Orange K-GL, Remazole Brilliant Yellow3-GL,

Prucian Blue MX-R, and Cibacron Blue FG3A) were selected on the basis of their market

demand and use in local textile industry. Solutions of all the dyes (0.01%) were prepared

in Kirk’s basal medium for screening of best fungus in triplicate 500 ml Erlenmayer

flasks. pH of each flasks was adjusted at 4.5 with M NaOH/Methyl Succinic Acid and

thetriplicates were autoclaved for 15 minutes. After autoclave, 5 ml of inoculum of each

of the five strains (Pleurotus ostreatsus IBL-02, Phanerochaete chrysosporium IBL-03,

Coriolus versicolor IBL-04, Ganoderma lucidum IBL-05, and Schizophyllum commune

IBL-06) were added in laminar air flow in triplicate sets of flasks prepared for individual

dye (table 3.3). The flasks were kept in shaking incubator (at120 rpm) for ten days. After

every 24 hours absorbance was noted by taking out 5ml of samples from triplicate

flasks.The decrease in absorbance was compared with the absorbance of the standards.

Decline in absorbance was indication of dye degradation.That was expressed in %

decoloriazation. The most decolorized Reactive dye Remazole Brilliant Yellow 3-GL by

Coriolus versicolor was finally selected for furher optimization process. After selecting

the best fungus and dye combination, four different nutrient media were used to check the

potential of C.versicolor for decolorization of Remazole Brilliant Yellow 3-GL. Medium

I (Kirk,s basal medium) was found to be better for decolorization of Reactive dye

(Remazole Brilliant Yellow 3-GL) . In this medium 100% dye was decolorized with in 7

days. In rest of the three media C.versicolor showed poor decolorization of the

dye.Therefore, medieum I was used in subsequent optimasation studies

3.5.1 Selection of Basal nutrient medium

Four different basal nutrient media (Asgher et al. 2006) were used to select out

the most suitable one giving maximum decolorization efficiency by G. lucidum IBL-6

and medium I was selected as the best one on the due to maximum percent

decolorization. Medium I (M-I) had the composition of Kirk’s basal salts medium (Tien

a& Kirk.1988) excluding varatryl alcohol and tween-80. It was composed of 0.22 g/l

ammonium tartarate; 0.2 g/l KH2PO4; 0.01 g CaCl2; 1 mg/l thiamine; 0.05 g/l


71

MgSO4.7H2O; 10 ml/l trace elements solution was added. Trace elements solution was

composed of (g/l): CuSO4, 0.08; MnSO4.4H2O, 0.05; ZnSO4.7H2O, 0.07 and Fe2 (SO4)3,

0.043.

3.5.2 Initial Medium pH

For optimization of pH, the Kirk’s nutrient medium containing 0.01% Remazole

Brilliant Yellow 3-GL was adjusted to varying initial pH value viz; 3.0, 3.5 4.0, 4.5 using

M NaOH/Methyl succinic acid (Table 2.1.4.). After inoculation, all flasks were incubated

in orbital shaker (120 rpm) at 30oC for 7 days, 5ml samples from triplicate flasks were

removed after every 24 hour, filtered and read on spectrophotometer in order to

determine the percent dye decolourization. Maximum percentage docolourization of the

dye was achieved at pH5.

Table 3.1.2. Composition of growth media for decolorization of Remazole brilliant

yellow 3-GLby C.versicolor *with varying pH

Components Treatments

T1 T2 T3 T4 T5 T6

Dye sol (mL) (0.01%) in Kirk’s basal medium

100 100 100 100 100 100

pH 3.0 3.5 4.0 4.5 5.0 5.5

*Incubation time, 7 days; temperature, 300 C

3.5.3 Effect of Incubation Temperature

The triplicate decolorization media was adjusted to initial pH 4 (optimum) and

incubated at 25, 30, 35, 40 and 45 0C temperature. At 300C strain showed maximum

potential for dye degradation.

Table 3.1.3. Composition of growth media for decolorization of Remazole brilliant

yellow 3-gl by C.versicolor *with varying incubation temperatures


T1 T2 T3 T4 T5 T6

Dye sol (mL) 100 100 100 100 100 100

pH 4 4 4 4 4 5.5

Temperature (00C) 25 30 35 40 45

*Incubation time, 6 days


72

3.5.4 Addition of Carbon Sources

The fungi produce extracelluar enzymes as secondary metabolites for

biodegradation of dyes. To enhance primary growth and decolorization of the dye

Remazole Brilliant Yellow 3-GL glucose, Glycerol, starch, wheat bran and Lactose

(1g/100mL) were used as carbon sources (Table 3.1.4). The experiment was conducted at

optimum pH and temperature. Glucose proved to be the best carbon source and it gave

optimum dye removal in 5 days.

Table 3.1.5. Composition of growth media * with different carbon sources


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

pH 4 4 4 4 4

Tempratue (0C) 30 30 30 30 30

Carbon Sources (1%) Glucose Glycerol Lactose Starch Wheat bran


3.5.5 Effect of varying concentrations of Glucose

The effect of varying concentration in the medium was investigated under

optimum conditions (table 3.1.5) of glucose varying concentration were used and

medium with 1% glucose was efficient among all.

Table 3.1.6. Composition of growth media * with varying concentrations of Glucose


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

pH 4 4 4 4 4

Tempratue (0C) 30 30 30 30 30

Glucose (%) 0.1 0.5 1.0 1.5 2.0



73

3.5.6 Effect of Additional Nitrogen Sources

Nitrogen is important component of growth medium for microorganism. Different

nitrogen sources like Ammonium oxalate, MGM (maize gluten meal)30%, MGM 60%

and corn steep liquor were applied to study the effect of nitrogen addition on lgnolytic

enzyme synthesis and dye degradation. Corn steep liquor showed maximum dye

decolorization efficiency under preoptimised conditions.

Table 3.1.7 Composition of growth media* with different Nitrogen sources


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

pH 4 4 4 4 4

Temprature (0C) 30 30 30 30 30

Glucose (1%) 1 1 1 1 1

Nitrogen Sources (0.1%)

Ammonium Oxalate

Corn Steep Liquor

Maize Glutein Meal

30%

Maize Glutein Meal

60%

Yeast Extract


3.5.7 Effect of varying concentrations of CSL

Since nature and concentration of nitrogen source affects the decolorizing

potential of WRF so varying concentrations of CSL were used to find out its best

concentration for maximum enzyme formation and decolorization of Remazole brilliant

yellow 3-GL by Coriolus versicolor.

Table 3.1.7. Composition of growth media * with varying concetration of CSL


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

pH 4 4 4 4 4

Temprature (0C) 30 30 30 30 30

Glucose (1%) 1.0 1.0 1.0 1.0 1.0

Corn Steep liquor (V/W%)

0.01 0.05 0.1 0.15 0.2

Incubation time, 4 days


74

3.5.8 Effect of Low Molecular mass Mediators

Role of low molecular mass mediators and fungal metabolite in lignolytic enzyme

production is highly mediators is highly significant in enzymes production as wel as dye

degradation. Different mediators like ABTS, H2O2, MnSO4, and veratry1 alcohol were

added. ABTS increased fungal capabilityn for enhanced LMEs secretion and to

decolorize Remazole brilliant yellow-3 GL mor than all other compounds used.

Table 3.1.8. Composition of growth media* with different Mediators


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

pH 4 4 4 4 4

Temprature (0C) 30 30 30 30 30

Glucose (1%) 1.0 1.0 1.0 1.0 1.0

Corn Steep liquor(1%) 1.0 1.0 1.0 1.0 1.0

Mediators (1mM) ABTS H2O2 MnSO4 Tween-80 Veratry1 alcohol

*Incubation time, 3 days 3.5.9 Effect of Metal Ions

Metal ions play important role in enzymatic decolorization of dyes. Some

essential metal ions (in the form of their salts) which are considered least toxic for WRF

like CaC12, Cd(NO3)2, CuSO4, FeSO4, MnSO4, and ZnSO4 were added in the preoptimized

dye decolorization medium.Decolorization patterns were different due to variation in

nature of metal ion and structure of the dye. Medium recieving CuSO4 exhibited

maximum rate of decolorization and it also reduced the incubation period up to 48 hours.


75

Table 3.1.9. Composition of growth media * with different metal ions


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

pH 4 4 4 4 4

Temprature (0C) 30 30 30 30 30

Glucose (1%) 1.0 1.0 1.0 1.0 1.0

Corn Steep liquor (1%) 1.0 1.0 1.0 1.0 1.0

Mediator ABTS (mM) 1.0 1.0 1.0 1.0 1.0

Metal Ions (1mM) CaC12 Cd(NO3)2 CuSO4 FeSO4 Zn SO4

*Incubation time, 2days; 3.5.10 Effect of different dye concentration

Dyes are toxic chemical compounds and are tolerated by different WRF cultures

to different extent.After optimization of different parameters explained above varying

concentrations of dye were used to check its effect on growth and and potential of the

fungus. Flasks containing 0.01% dye were found to be optimally decolorizaed with in 48

hours.

Table 3.1.10. Composition of growth media * with varying concentrations of Remazole brilliant yellow 3GL


T1 T2 T3 T4

Dye sol (mL) 100 100 100 100

pH 4 4 4 4

Temprature (0C) 30 30 30 30

Glucose(1%) 1.0 1.0 1.0 1.0

Corn Steep liquor(1%) 1.0 1.0 1.0 1.0

ABTS(mM) 1.0 1.0 1.0 1.0

Metal Ions(mM) CaC12 Cd(NO3)2 CuSO4 FeSO4

Dye concentration (g/100ml) 0.01 0.05 0.1 0.15



76

3.5.11 Dye Adsorption on Fungal Mycelia

In dye adsorption experiment only different concentrations of the dye were added

in distilled water and no nutrient was added. 5mL inoculum was added in each

flask.Adsorption flasks were used to check dye adsorbed on fungal mycelia

Table 3.1.11. Composition of adsorption flasks


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

pH 4 4 4 4 4

Temprature (0C) 30 30 30 30 30

Inoculum (mL) 1.0 1.0 1.0 1.0 1.0

Dye concentration 0.01 0.05 0.1 0.15 0.2

*Incubation time, 2 days; pH 3.5; temperature, (30±20C) .

3.6 DISPERSE DYES

3.6.1 Decolorization protocol


and each contained 100 ml of 0.01% solution of the Dispers Dyes prepared in basal




with 5 ml (5 % v/v) of spore suspension in laminar air flow (Dalton, ????) and the flasks

were incubated at 30oC for 7 days at 120 rpm in shaking incubator (Sanyo-GallenKemp

PLC, London, Japan). The incubation period reduced gradually with the optimization of





concentrations and lignolytic enzymes.


77

3.6.2 Dyestuff Analysis / Determination of Percent Decolourization










3.6.3 Screening of WRF on Disperse dyes

Four disperse dyes (Foron Turquoise SBLN-200, Foron Red RBLS, Foron Yellow

SE4G, and Foron Blue RDGLN) courteously provided by Clarient (Pvt) Ltd. Faisalabad,

Pakisatan were selected for this part of the research project. 0.01 % solution individual

dyes were prepared in 500 ml Erlenmayer flasks in Kirk,s basal salt medium. pH of each

flasks was adjusted at 4.5 and the flasks were autoclaved for 15 minutes at 1210C. After

cooling to room temperature, 5 ml of inocula of each of the five strains (Pleurotus

ostreatsus IBL-02, Phanerochaete chrysosporium IBL-03, Coriolus versicolor IBL-04,

Ganoderma lucidum IBL-05, and Schizophyllum commune IBL-05) were added in aseptic

arrangement in triplicate sets of flasks (Table 3.2.1). The flasks were placed in shaking

incubator (120 rpm) for incubation for ten days at 300C. After every 24 hours, 5 ml of

samples were taken out from triplicate flasks for reading absorbances on

spectrophotometer.The decrease in absorbance was compared with the absorbance of the

standard 0.01% solutions of the dyes. Decline in absorbance was a the criteria for dye

degradation.This was expressed in % decoloriasation. The most decolorized dye was

Foron Turqoise Blue SBLN200 was finally selected for furher optimization process using

Ganoderma lucidum.

3.6.4 Optimization Process for Decolorization of Foron Turquise SBLN 200 by

Ganoderma lucidum

Foran Turqoise SbLN200 was completely decolorized by Ganoderma lucidum by

Medium IV within seven days. After selecting best combination of media and fungus


78

different parameters were optimized At pH 4.5 and temperature 350C, effect of different

sources of nutrients like carbon source (wheat bran 1.5%), nitrogen source (maize gluten

60%) mediator (H2O2), metal ion (Mn SO4) and dye concentration (0.05%) were

thoroughly investigated .

3.6.5 Effect of media Composition

After selecting the best fungus and dye combination, four different nutrient basal

media (Asgher et al.2006) were used to select the best one giving highest decolorization

% by G.lucidm on Foron Turqoise SBLN200 (Table 3.2.2). Medium IV was the best

medium for Foron Turqoise SBLN-200. Rest of the three media showed poor efficiency

in decolorization of the dye.Therefore, medieum IV was the best choice. This medium

was nitrogen rich due to urea and deficient of veratryl alcohol (a redox mediator) and

Tween-80 (a non-ionic surfactant).It was composed of Urea 0.04; KH2 PO4 0.1; K2HPO4;

MgSO4.7H2O 0.5; Cac12 0.05; 10 ml/l trace elements solution was added. Trace elements

solution was composed of (g/l): CuSO4, 0.08; MnSO4.4H2O, 0.05; ZnSO4.7H2O, 0.07 and

Fe2 (SO4)3, 0.043. Incubation time was 7 days.

3.6.6 Effect of Additional Carbon sources

Different carbon sources play their role in fungal metabolism by supplying energy

to the fungus and carrying out metabolic activities. Glucose, Glycerol, Lactose, Starch,

and Wheat bran (1% w/v) were added to the medium IV.Wheat bran (1%), a cheap

agroindustrial waste maximally enhanced the decolorization of Foran Turqoise SbLN200

during investigation by G.lucidum.

Table 3.2.1. Composition of growth media * with different carbon sources for

decolorization of Foron Turquise SBLN200 by G.lucidum


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Carbon sources (1%) Glucose Glycerol Lactose Starch Wheat bran


3.6.7 Effect of varying concentrations of Wheat Bran

After optimization of different carbon sources, varying concentrations of wheat

bran were applied to investigate optimum level of W.B.Medium having 1% W.B showed


79

maximum decolorization.Choice of WB was based on its low cost and rich in cellulose

contents.WB showed same results within 5 days of incubation as were in preceeding

experiment.

Table 3.2.2 Composition of growth media * with varying concentrations of wheat

bran.


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Wheat Bran (w/v%) 0.1 0.5 1.0 1.5 2.0

*Incubation time, 5 days; pH, 4.5; Temperature, (35± 20C)

3.6.8 Effect of addition of different nitrogen sources

Nitrogen sources play important role in protein synthesis of fungal strain. So

different kinds of nitrogen sources coupled with selected carbon source influence the

extent as wel as rapidity of dis perse dyes decolorisation. Maize glutein meal (60%)

enhanced Foron Turquoise SBLN200.Nitrogen supplemented were Ammonium oxalate,

Corn steep liquor (CSL), Maize gluten meal (30%), Maize gluten meal (60%), and Yeast

extract. Maize gluten meal (60%) proved efficient decolorizer.

Table 3.2.4. Composition of growth media * with different nitrogen sources


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Wheat bran (w/v%) 1 1 1 1 1

Nitrogen sources (1w/v%)

Ammonium oxalate

Corn steep liquor(CSL)

Maize gluten meal (30%)


Yeast Extract

*Incubation time, 4 days ; pH, 4.5; Temperature, (35± 20C)

3.6.9 Effect of Varying Concentration of MGM 60%

Since nature and concentration of N source leave pronounced effects on

decolorization process so varying concentrations of MGM60% were supplemented to the

medium IV to investigate optimum level of the selected nitrogen source. After 4 days of

incubation maximum decolorization was experienced in triplicate set of flasks having

0.15% MGM60%.


80

Table 3.2.5. Composition of growth media * with varying concentrations of MGM

60%.


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Wheat bran (1%) 1 1 1 1 1

MGM 60% (w/v%) 0.01 0.05 0.1 0.15 0.2

Incubation time, 4 days ; pH, 4.5; Temperature, (35± 20C)

3.6.10 Effect of Low Molecular mass Mediators

Mediators act as cosubstrate for ligninases. So 1mM of a number of mediators

like ABTS, CuSO4, H2O2, MnSO4, Tween-80, and Veratry1 alcohol were used for

maximum LME’s production which, inturn, enhanced extent of dye decolorization.

Veratryl alcohol was found to be the key mediator during decolorization.It is a redox in

nature which reduced incubation time up to 72 hours.

Table 3.2.6 Composition of growth media * with Low Molecular mass Mediators


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Wheat bran (%) 1.0 1.0 1.0 1.0 1.0

Maize gluten meal 60% (g/100mL) 0.15 0.15 0.15 0.15 0.15

Mediators ABTS H2O2 MnSO4 Tween-08 Veratry1 Alcohol


3.6.11 Effect of Different Metal ions

Since a microorganism metabolism is influenced by metal ions so the addition of

different metal ion salts CaC12, Cd(NO3)2, CuSO4, FeSO4, MnSO4, and ZnSO4 were added

in presence of VA to increase further dye decolorization and reduced incubation period

up to 48 hours. FeSO4 increased efficiency of VA which played the role of redox

mediator.

Table 3.2.7 Composition of growth media * with different metal ions



81

T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Wheat bran (w/v%) 1.0 1.0 1.0 1.0 1.0

Maize gluten meal 60% (w/v %) 0.15 0.15 0.15 0.15 0.15

Veratryl Alcohol (mM) 1 1 1 1 1

Metal Ions(mM) CaC12 Cd(NO3)2 CuSO4 FeSO4 ZnSO4


3.6.12 Effect of varying concentrations of Dye

Change in dye toxicity level and its decolorization efficiency by fungus is affected

by structure and concentration of the dye. So after optimizing various parameters like

carbon sources, nitrogen sources, mediators and metal ions at 0.01% dye concentration

level, the maximum level of dye decolorization was investigated. The fungus showed

maximum decolorization in the medium containing 0.01% of the dye in 48 hours and

decolorization potential decreased with the increase in dye concentration.

Table 3.2.8 Composition of growth media * with varying concetrations of the dye


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Wheat bran (w/v%) 1.0 1.0 1.0 1.0 1.0

MGM 60% (w/v %) 0.15 0.15 0.15 0.15 0.15

VeratrylAlcohol(mM) 1 1 1 1 1

FeSO4 (mM) 1 1 1 1 1

Dye conc. (g/100mL) 0.01 0.05 0.1 0.15 0.2

*Incubation time, 2 days; pH, 4. 5; Temperature, (35± 20C)

3.6.13 Study of Dye Adsorption on Fungal Mycelia

It has been noted that some fraction of the dye is adsorbed on fungal mycelia

along with dye decolorization. Adsorption flasks were left for incubation just having

different dye concentrations and no nutrient was supplemented. The adsorption flasks

were provided optimum conditions of temperature and pH. 5 ml inoculum was added into


82

each adsorption flask and no nutrient was added. Adsorption trend was negligible in case

of Foron Turquoise SBLN200.

Table 3.2.9 Composition of adsorption flasks


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Inocculum (mL) 1.0 1.0 1.0 1.0 1.0

Dye concentration (%) 0.01 0.05 0.1 0.15 0.2

*Incubation time, 2 days; pH 4.5; temperature, (35±20C); 3.7 DIRECT DYES



and each contained 100 ml of 0.01% solution of the Direct Dyes prepared in basal





incubated at 30oC for 7 days at 120 rpm in shaking incubator (Sanyo-Gallen Kemp PLC,






concentrations and ligninases activities.

3.8 Dyestuff Analysis / Determination of Percent Decolorization








83




3.8.1 Screening of WRF on Direct Dyes

Solar Golden Yellow R, Solar Orange RSN, Solar Blue A, and Solra Red BA,

were choosen keeping in view their industrial uses 500 ml Erlenmayer flasks used in all

the experiments were performed in temperature controlled incubator shaker.Flasks were

prepared in triplicates and contained 100 ml of the Kirks Basal Media. Pure cultures of

white rot fungi were grown in presterialized slants.These were later on used for

inoculation under sterilized conditions. Temperature was controlled at 300C and 3.5 pH

for all the white rot species used for direct dyes. The triplicates were incubated for 10

days at120 rpm.The sealed flasks were oxygenated for a few minutes daily. 5ml of

samples were taken out daily and centrifuged at 120 rpm for 5 minutes. Double filteration

also gave satisfactory results. Clear supernatant was used for all analyses. Different

basidiomycetes showed variable potential for direct dyes degradation. Triplicates were

prepared and all necessary measures were taken which were used in screening Reacive

and Disperse dyes. Solar Golden Yellow R showed maximum decolorization in presence

of P. ostratus.Controlled flasks contained only dyestuffs solutions and nutrient media but

no fungal cultures.

3.9 OPTIMIZATION PROCESS FOR DECOLORISATION OF SOLAR

GOLDEN YELLOW R

3.9.1 Selection of Basal salt medium

Solar Golden Yellow R was maximally decolorizaed by Ploreutous ostreatus by

Medium III within seven days. It had composition (g/l); Urea 0.03; KH2PO4 0.2; MgSO4,

7H2O 0.01; CaC12 0.01. Trace elements solution was composed of (g/l): CuSO4, 0.08;

MnSO4.4H2O, 0.05; ZnSO4.7H2O, 0.07 and Fe2 (SO4)3, 0.043.Incubation period was 7

days.

This combination of dye and medium was studied by optimising diffent

parameters like carbon source, nitrogen source, mediator role and effect of metal ions,

dye stuffs concentration and dye adsorption on fungal mycelia..


84

3.9.2 Addition of Carbon Source

Carbon sources added for optimization of Solar Golden Yellow R were Glucose,

Glycerol, Lactose, Starch, and Wheat bran supplemented with the same concentration

(1% w/v) as in case of reactive and disperse dye.Wheat Bran was found the best carbon

source among all when their color removal efficiencies were compared and Disperse

dyes. However, maximum degradation was achieved in the presence of wheat bran.

Table 3.3.1. Composition of growth media * with different carbon sources for

decolorization of Solar Golden Yellow R by P.ostreatus.


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100



3.9.3 Effect of varying concentrations of Wheat Bran

The effect of varying concentrations (w/v %) of wheat bran were used to check its

optimum level. Medium sharing 1% wheat bran was the best decolorizer among all.

Table 3.3.2. Composition of growth media * with different varying concentrations

of wheat bran


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Wheat bran conc.(%) 0.1 0.5 1.0 1.5 2.0

*Incubation time, 5days ; pH, 3.5; Temperature, (30±20 C)

3.9.4 Effect of Nitrogen Sources

Nitrogen plays vital role in microorganism physiology. So different nitrogen

sources like Ammonium oxalate, Corn Steep Liquor (CSL), Maize Gluten Meal 30%,

Maize Gluten meal 60%, and Yeast Extract were added into medium III. It was found

that efficient decolorization occurred in medium containing MGM 60%.


85

Table 3.3.3. Composition of growth media * with different Nitrogen sources


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Wheat bran (1%) Glucose Glycerol Lactose Starch Wheat bran

Nitrogen sources (0.1%) Ammonium oxalate

Carbon steep liquor (Csl)

Maize gluten meal

(30%)

Maize gluten meal

(60%)

Yeast extract

*Incubation time, 4 days; pH, 3.5; Temperature, (30±20 C)

3.9.5 Effect of varying concentrations of Nitrogen sources

Since MGM60% was attractive nitrogen source for our fungal strain so its varying

concentrations (0.01%, 0.05%, 0.1%, 0.15% and 0.2%) were used to investigate its

optimum concentration level. Medium containing 0.15% MGM60% was shoed highest

decolorization of dye (Table 3.3.4).

Table 3.3.4. Composition of growth media * with varying concentrations of

nitrogen source


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Wheat bran (w/v %) 1 1 1 1 1

MGM60% (w/v %) 0.01 0.05 1.0 0.15 0.2

*Incubation time, 4 days; pH, 3.5; Temperature, (30±20 C)

3.9.6 Effect of low Molecular mass Mediators

Mediators act as cosubstrate during ligninases based dye decoloriztion.So

mediator’s role cannot be overlooked in WRF based dye degradation.ABTS, CuSO4,

H2O2, MnSO4, Tween-08, and Veratry1 alcohol like preceeding two dyes (Reactve and

Disperse) optimization processes were added to the direct dye solution. Medium having

H2O2 was the excellent dye decolorizer However, response of different mediators varied

due to different nature of dyes.


86

Table 3.3.5 Composition of growth media * with different mediators


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Wheat bran (w/v %) 1.0 1.0 1.0 1.0 1.0

MGM 60% (w/v%) 0.15 0.15 0.15 0.15 0.15

Mediators(1mM) ABTS H2O2 MnSO4 Tween-80 Veratry1 alcohol

*Incubation time, 2 days; pH, 3.5; Temperature, (30±20 C) 3.9.7 Effect of Metal Ions

Metal ions salts like CaC12, Cd(NO3)2, CuSO4, FeSO4, MnSO4, and ZnSO4 leave

pronounced effect on fungal physiology.But effect of metal ion varies from strain to

strain which depends upon its nature and concentration.With the exception of Pb and Hg

which are usually proved toxic for many strains rest of the mentioned have least toxic

effect.So these were added to enhance fungal strain efficiency for dye decolorization.

MnSO4 showed maximum efficiency in color removal and incubation period was reduced

to 48 hours.

Table 3.3.6 Composition of growth media * with different metal ions.


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Wheat bran (%) 1.0 1.0 1.0 1.0 1.0

MGM60%(%) 0.15 0.15 0.15 0.15 0.15

H2O2 (mM) 1.0 1.0 1.0 1.0 1.0

Metal Ions(1mM) CaC12 Cd(NO3)2 CuSO4 FeSO4 ZnSO4

*Incubation time, 2 days; pH 3.5; temperature, (300±2C) 3.9.8 Effect of different dye concentration

Dyes differ in nature due to their complex structures.So to check maximum

decolorization extent different concentrations were used. Triplicates having 0.01% dye

solution was decolorized within 48 hours completely.


87

Table 3.3.7 Composition of growth media * with different dye concentration.


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Wheat bran (w/v %) 1.0 1.0 1.0 1.0 1.0

MGM60%(w/v %) 0.15 0.15 0.15 0.15 0.15

H2O2 (mM) 1.0 10. 1.0 1.0 1.0

MnSO4(mM) 1.0 1.0 1.0 1.0 1.0

Dye conc. (g/100mL) 0.01 0.05 0.1 0.15 0.2

*Incubation time, 2 days; pH 3.5; temperature, (30±20C)

3.9.9 Effect of different dye adsorption

Extent of dye adsorption was cheked by providing 5 ml of inoculum and 0.01%

dye to the triplicates.No nutrient were to the Dye adsorption limit was checked by

Table 3.3.8 Composition of adsorption flasks.


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Inocculum (mL) 5.0 5.0 5.0 5.0 5.0

Dye conc. (g/100mL) 0.01 0.05 0.1 0.15 0.2

*Incubation time, 1 days; pH 3.5; temperature, (30±20C) 3.10 VAT DYES



and each contained 100 ml of 0.01% solution of the Cibanon Blue GFJ-MD prepared in

basal nutrient medium I. The pH of media was adjusted with M NaOH/Methyl Succinic

Acid to 4.5 (except the pH optimization experiment) and the flaks were sterilized (121oC)

in autoclave (Sanyo, MLS-3020U, Japan) for 15 minutes. The triplicates were inoculated


incubated at 30oC for 7 days at 120 rpm in shaking incubator (Sanyo-GallenKemp PLC,



88





concentrations and ligninases activities.

3.10.2 Dyestuff Analysis / Determination of Percent Decolorization










3.10.3 Development of method for Vat Dyes Solution Preparation

Vat dyes create seroiuos problem while making their solutions since these are

insoluble in water. In textile industries these solutions are prepared in sodium hydroxide

which is called leuco. But sodium hydroxide accelerates the pH up to 11-12 which is in

appropriate for white rot fungi in that its optimum growth level lies in the range of 4-7. In

order to get pH in this range use of HC1 caused repreciptiation which was otherwise

suitable for maintaining pH of Reactive, Direct and Disperse dyes solutions. 0.1 M NaOH

was used for making solution and 1.0 M Succinic acid to maintain its pH level in acidic

range without causing reprecipitation.

3.10.4 Screening for selection of best fungus and dye combination

Vat Dyes are mosylt used for costly fabrics. So these are not used so extensively

like Reactve, Direct, and Disperse.Like other dyes a group of four vat dyes (Cibanon Red

2B-DM, Cibanon Golden-Yellow PK-MD, Cibanon Blue GFJ–MD and Indenthrene)

were used in this project. 0.01 % solution of each dye was prepared in 250 ml shaken

flasks.pH of triplicate flasks was adjusted at 4.5 and the flasks were autoclaved for 15

minutes.After sterialisation process, 5 ml of inoculum of each of the strain was added in


89

triplicates. After every 24 hours absorbance was noted by taking out 5ml of samples.

Coriolus versicolor showed efficient decolorization in triplicates receiving Cibanon blue

GFJ-MD. Decrease in absorbance was compared with the absorbance of standards

prepared with 0.01% dye solution. Decline in absorbance was indication of dye

degradation.This was expressed as % decoloriazation.

3.11 OPTIMIZATION FOR DECOLORIZATION OF CIBANON BLUE GFJ-

MD BY CORIOLUS VERSICOLOR

3.11.1 Media optimisation

C. versicolor strain was grown in 4 different defined media (whose composition

has already been described) for Cibanon Blue GFJ-MD decolorization. Medium II

(modified Kirk’basal nutrient medium) proved effective for decolorization of the dye.It

had low nitrogen contents and without veratryl alcohol as wel as Tween-80. Its

composition was (g/L): 0.22; KH2PO4, 0.2; MgSO4.7H2O, 0.005; CaCl2, 0.01; thiamine, 1

mg/l; Trace elements solution was composed of (g/l): CuSO4, 0.08; MnSO4.4H2O, 0.05;

ZnSO4.7H2O, 0.07 and Fe2 (SO4)3, 0.043. Incubationperiod was 7 days.

3.11.2 Addition of Carbon Source

The fungi produced extracellular enzymes as secondary metabolities for

biodegradation of dyes. To enhance primary growth and secondary metabolism starch,

glucose, glycerol, lactose and wheat bran (1g/100mL) were used as carbon sources for

decolorization of the dye Cibanon blue GFJ-MD by Coriolus versicolor (Table 3.2.3.)

The experiment was conducted at optimum pH and temperature. Starch proved to be the

best carbon source.

Table 3.4.1. Composition of growth media * with different carbon sources for Cibanon Blue GFJ-MD


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100


*Incubation time, 5 days; pH 5; Temperature, (30±2 0C)


90

3.11.3 Effect of Varying Concentration of Glycerol

Glucose was selected as a most suitable corbon source, for efficient dye

decolorization. Varying concentrations of Glycerol were used, ranging from 0.1% - 2.0%

(Table 2.2.4.) to select the most suitable concentration of carbon supplement. The

experiment was conducted at optimim pH and temperature for 3 days. Glycerol proved

best decolorizing carbon source.

Table 3.4.2. Composition of growth media * with varying concentration of Glycerol


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Glycerol (%) 0.1 0.5 0.1 0.15 2.0

*Incubation time, 5 days; pH 5; Temperature, (30±2 0C)

3.11.4 Addition of Nitrogen Source

Nitrogen sources influence WRF cultures and their ligninases enzymes.So these

were used to investigate the effect of nitrogen on decolorization of Cibanon blue GFJ-

MD by Coriolus versicolor, Ammonium oxalate, maize gluten meal (30% & 60%), corn

steep liquor and yeast extract were used at 0.1% level (table 3.4.3.) Experiment was

conducted at optimum pH, temperature and carbon source for 5 days. But all nitrogen

sources fully inhibitd fungal growth and dye decolorization.

Table 3.4.3. Composition of growth media * with different nitrogen sources.


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Glycerol (1% w/v) 1% 1% 1% 1% 1%

Nitrogen souces (0.1%)

Ammonium oxalate

Corn steep liquor(CSL)



Yeast extract

* Incubation time, 4 days; pH 5; Temperature, (30±2 0C) 3.11.5 Effect of Mediators

Like reactive dyes vat dye decolorization is also enhanced by using mediators.

Various mediators like ABTS, H2O2, MnSO4, CuSO4 and Veratry1 alcohol were tried.

ABTS as a low molecular mass mediator enhanced the degradation of Cibanon blue GFJ-

MD and reduced incubation period up to 72 hours.


91

-Table 3.4.4. Composition of growth media * with different Mediators


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Glycerol (1%) 1.0 1.0 1.0 1.0 1.0

Mediators(1mM) ABTS H2O2 MnSO4 Tween-80 Veratry1alcohol

Incubation time, 3 days; pH 5; temperature, (30±2 0C) 3.11.6 Effect of Metal Ions

It has been found that metal ions play crucial role in enzyme formation directly or

indirectly. To check their effect on the vat dye decolorization different metal ions were

applied after the optimization of inducers.Highest decolorization was observed with in 48

hours in medium receiving CuSO4.

Table 3.2.5. Composition of growth media * with different Metal ions


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Glucose (W/V %) 1.0 1.0 1.0 1.0 1.0

ABTS (mM) 1.0 1.0 1.0 1.0 1.0

Metal Ions(1mM) ABTS CuSO4 H2O2 Tween-80 Veratry1 alcohol

*Incubation time, 2 days; pH 5; temperature, (30±2 0C) 3.11.7 Effect of different dye concentration

Solutions of different concentrations of vat dyes were used under pre optimized

conditions illustrated above to check maximum extent of decolorization. 0.01% dye was

degraded with in 48 hours.


92

Table 3.4.6. Composition of growth media * with different dye concentration


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Glucose (1%) 1.0 1.0 1.0 1.0 1.0

ABTS (mM) 1.0 1.0 1.0 1.0 1.0

CuSO4(mM) 1.0 1.0 1.0 1.0 1.0

Dye concentration (g/100mL)

0.01 0.05 0.1 0.15 0.2

*Incubation time, 2 days; pH 5; temperature, (30±2 0C) 3.11.8 Dye Adsorption on fungal mycelia

Dye decoloriztion and adsorption occur simoultaneously but not at equal rate. In

order to study these phenomena effect of dye adsorption was investigated, 5mL inoculum

was added in triplicates. Negligible level of dye adsorbed was noted under preoptimised

conditions of temperature and pH.

Table 2.4.7. Composition of adsorption flasks


T1 T2 T3 T4 T5

Dye sol (mL) 100 100 100 100 100

Dye concentration (w/v%) 0.01 0.05 0.1 0.15 0.2

*Incubation time, 2 days; pH 5; temperature, (30±2 0C)

3.11.9 Enzyme activity assays

To investigate the activities of LiP, MnP and Laccase involved in decolorization

mechanism, the contents of the decolorization triplicate flasks at the termination of each

optimization experiment were filtered. The filtrates (cell free extracts) were centrifuged

and the supernatants were collected carefully. These were later on assayed for the

determination of enzyme activities. The activity of LiP was monitored by following Tien

and Kirk, 1988 method which involves the oxidation of veratryl alcohol (4mM) to

veratraldehyde at 30oC in of 0.2mM sodium acetate buffer (pH 3) in the presence of

0.1mM H2O2.The reaction mixture contained 1mL of Sodium acetate buffer, 1.0 mL of


93

veratry1 alcohol, 500 ul. Of 0.1mM H2O2 and 100 uL of enzyme aliquote. Blank

contained 100 µL of distilled water instead of enzyme aliquot. The absorbance of the

sample and standard varatraldehyde was read at 420 nm.

MnP was assayed by the method of Wariishi et al. (1992) in a reaction mixture

containing 0.1ml of culture supernatant and 1.0 ml of 1M MnSO4,1.0 ml 50mM sodium

malonate buffer (pH 4.5) in the presence of 0.5ml 0.1mM H2O2. Manganese ions Mn+3

form a complex with malonate which absorbs at 270 nm (ε270 11590 M-1 cm-1). The

amount of enzyme required to cause a unit increase in absorbance in one min per ml of

the assay mixture was defined as one unit of MnP activity.

Laccase was assayed by a slight modification of the method of Shin and Lee

(2000) following the oxidation of 2,2´-azino-bis(3-ethyl-benzo-thiazoline-sulphonate)

(ABTS) at 420nm (ε420 36000 M-1 cm-1) in assay mixture containing 0.1ml enzyme filtrate

and 1 ml of 0.3 mM ABTS in 1 ml 50 mM sodium malonate buffer. One unit –of laccase

activity was defined as the amount of enzyme causing one unit increase in absorbance per

min per ml of the assay mixture.

3.12 STATISTICAL ANALYSIS

All the decolorization flasks were used in the form of triplicate set during

experiment.The data values have been given as mean (±) in chapter 4. Values are shown

as y-error bars in the figures. The data collected was statistically anaylsed by usisng

Analysis of variance (ANOVA) and Duncan Multiple Range Test (DMR) (Steel et al.,

1997).

94

Chapter 4

Results and Discussion 4.1 DECOLORIZATION OF REACTIVE DYES

4.1.1 Screening of White Rot Fungi on Reactive Dyes

Screening of five WRF cultures on the reactive dyes Drimarine G. Yellow K2R,

Drimarine Blue K2RL, Drimarine Orange KGL, Cibacron Blue P-3RGR, Prucion Blue

PX5R and Remazole Brilliant Yellow 3-GL has been presented in Table 4.1a. The

decolorization of almost all the dyes increased slowly with the passage of time and

different WRF showed variable decolorization of different dyes. Pleurotus ostreatus

showed complete (100%) decoloorization of Prucion Blue PX5R after 6 days followed by

76% and 68.5 % decolorization of Cibacron BlueP-3RGR and Drimarine Blue K2RL

Orange K-GL, respectively after 6 days. P.ostreatus could cause only 60% decolorization

Remazole Brilliant Yellow 3G at the end of 10 days trial and it showed poor

decolourization of the remaining two Drimarine dyes. Ganoderma lucidum showed best

performance on Remazole Brilliant Yellow 3GL with complete decolorization after 8

days followed by 62 % decolorization of Drimarine Golden Yellow K-2GL after 5days

and 59 % decolorization of Drimarine Orange K-GL after 6 days. Whereas, it did not

show any commendable performance on other reactive dyestuffs (Table 4.1a).

Schyzophyllum commune did not show effective results on Remazole Brilliant

Yellow 3GL and Prucion Blue PX5R. However, it could decolourize the Drimarine dyes;

65, 63, 62 and 56% decolorization of Drimarine Blue, Drimarine Golden Yellow,

Drimarine Orange and Cibacron Blue P-3RGR, respectively was achieved within 5-7

days. Coriolus versicolor was found to be ineffective on Drimarine dyes but it showed

very good results on Remazole Brilliant Yellow 3G and Prucion Blue PX5R causing

complete decolorization in 6 and 7 days, respectively. P. chrysosporium also showed

poor results on all Drimarine dyes; only 71 % decolorization of Drimarine Blue was

observed at the end of 10 days trial. However, it also gave better results on Remazole and

Prucion dyestuffs showing complete decolorization after 7 and 8 days, respectively.

Chapter 4 Results and Discussion

95

Table 4.1a Decolorization of reactive dyes by different white rot fungi in time

course study

Incubation Time

(Days)

WRF Cultures

Drimarene Blue K2RL

Drimarene Golden

Yellow K-2GL

Drimarene Orange K-

GL

Cibacron Blue P-3RGR

Remazol Brill.Yellow-

3GL

Prucion Blue PX-

5R

1

P.O. 15.7±1.25 11.3 ±1.12 1.89± 0.00 8.25 ± 0.15 0.00 0.00 16.6 0.25 G.L. 11.2 ±0.13 2.04 ± 0.60 1.29 0.01 1.55 0.35 0.00 0.00 2.82 0.85 S.C. 5.55 0.20 4.58 0.30 1.45 0.20 2.50 0.30 0.00 0.00 0.00 0.00 C.V. 3.20 0.40 2.40 0.20 2.21 0.10 1.30 0.20 14.0 1.70 5.15 0.65 P.C. 21.1 ± 0.25 5.98 0.01 2.08 0.03 18.2 0.38 0.00 0.00 9.09 0.59

2

P.O. 24.7 0.48 12.1 0.35 3.0 0.15 10.0 0.20 0.00 0.00 38.8 1.56 G.L. 20.7 0.35 15.2 0.30 2.95 0.20 4.1 0.10 0.00 0.00 11.3 1.25 S.C. 20.9 ± 0.60 19.6 0.40 3.240.20 18.8 0.40 0.00 0.00 0.00 0.00 C.V. 9.80 0.20 13.2 0.80 4.78 0.50 8.60 0.30 20.0 2.07 20.2 1.36 P.C. 23.9 0.54 18.9 0.13 6.12 0.18 20.0 0.15 1.00 0.24 13.2 0.98

3

P.O. 42.2 1.70 48.8 0.60 4.19 0.45 28.2 0.70 0.00 0.00 51.3 0.54 G.L. 43.6 0.40 35.7 0.30 3..9 0.85 8.59 0.35 5.00 1.24 29.5 0.89 S.C. 28.6 0.20 25.8 0.40 35.6 0.40 26.7 0.20 0.00 0.00 2.36 1.26 C.V. 18.3 0.60 20.1 0.55 3.2 0.30 30.6 0.10 41.0 3.20 48.5 1.59 P.C. 29.1 0.28 26.8 0.20 8.8 0.13 21.9 0.95 2.00 0.75 41.9 2.15

4

P.O. 60.4 0.30 50.3 ± 0.85 5.8 0.65 33.9 0.80 2.00 0.52 79.8 1.69 G.L. 64.9 0.70 60.5 0.35 44.10± 0.85 9.70 ± 0.80 38.0 2.11 30.9 0.54 S.C. 48.9 0.30 41.6 0.60 55.3 0.40 33.5 0.20 0.00 0.00 6.65 0.65 C.V. 26.6 0.50 22.3 0.30 40.5 0.10 72.3 0.30 79.0 1.90 69.1 0.89 P.C. 35.7 0.27 29.9 0.10 10.10 0.20 30.7 0.75 3.00 0.82 62.2 1.26

5

P.O. 69.2 ± 0.96 55.3 ± 1.65 07..5 0.40 58.7 1.50 5.00 1.04 93.7 0.98 G.L. 65.3± 0.85 61.5 ± 0.25 59.3 ± 1.02 10.6 0.30 64.0 1.75 35.2 0.54 S.C. 56.9 0.30 47.3 0.20 61.3 0.50 41.2 0.40 1.00 0.45 15.6 1.54 C.V. 35.4 0.20 32.1 0.50 46.3 0.10 26.7 0.30 96.0 2.85 86.7 1.98 P.C. 41.8 0.22 31.4 0.45 48.0 0.10 33.9 0.06 12.0 2.90 86.0 3.46

6.

P.O. 68.5 ± 1.26 55.0 ± 0.68 10.0 ± 0.46 76.3 ± 0.91 7.00 1.68 100 0.00 G.L. 59.1 ± 0.20 49.6 ± 0.40 10.9 ± 0.42 12.9 0.30 88.0 3.34 39.1 1.23 S.C. 65.6 0.60 55.2 0.60 62.3 ± 0.82 54 0.40 3.00 1.05 18.6 2.65 C.V. 43.1 0.10 39.3 0.50 48.7 0.30 83.5 0.20 100 0.00 94.1 1.65 P.C. 56.1 0.74 37.8 0.30 71.01 0.20 39.6 0.37 66.01.50 97.0 1.25

7

P.O. 60.3 0.25 59.3 ± 0.67 34.12 0.45 91.3 0.90 12.0 1.64 100 0.00 G.L. 59.5 ± 0.87 56.8 ± 0.15 40.1 ± 0.59 16.7 0.40 98.0 2.60 45.1 1.89 S.C. 65.2 ± 0.31 63.3 0.20 42.3 ± 0.70 56.4 0.10 7.001.25 21.5 1.53 C.V. 48.6 0.50 45.6 ± 0.40 49.2 0.60 84.3 0.10 100 0.00 98.5 0.56 P.C. 64.9 0.21 40.9 0.21 90.9 0.98 46.1 0.51 87.0 3.10 100 0.00

8

P.O. 64.7 0.60 46.3 ± 0.22 54.3 ± 0.30 100 0.00 100 0.00 100 0.00 G.L. 57.3 ± 0.21 62.3 ± 0.49 40.2 ± 0.59 21.5 0.40 100 0.00 50.7 1.65 S.C. 45.3 ± 0.30 42.3 ±1.19 40.8 ± 0.68 41.2 ± 0.58 9.00 2.30 23.6 2.10 C.V. 45.6 0.20 46.9 ± 0.30 49.9 ± 0.30 87.6 ± 0.10 100 0.00 100 0.00 P.C. 66.6 1.25 45.6 1.02 100.0 0.00 50.5 0.45 100 0.00 100 0.00

9

P.O. 68.8 0.40 43.9 ± 0.80 65.24 0.40 100 0.00 100 0.00 100 0.00 G.L. 60.1.± 0.80 45.2 ± 0.40 54.3 ± 0.15 32.9 0.20 100 0.00 52.1 1.87 S.C. 59.8 ± 0.80 40.0 ± 0.29 46.5 ± 0.49 45.6 0.50 10.0 3.10 27.9 0.95 C.V. 49.4 0.50 48.5 ± 0.29 50.2 ± 0.30 87.7 0.10 100 0.00 100 0.00 P.C. 70.1 0.93 47.7 0.28 100.0 0.00 51.9 1.53 100 0.00 100 0.00

10

P.O. 60.9 0.30 43.8 0.60 83.12 0.40 100 0.00 100 0.00 100 0.00 G.L. 60.2 ± 0.63 47.9 0.15 66.02 ± 0.15 43.9 0.40 100 0.00 53.5 1.78 S.C. 55.9 ± 0.26 48.9 0.10 100.0 ± 0.60 40.4 0.30 13.0 2.75 29.5 2.60 C.V. 43.6 0.46 29.8 0.60 51.01 0.50 87.6 0.40 100 0.00 100 0.00 P.C. 71.3 0.45 49.5 0.58 100.0 0.00 53.6 2.10 100 0.00 100 0.00


96

Overall, the P. chrysosporium-NFCCP cultures was the most effective on the all

dyestuffs tested followed by C. versicolor and P. Ostreatus (Table 4.1b). Complete

colour removals were observed with P. chrysosporium cultures within 6-8 of incubation

for all dyestuffs. C. versicolor culture showed better efficiency than the P. ostreatus on

Remazole Brilliant yellow and Procion Blue dyestuffs. On the other hand P. ostreatus

performed better on Procion Blue MX-R and Cibacron Blue FG3A. Decolorization of

some dyes took longer for some cultures as compared to others, and in some cases it was

incomplete even at the end 10 days study period. However, time taken by these local

isolates to achieve 100% decolorization compares favourably with reports on other white

rot fungi which required 7-20 days period for 90% decolorization of a diverse range of

synthetic dyes (Kirbs et al., 2000; Boer et al., 2004). C. versicolor culture could

completely (100%) decolorize only Remazole Brilliant Yellow and Prucian Blue in 7 and

8 days respectively. In this regard it compares favourably against some other C.

versicolor strains that required 9-15 days to achieve substantial dye decolourization (Fu

and Viraraghavan, 2001).

Physiological differences among the four WRF cultures may account for

differences in their decolorization abilities (Orth et al., 1993; Reddy, 1995; Chander et

al., 2004). The complex enzymatic systems responsible for dye degradation and the

conditions under which they are expressed also vary among the white rot fungi (Rogalski

et al., 1991; Nagai et al., 2002; Boer et al., 2004; Mazmanci and Ünyayar, 2005);

however, the relative rates of decolorization for the four reactive dyes cannot be easily

explained. Decolorization of a dye requires the destruction of the chromophore (Swamy

and Ramsay, 1999), the ease of which depends on the chemical structure of the dye,

relative position of substituents on the aromatic ring and their resulting interactions with

the azo bond (Pasti-Grigsby et al., 1992; Adosinda et al., 2001; Boer et al., 2004).

Further degradation involves aromatic ring cleavage which has also been found to be

dependent on the identity of the ring substituents with the presence of phenolic, amino,

acetamido, 2-methoxyphenol, or other easily biodegradable functional groups resulting in

a greater extent of degradation (Spadaro et al., 1992; Heinfling et al., 1997; Chander et

al., 2004). The relationship between chemical structure and decolorization rate of the


97

Table 4.1b Summary of decolorization of reactive dyes by white rot fungi

Reactive Dyestuffs WRF Cultures Maximum

Decolorization (%)

Incubation Time

(Days)

Drimarine Blue K2RL

Pleurotus ostreatus 69 5

Ganoderma lucidum 65 4

Schyzophyllum commune 67 6

Coriolus versicolor 49 7

Phanerchaete chrysosporium 71 10

Drimarine Golden Yellow K2R






Drimarine Orange KGL






Drimarine Brilliant Red K4BL






Remazol Brilliant Yellow 3GL






Prucion Blue PX5R







98

reactive dyes used in our study is clearly reflected in our results; different dyes were

decolorized to different extents by the same fungal culture. Individual dyes also showed a

different decolorization pattern for the four WRF cultures. Although no general pattern

has been established between the structure of dyes and decolorization, it can be inferred

from the research so far that the chemical structures similar to the natural substrates will

be decolorized faster and more efficiently than the other. Pasti-Grigsby et al.(1992)

showed that the nature and position of substituents on one of the aromatic rings of azo

dyes could markedly influence decolorization. Knapp et al. (1995) found that the dyes

that are structurally similar may still be differentially decolorized by the white rot fungi.

The reason was postulated to be due to electron distribution and charge density along

with steric distribution (Paszeznski etal.1991). However, the overall complexity of

structure alone is not an indicator of the difficulty of decolorization of a particular dye

(Spadaro et al., 1992; Toh et al. 2003; Maas and Chaudhari 2005)

4.1.2 Optimization of Remazole Brilliant Yellow 3-GL decolorization by Coriolus

versicolor

From the screening result it was concluded that R.B.Y was the maximally

decolorized dye by C. versicolor. Therefore, it was selected for further optimization

process.

Effect of different media compositins

The decolorization study was carried out by using four different defined media to

investigate the medium composition on decolorization of Remazole Brilliant Yellow-

3GL (RBY3-GL) C. versicolor. This experiment continued for seven days. Medium I

(Kirk,s basal salt medium having Veratryl alcohol and Tween-80) showed highest

decolorization (99.99%) on seventh day of incubation followed by 62.34, 10.0 and 12.0

% in medium II, III and IV, respectively. The change in pH was noted as 5.29, 4.19, 6.64,

and 6.91 in medium I, II, III, and IV, respectively (as presented in Table 4.2). Activities

of three lignolytic enzymes were quite dissimilar in all the four media. Lip activity profile

was lowest among all. Its activities were 124, 160, 26 and 19 IU/mL while MnP showed

254, 274, 32.00 and 21.00 IU/mL in medium I, II, III, and IV, respectively. Laccase was

prominent enzymeand its activities in medium I, II, III, and IV were noted as 319, 291,

73, and 31 IU/mL respectively (Fig.4.2).


99

Analysis of variance of the data showed that our results were significant (p ≤ 0.01

as shown in Appendex-1, Table 4.2). DMR test revealed that treatment means of different

media carrying same letters are not significant.

The sign of slope indicated that increase in decolorization was correlated with the

enzyme activities. The value of R2 indicated that dye decolorization was due to 93 %

variation in laccase and 83 % in MnP activities. Two parallel slopes in the figure (4.2b)

indicated that R2 for laccase (0.93) was higher than MnP (0.83). It clearly showed that

dcolorization may best be achieved through laccase production followed by MnP

synthesis.

The growth of the fungus, enzyme production, and dye decolorization is affected

by the culture conditions like pH, dye class, medium composition, shaking and presence

of heavy metals (Kaushik and Malik, 2008). It has been already demonstrated that

production of MnP enzymes by different white rot fungi is strongly dependent on growth

conditions (Rogalski et al., 2006). For Trametes versicolor, MnP and laccase have been

identified as the major lignolytic enzymes responsible for the degradation of recalcitrants

(Asther et al., 1988; Wang and Yu 1998). Several white rot fungi produce laccase and

manganese peroxidase but not apparently Lip, suggesting that they work in an oxidative

mechanism different from that of P.chrysosporium (Johansson and Nyman 1992). In a

previous study it was noted that rapid decolorization of RBBR in Kirk,s basal medium

correlates with the enhancement of laccase activity as wel as by MnP (Eichlerova et al;

2006).

Kirk,s basal medium degraded dye up to 99% within in seven days of incubation.

Fungal strain worked actively in this medium. Medium II was devoid of veratryl alcohol

and Tween -80 and showed lesser decolorization than medium I. Poorest decolorization

was noted in Medium III and IV containing urea. Urea being a rich source of nitrogen

inhibited the growth of fungus; its biomass and eventually resulted in negligible

decolorization.


100

Table.4.2 Decolorization* of Remazole Brilliant Yellow 3-GLBY C.versicolor using different basal nutrient media and its

lignolytic enzyme profile

Med

ium

Decolorization (%Mean ± S.E) Incubation Time (Days) Final pH

Enzyme Activity (IU/mL)

1 2 3 4 5 6 7 Lip MnP Lac

M-I 21.65 ± 0.40 37.57 ± 0.80 55.59 ± 0.81 78.93 ± 1.35 8535 ± 2.86 100 ± 0.00 99.99±0.01 a

5.29±0.09 b

124 ± 2.61 a

254 ± 3.76 a

319 ± 4.38 a

M-11 14.57 ± 0.91 30.85 ± 1.46 38.04 ± 2.06 41.72 ± 1.73 51.18 ± 1.53 55.97 ± 2.00 63.34 ±1.51 b

4.19±0.03 b

160±6.25 a

274 ±5.14 a

291±6.37 b

M-III 26.29 ± 1.26 29.32 ± 4.82 31.19 ± 1.17 19.25 ± 1.12 25.9 ± 1.83 18.00 ± 0.03 10 ±1.23 c

6.64±0.09 a

26±5.57 b

32±5.57 c

73 ± 4.62 c

M-IV 5.29 ± 0.31 7.21 ± 0.55 8.12 ± 0.12 11.00 ± 0.01 15.33 ± 0.10 16.00 ± 0.23 12 ± 0.24 c

6.91±0.20 a

19 ±5.24 b

21± 5.24 c

31±3.76 d

* pH, 4 ; Temperature (30±20C).


101

0

20

40

60

80

100

120

140

160

180

M-1 M-2 M-3 M-4

Medium

Dec

olo

riza

tio

n(%

)

0

50

100

150

200

250

300

350

En

zym

e A

ctiv

ity

(IU

/mL

)

Decolorization %LiPMnPLac

Fig.4.2a Effect of varying media compositions on decolorization of Remazole

Brilliant Yellow 3-GL by C.versicolor and its lignolytic enzyme production in 6 days.

y = 1.7713x + 156.37

R2 = 0.9223

y = 1.4834x + 135.05

R2 = 0.826

100

150

200

250

300

350

0 20 40 60 80 100 120

Decolorization (%)

En

zym

e ac

tivi

ty (

IU/m

L)

MnP

Lac

Linear (Lac)

Linear (MnP)

r (MnP) = 0.909**r (Lac) = 0.960**

Fig.4.2b. Relationship between dye decolorization and enzyme activities with different media compositions


102

Although there are ample evidences of Lip in presence of veratryl alcohol yet C.

versilcolor strain did not produce Lip in our experiment.The expression of LiP by C.

versicolor, like that by P. chrysosporium, requires N limitation, stationary cultures, high

O2 partial pressure, and the addition of detergents and veratryl alcohol (Johanson and

Nyman, 1987; Jonsson et al; 1987; Dodson etal; 1987; Kirk and Ferril, 1992). A previous

study (Wu et al.1996 ; Teker et al; 2001) revealed that there was no LiP activity detected

for the local and commercial T. versicolor strain when applied for decolorization of

Remazole Blue RR, Remazole Red RR and Remazole Yellow RR.

For laccase catalysed oxidation of phenolic dyes, a similar mechanism has been

proposed as like Lip (Chivukula and Ranganathan 1995). In the proposed mechanism

Laccase catalyses two sequential electron oxidation reactions to generate firstly a

phenoxy radical and then a carbonium ion in the phenol ring nucleophilic attack of water

then yield a diazine derivative and a quinone. Laccase based decolorization treatments

are potentially advantageous to bioremediation technologies since the enzyme is

produced in large amounts and often produced constitutively or requires less fastidious

inductions conditions than either Lip, or MnP (Schliephake et al; 1996; Thurston et al.

1994; Eggert et al. 1996; Hublick and Schinner1996; Pointing and Virjimoed 2000;

Compos et al., 2001).

The composition for growth and the optimum enzyme production and dye

degradation are not the same even for a given species and there are still greater

differences among the requirements of different species. This must be taken into

consideration in biotechnological applications of white rot fungi. The conditions must be

optimized with regard to the aim of application (Murgesan and Kalaichelvan, 2003).

Effect of initial pH

To study the influence of initial pH on dye decolorization triplicate sets of flasks

set at pH 3.0, 3.5, 4.0, 4.5 and 5.0 were inoculated with C. versicolor. The maximum dye

degradation (98.66 %) was observed in the medium maintained at pH 4 followed by

63.75, 72.66, 49.21, and 40.81 % dye removal at pH 3, 3.5, 5.0, 4.0, and 5.5 respectively.

At 5 very low level of degradation was noted (Table 4.3).The decolorization % increased

with increase in pH and peaked at pH 4. A further increased in pH gave low color

removal (Fig. 4.3a).


103

Table-4.3 Decolorization* of Remazole Brilliant Yellow 3-GL by C. versicolor and its lignolytic enzyme profile with varying pH levels

Varying pH

Level

Decolorization (% Mean ± S.E)

Incubation Time (Days) Final

pH

Enzyme Activity(IU/mL) on 6th Day

1 2 3 4 5 6 7 Mnp Lac

3 26.63±2.55 30.36±2.44 35.47±2.39 44.17±1.35 57.17±2.06 66.47±2.45 63.75±2.65 c

3.54±0.01 d

229 ±14.51 b

25±9.04 b

3.5 30.04±2.86 36.32±1.97 45.87±0.55 57.62±2.81 65.16±2.29 75.66±3.41 72.66±2.72

b 4.28±0.02

c 243±11.91

b 311±12.13

ab

4 33.63±1.61 50.30±1.12 62.26±2.36 67.05±1.64 81.55±1.93 100.0 ± 0.40 97.21±0.89 a

4.67±0.05 c

295±3.76 a

351±9.83 a

4.5 25.84±0.88 31.07±1.03 34.76±2.67 40.08±2.27 46.82±2.85 51.08±2.15 49.21±1.42

d 5.87±0.10

b 228±10.15

b 260±10.99

bc

5 17.92±1.03 20.87±1.12 23.74±1.05 26.91±0.85 34.15±1.63 42.64±2.16 40.81±2.17 e

6.28±0.25 a

209±11.84 b

251±9.03 c

* temperature (30±20C)


104

0

50

100

150

200

250

300

350

3 3.5 4 4.5 5

P H

Dec

olo

riza

tio

n (

%)

0

50

100

150

200

250

300

350

400

En

zym

e A

ctiv

ity

(IU

/mL

)

Decolorization %MnPLaccase

Fig.4.3a Effect of varying pH on decolorization of Remazole Brilliant Yellow 3-GL by C. versicolor and its lignolytic enzyme activities production in 6 days

y = 1.9146x + 169.24

R2 = 0.8905

y = 1.5728x + 137.75

R2 = 0.7819

100

150

200

250

300

350

400

40 50 60 70 80 90 100 110

Decolorization (%)

En

zym

e ac

tivi

ty (

IU/m

L)

MnPLacLinear (Lac)Linear (MnP)

r (MnP) = 0.901**r (Lac) = 0.958**

Fig.4.3b: Relationship between dye decolorization and enzyme activities with varying pH


105

Change in pH was 3.54, 4.28, 4.67, 5.86 and 6.28 in medium with pH 3, 3.5, 4,

4.5 and 5 respectively. Maximum pH change occured in the medium having pH 5 and it

increased up to 6.

Laccase was found as major enzyme and manganese peroxidase activities were at

secondary level. Maximum laccase activity was 351 IU/mL at pH 4 . Laccase activity

profile of the medium maitained at was 296, 312, 260 and 251 IU/mL and Mnp activities

were 230, 243, 295, 228 and 209 IU/mL at pH 3, 3.5, 4, 4.5 and 5 respectively. This

suggests the principle role of lccase in decolorization of Remazole Brilliant Yellow 3-

GL.

Analysis of variance of the data (Appendex-1, Table 4.3) also showed that our

results were significant (p ≤ 0.01). DMR test revealed that treatment means of varying pH

carrying same letters are not significant.

The sign of slope (Fig.4.3b) indicated that increase in decoloriztion was positively

correlated with the enzyme activities. The value of R2 indicated that dye decolorization

was due to 89% variation in laccase and 78 % in MnP activities. Two parallel slopes

indicated that R2 for laccase (0.89) was higher than for MnP (0.78) which showed major

role of laccase in decolorization of Remazole Brilliant Yellow 3-GL by C. versicolor.

Optimum growth conditions differ from species to speciecs and even same strain

in different environment show quite dissimilar behaviour. Fluctuation in pH from its

optimum level (either below or high) causes fungal growth inhibition leading to lower

enzyme production and minimum dye decolorization. Generally it is observed that

lignolytic fungi work efficiently in acidic pH range. The pH optima for various WRF lies

in the range of 4-6 depending upon the nature of medium components as well as dye

taken in decolorization medium(Kapdan et al; 2000; Jolivalt et al; 2006 and and Hai etal;

2006). Dye decolorization by WRF increases increases in the beginning but decreases as

pH change exceeds from 4-4.5 (Banat etal;1996; Asgher et al; 2006; Kapdan and Kargi

(2002) worked on decolorization of textile dyes and found pH 4.5-5.0 as optimum for

different fungal strains.


106

Effect of Temperature

Coriolus versicolor showed maximum decolorization (99.02 %) in the flasks

incubated at 300C followed by 70.81, 58.82% decolorization at 250C, 350C, 400C, and

450C respectively with in 5 days of incubation (Table 4.4). At temperature 400C and

above very poor decolorizatin was observed in table 4.4. Change in optimum pH (4) was

4.22, 4.48, 4.59, 4.95 and 5.49 in medium incubated at 250C, 300C, 35 0C, 400C and 450C

respectively. Minimum decolorization was observed at temperature 450C where pH level

changed to 4.4.

MnP activity profile was 241, 265, 212, 223 and 232 IU/mL; while laccase

activities were 358, 371, 338, 298 and 282 IU/mL at 250C, 300C, 350C 400C and 450C

respectively. Laccase maximum activity was noted at 300C. Laccase activity was 370

IU/mL and manganese peroxidase was 211 IU/mL. Minimum Laccase activity was

282.67 IU/mL at 45 0C. However, manganese peroxidase showed maximum activity (358

IU/mL) at 250C (Table 4.4).

Analysis of variance of the data also showed that our results were significant (p ≤

0.01) as presented in Appendex-1, Table 4.4). DMR test revealed that treatment means

with varying temperatures carrying same letters are not significant .The sign of slope

indicated that increase in decoloriztion was correlated with the enzyme activities. The

value of R2 indicated that decolorization of Remazole Brilliant Yellow 3-GL5 was due to

9% variation in laccase and 78% in MnP activities. Two parallel slopes (Fig-.4.4a)

indicated that R2 for laccase (0.59) was lower than for MnP (0.78) showing that

dcolorization at 300C may best be achieved through MnP production followed by laccase.

The temperature optimum for the growth of most of white rot fungi and activity of

ligninases enzymes are around 300 (Bohemer et al; 2006; Hai et al; 2006). Temperature

optima were found to vary between 25-37 0C for various WRF cultures (Swamy and

Ramsay, 1999b; Tekere 2001; Toh et al., 2003; Chander and Arora 2006). Novotany et

al., 2004 reported complete decolorization of different dyes by WRF at 28 0C with in 10

days.


107

Table 4.4 Decolorization of Remazole Brilliant Yellow 3-GL by C. versicolor and its lignolytic enzymes at different

Temperatures under optimum conditions*

Temperature (0C)

Decolorization (% Mean ± S.E) Incubation Time (Days)

Final Enzyme

Activity(IU/mL) on 5th Day

1 2 3 4 5 6 pH MnP Lac

25±2 46.21±1.58 52.98±1.77 58.06±2.00 63.87±2.67 70.81±70.81 66.39±2.60 b

4.22±0.03 c

2416±6.09 b

358±4.06 a

30±2 55.29±1.55 62.05±1.71 72.94±2.95 82.58±2.07 99.02±0.07 96.99±0.11 a

44.48±0.02 c

265±1.77 a

371±1.86 a

35±2 31.64±2.20 36.63±1.94 43.31±2.58 52.93±2.52 58.82±2.58 55.90±2.39 c

4.59±0.30 bc

212 ±11.58 c

338±3.48 b

40±2 27.35±1.28 33.54±2.34 51.76±2.03 54.70±2.38 57.19±2.69 53.79 ±1.97 cd

4.95±0.02 b

223±4.38 c

298±6.57 c

45±2 14.87±0.87 21.84±1.60 28.14±0.62 42.70±1.59 51.49±1.56 49.18±1.27 d

5.49±0.06 a

232±7.52 bc

283±4.42 d

* Medium, I; pH, 4.


108

0

50

100

150

200

250

300

25 30 35 40 45

Temperature (0C)

Dec

olo

riza

tio

n (

%)

0

50

100

150

200

250

300

350

400

En

zym

e A

ctiv

ity

(IU

/mL

)

Decolorization%MnPLaccase

Fig.4.4a Effect of varying temperatures on decolorization of Remazole Brilliant Yellow 3-GL by C.versicolor and lignolytic enzyme production in 5 days

y = 1.2342x + 257.16

R2 = 0.5903

y = 1.1472x + 156.85

R2 = 0.7899

100

150

200

250

300

350

400

40 50 60 70 80 90 100

Decolorization (%)

Enzy

me

acti

vity

(IU

/mL)


r (MnP) = 0.810**r (Lac) = 0.804**

Fig.4.4b: Relationship between dye decolorization and enzyme activities with varying temperatures


109

Kapdan and Kargi (2002) studied the decolorization of Textile dyes by C.

versicolor and got best results at 28 0C. The optimum temperature for growth and dye

decolorization studies with T. versicolor have been reported as 300C (Tien and Kirk,

1988; Yung and Yu, 1997; Wong andYu, 1999).

Effect of Additional Carbon

(i) Effect of different carbon sources

Different types of carbon sources were used as supplements to enhance the

ligninases production by the fungus in secondary metabolism for consequent dye

degradation. Decolorization on 4th day of incubation was 81.97, 98.56, 64.12, 17.25,

88.01 and 79.13 and 25.10 % in Glucose, Glycerol, lactose, starch and wheat bran

containing flasks respectively. MnP activities were 262, 297, 250, 239 and 252 IU/mL;

while laccase activities were 378.0, 543, 353, 347, 328, 337 IU/mL, respectively.

Maximum decolorization (98.56 %) was obtained after 4th day of incubation in medium

receiving glucose and decreased slightly after 5 days of incubation while control flasks

had shown highest decolorization (98.9%) on 5th day of incubation.

Out of the different sources used glucose enhanced dye decolorizations by C.

versicolor. Whereas all other showed fungal growth inhibition leading to slower enzyme

formation and dye removal (Fig.4.5a)

Change in pH from optimum level 4 was 4.66, 4.63, 4.39, 7.98, 6.77 in Glucose,

Glycerol, lactose, Starch, wheat bran containing flasks, respectively. It shows that trend

towords basic pH leads to poor dye degradation.

Maximum laccase activity (388 IU/mL) was also found in the flasks containing

glucose as a carbon source. The activities shown by glycerol, lactose, starch and wheat

bran were 353.00 IU/ml, 346, 67IU/mL, 328.33IU/m 337.00 IU/mL respectively. MnP

activity was maximum (297 IU/mL) in glycerol followed by 297, IU/mL, 261.67%

IU/mL, 252 IU/mL and 250 IU/mL, 238.67 IU/mL in glycerol, glucose, wheat bran,

lactose and starch respectively.

Analysis of variance of the data (Appendex-1, Table 4.5) showed that our results

were significant (p ≤ 0.01). DMR test reveals that treatment means with different carbon

sources carrying same letters are not significant (Table 4.5a).


110

Table 4.5 Decolorization of Remazole Brilliant Yellow 3-GL by C. versicolor and its lignolytic enzyme profile at with

different carbon sources under optimimum conditions*

Carbon Sources (1%)

Decolorization (% Mean ±S.E) Incubation Time (Days) Final

p H

Enzyme Activity(IU/mL)

on 4th Day

1 2 3 4 5 MnP Lac

Control 54.98±1.69 61.88±1.54 71.96±2.32 81.97±2.11 98.9±0.04a

4.51±0.03c

272±4.25a

377±4.12 b

Glucose 69.02±1.66 76.28±0.69 86.89±0.58 98.56±4.65 96.17±0.36 a

4.66± 0.01c

362±5.46a

543±1.53 a

Glycerol 38.23±1.05 42.15±3.17 50.25±2.03 64.12±2.59 61.50±1.98 b

4.63±0.07 c

297±9.55 a

353±3.79 b

Lactose 24.98±1.03 31.47±2.39 24.12±0.97 17.25±0.56 10.12±0.97 d

4.39±0.01 d

250±8.40 c

346±6.23 ab

Starch 49.6±1.00 57.6 ±1.65 70.83±1.19 88.09±1.23 92.12±1.05a

7.98±0.01 a

239±4.34 d

328±25.07b

Wheat Bran 52.63±0.37 59.58±2.40 63.16±0.16 79.13±0.12 85.1±1.01a

6.77±0.06 b

252±24.04cd

337 ±28.22ab

* Medium, I; pH, 4 ; Temperature (30±20C)


111

0

50

100

150

200

250

300

350

400

Control Glucose Glycerol Lactose Starch WheatBran

Carbon Source (1%)

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

En

zym

e A

ctiv

ity

(IU

/mL

)

Decolorization%MnPLaccase

Fig.4.5a Effect of different carbon sources on decolorization of Remazole Brilliant Yellow 3-GL by C. versicolorand its lignolytic enzyme production

y = 0.3365x + 247.72

R2 = 0.2962

y = 0.5135x + 332.46

R2 = 0.4978

100

150

200

250

300

350

400

450

50 60 70 80 90 100

Decolorization (%)

Enzy

me

acti

vity

(IU

/mL)

MnPLacLinear (MnP)Linear (Lac)

r (MnP) = 0.461r (Lac) = 0.601*

Fig.4.5b: Relationship between dye decolorization and enzyme activities with

different carbon sources


112

The sign of slope indicated that increase in decoloriztion was correlated with the

enzyme activities. Two parallel slopes in the figure (4.5b) indicated that R2 for laccase

(0.50) was higher than for MnP (0.29). It clearly showed that dcolorization may best be

achieved through laccase production followed by MnP synthesis.

ii) Effect of varying concentrations of Glucose

After having found glucose as best carbon source for decolorization of Remazole

Brilliant Yellow 3-GL by C. versicolor, effect of varying concentrations of glucose was

also investigated in order to select most suitable concentration in the medium. Dye

decolorization was 36.81, 51.32, 96.02, 72.07, and 61.90 % in media receiving 0.1, 0.5,

1.0, 1.5, and 2.0 % glucose respectively in 4 days.

It was observed that dye decolorization was maximum in the presence of 1%

glucose as a supplementary carbon source and its decline commenced with further

increase in its concentration (Table 4.6). At 1.0 % glucose level, laccase also exhibited

maximum activity (540 IU/mL) and dye decolorization was highest at this concentration

(Fig.4.6a). pH level shifted from optimum level to 4.62. MnP activity was 164, 296, 365,

136 and 174 IU/mL where as laccase activity was 311, 371, 542, 486 and 463 IU/mL in

media receiving 0.1, 0.5, 1.0, 1.5, 2.0 % glucose, respectively.

Analysis of variance of the data (Appendex-1, Table 4.6) also showed that our

results were significant (p ≤ 0.01). DMR test revealed that treatment means with varying

concentrations of glucose carrying same letters were not significant (Table 4.6).

The sign of slope (Fig. 4.6b) indicated that increase in decoloriztion was correlated

with the enzyme activities. The value of R2 indicated that dye decolorization was due to

88% variiation in laccase and 28% in MnP activities.Two parallel slopes in the Figure

(4.6b) indicated that R2 for laccase (0.89) was higher than for MnP(0.29) clearly showing

that dcolorization was achieved mainly through laccase activity followed by MnP

synthesis.

It has been reported in literature that additional carbon sources increase the fungal

growth and enzyme activity to get maximum dye decolorization (Robinson et al, 2003;

2006; Selvam et al; 2006; Sanghi et al., 2006 Chander and Arora, 2007).


113

Table 4.6 Decolorization of Remazole Brilliant Yellow 3-GL by C. versicolor and its lignolytic enzyme profile with varying

concentrations of glucose under optimum conditions*

Concentrations of Glucose

(%)

Decolorization (% Mean±S.E) Incubation Time (Days)

Final pH

Enzyme Activity(IU/mL)

on 4th Day 1 2 3 4 5 MnP Lac

0.1 57.29±2.20 64.21±2.89 74.77±2.20 88.85±2.58 96.81±2.53 b

4.30±0.06 c

264±6.81 cd

381±11.23 d

0.5 62.77±2.46 82.17±3.62 78.06±3.69 93.5±3.50 98.32±3.21 d

4.36±0.04 c

296±8.68 b

371±3.47 b

1 70.05±1.88 85.89±2.47 87.1±0.78 98.06±0.09 96.02±0.44 a

4.62 ±0.07 b

365±17.69 a

542 ±10.77 a

1.5 52.25±1.42 75.82±2.42 66.79±1.04 59.17±2.28 72.07±1.21 b

4.61±0.02 b

236±11.06 d

486±9.61 b

2 43.93±1.47 31.97±1.45 31.14±0.65 43.72±2.69 51.90±0.52 c

4.90±0.06 a

174±8.77 c

463±6.75 b

* pH, 4 ; Temperature (30±20C)


114

0

20

40

60

80

100

120

0.1 0.5 1 1.5 2

Glucose concentration (1%)

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

700

800

900

1000

En

zym

e A

ctiv

ity

(IU

/mL

)

Decolorization (%)MnPLac

Fig.4.6a Effect of varying concentrations of glucose on decolorization of

Remazole Brilliant Yellow 3-GL by C.versicolor and its lignolytic enzyme production in 4 days

y = 3.6013x + 171.93

R2 = 0.8863

y = 2.3293x + 92.395

R2 = 0.287

100

150

200

250

300

350

400

450

500

550

600

25 35 45 55 65 75 85 95 105

Decolorization (%)

Enzy

me

acti

vity

(IU

/mL)


r (MnP) = 0.522*r (Lac) = 0.784**

Fig.4.6b: Relationship between dye decolorization and enzyme activities with varying concentrations of glucose


115

Glucose showed maximum decolorization and maximum enzymes activities were

also noted in this set of flasks. It is due to its easily metabolisable nature. On the other

hand starch showed very poor performance. It is polysaccharide and show insignificant

potential in decolorization of Remazole Brilliant Yellow 3-GL. In previous study on

decolorization of Anthraquinone based Remazole Briliant Blue R by a Trametes species

(RVAN2) it was noted that glucose was the best carbon source for RBBR decolorization

(Verde et al., 2007).

Decolorization was not stimulated by carbon limitation. Several factors indicated

that dye decolorization required a minimum amount of glucose. The cessation of

decolorization after successive dye decolorization corresponded to glucose depletion and

decolorization was restored by replenishing glucose. Swamy and Ramzy (1999) reported

that glucose-fed batch cultures maintained high rates of decolorization for more than

twice as long as unfed batch cultures. Furthermore, decolorizing cultures consumed

glucose at higher rates than those to which no dye was added.

Glucose is thought to play multiple role in decolorization which might be: (a) the

generation of H2O2 required for extracellular peroxidase activity and/or (b) the generation

of Mn+3 complexing agents necessary for MnP activity (Kirk and Ferril,1987; Gold et al;

1988).The activity of the extracellular peroxidases, such as LiP and MnP, requires H2O2

as a cosubstrate (Swamy and Ramsy, 1999). In many species of white rot fungi, enzymes

such as glucose-1-oxidase and glucose-2-oxidase are responsible for H2O2 system (Zhao

and Janse,1996) generation and are expressed as a part of the lignin degrading system .

The preferred substrate for this enzyme was D-glucose, the primary C source in this

study. It is possible that glucose is the substrate for endogenous H2O2 generation during

dye degradation.

A critical glucose concentration is required for decolorization of dye by WRF

below this value, decolorization rates rapidly decline with no decoloration at 0 g/l glucose

Swamy and Ramsy (1999) have reported that the requirement for glucose appeared to be

associated with a rate limiting step in the decolorization mechanism rather than

accelerated metabolic activity. But


116

Our strain of C. versicolor was supplemented with 1g/100mL glucose for

decolorization of RBY3GL and it also gave best results. High or low carbon source

concentration depends on the nature of dye being degraded.

Effect of Additional nitrogen

i) Effect of different nitrogen sources

Different nitrogen sources like ammonium oxalate, corn steep liquor, maize

glutein meal (30%,) maize glutein meal (60%) and yeast extract were used as nitrogen

additives (0.1%) in the medium to study their effects on decolorization of dye Remazole

Brilliant Yellow 3-GL by C.versicolor. Decolorization was 98.80, 48.67, 98.95, 35.77,

70.0 and56.26% in medium containing CSL, MGM 30%, MGM 60% and yeast extract

respectively. Enzyme Activities of MnP were 312, 360 103, 276 and 228 IU/mL where as

laccase profile was 544, 443, 618, 228, 370 and 348 IU/mL in CSL, MGM 30%, MGM

60% and yeast extract, respectively.


were significant (p ≥ 0.01). DMR test revealed that treatment means with different

nitrogen sources carrying same letters were not significant (Table 4.7).


enzyme activities. The value of R2 indicated that 96 % variation in laccase and 47 % in

MnP activities was responsible for the dye decolorization.Two parallel slopes (Figure

4.7b) indicated that R2 for laccase (0.96) was higher than for MnP (0.47) showing the

major role of laccase followed by MnP in decolorization mechanism.

Corn steep liquor enhanced dye decolorization to 96.17% in 3 days as compared

to control flasks giving maximum result in 4 days. All other nitrogen sources showed

maximum inhibition of enzyme activities as well as dye color removal.


117

Table-4.7 Decolorization of Remazole Brilliant Yellow-3GL by C. versicolor and its lignolytic enzyme profile using

different nitrogen sources under optimimum conditions*

Nitrogen sources (0.1%)

Decolorization (% Mean ± S.E) Incubation Time (Days) Final

pH

Enzyme Activity (IU/mL) on 3rd Day

1 2 3 4 Mnp Lac

Control 70.12±2.36 86.89±1.36 98.8±0.32 96.81±0.56 a

4.31±0.12 c

369±12.32 a

544±11.6 b

Ammonium Oxalate

29.35±14.43 42.85±17.640 48.67±20.23 45.6±2.95 c

5.23 ±0.03 a

312±13.03 b

443±22.68 c

Corn Steep Liquor(CSL)

73.72±3.45 88.68±4.34 98.95±0.09 96.17±0.60 a

4.95±0.05 c

360±30.20 a

618±19.31 a

Maize Glutein Meal60%

25.70±2.78 30.98±2.73 35.77±2.26 33.14 ±1.99 d

4.66 ±0.01 c

103±21.20 d

228±17.19 e

Maize Glutein Meal 30%

57.89±3.99 63.68±2.20 70.10±4.52 67.51±4.62 d

5.06±0.04 b

276±11.06 b

370±26.89 d

Yeast Extract 43.73±2.73 49.92±2.49 56.26±3.05 52.84 ±3.49 c

4.69 ±0.02 c

228±23.81 c

348±16.76 d

* Medium, I; pH, 4 ; Temperature (30±20C) ; Glucose, 1%.


118

0

20

40

60

80

100

120

0

Amm

onium

oxala

te

Corn

Steep

Liqu

or

M G

M(3

0%)

M G

M (6

0%)

Yeast

Extrac

t

Nitrogen source(%)

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

700

800

900

En

zym

e A

ctiv

ity

(IU

/mL

)

Decolorization(%)MnPLac

Fig.4.7a Effect of different nitrogen sources on decolorization of Remazole Brilliant Yellow 3-GL by C. versicolor and its lignolytic enzyme production in 3 days

y = 7.8186x + 91.209

R2 = 0.9652

y = 1.1327x + 269.25

R2 = 0.4713

100

200

300

400

500

600

700

800

900

25 35 45 55 65 75 85 95 105

Decolorization (%)

En

zym

e ac

tivi

ty (

IU/m

L)


r (MnP) = 0.669**r (Lac) = 0.928**

Fig.4.7b Relationship between dye decolorization and enzyme activities with different nitrogen sources.


119

ii) Effect of varying concentration of CSL

In order to optimize concentration of best nitrogen source, varying concentrations

(0.01, 0.05, 0.1, 0.15 and 0.2g/100mL) of CSL were supplemented to the medium. Dye

decolorization was 95.69, 98.18, 97.13, 90.76, and 54.61% in medium supplemented with

0.01, 0.05, 0.1, 0.15, 0.2 CSL respectively. Maximum dye decolorization was observed in

the medium containing 0.1 % CSL like the previous experiment after 72 hours. Final pH

of the media having 0.01, 0.05, 0.1, 0.15, 0.2 CSL was 5.26, 5.50, 5.85 and 6.72

respectively. MnP profile was 274, 357, 188, and 259 IU/mL; while laccase activities

were 566, 809, 542 and 246259 IU/mL in medium sharing 0.01, 0.05, 0.1, 0.15, 0.2 CSL,

respectively IU/mL (Table.4.8).

Analysis of variance of the data (Appendex-1, Table 4.7) also showed that results

were significant (p ≥0.01). DMR test revealed that treatment means with varying

concentrations of CSL carrying same letters were not significant (Table 4.8).


enzyme activities. The value of R2 indicated that dye decolorization was outcome of 85 %

variation in laccase and 50 % in MnP activities. Two parallel slopes in the Figure (4.8b)

indicated that R2 for laccase (0.85) was higher than for MnP (0.50) showing that

decolorization was faster through laccase followed by MnP synthesis.

It is commonly experienced that different nitrogen sources have different

influence on different strains. Some white rot grow better in limited supply of nitrogen in

the medium while some other show better decolorization in nitrogen sufficient

medium.(Robinson etal., 2003).The extent of

Moreover, addition of nitrogen beyond optimum level leads to rapid decrease in

dye decolorization (Radha et al., 2005) Kamie et al., 2006 also observed that low

nitrogen contents show higher dye degradation. Ramsy et al; (2006) also used the

nitrogen free Kirk,s medium with 1g/L glucose to get best results. Gao et al. (2006) also

investigated production of lignolytic enzyme production under limited nitrogen condition.

The high nitrogen content of the media enhanced the lignolytic enzyme, laccase, LiP, and

MnP. Several researchers have pointed out that medium supplemented with high nitrogen

allow high titres of enzymes (Verde et al; 2007).


120

Table-4.8 Decolorization of Remazole Brilliant Yellow-3GL by C.versicolor and its Lignolytic enzyme profile using varying

concentrations of CSL under optimimum conditions*

Varying Concentrations

of CSL (%)


Final EnzymeActivity(IU/mL)

on 3rd day

1 2 3 4 pH MnP Lac

0.01 71.27±14.42 42.63±15.56 49.93±17.81 45.69±19.61 c

4.95±1.27 d

262±67.67 b

339±65.57 d

0.05 67.09±27.22 85.349.31 89.97±52.01 98.18±8.80 bc

5.26 ±0.06 c

374±10.10 a

596±28.92 a

0.1 73.67±1.94 89.01±1.64 98.96±0.08 97.13 ±0.99 a

5.50±0.03 c

357±24.82 a

609±18.61 a

0.15 67.51±2.38 72.11±2.11 89.26±1.08 70.76±90.76 b

5.85±0.02 b

288±4.62 b

542±15.33 b

0.2 50.83±2.36 55.95±2.76 58.77±1.25 54.61±2.17 bc

6.72±0.05 a

259±9.18 b

346±21.64 c

* Medium, I; pH, 4 ; Temperature (30±20C) ; Glucose, 1%.


121

0

50

100

150

200

250

300

350

400

0 0.01 0.05 0.1 0.15 0.2

CSL Concentration (%)

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

700

800

900

1000

En

zym

e A

ctiv

ity

(IU

/mL

)


Fig.4.8a Effect of varying concentrations of CSL on decolorization of Remazole Brilliant Yellow 3-GL by C. versicolor and lignolytic enzyme production in 3 days

y = 7.7452x + 37.616

R2 = 0.8575

y = 2.4547x + 88.556

R2 = 0.5014

100

200

300

400

500

600

700

800

900

40 50 60 70 80 90 100

Decolorization (%)

Enzy

me

acti

vity

(IU

/mL)


r (MnP) = 0.673**r (Lac) = 0.859**

Fig.4.8b: Relationship between dye decolorization and enzyme activities with varying concentrations of CSL


122

Mineralization studies have revealed that most of the dyes investigated were

degraded extensively only in a certain range of nitrogen concentrations in P.

chrysosporium, Spadro et al; 1995 and Trametes versicolor, Kapdan 2000; Spadro et al;

1995; Hattaka 1994; Swamy and Ramsay, 1999b.

In a research study carried out by Tekere et al., 2001, using strains like Trametes

pocos, T. singulata, and T. elegans concluded that these had their highest manganese

peroxidase activity under conditions consisting of both high carbon and nitrogen

concentrations in the medium. Culture conditions combinig high nitrogen and high

carbon levels gave the highest laccase activity for Trametes versicolor, T.elegans ansd D.

concentrica.

Effect of mediators

Laccase was the major enzyme actively involved in the Remazole Brilliant

Yellow 3-GL decolorizatio by C. versicolor followed by MnP. A variety of mediators

that can be used to enhance the catalytic efficiency of ligninasesconjuction with laccase

have been reported in the literature. In our research ABTS, CuSO4, H2O2, MnSO4,

Tween-80, and Veratryl alcohol were used as low molecular mass mediators (1mM) on

account of their potential to influence the entire mechanism of reaction as well as enzyme

activities. ABTS receiving flasks showed maximum dye decolorization (99.52%) after 48

hours while 91.64. % decolorization in controlled flasks (without mediators) was

observed after 72 hours of incubation. Decolorization potential declined up to 98.90%

after 72 hours in experinmental flasks. H2O2, MnSO4, Tween-80, and Veratryl alcohol

caused 99.52, 81.13, 98.27 and 86.70% respectively (Table 4.9). Laccase activities noted

by using ABTS, H2O2, MnSO4, Tween-80, and Veratryl Alcohol were 758, 655, 552, 572

and 592 IU/mL while MnP activities were 387, 346, 463, 349 and 274 IU/mL

respectively (Fig.4.9a). Control flasks had 359, 825 IU/mL for MnP and laccase

respectively. Activities were calculated after 72 hours of incubation time. ABTS and

MnSO4 enhanced ligninases production and action in dye degradation.

Analysis of variance of the data (Appendex-1, Table 4.9b) showed that our results

were significant (p ≥ 0.01). DMR test reveals that treatment means with different

mediators carrying same letters are not significant (Table 4.10).


123

Table-4.9: Decolorization of Remazole Brilliant Yellow 3-GL by C.versicolor using low molecular mass mediators and its

lignolytic enzymes profile under optimimum conditions*

Mediators (1mM)


Final pH

Enzyme Activity(IU/mL) after 48 hours

1 2 3 MnP Lac

0 73.14±1.25 85.9±0.87 98.64 ±0.78

a 4.65±0.06

a 359±12.45

c 605±11.26

b ABTS

83.94±1.65 99.52±0.62 98.90±0.90 a

2.77±0.03 c

387±1.45 d

758±1.20 a

H2O2 71.05±2.88 81.13±1.13 88.25±1.43

b 3.78±0.11

b 366±17.92

a 552±2.61

c MnSO4

81.46±2.31 98.27±2.62 87.99±3.15 b

3.56±0.02 b

346±14.76 b

572±2.61 c

Tween-80 75.14 ±2.48 94.26±2.34 89.62±1.79

b 3.72±0.02

b 349±15.12

c 592±5.37

b Veratryl Alcohol

69.14.±1.13 86.70±1.98 90.12±3.04 ab

3.66± 0.03 b

274±9.84 a

285±18.79 d

* Medium, I; pH, 4 ; Temperature (30±20C) ; Glucose, 1% ; CSL, 0.1%.


124

0

20

40

60

80

100

120

0 ABTS H2O2 MnSO4 Tween-80 VeratrylAlcohol

Mediators (1mM )

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

700

800

900

1000

En

zym

e A

ctiv

ity

(IU

/mL

)


Fig.4.9a Effect of low molecular mass mediators on decolorization of Remazole

Brilliant Yellow 3-GL by C. versicolor and lignolytic enzyme production

y = 15.87x - 666.96

R2 = 0.9017

y = -6.2116x + 899.35

R2 = 0.9146

100

200

300

400

500

600

700

800

900

1000

45 55 65 75 85 95 105

Decolorization (%)

En

zym

e ac

tivi

ty (

IU/m

L)


r (MnP) = -0.956**r (Lac) = 0.950**

Fig. 4.9b: Relationship between dye decolorization and enzyme activities with low molecular mass mediators in 28 hours


125


enzyme activities. The value of R2 showed that dye decolorization was due to 90%

variation in laccase and 91 % in MnP activities. Two parallel slopes (Fig.4.9b) indicated

that R2 for for MnP (0.91) was higher than laccase (0.90). It clearly showed that

decolorization may best be achieved through MnP production followed by laccase

synthesis.

Enhancement of enzyme activities and decolorization of Remzole Brilliant

Yellow It is worth to consider that several researchers compared fungal laccases with a

variety of redox mediators and concluded that the redox potential of laccases varied with

the change in laccase source. This emphasizes the need/or nature of the redox mediator

for the degradation of particular dye to occur (Couto et al; 2007). Sores et al. (2001) have

reported decolorization of RBBR using a combination of redox mediator, laccase, and

nonionic surfactants.They postulated that mediator was a requisite for laccase catalysed

decolorization. A Brazillian strain, P. pulmonarius, producing only laccase was able to

decolorize dyes of all kinds and ABTS role in dye decolorization by enhancing enzyme

production is confirmed. It proved as a best mediator among all among all used in our

study in different triplicates.

It is hypothesized here that once mediators have been oxidized through the action

of laccase, they may indiscriminately react with other compounds thereby having the

potential to enhance the conversion of substrates by providing laccase with an additional

mechanism of attack. In addition, since the catalytic reactions have impact on enzyme

activities, it is possible that mediators would change the nature of the interactions

between the enzyme, substrate, and oxidation products and may tend to influence the

degree of inactivation (Kim and James 2006).

The rather broad substrate specificity of laccase may be additionally expanded by

addition of redox mediators, such as ABTS, 1-hydroxybenzotriazole, or compounds

secreted by lignolytic fungi.(Bourbonnais etal1995; Egger et a1; 996; Johannes and

Majcherczk 2000; Reyes et al;1999 ; Wong and Yu.1999).

It has been reported in previous study by Hofrichter et aI; (1998) that once a small

quntity of Mn (III) was added it reduced the lag period considerably which indicated that


126

it was able to initiate the reaction of MnP and therefore its own formation. MnP catalyses

the oxidation of Mn+2 to Mn+3 which is mainly involved in the oxidation of many

phenolic compounds(Warrishi etal; .1998). In presence of 0.5 m M of Mn and 2% of

Tween-80 Reactive dye decolorization by Phanerochaetae sordida reached up to 90.5%

in 48 hour activities of MnP in this way synergetic effect of both resulted faster dye

decolorization(Horazno et al; 2003).

Tween -80 acts as a surfactant which produces many organic acids which, in turn,

play the role of chelators and thus enhances enzyme activities which consequently

accelerate dye decolorization. Veratryl alcohol (3,4-dimethoxy benzyl alcohol),a

secondary metabolite of several WRF(de Jong et al; 1994), after its oxidation to the VA

cation radical (VA) by LiP, acts as a mediator for the degradation. Since in our research

there are insignificant activities of Lip so VA role in dye decolorizatioin was not that

appreciable. It has been noticed that VA is one of the most inactivating agents for laccase

which might be due to degradation of aromatic amino acids of laccase by the free radical

of VA (Couto et al; 2007).

Effect of Metal Ions

Keeping in view the significant role of metal ion on C. versicolor potential for

dye degradation was further checked.1mM solutions of different metal salts were applied.

These included mainly CaCl2, Cd(NO3)2, CuSO4, FeSO4, and ZnSO4. Dye solution

containing copper ions showed maximum (99.20%) while controlled set showed 83.6%

during decolorization with in 48. After 72 hours there was slight decline in experimental

flasks and increase in decolorization in control flaks (Table 4.10). Cadmium ranked

second with 82.51%. CaCl2 showed minimum (53.14%) decolorization. Zinc ion

influenced moderately (59.39 %) on decolorizing efficiency of C. versicolor. Fe with

71.32% decolorization respectively were best competitors of Cu and Cd (Fig 4.10a).

There was no significant change from optimum level. pH profile was 374, 4.15, 3.87,

3.93, and 3.89 in medium receiving CaCl2, Cd(NO3)2, CuSO4, FeSO4, and ZnSO4 ions

respectively. Most of the metal ions worked efficiently in acidic medium. MnP activities

were 362, 221, 383, 270 and 470 IU/mL while laccase activities profile was 563, 402,


127

Table 4.10 Decolorization of Remazole Brillaint Yellow 3-GL by C.versicolor and its lignolytic enzyme profile using

different metal ions under optimimum conditions*

Metal Ions (1mM)

Decolorization (% Mean ±S.E)

Incubation Time (Days) Final pH

Enzyme Activity (IU/mL) after 24 hours

1 2 MnP Lac

0 83.6±0.78 99.45±0.89 a

3.72±0.04 d

269±11.23 c

641±9.10 b

CaCl2 49.62±17.43 53.14±2.22 d

3.74±0.03 d

362±8.20 c

563±3.29 c

Cd(NO3)2 84.98±2.09 82.51±1.98 b

4.15±0.04 a

421±13.24 b

402±38.91 d

CuSO4 99.20±0.53 96.35±0.28 a

3.87±0.04 bc

383±5.05 b

797±10.49 a

FeSO4 75.38±1.38 71.32±0.90 c

3.93±0.02 b

270±11.00 c

474 ±9.50 e

ZnSO4 65.80±1.82 59.39±4.16 d

3.89±0.02 bc

470±10.98 a

683 ±7.23 b

* Medium, I; pH, 4 ; Temperature (30±20C) ; Glucose, 1% ; CSL, 0.1% ;Meditor, ABTS (1mM).


128

0

100

200

300

400

500

600

0 CaCl2 Cd(NO3)2 CuSO4 FeSO4 ZnSO4

Metal Ions

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

700

800

900

En

zym

e A

ctiv

ity

(IU

/mL

)

Decolorization(%)MnPLac

Fig.4.10a Effect of different metal ions on decolorization of Remazole Brilliant

Yellow 3- GL and lignolytic enzymes production by C.versicolor 24 hours

y = 7.0989x + 159.52

R2 = 0.4136

y = 1.2392x + 358.11

R2 = 0.0681

100

200

300

400

500

600

700

800

900

1000

1100

45 55 65 75 85 95 105

Decolorization (%)

Enzy

me

acti

vity

(IU

/mL)


r (MnP) = 0.261r (Lac) = 0.643**


different metal ions


129

797, 474 and 683 IU/mL in triplicate flasks receiving CaCl2, Cd(NO3)2, CuSO4, FeSO4,

and ZnSO4 respectively. Overall, none of the metal ions significantly enhanced ligninases

synthesis by C. versicolor and dye degradation except CuSO4, rather all of them found

inhibitory to the process.


were significant (p ≥ 0.01). DMR test reveals that treatment means with different metal

ions carrying same letters are not significant (Table 4.10).


enzyme activities. Two parallel slopes in the Figure (4.10b) indicated that R2 for laccase

(0.64) was higher than for MnP(0.012) clearly showed that decolorization may best be

achieved through laccase production followed by lower activities of laccase. Laccase

showed maximum (797 IU/mL).

Heavy metals can affect the growth (Baldarinan and Gabriel, 1997; Mandal et al.,

1998; Pointing et al; 2000), the extra cellular enzyme production (Baldrian et al; 2000)

and the dye decolorizing abilities of WRF. Laccase formation is influenced by various

physiological factors and can be enhanced by the addition of organic compounds and

metal ions. The highest laccase activity (697 IU/mL) was obtained by using copper

(2mM) and it increased up to 800 IU/mL by using 3.5 mM CuSO4 (Lorenza et al. 2006) .

Our strain produced 797 IU/mL of laccase in 48 hours with 1mM CuSO4 indicating

better performance and efficient decolorization ability of C. versicolor Remazole

Brilliant Yellow 3-GL.

Some metal ions are essential for the fungal metabolism, where as others have no

known biological role. Both essential and non-essential heavy metals are toxic for fungi.,

when present in excess where as fungi have metabolic requirement for trace metals, the

metals are toxic when at concentrations only a few times greater than those required

(Hughes and Pool l991) The metals necessary for fungal growth include copper, iron,

cadmium, manganese, molybdenium, zinc and nickel. Non essential metals commonly

encountered include chromium, lead, mercury and silver (Gadd etal1993). Since the

effect of heavy metals on the activity of extracellular lignin degrading enzymes has been


130

described it is clearly that the presence of metals also affect the biotechnological

processes based on their activity (Baldrian, 2004). Enzyme activities were directly

correlated with the metal ion effects. The metal ions like copper which left pronounced

effect on decolorization also gave maximum laccase activities. Both showed maximum

ativity of laccase. pH change in CuSO4 containing flasks was 0slightly higher than

optimum level. The positive effect of copper addition on the production of laccase has

also previously been observed in Ceriporiopsis subvermispora, (Salas et al., 1995;

Karahaniasn et al., 1998), T. vericolor, (Collins and Dobson 19970), Marasmius

quercophilus, (Farnet et al 1999), P. ostratus, (Palmieri et al; 2000), P.chrysosporium,

(Dittmer et al; 1997), T. pubbescens, T. multicolor, T. hirsuta, T. gibbosa, T. suaveolens,

G. applaantum, Polyporus ciliatus and Pinus tigrinu, (Galhaup and Haltrich 2001), P.

sjarcaju, (Soden and Dobson 2001) and T. trogii, (Levin et al; 2002) .

Addition of Cu caused not only increase in enzyme production but also the

stabilizsation effect of this metal which induces transcription of laccase gene (Collin and

Dobson, 1997). T. versicolor showed enhanced decolorization in presence of (0.1mM)

Cu+2 (Baldrian and Gabriel 2002) but inhibition was observed at its higher concentration

(Kaushik and Malik, 2008).

Laccase is a blue copper enzyme which catalyses the one electron oxidation of

various aromatic compounds especially phenols and aniline while reducing molecular

oxygen to water (Gianfreda and Alic1993). The present study also suggests that heavy

metal salts could be used to enhance the enzyme production and and stability during the

large scale production and its application in bioremediation processes.

Role of iron was not inhibitiory during decolorization of Remazole Brilliant

Yellow 3-GL Incase of RBBR severe inhibitin was observed in the presence of Fe (more

than 25% inhibition).This could be explained due to instability of laccase in the presence

of iron at high concentration i.e 10mM (Rodriguez Cuoto et al.2005). In case of

Remazole Brilliant Yellow 3-GL no such observation was. It might be due to use of low

concentration (1Mm) of metal ions.


131

Effect of varying concentrations of dye

The effect of varying concentrations of RBY-3GL on decolorizing ability of C.

versicolor was investigated. The dye concentration range was 0.01-0.2 g/100mL. Highest

deecolorization (96.60%) under optimum conditions was observed in the medium

receiving 0.01% dye after 24 hours. Medium receiving 0.01, 0.05, 0.1, 0.15 and 0.2 % of

the dyer showed 81.75, 49.73, 15.67 and 9.67 %respectively (Table 4.11a). As the

concentration increased there was pronounced inhibition in terms of enzyme activity and

dye degradation. Enzyme activities were noted as: 389, 349, 333, 286 and 236 IU/mL for

MnP while 1019, 976, 531, 338 and 261 IU/mL for laccase in 0.01, 0.05, 0.1, 0.15, and

0.2% respectively (Fig.4.11a).

DMR test reveals that treatment means with nitrogen sources carrying same

letters are not significant (Table 4.11). Analysis of t variance of the data (Appendex-1,

Table 4.12) showed that our results were significant (p ≥ 0.01).


enzyme activities. The value of R2 indicated thatdye decolorization was due to 95%

variation in laccase and 90 % in MnP activities were due to decolorization. Two parallel

slopes (Fig.4.12) indicated that R2 for laccase (0.95) was more than for MnP (0.90)

showing major contribution of laccase followed by MnP synthesis.

C. versicolor is one of the most commonly used strain for dye decolorization. In

previous studies it was reported that C. versicolor decolorized 14.90, 37.80, 13.45, 5.65,

27.28 and 49.0 mg/L of Indigo Carmin, Reactive Blue5, Acid Blue 25, and Acid Black 45

respectively at the end of 9 day of incubation (Erkurt et al., 2007). Erkut et al. (2007)

reported that Casndida kruesi and Pseudozyma rugulosa decolorized 99 % of 200 mg/L

Reactive Brilliant Red k-2bp in 24 hours. But as the dye concentration increased up to

1000 mg/L, decolorization potential decreased to 20%.

Our strain of C. versicolor showed better decolorization (99.71%) of Remazole

Brilliant Yellow 3-GL at 0.01g/100mL dye concentration as compared to previous studies

of Aksu et al. (2007b).They observed 92% decolorization of Rmazole BlackB (azodye) at

initial concentration. It shows that dye degradation rate becomes slower with increase in

dye concentration. So there is inverse relationship between dye concentration and

decolorization %, the higher the dye concentration lower will be the enzyme activities

and slower the decolorization rate.


132

Table 4.11 Extent of decolorization of varying concentrations of Remazole Brilliant Yellow 3-GL C. versicolor and its

lignolytic enzyme profile under optimimum conditions *

Varying Dye

Concentrations (%)

Decolorization (% Mean±S.E) Incubation Time (Days)

Time (Days) Final pH


1 2 MnP Lac

0.01 99.6±0.30 96.71±0.15 a

5.30±0.68 a

389±0.33 a

795±0.97 a

0.05 81.76±2.27 79.85 ±1.79 b

4.61±0.02 d

349±5.37 b

676±7.78 a

0.1 49.73±2.40 49.51±2.46 c

4.60±0.05 d

333±10.60 b

531±9.95 b

0.15 15.67±2.26 15.73±2.10 d

4.65±0.03 c

286±5.30 c

338±26.28 c

0.2 9.67±1.19 9.54±1.18 e

4.71±0.02 b

236±9.30 d

261±14.85 d

* Medium, I; pH, 4 ; Temperature (30±20C) ; Glucose, 1% ; CSL, 0.1%,Meditor, ABTS1mM) ; Metal Ion, CuSO4 (1mM).


133

0

50

100

150

200

250

300

350

400

450

0.01 0.05 0.1 0.15 0.2

Dye Concentration (%)

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

700

800

900

En

zym

eAct

ivit

y (I

U/m

L)

Decolorization %MnPLac

Fig.4.11a: Effect of varying concentrations of Remazole Brilliant yellow 3-GL decolorization by Coriolus versicolor and lignolyitic enzymes production in 24 hours

y = 8.8413x + 195.17

R2 = 0.9524

y = 1.1231x + 272.8

R2 = 0.9031

100

200

300

400

500

600

700

800

900

1000

1100

0 20 40 60 80 100 120

Decolorization (%)

En

zym

e ac

tivi

ty (

IU/m

L)


r (MnP) = 0.937**r (Lac) = 0.984**

Fig.4.11b: Relationship between dye decolorization and enzyme activities with varying concentrations of the dye


134

Adsorption of dye on fungal mycelia

Adsorption flasks had no nutrients and received only 100 ml of dye solution in

distilled water and 5 ml inoculum. Negligible adsorption % was noted in lowest dye

concentration. The flasks receiving 0.01, 0.05, 0.1, 0.15 and 0.2% dye showed 0.04, 0.06,

0.11, 0.42, and 0.46% adsorption in 24 hours (Table12)

DMR test revealed that means of treatment values of nitrogen sources carrying

same letters are not significant (Table 4.12). Analysis of the variance of the data

(Appendex-1, Table 4.12) showed that our results were significant (p ≥ 0.01).

In a previous study it was noted that WRF decolorization can be achieved from

both adsorption at fungal mycelium and /or biotransformation of the dye molecule (Fu

and Viraraghavn, 2001). During the study of ABU 62 decolorization was carried out

using six different WRF strains C. polyzona, P. tephropora, P. ochroleuca, P. sanguineus

MUCL38531, P. sanguineus MUCL 41582 and T. versicolor. The decolorization of

Abu62 dye was mainly due to biotransformation instead of adsorption on mycelia. Since

there was 10% desorption of initial dye concentration at the termination of experiment

(Vanhulle et al., 2008).

The reactive dyes are highly water soluble polymeric aromatic molecules, which

means that their adsorption to solid is relatively poor (Nilsson et al. 2006). Microscopic

and spectroscopic studies of fungal mycelia revealed that the decolorization is brought

about by microbial metabolism not sorption (Erkurt et al; 2007; Mazmanci et al; 2005

and Unyayar et al; 2005) also reported a similar result. Blanquez et al; (2008) also

suggested that ultimate color removal was due to decolorization process not because of

adsorption which occurred normally on biomass of fungal strain at initial stage.

However, it depends on the dye concentration level and fungal culture age. With

lowest dye concentrations (0.01) there was negligible dye adsorption while at higher

concentrations, dye adsorption increased and maximum (0.59%) was noted with 0.2%

dye concentration.


135

Table 4.12 Adsorption of Remazole Brilliant Yellow 3-GL on fungal mycelia

under optimimum conditions*


Decolorization (% Mean±S.E) with Incubation Time (Days)

Final pH

0.01

1 2 4.67±0.02

a 0.04±0.00

0.002±0.00 a

0.05 0.06±0.00

0.005±0.00 b

4.51±0.6 c

0.1 0.11±0.02

0.06±0.01 b

4.60±0.02 b

0.15 0.42±0.03

0.14±0.03 b

4.59±0.01 b

0.2 0.48±0.01

0.19±0.6 a

4.43±0.33 d

* pH, 4 ;Temperature (30±20C).

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.5 1 1.5 2

Dye concentration

Dec

olo

riza

tio

n (

%)

Fig.4.12a Effect of varying dye concentrations on adsorption by fungal mycelia


136

Wong and Yu (1999) investigated adsorption and degradation of synthetic dyes

with T. versicolor. The dye adsorption reported was 17-150 mg/L but the adsorbed dye

molecule could be degraded by enzymes produced within 7 days. In present study

insignificant dye adsorption was found at optimum dye concentration, thus confirming

the enzymatic dye degradation as the major route of color removal.

In a comparative study (static vs agitated) carried out by Swamy and Ramsay

(1991) using T. versicolor for dye decolorization, no mycelial sorption of dye was

detected in agitated cultures. In fact liquid state culture enhances decolorization process.

Our results are in line with these findings. The superior performance of the agitated

cultures may be due to increased mass and oxygen transfer between the cells and the

medium due to mixing. Biodegradation by white rot fungi has been attributed to the

extracelluler activity of oxidative enzymes (Field et al.1993).These enzymes in

T.versicolor and P.chrysosporium have been found to function optimally at 100 % O2

.(Archibald etal. 1990; Kirk etal. 1978). Adsorption of dye in solid cultures is more than

liquid ones. This is due to formation of “ mycelial mats” at the surface in static cultures

which causes limited supply of O2 transfer beneath the cells and in the medium. Oxygen

limitation is likely to inhibit the oxidative enzymes and prevent dye decolorization

(Swamy and Ramsay1991). In addition, decolorization of RBBR by P. ostreatus showed

that color rmoval from the cultivation medium was due to dye transformation rather than

adsorption of the dye on the fungal mycelium. Moreover, changes in absorption spectra

indicate that decolorization is not a single step process instead involves complex

mechanism (Palmieri et al; 2005).

4.2 DECOLORIZATION OF DISPERSE DYES

4.2.1 Screening of White Rot fungi on disperse dyes

Screening of all white rot fungi on disperse dyes Foron Turquoise SBLN-200,

Foron Blue RDGLN, Foron Red RDRBLS and Foron Yellow SE4G was carried out and

decolorization results have been presented in Table 4.13a. P.ostreatus and G. lucidum

showed maximum decolourization of all Foron dyes on 6th day of incubation. P.

ostreatrus caused a color removal of 63.91, 70.2, 60.90 and 40.3% for Foron Turquoise,

Foron Blue, Foron Red and Foron Yellow, respectively. Whereas G. lucidum removed

91.86, 76.1, 84.1 and 64.7% of the dyes in 7 days respectively.


137

Table 4.13a. Decolorization of Disperse dyestuffs by different white rot fungi

Incubation Time

(Days)

WRF Cultures

Decolorization (%)Foron Turquiose

SBLN- 200 Foron Blue

RDGLN Foron Red RDRBLS

Foron Yellow SE4G

1

P.O. 2.15 0.15 2.75 0.17 2.54 0.10 1.15 0.15 G.L. 15.10 0.30 2.04 0.60 3.55 0.55 1.55 0.35 S.C. 5.55 0.20 4.58 0.30 6.20 0.20 2.50 0.30 C.V. 3.20 0.40 2.40 0.20 4.10 0.10 1.30 0.20 P.C 30.6 0.10 20.2 0.20 25.9 0.30 15.4 0.20

2

P.O. 14.7 0.48 12.1 0.35 13.0 0.145 10.0 0.20 G.L. 26.72 0.35 15.2 0.30 15.9 0.20 10.1 0.10 S.C. 20.9 0.60 19.6 0.40 23.4 0.20 18.8 0.40 C.V. 9.80 0.2 0 13.2 0.80 11.9 0.50 8.60 0.30 P.C. 24.3 0.50 32.5 0.80 37.0 0.75 28.9 0.30

3

P.O. 42.2 1.70 48.8 0.60 38.6 0.45 28.2 0.70 G.L. 36.76 0.40 35.7 0.30 38.9 0.85 28.9 0.35 S.C. 28.6 0.20 25.8 0.40 35.6 0.40 26.7 0.20 C.V. 18.3 0.60 20.1 0.55 19.6 0.30 15.6 0.10 P.C. 32.8 0.30 40.6 0.55 56.2 0.55 36.7 0.10

4

P.O. 51.4 0.30 54.3 0.85 50.8 0.65 33.9 0.80 G.L. 58.19 0.70 41.5 0.35 55.7 0.80 41.7 0.80 S.C. 48.9 0.30 41.6 0.60 55.3 0.40 33.5 0.20 C.V. 26.6 0.50 22.3 0.30 20.5 0.10 18.3 0.30 P.C. 42.9 0.10 54.7 0.30 71.2 1.10 44.5 0.30

5

P.O. 58.9 0.4 0 76.4 0.80 64.7 0.40 43.7 1.50 G.L. 65.54 0.80 51.9 0.25 49.4 0.40 46.6 0.30 S.C. 56.9 0.30 47.3 0.20 51.3 0.50 41.2 0.40 C.V. 35.4 0.20 32.1 0.50 31.3 0.10 26.7 0.30 P.C. 54.4 0.10 65.3 0.50 74.9 0.59 55.3 0.30

6

P.O. 63.91 0.60 70.2 0.10 60.9 0.40 39.3 0.90 G.L. 82.98 0.20 63.2 0.40 71.9 0.40 56.9 0.30 S.C. 65.6 0.60 55.2 0.60 67.6 0.80 49.3 0.40 C.V. 43.1 0.10 39.3 0.50 42.7 0.30 34.5 0.20 P.C. 61.12 0.20 76.1 0.65 76.2 0.98 60.9 1.56

7

P.O. 60.3 0.25 62.8 0.60 54.8 0.45 34.3 0.90 G.L. 91.86 0.80 76.1 0.15 84.1 0.50 64.7 0.40 S.C. 70.2 0.30 63.3 0.20 75.6 0.70 56.4 0.10 C.V. 48.6 0.50 50.6 0.40 48.9 0.60 41.3 0.10 P.C. 83.18 0.20 79.9 0.98 80.5 1.23 64.2 2.1

8

P.O. 54.7 0.60 62.8 0.20 58.4 0.30 30.4 0.20 G.L. 91. 87 0.00 79.00 0.40 86.7 0.50 68.5 0.40 S.C. 65.6 0.30 59.4 0.10 71.3 0.60 51.3 0.50 C.V. 45.6 0.20 40.9 0.30 42.7 0.30 37.6 0.10 P.C. 88.34 0.03 78.2 1.75 84.5 2.50 67.2 0.79

9

P.O. 48.8 0.40 50.9 0.80 44.7 0.40 23.0 0.55 G.L. 91.89 0.00 81.4 0.40 86.8 0.15 76.9 0.20 S.C. 59.8 0.40 54.3 0.30 65.6 0.40 45.6 0.50 C.V. 39.4 0.50 35.5 0.20 36.2 0.30 39.6 0.10 P.C. 89.16 0.00 86.6 2.26 87.8 1.94 71.9 1.35

10

P.O. 40.9 0.30 43.8 0.60 37.5 0.40 18.4 0.30 G.L. 91.94 0.00 81.9 0.15 89.1 0.15 76.9 0.40 S.C. 54.3 0.20 48.9 0.10 58.7 0.60 40.4 0.30 C.V. 33.6 0.70 29.8 0.60 31.3 0.50 32.5 0.40 P.C. 90.12 0.00 88.9 1.56 89.8 2.60 74.0 1.56


138

G.lucidum had maximum (91.86%) color removal efficiency on Foron Turquize

in 7 days. However, it also effectively decolorized of Blue, Red, and Yellow dyes. S.

commune also showed reasonable potential on Turquize, Blue and Red dyes. However, C.

versicolor showed comparatively poor performance showing 41-51 % dye decolorization

of disperse dyes after 7 days. The combinations of fungi and dyestuffs based on the

results in terms of maximum decolourization in 10 days initial trial have been shown in

Table 4.13a.

Synthetic dyes are released in waste water from different sources like textile

industries, leather industry, pharmaceuticals and foodstuffs manufacturers. But Disperse

dyes are usually discharge mainly from textile manufacturing units. Their decolorization

is mandatory prior to discharge in wastewaters (Azmi et al., 1998).This study illustrates

that indigenous white rot strains have immense potential for textile dyes degradation and

can be considered as a good dye decolorizers. Decolorization ability is widely accepted

on account of LME system in white rot fungi (Reddy1995).

It is difficult to explain difference in decolorization % of different dyes. The

process involves breaking of chromophore. In some cases poor decolorization might be

due to complexity of dyes’ chromphore but the structural complexity is not solely

responsible for the slowness or low level of dye decolorization process.From the

screening results it was concluded that ForonTurquoise was maximally decoloriszed form

optimization process.

4.2.2 Optimisation of Foron Turquoise SBLN-200 decoloriszation by G.lucidum

The selected combination of fungus and dye from screened experiment was used

for process ptimisation.

Effect of different media compositions

Four different nutrient media compositions were used to select a simple and

suitable meium for decolorization of Foron Turquoise SBLN-200 by G.lucidum. Medium

IV was found to be the best among all the media. The decolorization was 86.91, 55.47,

69.23 and 98.04 % in medium I, II, III and IV respectively (Table 4.14). On 7th day of

incubation.


139

Table.4.14 Decolorization of Foron Turquoise SBLN-200 by Ganoderma lucidum and its lignolytic enzyme using different

basal nutrient media

Med

ium

Decolorization (% Mean ±S.E) Incubation Time (Days)

Final Enzyme Activity

(IU/ mL)

1 2 3 4 5 6 7 pH LiP MnP Lac

M-I 16.19±0.67 27.89±0.90 43.99±0.67 58.97±1.10 65.71±1.47 78.52±0.81 86.91±0.90

b 2.42± 0.10

b 282±3.76

b 115±2.65

c 116 ±3.18

c

M-II 6.53±0.93 21.13±1.12 35.35±0.95 47.61±0.83 49.94±1.21 53.48±1.47 55.47 ±2.02

d 2.45± 0.06

b 119± 6.74

c 108 ±6.75

d 102± 7.54

c

M-III 8.05±0.50 19.79±0.87 36.79±0.91 45.98±1.49 55.08±3.20 61.13±1.13 69.23± 1.79

c 4.41± 0.12

a 166 ± 3.76

d 185± 4.59

b 145 ±2.97

b

M-IV 22.86±1.77 36.74±0.89 58.49±15.26 77.90±2.05 84.84±3.04 91.74±2.11 99.65 ±3.49

a 4 .77±0.11

a 390±1.45

a 222±3.06

a 160 ±2.34

a

* pH, 4 ; temperature (30±20C).


140

0

100

200

300

400

500

600

700

M-1 M-2 M-3 M-4

Medium

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

700

En

zym

e A

cti

vity

(IU

/mL

)

Decolorization (%)LiPMnPLac

Fig. 4.14a Effect of varying media compositions on decolorization of Foron

Turquoise SBLN-200 by Ganoderma lucidum and its lignolytic enzyme production in 7 days

y = 4.7571x + 158.37

R2 = 0.4252

y = 4.0157x + 154.3

R2 = 0.7352

y = 4.0155x + 9.4735

R2 = 0.8594

100

200

300

400

500

600

700

30 40 50 60 70 80 90 100

Decolorization (%)

En

zym

e ac

tivi

ty (

IU/m

L)

LipMnPLacLinear (Lip)Linear (MnP)Linear (Lac)

r (Lip) = 0.652*r (MnP) = 0.857**r (Lac) = 0.927**

Fig 4.14b: Relationship between dye decolorization and enzyme activities with varying media compositions


141

Final pH on 7th day was 1.42, 1.45. 4.41 and 4.77 in media I, II, III and IV

respectively. pH shifted towprds more acidic range in medium I and II while it remained

in less acidic in media II and IV. Activities of LiP were 419, 229, 564 and 621 IU/mL .

MnP activities noted were 325, 298, 485 and 522 IU/mL; laccase activity profile was

325, 298 and 485 IU/mL in medium I, II, III and IV respectively. Lowest activities of the

three ligninases enzymes were in modified Kirk’s Basal Medium (M-2) and highest were

noted in M-IV (Fig.4.14a).

Analysis of variance of the data (Appensedex-1Table 4.14b) showed that our

results were significant (p< 0.01). DMR test shows that treatment means of different

media carrying same letters have significant difference (p< 0.01). The sign of slope

indicated that increase in decoloriztion was positively correlated with the enzyme

activities. The value of R2 indicated that dye decolorization was due to 42% variation in

LiP, 73 % in MnP and 86 % in laccase activities. Three parallel slopes (Fig.4.14b)

indicated that R2 for laccase was (0.86) highest than MnP(0.85) and Lip (0.65) among

three enzymes. It clearly showed that dcolorization may best be achieved through laccase

production followed by MnP and Lip formation.

Medium IV was selected as the best medium for dye decolorization due to

G.lucidum. Since medium It was costly due to Veratryl Alcohol and Tween -80 while

remaining media were without these two components. A common feature of the medium

III and IV was presence of urea (a rich source of nitrogen). The choice for an alternative

medium was the only way to cope with both problems; cost effectiveness as well as faster

dye degradation.

In a study on Cibacron turquoise P-GR it was observed that decolorization by G.

lucidum successfully completed in Medium II which was also lacking Tween-80 and

Veratryl Alcohol like M-IV (Hafiz et al; 2007). Kapdan et al; (2000) noted best

decolorization of Everzole Turquiose Blue G in medium III using Coriolus versicolor

MUCL. It has also been investigated by D’Souza et al; (1999) found only laccase activity

in G. lucidum cultures with complete absence of complete absence of LiPs and MnPs.

But it has been wel documented that nitrogen levels, aeration and other factors in the

mediu influence the LME production by white rot fungi (Kirk, and Farrell 1987; Buswell

and Odier 1987; Boominathan and Reddy 1992; Van der et al 1993 Buswell, et al;1995).

As LME production is influenced by multiple factors so dye decolorization is also

affected by the different media composition.


142

Composition of medium played critical role in inducing genes responsible for

enzyme production. (D’Souza, and, Reddy 1999). Appearently G.lucidum has genetic

potential for LiP. Their production or inhibition is highly dependent on nature of

culturing media and even presence or absence of any single component

Our indegnous strain of G. lucidum also produced all three types of LMEs (LiP,

MnP and laccase) while maintaining culture conditions in medium IV which was N-rich

due to the presence of urea.

It has been reported that Ganoderma lucidum produce all three types of lignolytic

enzymes. Although LiP shows maximum activity in dye decolorisation yet the role of

MnP and laccase in dye decolorization cannot be overlooked.A recent study carried out

on G. lucidum by Hafiz etal; (2007) showed that indigo and azo dyes werewel substrates

decolorized by MnP and LiP and the reaction by MnP was faster.

Effect of Additional carbon Sources

i) Addition of different carbon sources

Carbon sources play vital role in fungal metabolic activities. Different carbon

sources were used to check their potential in color removal by Ganoderma lucidum in

medium IV. Color removal was the highest (96.78 %) with wheat bran followed by

glucose (88.84 %), starch (85.05 %), glycerol (43.88 %) and lactose (33.33%) after 5 ays

of incubation. Lowest decolorization occurred in lactose. Drift from optimal pH (4.5)

level was 0.78, 1.58, 0.48, 1.78 and1.19 in glucose, glycerol, lactose, starch, and wheat

bran containing media, respectively. Wheat bran enhanced decolorization and pH shifted

towards more acidic range. Experimental flasks showed maximum decolorization

(90.11%) on 5th day while controlled flasks decolorized the same dye up to 75.71%

(Table 4.16a). Enzyme activities after the addition of carbon sources showed variable

trends. Activities of LiP were 416, 275, 255, 398, and 419 IU/mL and MnP activities

profile noted was as: 338, 236, 228, 332, and 348 IU/mL and laccase activities in media

containing glucose, glycerol, lactose, starch and wheat bran were 190,147,118, 169, and

202 IU/mL respectively (Fig.4.15a). By the addition of carbon sources incubation period

decreased to 6 days.


143

Table.4.15 Decolorization of Foron Turquoise SBLN- 200 BY Ganoderma lucidum and its lignolytic enzyme profile using

different carbon sources under optimum conditions*

Carbon sources (1%)


pH


1 2 3 4 5 6 Lip MnP Lac

0 22.9± 0.89 35.96±0.12 58.61±0.45 77.16±0.89 84.88±0.88 91.64± 1.01

a 4.72 ±0.03

a 391± 11.23

b 296±14.25

c 176±11.29

c

Glucose 41.80±1.73 48.60±1.98 60.66±1.56 88.84±1.84 90.11±1.89 88.84 ±1.76

b 3.72±0.06

b 416±3.25

b 338 ±3.17

b 190±3.18

b

Glycerol 23.51±2.02 31.7±2.80 39.24±1.70 46.30±2.83 49.71±1.23 43.88±2.24

d 2.92±1.01

d 275±7.58

c 236 ±5.61

d 147±3.85

c

Lactose 19.09±1.16 16.10±1.64 26.1±1.74 36.45±2.48 45.36±0.45 33.33±1.73

c 4.02±2.72

a 255±4.36

d 229 ±8.12

d 118 ±4.81

d

Starch 55.91±1.62 67.32±2.05 79.04±3.67 79.91±1.97 86.11±0.77 85.05±2.19

b 2.72±0.01

d 398±8.40

b 332±7.65

b 169±5.85

b

Wheat Bran 53.64±2.79 77.29±1.83 86.18±3.13 90.64±1.38 96.78±0.98 92.04±1.54

a 3.31±0.07

c 469 ±10.42

a 348±8.92

a 202±6.77

a

* pH, 4.5 ; temperature (35±20C); medium IV


144

0

50

100

150

200

250

300

350

400

450

0 Glucose Glycerol Lactose Starch Wheat Bran

Carbon Source(1%)

Dec

olo

riza

tio

n (

%)

0

50

100

150

200

250

300

350

400

En

zym

e A

ctiv

ity

(IU

/mL

)

Decolorization (%)LiPMnPLaccase

Fig.4.15a: Effect of different carbon sources on decolorization of Foron Turquoise SBLN-200 by Ganoderma lucidum and lignolyitic enzymes production

y = 6.5578x + 250.77

R2 = 0.8279

y = 4.3978x + 247.97

R2 = 0.7203

y = 1.4492x + 246.63

R2 = 0.4644

0

100

200

300

400

500

600

700

800

900

1000

30 40 50 60 70 80 90 100

Decolorization (%)

En

zym

e ac

tivi

ty (

IU/m

L)


r (Lip) = 0.910**r (MnP) = 0.849**r (Lac) = 0.681**

Fig.4.15 b: Relationship between dye decolorization and enzyme activities with different carbon sources


145

Analysis of variance of data (Appendex-1; Table 4.15a) shows that our results

were significant (p>0.01). DMR test showed that means of treatment values of enzyme

activities showed significant difference (Table 4.16a).The sign of slope indicated

(Fig.4.16b) that increase in decolarization was correlated with the enzyme activities. The

value of R2 indicated that dye decolorization was due to 83 % variation in LiP, 72 % in

MnP and 46 % in laccase activities. Three parallel slopes in the figure (4.15b) indicated

that R2 for LiP(0.82) was the highest among three enzymes showing that dcolorization

may have been achieved through Lip production followed by MnP and laccase formation.

ii) Effect of varying concentrations of wheat bran

After selecting wheat bran as the cheapest carbon source, varying concentrations

of wheat bran were applied 0.1 to 2.0 % were added difference of 0.5 % were added.

Foron Turquise SBLN-200 decolorization by G.lucidum. After four days of incubation

time decolorization was 76.01, 83.86, 90.41, 98.11 and 81.36 % in flasks receiving 0.1,

0.5, 1.0, 1.5 and 2.0 % wheat bran after 5 days of incubation (Table 4.16). Medium

supplemented with 1.5% wheat bran showed highest dye decolorization (98.81%) in 4

days. Controlled flasks showed 94.98 % decolorization within same incubation time. pH

profile was 5.98, 6.11, 5.85, 5.63 and 4.94 respectively (as presented in Table 4.16a). LiP

activities observed were 248, 274,298, 318 and 269 IU/mL; MnP activities were 291,

325, 354, 403 and 321 IU/mL while laccase profile was as 248, 274, 298,318 and 294

respectively (Fig.4.16a). Analysis of variance of the data (Appendex-1; Table 4.16b)

shows that our results were significant (p>0.01). DMR test shows that treatment means of

varying concentrations of carbon sources carrying same letters are not significant

(p>0.05, Table 4.16a). The sign of slope indicated (Fig.4.16b) that increase in

decoloriztion was correlated with the enzyme activities. The value of R2 indicated that the

dye decolorization took place due to 66% variation in LiP, 74% in MnP and 40% in

laccase activities. R2 for MnP (0.74) indicated that decolorizatin was mainly caused by

MnP activities followed by LiP and laccas in the presence of 1.5% wheat bran. It clearly

showed that dcolorization was mainly caused by activities followed by Lip and laccase in

the presence of wheatbran production followed by MnP and accase synthesis


146

Table.4.16 Decolorization of Foron Turquoise SBLN- 200 by Ganoderma lucidum and its lignolytic enzyme profile using varying concentrations of wheat bran under optimum conditions*

Concentrations of Wheat Bran

(%)


Final


1 2 3 4 5 pH Lip MnP Lac

0.1 38.01±1.64 59.24±5.53 67.18±4.81 76.01±3.65 81.29±3.73 b

5.98±0.04 ab

402±17.83 d

291±4.34 d

248±3.18 d

0.5 46.41±2.62 56.73±6.84 64.5±3.02 83.86±3.01 80.49±3.22 c

6.11±0.01 a

442 ±14.42 c

325±3.06c

274±7.89 c

1 52.88±3.34 76.66±3.19 86.91±1.42 90.41±8.41 94.01±0.91 a

4.65±0.06 b

455± 12.10 b

354±5.37 b

298±2.41 b

1.5 49.91±3.67 60.66±4.45 86.71±3.19 98.11±1.04 98.88±1.13 a

5.63±0.01 b

496±15.67 a

398±2.03 a

308±3.48 a

2 62.16±2.90 73.69±3.89 77.73±4.12 83.36±2.96 81.14±2.30 b

5.09±0.03 c

436 ±12.97 a

321±3.15 c

269±8.52 d

*MediumIV;pH,4.5;temperature(35±20C);


147

0

100

200

300

400

500

600

0.1 0.5 1 1.5 2

Concentration of Wheat Bran (%)

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

700

800

En

zym

e A

ctiv

itie

s (I

U/m

L)


Fig.4.16a: Effect of varying concentrations of wheat bran on decolorization of Foron Turquoise SBLN-200 by Ganoderma lucidum and lignolyitic enzymes production in 4 days.

y = 8.7798x + 94.264

R2 = 0.6606

y = 4.5297x + 282.99

R2 = 0.7362

y = 1.59x + 202.66

R2 = 0.4076

0

200

400

600

800

1000

1200

50 60 70 80 90 100

Decolorization (%)

Enzy

me

acti

vity

(IU

/mL)


r (Lip) = 0.813**r (MnP) = 0.858**r (Lac) = 0.638*

Fig.4.16b: Relationship between dye decolorization and enzyme activity with varying concentrations of wheat bran


148

White rot fungi fungi utilize easily available carbon sources at preliminary stages

of growth and produce lignolytic enzymes and in secondary metabolism and extracellular

enzymes for biodegradation of dyestuffs at minimal concentrations of carbon and

nitrogen. The most commonly useable source by white rot fungi is glucose (Kapdan etal

2000). But wheat bran has dual edge over rest of the carbon sources on account its cost

effectiveness as well as highest decolorization potential for Foron Turquoise SBLN 200

by G. lucidum.The presence of wheat bran in culture medium of Ganoderma lucidum has

also previously been found responsible for the production of LiP, MnP and lacase and

increase their activities (Silve et al., 2005). Wheat bran due to low cost and dye

degrading potential was experienced as a promising substrate for dye removal. G.

lucidum and ligninases enzymes degraded dye adsorbed on its particles. Degradation of

the dye could be attributed more to the involvement of G. lucidum extracellular enzymes

and less to adsorption factor.Wheat bran is an inexpensive agrobased waste. Its contents

such as cellulose and starch which are polysaccharide are useful as carbon source for

fungal growth. Wheat bran posses two different properties; firstly, as a good adsorbent

and secondly, as a good source for production as well as working of ligninases

machinery. During utilization of wheat bran pH also affects on adsorption capacities of

this carbon source. The pH affects surface binding sites of the adsorbent as well as the

availability of the adsorbate compound.

Effect of additional nitrogen sources

i) Effect of different nitrogen sources

After selection and optimization of carbon sources (0.1%) like ammoium oxalate,

Maize Glutein Meal 30 %, Maize Glutein Meal 60%, Corn Steep Liquor and yeast extract

were supplemented to the flasks to study their inhibitory or enhancing effect on

decolorization rate of the dye. Dye decolorization was found to be 45.31, 36.12, 66.12,

99.11 and 36.51 % in ammoium oxalate, Corn Steep Liquor, Maize Glutein Meal 30 %,

Maize Glutein Meal 60%, and yeast extract receiving flasks respectively after 3 days of

incubation.

It indicated that dye decolorization and enzyme synthesis by G.lucidum was

inhibited by all nitrogen sources except 60%MGM. Controlled medium devoid of

additional nitrogen showed maxium (90.71 %) after 72 hours. Drift in pH observed in

medium was 5.63, 7.09, 7.08, 6.15, 5.87 and 7.74 respectively. An increase in pH was the

result of rich nitrogen sources added and urea already present in the nutrient medium.


149

Table. 4.17 Decolorization of Foron Turquise SBLN- 200 by Ganoderma lucidum and its lignolytic enzyme profile using

different nitrogen sources under optimimum conditions*



pH

Enzyme Activity(IU/mL) on 3rd Day

1 2 3 4 Lip MnP Lac

0 79.23±1.23 91.02±0.89 90.71±0.96 98.89±0.87 a

5.63±0.3 d

466±10.23 a

356±9.32 b

308±12.02 b

Ammonium Oxalate

26.99±5.44 40.11±10.59 45.31±12.84 54.06±4.40 c

7.09±0.02 b

379±18.60 b

291±6.66 a

149±3.56 e

Corn Steep Liquor

19.11±2.30 27.04±2.65 36.12±3.26 39.73±3.21 e

37.08±0.02 b

318±36.10 c

249±10.0 d

131±1.85 e


49.11±1.66 58.65±5.42 66.12±4.12 72.51±3.97 b

5.15±0.07 d

408±11.50 b

309±11.52 d

176 ±2.03 c


50.21±1.21 76 .11±1.01 99.11±0.66 98.76±1.08 a

5.87±0.04 c

526±4.87 a

486±4.63 c

396 ±4.48 a

Yeast Extract 15.32±2.28 28.88±1.70 36.51±2.03 35.75±1.65 c

7.74±0.3 a

308±2.60 d

273±2.61 e

199 ±3.18 d

* Medium IV; pH, 4.5 ; temperature (30±20C) ; Wheat Bran, 1.5%; medium IV.


150

0

20

40

60

80

100

120

0 AmmoniumOxalate

Corn steepLiquor

Maize GluteinMeal 30%


Yeast Extract

Nitrogen Source (0.1%)

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

En

zym

e A

ctiv

ity

(IU

/mL

)


Fig.4.17a: Effect of different nitrogen sources on decolorization of Foron Turquoise SBLN-200 by Ganoderma lucidum and lignolyitic enzymes production in 3 days.

y = 4.0083x + 350.71

R2 = 0.9063

y = 1.9941x + 285.91

R2 = 0.929

y = 1.4005x + 9.0554

R2 = 0.6826

0

100

200

300

400

500

600

700

800

900

20 30 40 50 60 70 80 90 100

Decolorization (%)

Enzy

me

acti

vity

(IU

/mL)


r (Lip) = 0.952**r (MnP) = 0.964**r (Lac) = 0.826**

Fig. 4.17b: Relationship between dye decolorization and enzyme activities with different nitrogen sources.


151

Activities of Lip, MnP and laccase were lower in all nitrogen supplemented media

as compared to control except with MGM 60% (Fig.4.17a).

Analysis of variance of the data (Appendex-1, Table 4.17b) showed that our

results were significant (p< 0.01). DMR test showed that treatment means of nitrogen

sources with same letter are not significant (Table 4.17). The sign of slope (Fig.4.17b)


activities.The value of R2 indicated that decolorization was due to 90 % variation in LiP,

93 % in MnP and 68 % in laccase activities. R2 for MnP was (0.92) highest among three

enzyme indicating major role of MnP in dcolorization followed by LiP and laccase

synthesis in the presence of 60% MGM.

Effect of varying concentrations of MGM60%

Varying concentrations of MGM 60% were used tofind out the optimum level of

MGM60%. Maximum dye decolorization (99.22%) was observed in 0.15 % MGM60%

containing flasks. Higher concentrations of MGM60% showed inhibition of ligninases

and dye removal also observed. pH change was 4.35, 4.95, 5.15, 6.29 and 6.36 in the

media with different MGM60% concentrations as presented in the Table (4.19a). LiP

profile with varying concentrations of MGM 60 % was as: 382, 547, 928, 933 and 584

IU/mL; for MnP it was 361, 380, 385,197 and 348; 362, 426, 777, 635 and 560 IU/ mL,

and laccase activities were calculated as; 361, 380, 385, 197, 348 IU/mL in medium

receiving 0.01, 0.05, 0.1, 0.15 and 0.2% MGM60% respectively (Fig 4.19b). DMR test

shows that treatment means of varying concentrations of MGM60% vlues shows

significant difference between enzyme activities as shown in the (Table 4.19a).The

analysis of variance of the data (Appendex-1, Table 4.19b) shows that our result were

significant (p< 0.01). DMR test shows that our results were significant (≤p0.01). The sign

of slope (Fig.4.18b) indicated that increase in decoloriztion was correlated with the

synthesis synthesis of ligninases enzymes. The value of R2 indicated that dye

decolorization was caused by 74% variation in LiP, 87 in MnP and 11 % in laccase

activities. Three parallel slopes (Figure 4.19b) indicated that R2 for MnP (0.87)was

highest among the three enzymes indicating that decolorization may be result of mainly

MnP action on dye, followed by LiP and lasccase synthesis.


152

Table.4.18 Decolorization of Foron Turquiose SBLN- 200 by Ganoderma lucidum and its lignolytic enzyme profile using

varying concentrations of Maize Glutein Meal 60% under optimum conditions*

Maize Gluten Meal 60%

(%)



(IU/mL)

1 2 3 4 pH Lip MnP Lac

0.01 66.34±1.67 73.42±2.70 82.11±2.74 81.66±2.62 ab

0.02±0.02 d

422 ±3.79 c

341±18.12 c

291±8.42 d

0.05 73.82±1.68 77.93±1.73 80.25±3.75 90.90±1.86 c

4.95±0.01 c

402±9.42 d

374±6.67 b

259±5.49 b

0.1 80.68±1.04 86.90±2.10 99.22±2.90 98.80±2.03 a

5.15±0.01 c

527±13.59 a

435±9.83 a

328±5.60 a

0.15 74.12±1.10 84.92± 91.23±0.98 89.12±0.97 a

6.29±0.02 b

490±2.33 b

371±7.22 b

297±5.90 c

0.2 62.12±1.09 78.15± 0.89 78.31.36±0.89 80.36±0.89 b

6.36±0.03 a

419±10.12 c

327±10.09 d

221±5.11 e

* Medium IV; pH,4.5 ; temperature (35±20C) ; Wheat Bran, 1.5%.


153

0

100

200

300

400

500

600

700

0.01 0.05 0.1 0.15 0.2

Concentration of 60% MGM (%)

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

700

800

En

zym

e A

ctiv

ity

(IU

/mL

)


Fig.4.18a: Effect of varying concentrations of MGM60% on decolorization of Foron Turquoise SBLN-200 by Ganoderma lucidum and lignolyitic enzymes production in 3 days

y = 6.1032x + 227.65

R2 = 0.7429

y = 4.6042x + 221.85

R2 = 0.8745

y = 0.2386x + 358.07

R2 = 0.1105

0

100

200

300

400

500

600

700

800

900

1000

20 30 40 50 60 70 80 90 100

Decolorization (%)

Enzy

me

acti

vity

(IU

/mL)


r (Lip) = 0.862**r (MnP) = 0.935**r (Lac) = 0.332

Fig.4.18b: Relationship between dye decolorization and enzyme activities with varying concentrations of MGM 60%.


154

The most widely sources for fungal decolorization of dyestuffs are ammonium

salts such as ammonium tartarate or chloride (Prouty, 1990; Gogna et al., 1992; Reid and

Seifert, 1982). G. lucidum showed better rate of decolorization in the medium

supplemented with MGM 60% when wheat bran was used as a carbon source.

Because ammonium salts are expensive and cannot be used for waste water

treatment so alternative cheap sources like MGM 30% and MGM 60% were utilized for

this purpose. MGM 60% showed more than 99.11 % decolorization after four 3 days of

incubation periods. Interestingly, G. lucidum has been found to exhibit fastest dye

decolorization rate under high nitrogen environment when cellubiose or glucose was used

as carbon sources indicating stimulation of LME under high N-conditions(Leung and

Pointing 2002). Previous studies also show that Nitrogen enriched media to cause better

decolorization by Ganoderma lucidum with higher lignolytic activities as compared to

nitrogen deficient media (Kaal et al., 1995; D’Souza et al., 1999; Revankar and Lele,

2007). However, addition of nitrogen source beyond optimum level leads to poor dye

decolorization (Radha et al., 2005)

The type and amount of nitrogen in the media affects dye degradation by

changing the enzyme profile of WRF. Studies on the effects of nitrogen concentration on

the dye degragdation were performed by Hatvani and Mec(2002) used ammonium

chloride, peptone and malt extract(1 mM) and acheived complete decolorization of Poly

R -478 by Lentinus edodes in more than two weeks. For several species lignolytic

enzyme activities is suppress instead of stimulation in the presence of high nitrogen

concentrations (25-60mM). However, due to difference in environmental conditions and

fungal physiology our G.lucidum strain effectively deolorized the Foron turquoise

SBLN200 in nitrogen rich media only in 3 days.

Effect of mediators

Several organic and inorganic compounds, thiols and phenols, aromatic

derivatives, N-hydroxy compounds and ferrocyanide have been used as an effective

mediators of ligninases (Couto and Sanroman, 2007). Such low molecular mass aromatic

compounds have been reported to enhance LME production by white rot fungi (Bollag et


155

Table.4.19 Decolorization of Foron Turquise SBLN- 200 by Ganoderma lucidum and its Lignolytic enzyme profile using low

molecular weight mediators under optimimum conditions*.

Mediators (%)


Final Enzyme Activity(IU/mL)

after 48 Hours

1 2 3 pH Lip MnP Lac

0 80.81±1.24 90.47±86.47 99.16±0.91 a

5.22.±0.03 a

482±16.91 c

474±7.12 b

216±7.19 b

ABTS 67.14±16.60 74.13±18.56 90.38±1.75 c

4.12±0.04 b

444±12.61 e

393± 11.16 c

322±6.37 a

H2O2 70.69±3.03 88.68±2.50 88.12 ±3.14 b

4.38±0.01 b

468 ±13.76 b

398± 16.23 c

182 ±132 c

MnSO4 83.39±2.01 90.68±2.19 96.14±3.47 ab

3.94±0.07 c

505±12.61 cd

476± 4.34 b

223 ±2.34 b

Tween-80 76.10±3.33 88.91±2.27 90.90±1.47 ab

4.11 ±0.03 b

475±2.31 b

441±13.22 c

219±2.03 d

Veratryl Alcohol

88.96±0.04 99.96±1.02 99.65±1.64 a

3.75±0.01 d

590±1.73 a

481±13.79 a

132 ±6.67 e

* Medium IV; pH, 4.5 ; temperature (35±20C) ; Wheat Bran, 1.5% ; MGM60% (0.15%)


156

0

100

200

300

400

500

600


Mediators (1mM)

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

En

zym

e A

cti

vity

(IU

/mL

)%)


Fig.4.19a: Effect of low molecular mass mediators on decolorization of Foron Turquoise SBLN-200 by Ganoderma lucidum and production of lignolyitic enzymes

y = 5.425x + 495.88

R2 = 0.9126

y = 3.2317x + 400.01

R2 = 0.8314

y = -4.8721x + 652.66

R2 = 0.9312

0

200

400

600

800

1000

1200

35 45 55 65 75 85 95

Decolorization (%)

Enzy

me

acti

vity

(IU

/mL)


r (Lip) = 0.955**r (MnP) = 0.912**r (Lac) = -0.965**

Fig.4.19b: Relationship between dye decolorization and enzyme activity with low molecular mass mediators


157

al,1984, Bominathan and Reddy, 1992; Buswel and Odier, 1987; Kirk and Ferril,

1987; Schlosser etal,1997). ABTS, CuSO4, H2O2, MnSO4, Tween-80 and Veratryl

alcohol were used as low molecular weight mediators. Declorization was 61.38, 43.26,

74.12, 86.14, 87.90 and 96.47 % and pH was 4.12, 3.96, 4.38, 3.94, 4.11 and 3.75, 4.12

after 2 days in ABTS, CuSO4, H2O2, MnSO4, Tween-80 and Veratryl alcohol containig

flasks respectively. Activities of LiP were 444, 468, 505, 475 and 590 IU/mL and MnP

activities were 393, 398, 476, 441and 481 IU/mL while laccase activities were 406, 364,

371, 349 and 290 IU/mL in ABTS, CuSO4, H2O2, MnSO4, Tween-80 and Veratryl

alcohol. Experimental flasks showed maximum (99.96%) decolorization after 48 hours.

Control flasks showed decolorization (99.11 %) after 72. Enhanced decolorization of

Foron Turquise SBLN200 by G.lucium after 72 hours of incubation and higer ligninases

activities by the addition of veratryl alcohol suggested the role of ligninases synthesis by

G.l involved in the degradation of Foron Turquise SBLN200. Analysis of variance of the

data showed that our results were significant (p< 0.01 as shown in the Table 4.19b of

Appenedex-1). DMR test showed that treatment means of activities with low molecular

mass mediatora carrying same letters were not significant (p< 0.01 Table 4.19a).

The sign of slope indicated that decoloriztion had positive correlation with

enzyme activities. The value of R2 indicated that 91% variation in LiP, 83 in MnP and 93

% in laccase activities was due to decolorization while remaining 9, 17 and 7 % variation

in LiP, MnP and laccase activities respectively may be due to other unknown factors.

Three parallel slopes in theindicated that R2 for laccase(0.93) (Fig. 4.20b) highest among

the three enzymes. It clearly showed that higher dcolorization may best be achieved

through laccase production followed by LiP and laccase synthesis.

Role of veratryl alcohol in dye degradation process closely resembles with the role

played during lignolysis. Major role of veratryl alcohol includes: induction of lignolytic

enzyme, stabilization of LiP, charge transfer mediation, formation of ROS and


158

acting as substrate for H2O2 generating species (Akene and Agathos 2001).In a Study

carried out by Jaouani etal; (2006) it has been reported that veratryl alcohol was the best

inducer for all three enzymes during dye decolorization.

The model substrate for LiP is Veratryl Alcohol (3,4-dimethoxybenzyl alcohol)

which is also secreted by the white rot fungi. VA has been suggested to participate in the

LiP reaction mechanism in protecting LiP from inactivation by H2O2 (Villi et al., 1990)

or by acting as a diffusible redox mediator between the enzyme and the substrates. That

cannot approach the redox centre (Harvey et al., 1986).

A redox mediator should have three properties: firstly, it should be able to

produce a free radical afte being oxidized by single electron from enzyme., secondly, the

radical generated should be so stable that that it can diffuse and inteact with the target

compound; thirdly; it appropriate redox potential should be in appropriate range (Tinoco

et al;2007). VA have all these properties which make it essential for LiP. More recent

work indicates that LiPI and VA react to form a LiP II -VA•+ complex with a redox

potential and life time in accordance with the reaction catalysed by LiP (Kindara et al.,

1996).

Effect of Heavy Metals

The production of LME of WRF is influenced by the metal ions on the level of

transcriptional and translational level regulation (Baldrin 2003). So low concentrations of

essential metal ion is necessary for the development of extracellular enzyme

system.Different metal ions (1mM) were added to decolorization medium under

preoptimized conditions. The metal ion salts used were: CaCl2, Cd(NO3)2, Cu SO4,

FeSO4, MnSO4, and ZnSO4. Decolorization was 42.42, 63.37, 78.87,99.78 and 46.17%

after 24 hours Change in pH was 3.78, 4.36, 3.99, 3.64, 3.78 and 4.26 in CaCl2,

Cd(NO3)2, Cu SO4, FeSO4, MnSO4, and ZnSO4 (Table 4.20a). Activities of LiP were 345,

370, 416, 640 and 433 IU/mL MnP activities were 285, 391,396,541 and 361IU/mL

while laccase exhibited 207,259,490, 345, and 275 IU/mL in the presence of metal ions

(Fig.4.21a).It was noted that all metal ions caused strong inhibition of enzyme synthesis

and dye decolorization but to variable extent.FeSO4 showed showed almost no effect on

the dye decolorizationactivities of G.lucidum. DMR test showed that treatment means of

enzyme activities with metal ions carrying same letters were not significant (p<0.01

Table 4.21a). Analysis of variance of the data presented in Table 4.21b of Appendex-1).

showed that our results were significant (p <0.01).


159

Table. 4.20 Decolorization of Foron Turquoise SBLN- 200 by Ganoderma lucidum and its lignolytic enzyme profile using

different metal ions under optimimum conditions*

Metal Ions (1mM)


pH

Enzyme Activity (IU/mL) after 24 Hours

1 2 Lip MnP Lac

0 88.98±0.65 99.49 ±0.58 a

3.78±0.06 c

448±14.89 a

428±12.65 b

356± 9.36 b

CaCl2 42.42± 34.40±1.19 c

4.36 ±0.04

a

345±5.26

d 385 ±4.70

c 207± 6.25

b

Cd(NO3)2 63.37±4.02 58.51±4.43 c

4.16±0.01 ab

370±8.19 c

371 ±5.69 c

410± 18.12 a

CuSO4 78.87±2.53 75.34±2.49 b

3.64±0.26 b

416±2.60 b

396±6.89 b

496± 6.23 a

FeSO4 99.78±0.23 98.89±0.73 a

3.78±0.09 ab

640 ±6.98 a

541±10.67 a

445± 4.36 c

ZnSO4 46.17±2.43 43.96±2.57 4.26±0.04 a

353±4.33 d

3 61±4.34 d

275±4.36 b

* Medium, IV; pH, 4.5 ; temperature (35±20C) ; Wheat Bran, 1.5% ;MGM60%, (0.1%) ; Mediator, Veratryl Alcohol (1mM)


160

0

20

40

60

80

100

120

140


Metal Ions (1mM))

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

700

En

zym

e A

ctiv

ity

(IU

/mL

)


Fig.4.20a: Effect of metal ions on decolorization of Foron Turquoise SBLN-200 by Ganoderma lucidum and lignolyitic enzymes production

y = 4.9533x + 538.7

R2 = 0.951

y = 3.9733x + 318.69

R2 = 0.8771

y = 3.9376x + 77.023

R2 = 0.8831

0

200

400

600

800

1000

1200

30 40 50 60 70 80 90 100

Decolorization (%)

En

zym

e ac

tivi

ty (

IU/m

L)


r (Lip) = 0.975**r (MnP) = 0.937**r (Lac) = 0.940**

Fig.4.20.b: Relationship between dye decolorization and enzyme activities with different metal ions


161

The increase in decoloriztion had positive correlation with the enzyme activities.

The value of R2 indicated that 95% variation in LiP, 88 % in MnP and 88 % in laccase

activities was due to decolorization factors. Figure (4.21b) indicated that R2 for LiP(0.95)

the highest among the three enzymes showing that dcolorization was mainly caused

through LiP action followed by MnP and laccase synthesis in the medium.

Concentration of heavy metal ion is strongly dependent on the nature of fungal

strain and media composition. In a previous report (Baldrian, 2003) Ganoderma lucidum

toxicity of heavy metal decreased in the order Hg>Cd >Cu>U>Pb >Mn=Zn. The

decrease of fungal growth rate is sometimes accompanied by the increase in lag phase

and morphological changes of the growing mycelium.

Metal ions are thought to be an important group of enzymatic activity modulators

and some times leave a positive impact on enzymatic activity and treatment efficiency

(Kim and Nicell 2006). Heavy metals present in the environment interact with

extracellular enzymes of fungi. However, to cause a physiological response, heavy metals

must be taken up by the fungus. The uptake from the liquid environment is the simplest

solution not only in the laboratory conditions, but also in the water containing substrates.

White rot fungi can concentrate metals taken up from substrates in their mycelia. After

uptake of heavy metals these enter the fungal cell, thus affecting both individual reactions

as well as metabolic process. Growth is the most popular complex phenomenon that is

studied from the view point of heavy metal toxicity (Baldrin, 2003).

Toxicity of metal up to a certain limit is alleviated by the defence system of fungi

developed after different physiological changes. The defence system is based on

immobilization of heavy metals using extracellular and intracellular chelating

compounds.One of the typical chelators produced by the white rot fungi is oxalates.The

production of oxalaic acid by white rot fungi provides a means of immobilizing soluble

metal ions or as insoluble oxaltes, thus decreasing bioavailibilty and increasing tolerance

to these metals. Metal oxalates can also be formed by Ca, Cu, Cd, Co, Mn, Sr and Zn

(Sayar and Gadd 1997).


162

Effect of varying concentrations of the dye

In order to determine the maximum dyestuffs concentration tolerated by

G.lucidum experiment with varying dye concentrations was carried out. Dye

decolorization medium having 0.05, 0.1, 1.5 and 2.0 dye was 99.29, 81.90, 49.98, 24.53

and 10.53 % respectively (Table 4.21a) after 48 hours of incubation.Variation in pH from

optimum limit was 5.05, 4.98, 4.88, and 5.23 respectively.The LiP profile was 636,578,

445 and 377 IU/mL. MnP activities were 554, 601, 277, 51 and 15 while laccase

activities calculated were 382, 289, 167, 140 and 110 IU/mL with 0.01, 0.05, 0.1, 1.0 and

1.5 % dye concentration respectively (Fig.4.22a).

Analysis of variance of the data (Table 4.22b of Appendex -1) showed significant

(p < 0.01) effect of dye concentration on dye decolorization. DMR test reveals that

treatment means of activitie sharing same letters are not significant (p < 0.01).

Decoloriztion was found to be correlated with the enzyme activities. The value of R2

indicated that dye decolorization was due to 96% variation in LiP, 86% in MnP and 85%

in laccase activities (Fig.4.22 b). R2 for LiP (0.96) was the highest than those of MnP

(0.85) and Laccase (0.84) clearly indicating that dcolorization may best be achieved by

LiP followed by those of MnP and Laccase.

At the end of incubation period (48hours), there was nearly100 % color removal

in flasks with 0.01% dye solution. However, effecincy decreased with increasing dyestuff

concentration. The dyestuffs removal was significant in 0.05 %. However, it decreased

gradually with increase in dye concentration of dye due to increase in toxicity caused by

metabolites produced during dye transformation before decolorization.


163

Table.4.21 Decolorization of Foron Turquoise SBLN- 200 by Ganoderma lucidum and its lignolytic enzyme profile using

varying concentrations of the dye under optimimum conditions*


(%)


Final Enzyme Activity (IU/mL)

after 24 Hours

1 2 pH Lip MnP Lac

0.01 99.29±0.31 99.02 ±0.51 a

4.84 ±0.12 b

636 ±11.16 a

531±11 a

382±4.88 a

0.05 81.9±2.17 80.86±1.29 a

5.05±0.11 ab

578±3.79 b

496±4.92 b

289 ±2.43 b

0.1 49.88±2.94 49.73 ±1.83 b

4.98 ±0.04 ab

445±2.08 c

377±3.06 c

167 ±5.93 c

0.15 24.53±2.06 23.81±3.64 c

4.88±0.03 b

377±8.10 d

203±9.18 c

140±2.12 c

0.2 10.53±2.45 9.74±1.81 d

5.23 ±0.02 a

236±1.03 e

161±27.21 d

110±1.19 d

* Mediu, IV; pH, 4.5 ; temperature (35±20C) ; Wheat Bran, 1.5% ;MGM60%, (0.1%) ; Mediator, Veratryl Alcohol (1mM)


164

0

20

40

60

80

100

120

0.01 0.05 0.1 0.15 0.2


Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

700

En

zym

e A

ctiv

ity

(IU

/mL

)


Fig.4.21a: Effect of varying concentrations of dye on decolorization of Foron

Turquoise SBLN-200 by Ganoderma lucidum and its lignolyitic enzymes production in 24 hours

y = 6.9384x + 352.03

R2 = 0.9621

y = 3.8206x + 288.77

R2 = 0.8595

y = 2.6484x - 8.8617

R2 = 0.8479

100

200

300

400

500

600

700

800

900

1000

1100

0 20 40 60 80 100

Decolorization (%)

En

zym

e ac

tivi

ty (

IU/m

L)


r (Lip) = 0.981**r (MnP) = 0.927**r (Lac) = 0.921**

Fig.4.21b: Relationship between decolorization and enzyme activity with varying concentrations of the dye in 24 hours


165

Kaushik and Malik (2008) reported the maximum (Turquoise Blue, phthalocyanin

dye) concentration tolerated by C. versicolor. Decolorization efficiency of the fungus was

almost100 % between dyestuffs concentrations of 100-250 mg/L, respectively (in3 days)

whereas 80% coor removal was noted with dye cocentrations ranging from 700-

1200mg/L(9 days). Our strain showed better results while comparing dye removal rate,

dye concentration and incubation period.

Adsorption of Foron Turquoise SBLN-200 by fungal mycelia of Ganoderma lucidum

Initially dye adsorption was shown by fungal mycelia of the Foron dye but to a

lower extent. Dye adsorbed on mycelia was 0.05, 0.12, 0.49, 0.52, and 0.61% in the

media having 0.01, 0.05, 0.1, 0.15, and 0.2 % dye respectively. However, with he passage

of time adsorbed dye was also removed and degraded by the fungal enzymes.The culture

media were not supplemented with any kind of nutrients. Analysis of variance of the data

also showed our results were non significant (p < 0.05, Table 4.22 of Appendex-1). DMR

revealed that adsorption % had significant difference (p < 0.01 in Table 4.23a).

The complete decolorization of Amaranth was observed by fungal cultures

without adsorption on mycelia (Knap etal 1995). However, it has been observed in

stationary cultures where dye decolorization occurred due to sorption since formation of

mat at the surface restricts the supply of oxygen to the cell beneath the surface and in the

medium which inhibits role of oxidative enzymes and diminishes decolorization

process(Swamy and Ramsy 1991).The superior performance of the liquid state

fermentation is due to physiological state of the fungi as pellets increase transfer of mass

and oxygen between the cells and the medium due to mixing (Murugesan and

Kelaichelvan 2003).In a recent research carried out by Parshgetti et al; (2007) dye

decolorization by A. ochraceus that higher concentration of dye left pronounced effect on

adsaorption as well as decolorization. This result manifests that decolorization was

inversely related to the concentration of dye.


166

Table.4.22 Adsorption of Foron Turquoise SBLN-200 on fungal mycelia at

optimum pH and temperature*

Dye concentrations (%)

Adsorption (% Mean ±S.E) Incubation Time (Days)

Final pH 1 2

0.01 0.05±0.02 0.001±0.00

e 4.07±0.04

d

0.05 0.12±0.06 0.002±0.00

d 4.76±0.29

b

0.1 0.49±0.09 0.02±0.00

c 5.10 ±11

c

0.15 0.52±0.012 0.04±0.01

b 6.56±0.09

b

0.2 0.61±0.19 0.06±0.01

a 7.74±0.04

a

*pH, 4.5;Temperature, (35±20C),

0

0.2

0.4

0.6

0.8

1

1.2

0.01 0.05 0.1 0.15 0.2


Ad

sorp

tio

n (

%)

Fig.4.22a. Dye adsorption on fungal mycelia of Foron Turquoise SBLN200 with varying concentrations of the dye


167

4.3 DECOLORIZATION OF DIRECT DYES

4.3.1 Screening of White Rot fungi on direct dyes

Four Direct dyes Solar G. Yellow, Solar Blue A, Solar Orange RSN and Solar

Red BA were subjected to decolorization by five WRF strains and the results of 10 days

time course study have been shown in Table 4.23a. Different direct dyes were

decolorized to variable extent by all strains. Ganoderma lucidum showed poor

decolorization on the direct dyes. It deciolorized maximally (58.91%) Solar Blue A in 6-

days while no significant decolorization of other dyes was observed at the end of trial.

Pleurotus ostreatus decolorized all the dyes very efficiently. It showed maximum

decolorization (87.47%) on Solar G. Yellow followed by 74.0% decolorization of Solar

Red BA, 66.1% decolorization of Solar Blue A and 58.70% Solar Orange RSN was

poorly degraded by G. lucidum in 6-days. However, Solar G. Yellow R was decolorized

up to 93.10% on 7th day by P.ostreatus.

S. commune also decolorized all the dyes efficiently giving maximum

decolorization result (72%) on Solar Blue A followed by 66.1% decolorization on Solar

G. Yellow, 61.90% decolorization on Solar Orange but it caused poor decolorization of.

RSN Solar Red BA. C. versicolor showed very poor decolorization on all direct dyes. P.

chrysosporium showed maximum decolorization (71.36%) on Solar G. Yellow followed

by 53.62% decolorization of Solar Red BA and no commendable results were found on

other dyes.The selected best fungus-dye combinations in time course decolorization

studies by are given in Table 4.23a.

Screening experiment revealed that Pleurotus ostreatus decolorized all the Direct

dyes maximum within 6 days of incubation. So P. ostreatus was selected as the best

strain in our study. Decolorization started after an initial lag phase of 48 hours and most

of the color loss occurred after 72 hours and further decolorization continued with a much

slower rate throughout the rest of the days. Decolorization and degradation of four direct

dyes was noted by the diminution of absorbance at maximum wavelength of the

respective dyes which indeed happened due to destruction of chrmophores.Generally

researchers involved in this area consider complexity of structure as great hindrance in

decolorization of a particular dye. However, this is not the only factor, several other

factors like environment and fungal physiology also play critical role in degradation.


168

Table 4.23a Decolorization of direct dyes by different white rot fungi in time

course study

Incubation

Time (days)

White rot cultures

Dyes

Solar G. Yellow R

Solar Orange RSN

Solar Blue A Solar Red BA

1

P.O. 26.3 ± 0.33 26.2 ± 0.45 18.3 ± 0.34 4.86 ± 0.69 G.L. 14.8 ± 0.13 11.9 ± 0.60 13.55 0.55 2.1 0.35 S.C. 15.9 0.20 11.9 0.30 6.20 0.20 14.2 0.30 C.V. 3.20 0.40 2.40 0.20 4.10 0.10 1.30 0.20 P.C. 12.1 ± 0.25 5.98 0.01 8.03 0.03 12.2 0.38

2

P.O. 39.0 1.30 23.38 0.11 12.0 0.28 16.4 0.78 G.L. 26.2 0.35 23.1 0.30 15.9 0.20 11.0 0.10 S.C. 22.3 ±0.60 19.6 0.40 23.4 0.20 17.8 0.40 C.V. 9.80 0.20 13.2 0.80 11.9 0.50 8.60 0.30 P.C. 46.9 0.54 18.9 0.13 10.8 0.18 20.0 0.15

3

P.O. 53..2 0.91 27.8 0.41 38.5 0.64 27.8 0.18 G.L. 26.2 0.40 38.1 0.30 28.9 0.85 17.3 0.35 S.C. 36.2 0.20 36.0 0.40 35.6 0.40 22.7 0.20 C.V. 18.3 0.60 20.1 0.55 19.6 0.30 15.6 0.10 P.C. 29.1 0.28 26.8 0.20 12.8 0.13 34.9 0.95

4

P.O. 65.4 0.24 34.2 ± 0.56 31.1 0.09 19.1 0.32 G.L. 31.82 0.70 29.2 0.35 43.4 ± 0.85 37.2 ± 0.80 S.C. 59.6 0.30 34.3 0.6 35.3 0.40 28.2 0.20 C.V. 26.6 0.50 22.3 0.30 20.5 0.10 18.3 0.30 P.C. 35.7 0.27 29.9 0.10 25.9 0.20 40.7 0.75

5

P.O. 71.61 ± 0.88 43.1 ± 0.92 51.3 0.19 28.9 0.27 G.L. 34.12± 0.85 34.0 ± 0.25 43.3 ± 1.02 26.8 0.30 S.C. 58.5 0.30 41.0 0.20 61.3 0.50 29.8 0.40 C.V. 35.4 0.20 32.1 0.50 31.3 0.10 26.7 0.30 P.C. 41.8 0.22 31.4 0.45 38.0 0.10 33.9 0.06

6

P.O. 87.47 ± 1.01 74.0 ± 0.37 66.1 ± 0.99 58.7 ± 0.72 G.L. 39.4 ± 0.20 37.1 ± 0.40 58.91 ± 0.42 35.5 0.30 S.C. 58.9 0.60 41.4 0.60 62.3 ± 0.82 42.4 0.40 C.V. 43.1 0.10 39.3 0.50 42.7 0.30 34.5 0.20 P.C. 56.1 0.74 37.8 0.30 39.9 0.20 55.6 0.37

7

P.O. 93.10 0.93 70.1 ± 0.61 69.3 0.35 60.1 0.30 G.L. 41.2 ± 0.87 31.2 ± 0.15 56.1 ± 0.59 48.1 0.40 S.C. 66.1 ± 0.31 39.9 0.20 42.3 ± 0.70 48.9 0.10 C.V. 48.6 0.50 45.6 ± 0.40 48.9 0.60 41.3 0.10 P.C. 64.9 0.21 40.9 0.21 42.9 0.98 69.1 0.51

8

P.O. 93.11 0.21 73.1 ± 0.93 74.21 ± 0.56 68.0 0.30 G.L. 46.5 ± 0.21 30.3 ± 0.49 54.2 ± 0.59 54.0 0.40 S.C. 77.7 ± 0.30 39.2 ± 1.19 40.8 ± 0.68 58.2 ± 0.58 C.V. 45.6 0.20 46.9 ± 0.30 48.7 ± 0.30 43.6 ± 0.10 P.C. 66.6 1.25 45.6 1.02 44.3 0.95 66.5 0.45

9

P.O. 93.14 0.00 79.12 ± 0.17 78.1 0.91 67.0 1.03 G.L. 47.14 ± 0.80 30.2 ± 0.40 52.3 ± 0.15 51.9 0.20 S.C. 76.79 ± 0.80 38.1 ± 0.29 46.5 ± 0.49 57.7 0.50 C.V. 39.4 0.50 48.5 ± 0.29 57.2 ± 0.30 39.6 0.10 P.C. 70.1 0.93 47.7 0.28 52.3 0.35 68.9 1.53

10

P.O. 93.15 0.00 85.0 0.12 78.0 0.46 71.1 0.71 G.L. 47.79 ± 0.12 29.0 0.15 43.1 ± 0.15 51.6 0.40 S.C. 76.22± 0.26 36.5 0.10 78.7 ± 0.60 57.2 0.30 C.V. 33.6 0.46 29.8 0.60 31.3 0.50 32.5 0.40 P.C. 71.3 0.45 49.5 0.58 44.4 1.34 53.6 2.10


169

It has been reported by Toh et al; 2003; Mass and Chaudhry 2005 that complexity

of dye structure was not an absolute factor in dye decoorization process. In a previous

study carried out by Reddy et al. (1992) and Chander et al., (2004) physiological

difference among the WRF cultures were considered responsible for decolorizing

abilities. Moreover, patteren of expression of complex LME system of the white rot fungi

vary from starain to strain (Nagi et al., 2002; Boer et al., 2004; Mazmanci and Unyayar et

al., 2005). Study of Spadro et al., (1992) and Chander et al., (2004) revealed that

degradation process dependent on aromatic ring structure, its pattern of cleavage and

number, nature, and position of substtituents attatched to it. Knapp et al. (1997) findings

support the view that structurally similar dyes may even degrade to different extent.

After screening of the white rot fungi on decolorization of Direct dye it was found

that Pleurotus ostreatus worked most efficiently on Solar Golden Yellow RSN and it was

investigated for further decolorization.

4.3.2 Optimisation Of Solar Golden Yellow R decolorization by Pleurotus ostreatus

The selected combination of the direct dye and fungus was used for optimization

process.

Effect of Media Composition

The Dye was subjected to different media to check fungal potential . Medium III

showed best result among all the media. Medium III differs from Kirk’s basal medium

due to absence of Tween-80 and Veratryl Alcohol and presence of urea. A slight

difference in medium III and IV is because of different urea contents.

After seven days of incubation time the decolorization was 54.46, 54.11, 98.86

and 73.50 % in M-I, M-II, M-III, and M-IV respectively (Table 4.24). Change in pH from

4.5 was 6.91, 6.92, 7.45, and 7.28. Ligninases enzyemes profile indicated maximum

activities of MnP and laccase while lowest level of LiP was found in all the media.Lip

activities were 61, 58, 95 and 85 IU/mL. MnP activities were 163, 158,376, and 295

IU/mL and laccase activities were 140,138, 266, and 247 IU/mL in M-I, M-II, M-III, and

M-IV respectively. Maximum decolorization was shown by MnP (Fig.4.15a). Analysis of

variance of the data (Appendex-1 Table 4.24) shows that our results are significants

(p≥0.01). DMR test revealed that treatment means of different media carrying same

letters have significant difference. The sign of slope indicated that increase in


170

Table.4.24 Decolorization* of Solar Golden Yellow R by Pleurotus ostreatus and its lignolytic enzyme profile using different


Med

ium

Decolorization (%Mean ± S.E) Incubation Time (Days)


(IU/mL)

1 2 3 4 5 6 7 pH LiP MnP Lac

M-I 12.36±3.63 17.00±4.86 23.42±3.90 31.66±2.44 38.50±1.75 43.61±2.24 54.46±3.29

c 4.91±0.32

d 61±6.90

b 163±6.50

c 140±8.22

c

M-II 15.37±1.94 21.12±1.43 28.34±1.42 35.5±3.38 41.96±2.59 48.18±3.77 54.11±3.65

c 4.96±0.07

c 58±2.08

b 158±7.23

d 138 ±5.55

c

M-III 32.72±0.41 36.57±1.53 43.52±1.93 59.25±1.35 68.87 ±2.73 81.13±2.51 98.86±0.57

a 5.13±0.02

b 95±8.45

a 376 ±14

a

266± 2.52

a

M-IV 31.31±1.98 38.75±1.49 49.42±2.62 57.95 66.21 71.72±3.32 73.50

b 5.28±0.05

a 85 ±7.56

b 295±11.23

b 247 ±2.03

b



171

0

20

40

60

80

100

120

M-I M-II M-III M-IVMedium

Dec

olo

riza

tio

n (

%)

0

50

100

150

200

250

300

350

En

zym

e A

ctiv

ity

(IU

/mL

)

Decolorization (%)LiPMnPLac

Fig.4.24a: Effect of varying media compositions on decolorization of Solar Golden Yellow R by Pleurotus ostreatus and its ezyme production in 7 days

y = 1.2282x + 174.49

R2 = 0.401

y = 1.7683x + 160.73

R2 = 0.4098

100

150

200

250

300

350

400

40 50 60 70 80 90 100

Decolorization (%)

En

zym

e ac

tivi

ty (

IU/m

L)


r (MnP) = 0.640*r (Lac) = 0.633*

Fig .4.24b: Relationship between dye decolorization and enzyme activities with varying media compositions


172

decoloriztion was positively correlated with the enzyme activities. The value of R2

indicated that decolorization was due to 59% variation in MnP, 60% in laccase. Two

parallel slopes (Figure 4.24b) indicated that R2 for laccase (0.41) was slightly higher than

MnP (0.40) indicating laccase slightly more efficient than MnP in dcolorization of Solar

Golden Yellow R.

In a study carried out by Asgher et al., (2008), G. lucidum decolorized Solar

golden yellow 77% on 7th day of incubation in Kirks basal salt medium which was

moderately rich in nitrogen. But our P.ostreatus strain showed better performance in

Medium III which was rich in N due to urea. Moreover, our results can be compared to

our initial report (Asgher et al. (2006b) showing 100% decolorization of Drimarene

orange K-GL (0.2 g/L) by P. chrysosporium in 8 days. Pasti-Gribsby (1992) found that

P. chrysosporium required 3-4 days to cause a substantial decolorization of different

azo dyes.

The study done by Asgher etal.2008 showed that S. commune IBL-06 produced

all the three lignolytic enzymes but maximum activity was noted for MnP followed by

laccase after complete optimization. There were negligible activities of LiP in medium I.

So nature of medium, nature and physiology of fungal strain as wel as stress condition

affect the overall performance of the fungus.

Presence of extracellular peroxidases in P.ostreatus and their involvement has

been confirmed by many studies. It has been reported in previous research by Kim and

Shoda199; Shin etal,1997;Viyas and Molitoris 1995; that P.ostreatus and Geotrichum

candidum produce extracellular peroxide-dependent peroxidase.Extracellular peroxidases

of P.ostreatus with a Mn2+-binding site have been described and these enzymes can

efficiently oxidize Mn2+ to Mn3+ which is then chelated with organic acids. The chelated

Mn3+ diffuses freely from the active site of the enzyme and can oxidize secondary

substrates (Mester and Field, 1998). Some of them also can utilize phenolic compounds

to complete their catalytic cycles and others can directly oxidize non-phenolic and/or

high-molecular weight compounds and are known as hybrid-type or versatile peroxidases

(Camarero et al., 1999, Ha et al., 2001). Although the efficiency of enzymatic

decolorization using MnP can be enhanced by optimizing the concentrations of hydrogen

peroxide and manganese ions as well as the amount of enzyme, the pH and the reaction

temperature for each dye (Mielgo& Moreira 2003). Enzymatic decolorization might be


173

also improved in the presence of surfactants, such as Tween 80 (Harazono and

Nakamura, 2005).

Effect of Additional Carbon Sources

i) Effect of different carbon sources

Carbon source is a requisite for metabolic activities of a microorganism. So

different carbon sources were used as additives for energy supply to the medium which,

inturn, enhanced the dye decolorization rate. For this purpose glucose, glycerol, lactose,

starch and wheat bran were taken up to investigate the potential of P.ostratus for

decolorizing the dye. Decolorization was 85.90, 63.91, 19.88, 91.65 and 98.51% in

glucose, glycerol, lactose, starch and wheat bran respectively on 5tth day. Change in pH

was 2.80, 3.12, 3.18, 2.57 and 2.57 (Table 4.25). The controlled flasks showed maximum

decolorization (81.52%) on 6th day of incubation.

Activities of MnP were 370, 350, 173, 390, and 425 IU/mL while laccase

activities noted were 273, 260, 129, 234 and 346 IU/mL in glucose, glycerol, lactose,

starch, and wheat bran respectively (Fig.4.25a). Activities of laccase and MnP were 358

and 218 IU/mL in controlled medium. Analysis of variance of the data (Appendex-1,

Table 4.26b) shows that our results were significant (p< 0.01). DMR test reveals that

treatment means of enzyme activities with carbon sources sharing same letters are not

significant (p< 0.01). The sign of slope indicated that increase in decoloriztion was

positively correlated with the enzyme activities. The value of R2 indicated that

decolorization was due to 95 % variation in MnP, 96% variatin in laccase activities.Two

parallel slopes in the (Figure 4.25b) indicated that R2 for MnP (0.95) and laccase (0.96)

clearly indicated that dcolorization may be achieved through laccase production followed

by MnP synthesis.


174

Table-4.25 Decolorization of Solar Golden Yellow R by Pleurotus ostreatus and its lignolytic enzymes profile using different

carbon sources under optimum conditions*

Carbon Sources

(1%)

Decolorization (%Mean ± S.E) Incubation Time (Days) Final

pH

Enzyme Activity (IU/mL) on 5th Day

1 2 3 4 5 6 MnP Lac

0 32.16± 1.65 37.12± 1.45 44.85± 1.65 58.69± 1.87 67.98± 2.74 81.52 ± 2.78 b

6.3± 0.36 a

358 ± 11.45 d

248±14.55 d

Glucose 16.9± 1.36 42.32± 7.89 63.16± 9.13 76.83± 2.16 81.95± 16.29 85.90± 3.88 b

2.80±0.23 ab

370 ± 3.53 b

273± 0.28 b

Glycerol 13.65± 2.36 30.67± 3.69 51.11± 1.78 58.42± 0.89 63.19± 4.13 61.91± 4.47 c

3.12±0.02 b

350± 3.18 c

260± 4.62 c

Lactose 44.87 75.64± 4.83 84.50± 1.82 87.68 19.43± 1.37 17.88± 1.30 d

3.18 ± .01 b

173± 2.73 e

129 ± 2.89 e

Starch 48.56± 1.14 75.64 81.97± 2.38 88.69± 0.55 91.65± 0.88 90.65±1.61 a

2.57± .02 b

390 ±1.45 c

234± 1.73 d

Wheat Bran 60.47± 1.02 85.96± 1.23 89.27± 0.97 93.81± 1.35 98.51± 0.63 96.51± 0.93 a

2.57±0.01 b

425±4.64 a

346± 3.18 a


175

050

100150200250300350400450500

0 Glucose Glycerol Lactose Starch Wheat Bran

Carbon source (1%)

De

colo

riza

tio

n (

%)

0

50

100

150

200

250

300

350

400

En

zym

e A

ctiv

ity

(IU

/mL

)


Fig.4.25a Effect of different carbon sources on decolorization of Solar Golden Yellow R by Pleurotus ostreatus and its lignolyitic enzymes aproduction in 5days

y = 3.3664x + 179.89

R2 = 0.9508

y = 4.8224x - 111.07

R2 = 0.9622

100

150

200

250

300

350

400

450

500

550

50 60 70 80 90 100

Decolorization (%)

Enzy

me

acti

vity

(IU

/mL)


r (MnP) = 0.975**r (Lac) = 0.982**

Fig. 4.25b: Relationship between dye decolorization and enzyme activities with

different carbon sources.


176

Effect of varying concentrations of wheat bran

Since stress conditions affet greatly decolorization process so varying

concentration of wheat bran were used to find out optimum level of WB. Wheat bran

(1%) showed excellent result in terms of dye decolorization as wel as enzyme activities in

presence. So in a further experiment varying concentrations of wheat bran were used.

Decolorization was 91.50, 98.81, 53.82 and 71.44% in 5 days of incubation. Drift in pH

was 3.81, 3.95, 4.15, 4.22, 4.35 in 0.1, 0.5, 1.0, 1.5 and 2.0 % wheat bran respectively as

shown in the Table.4.26a. Activities of MnP were 397, 428, 362 and 334 IU/mL and

laccase actiivites profile noted was 268, 348, 267 and 240 IU/mL in 0.1, 0.5, 1.0, 1.5 and

2.0 % wheat bran respectively (Fig.4.17a). Activities of MnP and laccase in controlled

flasks were 373 and 233 IU/mL. Analysis of variance of the data (Appendex- 1, Table

4.27b) shows that our results are significant (p < 0.01). DMR test reveals that treatment

means of activites with varying concentrations of wheat bran carrying same letters are not

insignificant (p>0.01). The sign of slope (Fig.4.26b) indicated that increase in

decoloriztion was positively correlated with the enzyme activities. The value of R2

indicated that dye decolorization was due to 49 % variation in MnP and 53 % variation in

laccase activities. Two parallel slopes in the (Figure 4.26b) indicated that R2 for

MnP(0.84) was lowe than R2 for laccase (0.92) indicating the major role of laccase in dye

degradation. It clearly showed that dcolorization was mainly caused by laccase activities

followed by MnP synthesis in the presence of 1% wheat bran..

Biodegradation of dyes by white rot fungi like is a cometabolic process requiring

carbon as a primary substrate which is source for energy and assists in expression of

secondary metabolism. Moreover, ligninases enzymes are produced by the fungus during

the idiophase in presense of some stress condition which may be induced by different

environmental triggers, e.g carbon or nitrogen (Aken and Agathos 2002).The highest

MnP activities were found when P. ostreatus was grown on medium with wheat bran.

Starch and lactose also ensured comparatively higher activities than pure carbohydrates

like glucose and glycerol. So wheat bran was found to be the most appropriate growth

substrate for manganese peroxidase secretion in culture medium.


177

Table 4.26 Decolorization of Solar Golden Yellow RSN by Pleurotus ostreatus and its lignolytic enzyme profile with varying

concentrations of wheat bran optimum conditions*

Wheat Bran (%)

Decolorization (%Mean ± S.E) Incubation Time (Days)


(IU/mL) on 5th Day

1 2 3 4 5 6 pH MnP Lac

0.1 59.65±1.34 22.96±3.62 66.41±4.37 72.20±3.10 87.88±3.41 95.88±2.98 ab

3.81±0.02 d

373±10.28 c

233 ±7.10 d

0.5 64.97±1.65 71.70±2.20 79.06±2.44 86.83±1.21 91.50±2.76 93.76±2.89 b

3.95±0.02 c

397±10.25 b

268±12.91 b

1 71.52±1.42 84.88±1.96 88.77±0.83 92.85±0.91 98.81±0.24 97.64±0.77 a

2.67± 0.01 b

428±14.36 a

348±11.23 a

1.5 53.34±2.34 62.36±3.77 78.64±3.30 88.18±2.25 87.82±2.29 86.51±2.45 c

4.22 ±0.03 b

362±11.11 d

267 ±18.45 b

2 48.78±2.56 56.84±2.98 61.14.14±3.82 70.96±3.48 80.44±3.86 79.62 ± 40.62 d

4.35±0.04 a

334 ±8.15 e

240 ±14.53 b

* Medium III; pH, 3.5 ; temperature (30±20C).


178

0

20

40

60

80

100

120

0.1 0.5 1 1.5 2

Concentration of Wheat Bran(%)

Dec

olo

riza

tio

n (

%)

0

50

100

150

200

250

300

350

400

450

500

En

zym

e A

ctiv

ity

(IU

/mL

)


Fig.4.26a Effect of varying concentrations of wheat bran on decolorization of Solar Golden Yellow R by Pleurotus ostreatus and lignolytic enzymes production in 5 days

y = 4.9438x + 162

R2 = 0.5347

y = 1.893x + 209.52

R2 = 0.4945

100

200

300

400

500

600

700

800

25 35 45 55 65 75 85 95 105

Decolorization (%)

En

zym

e ac

tivi

ty (

IU/m

L)


r (MnP) = 0.901*r (Lac) = 0.703**

Fig. 4.26b: Relationship between dye decolorization and enzyme activities with varying concentrations of wheat bran


179

Glucose which normally provides excellent mycelial growth gave lowest level of laccase

activity by P.ostreatus in this medium.

Generally, it is found that Basdiomycetes fungi cultivation in many cases

stimulate ligninases enzyme secretion even without supplementation of specific inducers

to the culture medium (Kapich et al. 2004; Mikiashvili etal. 2005). Moreover, in a

previous research wheat straw ensured higher MnP activity from P. ostreatus than non

lignocelulosic carbon sources. So in our research work wheat bran being lignocellulosic

waste also increased MnP activity which supports our carbon source efficiency during

dye decolorization.

Kapdan and Kargi; (2002) found that Everzol turquoise blue G was decolorized

(77%) at optimum conditions by Coriolus versicolar in presence of 0.5 g/100ml

glucose as additional carbon source and Glucose levels of 0.2% could cause only 65%

color removal.

Effect of Additional Ntrogen Sources

i) Addition of different nitrogen Sources

Nitrogen is not only necessary for carrying on metabolism, in general, but also for

the synthesis of the lignolytic system. So different nitrogen source (0.1%) like

ammonium oxalate, CSL, MGM60%, MGM30% and yeast extract were added to

increase decolorozation rate after the selection of wheat bran as a best energy source.

Decolorization tendency was observed as 44.99, 49.92, 59.36, 98.07 and 53.07% on 4th

day of incubation (Table 4.27). Drift in pH was 3.75, 4.12, 2.50, 3.35, 4.09 in ammonium

oxalate, CSL, MGM60%, MGM30% and yeast extract respectively. Activities of MnP

were 231, 239, 266, 505 and 228 IU/mL while laccase activitiesprofile was 190, 219,

236, 430 and 279 IU/mL in ammoniumoxalate, CSL, MGM60%, MGM30% and yeast

extract respectively (Fig.4.27a) on 4th day of incubation. Controlled flasks showed

maximum decolorization (97.47%) on 5th day and activities of MnP and laccase were

424 IU//mL and 347 IU//mL respectively.

Analysis of variance of the data (Apendex-1, Table 4.27) shows that our results

are significant (p < 0.01). DMR test reveals that treatment means of activities with

different nitrogen have significant difference (p<0.01) (Table 4.28).


180

Table 4.27 Decolorization of Solar Golden Yellow R by Pleurotus ostreatus and its lignolytic enzymes profile using different

nitrogen sources optimum conditions*



pH

Enzyme Activity (IU/mL) on 4th Day

1 2 3 4 5 MnP Lac

0 60.8± 1.45 85.01± 1.78 87.89± 2.03 92.14± 1.89 97.47± 1.52 a

2.69± 0.04 d

424± 14.36 b

347± 12.45 b

Ammonium Oxalate 28.84± 2.65 44.60± 1.66 32.58± 1.28 44.99± 1.46 46.26±4.21 c

3.75 ± .07 b

231± 3.45 cd

190± 4.90 e

Corn Steep Liquor 73.97± 2.34 20.17± 9.35 39.13± 2.26 49.92± 2.42 46.94± .06 c

4.12± 0.07 a

239 ± 5.55 d

219 ± 2.08 d

Maize Gluten Meal (30%) 38.39± 1.36 47.84± 2.64 53.49± 3.51 59.36± 4.53 56.56± .04 b

2.50 ± .05 d

266± 9.61 e

236± 4.06 b

Maize Gluten meal (60%) 60.25± 1.87 87.92± 1.94 89.99± 1.80 97.07± 3.09 97.43± .24 a

3.35± 0.05 c

505± 7.23 a

430± 5.79 a

Yeast Extract 26.52± 1.79 38.33 46.27± 3.32 53.02± 3.89 55.71±4.31 b

4.09± 0.04 a

228± 5.30 a

279± 4.34 c

* Medium, III; pH, 3.5 ; temperature (30±20C); wheat Bran, 1.0%


181

0

100

200

300

400

500

600

0 AmmoniumOxalate

CornSteepLiquor


Mazie GluteinMeal60%

Yeast Extract

Nitrogen Source (%)

Dec

olo

riza

tio

n (

%)

0

50

100

150

200

250

300

350

400

450

500

En

zym

e A

ctiv

ity

(IU

/mL

)Decolorization (%)MnPLac

Fig. 4.27a: Effect of different nitrogen sources on decolorization of Solar Golden

Yellow R by Pleurotus otreatus and its lignolyitic enzymes aproduction in 4 days

y = 5.4884x + 181.38

R2 = 0.8424

y = 2.6419x + 105.28

R2 = 0.9188

100

200

300

400

500

600

700

800

25 35 45 55 65 75 85 95 105

Decolorization (%)

En

zym

e ac

tivi

ty (

IU/m

L)


r (MnP) = 0.918**r (Lac) = 0.959**


different nitrogen sources.


182

The sign of slope (Figure 4.28b) indicated that increase in decoloriztion was

correlated with the enzyme activities. The value of R2 indicated that decolorization was

due to 84% variation in MnP and 92% in laccase activities. Figure 4.28b indicated that R2

for laccase (0.92) was higher than R2 for MnP (0.84) indicating the major role of laccase

in decolorization in the presence of MGM,60%.

Effect of varying concentrations of MGM 60%

Since different concentrations of MGM60% exert different physiological effects

on fungi so varying concentrations of MGM60% were used to investigate the optimum

level of MG60% in decolorization of Solar Golden Yellow R. On 3rd day of incubation

dye decolorization noted was 83.01, 86.71, 97.26, 99.75 and 76.16% in media

supplemented with 0.01, 0.05, 0.1, 0.15 and 0.2 % MGM60% respectively (Tabl 4.28).

The change in pH was 3.88, 3.94, 4.14, 4.24, 4.35 in flasks having 0.01%, 0.05%, 0.1%,

0.15 %, and 0.2% of MGM 60% respectively. MnP activities were 494, 488, 478, 566

and 486 IU/mL while laccase activities were 371, 3765, 490, 492, and 364 IU/mL. DMR

test shows that treatment means of activities with MGM60% concentrations sharing same

letters are not significant. Analysis of variance of the data (Appendex -1, Table 4.28b)

shows that our results are significants (p < 0.01). The sign of slope indicated that increase

in decoloriztion was correlated with the enzyme activities.The value of R2 indicated that

dye decolorization was due to 61 % variation in MnP and 67% in laccase activities. Two

parallel slopes (Figure 4.28b) indicated that R2 for for laccase (0.92) was higher than R2

of MnP (0.84) clearly showing that dcolorization was better through laccase activies than

by MnP synthesis.

Our choice for use of N-rich source is supported by a previous study carried out

by D,suoza Ticklo (2006), who used glutamic acid, CSL, Beef Extract and glycine during

decolorization of textile effluents by a strain NIOCC≠2a isolated from mangrove wood.

In a recent study by Bhatti etal, (2008) it has been reported that Solar Orange

RSN (a direct dye) was effectively decolorized by G.lucidum IB-L05 in presence of

maize seed meal which is a N- rich source. Nitrogen sources showed variable influence

on dye decolorization but the effect of MGM60% was pronounced among all indicating

that the dye could be effectively decolorized in presence of N-rich media. MGM60%

stimulated the MnP and laccase synthesis. More over, 0.015% of MGM60% showed


183

maximum (98.57%) dye decolorization as well as Mnp and laccase synthesis on 3rd day

of incubation. In presence of 0.1% of MGM60% dye decolorization was 97.23% on 4th

day. N-rich medium.

Despite the presence of urea in Medium III, addition of MGM 60% did not inhibit

growth of fungus and secretion of ligninases enzymes. On the other hand, when same dye

was subjected to decolorization by S.commune using Kirk,s Basal medium, there was

87% decolorization in presence of peptone (Asgher etal; 2008) which indicted better

performance than our strain. But the superior role of S.commune might be due to

difference in media composition and physiology of strain. Ammonium oxalate played

maximum inhibitory role as compared to rest of the nitrogen sources (Fig. 4.28b).

In literature there are contradictory evidences about the effects of nitrogen sources

(in terms of nature and concentration) on the lignolytic enzyme production. Regardless of

these controversies, nature and concentration of of nitrogen sources are powerful

nutrition factors in regulating lignolyting enzyme production by wood rotting

basidiomycetes (Galhaup et al. 2002; Mikishiashvilli et al., 2005). Nitrogen rich media

gave the highest laccase activity in Lentinula edodes, Rigidoporus lignosus, and Trametes

pubescens while N-limited condition enhanced the enzyme production in P.cinabarinus,

P.sanguineus, and Phlebia radiate (Mester and Field 1997; Giana Freda et al.1999;

Galhaup et al; 2002).

High nitrogen contents increase the biomass formation and the respiration rate,

which promotes a rapid depletion of the energy sources at the expense of secondary

metabolism (Kirk et al. 1978). The nitrogen metabolism competes with the metabolism of

lignin through the requirement for the same cofactors (Buswel & Codier 1987). Variable

role played by different nitrogen sources in dye decolorization process depends on the

chemical nature of the dye to be degraded and nature of fungal strain and conditions of

optimization. There are no significant reports available in literature about the use of

MGM60% as a nitrogen source for white rot fungi but enhanced the production of MnP

and laccase when used during decolorization of Solar Golden Yellow by P.ostreatus.


184

Table 4.28 Decolorization of Solar Golden Yellow R by P.ostratus and lignolytic enzymes profile with varying

Concentrations of Maize Glutein Meal 60% under optimum conditions*


(%)


pH

Enzyme Activity (IU/mL) on 3rd Day

1 2 3 4 MnP Lac

0.01 60.27± 1.21 78.05± 2.59 83.01± 2.11 81.36 ± 1.34 c

3.88± 0.01 d

494± 15.64 b

371± 2.73 c

0.05 66.09± 2.56 83.24± 1.05 86.71± 1.58 90.40± 1.44 b

3.94± 0.01 d

488± 7.27 c

376± 2.08 b

0.1 61.41± 2.62 74.54± 3.05 88.90± 5.01 97.23± 5.54 a

4.14± 0.03 c

478± 12.43 c

490± 5.51 bc

0.15 76.16± 0.98 88.24± 10.93 99.75± 1.21 98.57 ± 0.28 a

4.24± 0.01 b

566± 5.87 a

490± 6.09 a

0.2 61.75± 2.10 71.15± 2.00 76.16± 1.38 86.63± 1.39 c

4.35± 0.04 a

486± 6.57 bc

364± 6.81 c

* Medium, III; pH, 3.5 ; temperature (30±20C) ; Wheat Bran, 1.0%.


185

0

20

40

60

80

100

120

0.01 0.05 0.1 0.15 0.2

MGM60 % concentration (%)

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

700

En

zym

e A

ctiv

ity

(IU

/mL

)


Fig. 4.29a: Effect of varying concentrations of MGM 60% on decolorization of

Solar Golden Yellow R by Pleurotus ostreatus and lignolytic enzymes production in 3 days

y = 4.24x + 236.21

R2 = 0.6171

y = 2.026x + 132.92

R2 = 0.6702

100

200

300

400

500

600

700

800

40 50 60 70 80 90 100

Decolorization (%)

Enzy

me

activi

ty (IU

/mL)


r (MnP) = 0.786**r (Lac) = 0.819**

Fig. 4.29b: Relationship between dye decolorization and enzyme activities with

varying concentrations of MGM 60%


186

Effect of low molecular mass mediators

Significance of mediators in stimulating production of ligninases enzymes and

dye decolorization process cannot be overlooked. ABTS, CuSO4, H2O2, MnSO4, Tween-

80, and Veratryl Alcohol were used as low molecular weight mediators. Variable trend

was found in the decolorization of Solar Golden Yellow R by the addition of mediators.

ABTS, H2O2, MnSO4, Tween-80 and Veratryl Alcohol showed decolorization 78.36,

99.12, 89.18, 86.61 and 81.60% respectively after 48 hours of incubation. Drift in pH was

6.02, 6.03, 5.80, 5.44 and 4.11 in ABTS, H2O2, MnSO4, Tween-80 and Veratryl Alcohol

(Table 4.28). Activities of MnP were 175, 622, 610, 594 and 285 IU/mL while laccase

profile was 354, 450, 398, 378 and 281 IU/mL in ABTS, CuSO4, H2O2, MnSO4, Tween-

80 and Veratryl Alcohol respectively. MnP showed highest activities in the medium

recieving H2O2 and the lowest in presence of ABTS (Fig.4.29a). Experimental flasks

showed decolorization (99.12%) after 48 hours where asmaximum decolorization

(98.18%) in controlled flasks was noted after 72 hours. Analysis of variance of the data

(Appendex-1, Table 4.28) shows that our results are significant (p<0.01). The sign of

slope (Fig.4.29b) indicated that increase in decoloriztion was correlated with the enzyme

activities. DMR test shows that treatment means of activities with low molecular mass

mediators sharing same lettes are not significant (p≤0.05). The value of R2 indicated that

decolorizatization was due to 91 % variation in MnP activies and 92% in laccase

activities. Two parallel slopes in the (Figure 4.29 b) indicated that R2 for MnP(0.91) and

for laccase (0.92) clearly shows that dcolorization may be achieved through laccase

production followed by MnP synthesis.

The efficiency of enzymatic decolorization using MnP can be enhanced by

optimizing the concentrations of hydrogen peroxide and manganous ions as well as the

amount of enzyme, the pH and the reaction temperature for each dye (Mielgo Moreira

2003). Some reports illustrate that MnP require H2O2 (Ollika et al., 1993; Meilgo et al.,

2003). However, MnP from Bjerkandera adusta, DSM 11310, Pleurotus eryngii

ATCC90787 decolorized four azo and thalocyanine dyes in Mn+2 independent reactions

in which the enzymmes are found to interact directly with dye (Heinfleing et al. 1998).

Enzymatic decolorization might be also improved.


187

Table 4.29 Decolorization of Solar Golden Yellow R by Pleurotus ostreatus and its lignolytic enzymes profile using low

molecular mass mediators under optimum conditions*

Mediators (1mM)


pH


1 2 3 MnP Lac

0 76.14±1.25 87.94±2.01 98.28 ±2.13 a

4.16±0.04 d

540±12.24 c

391±8.75 b

ABTS 67.9±0.87 78.36±17.54 83.96±1.10 c

6.02±0.06 a

175±6.18 e

354 ±6.44 e

H202 84.12±1.87 99.12±0.87 98.91±1.47 a

6.03±0.03 a

622 ±18.03 a

450 ±3.18 a

MnSO4 82.58±1.17 89.18±1.13 92.5±9.18 b

5.80±0.04 b

610 ±8.67 a

398±6.23 b

Tween-80 75.65 86.61±1.24 86.16±1.44 b

5.44±0.2 c

594±16.97 b

378±9.36 c

Veratryl Alcohol 64.14±1.69 81.6±21.32 5 0.16±1.54 c

4.11±0.08 d

285±8.45 d

281±6.03 d

* Medium, III; pH, 3.5 ; temperature (30±20C) ; Wheat Bran, 1.0% MGM (60%), 0.15%.


188

0

20

40

60

80

100

120


Mediators (1mM)

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

700

En

zym

e A

ctiv

ity

(IU

/mL

)


Fig.4.29a: Effect of low molecular mass mediators on decolorization of Solar Golden Yellow R by Pleurotus otreatus and lignolytic enzymes production in 2 days

y = 12.42x - 271.09

R2 = 0.9145

y = 4.4451x + 147.99

R2 = 0.9234

100

200

300

400

500

600

700

800

900

1000

1100

45 55 65 75 85 95 105

Decolorization (%)

Enzy

me

acti

vity

(IU

/mL)


r (MnP) = 0.956**r (Lac) = 0.961**

Fig.4.29b: Relationship between dye decolorization and enzyme activities with low molecular mass mediators


189

in the presence of surfactants, such as Tween-80 (polyoxyethylenesorbitan monooleate)

which is a source of unsaturated fatty acids (Harazono and Nakamura 2005). Our findings

show that strain improvement or physiological improvement of white rot fungi should be

sought in the presence of H2O2 in combination with Tween-80 for increasing the

production of peroxidases efficiencies or activites. Synergetic effect of H2O2 and Tween-

80 might play more effective role in dye decolorization than indivisual role. Likewise

accumulative effect of ABTS and CuSO4 enhanced laccase potential for Solar Golden

Yellow R. Our research lies in line with the findings of Kotterman et al.(1996) who

worked on polycyclic aromatic hydrocarbon degradation by white rot fungi.

MnP catalyzes oxidation of Mn+2 to Mn+3, which oxidizes a variety of phenolic

substrates. However, it has been found that not only lignolytic enzymes are responsible

for lignin and organopollutants degradation but there is significant role of H2O2 and free

radicals in this process (Kapich etal, 1999; Tanaka et al, 1999). Two main peroxidases

(LiP and MnP) involved in degradation process require H2O2 as a cosubstrates (Zhao and

Janse 1996). In WRF a number of enzymes like glucose-1-oxidase, glyoxal oxidase,

veratryl alcohol oxidase are source of H2O2 generation.

Synthetic dye decolorization is strongly influenced by the generation of

extracellular H2O2. However, too high or low H2O2 level inhibits decolorization

efficiency (Eichlerova et al., 2006). There might be no benefit of hyperproduction of

manganese peroxidase unless there is enhancement of endogenous H2O2. H2O2 also

influences even activity and decolorization potential of laccase. P.ostreatus CCBAS473

in presence of 1.58 oµM f H2O2 decolorised Orange G up to 86.6% and 51.1% while

activities of laccase and Mnp shown by this strain were1.58 and 0.7µM//mL and

respectively. Pleurotus eryngii CCBAS 471 decolorised the same dye up to 40.0% and

0.00% with laccase (108.6mu/ml) and MnP (1.0 µM/mL) in the presence of 0.46 µM of

H2O2. Pleurotus citrinopileatus CCBAS 691 having 1.72 uM of H2O2 showed 0.00%

decolorization of the dye despite the 1.73 IU/mL and 5.5 IU/mLlaccase and MnP

activities (Eichlerova et al. 2006).

It has been reported that addition of Mn2+ results in significant changes in MnP

activity secreted by P. ostreatus (Giardina, et al; 2000; Cohen et al, 20). Addition of

MnSO4 enabled detection of MnP activity extracellularly. In a research of Brown et al.


190

(1991) it was found that Mn was a naturally occurring metal ion in wood and participated

in the lignin degrading processes of white rot fungi. Mn is not only a substrate and

important diffusible mediator produced by MnP but it also regulates the enzyme

production by inducing gene transcription as was observed in P. chrysosporium.

Increasing concentrations of Mn resulted in higher levels of MnP mRNA, MnP protein

synthesis and enzyme activity in many white rot fungi (Li et al; 1995; Piri and Gold,

1991; Bonnarmen and Jeff’ries 1990). The enzymes in the culture supernatant of P.

ostreatus were also enhanced by the extra addition of Mn salt which supports our

research findings. In this study we have also observed that Mn salt addition in nutrient

medium helped to trigger the production of MnP. Manganese peroxidase (MnP) has a

Mn2+ binding site and oxidizes Mn2+ to Mn3+, in the presence of H2O2. The Mn3+ is then

stabilized by organic chelators in the culture filtrates to oxidize phenolic substrates

(Wariishi and Gold 1989). MnP withdraws one electron from phenolic compounds to

generate a phenoxy radical (Wariishi and Gold 1988) although it also oxidizes non-

phenolics in combination with appropriate redox mediators or by initiating free radical

chain reactions of unsaturated fatty acid (Watanabe et al. 20001).

Effect of different metal ions

Metal ions are essential for fungal physiology as wel as dye degradation process.

Metal ions necessary for improving fungal efficiency were supplemented in the dye

solutions in the form of their salts. CaCl2, Cd(NO3), CuSO4, FeSO4 and ZnSO4 were

added separately in the flasks. After 48 hours of incubation, decolorization was found as

47.55, 83.98, 90.21, 99.10 and 62.73% in CaCl2, Cd(NO3), CuSO4, FeSO4 and ZnSO4

respectively after 24 hours of incubation time (Table.4.30). Controlled flasks showed

98.16 % decolorization after 48 hours. Change in pH was 3.58, 3.58, 3.71, 3.73 and 4.03

respectively. MnP activities were 467, 597,388, 688 and 464 IU/mL while laccase

activities were 437, 443, 496, 377 and 324 in flasks recieving CaCl2 Cd(NO3)2, CuSO4

FeSO4 and ZnSO4 IU/mL respectively. MnSO4 played major role in decolorizing the dye

under optimum conditions (Fig .4.30a). Analysis of variance of the data (Appendex -1,

Table, 4.20b) shows that our results were significants (p < 0.01). DMR test shows that

means of activities with metal ions sharing same letters are not significant (p < 0.01,


191

Table 4.30 Decolorization of Solar Golden Yellow R by Pleurotus ostreatus and its lignolytic enzyme profile using different

metal ions under optimum conditions*

Metal Ions (1 mM)

Decolorization (%Mean± S.E) Incubation Time (Days) Final

pH


1 2 MnP Lac

0 83.84± 1.32 98.16 ± 1.63 a

6.04± 0.02 a

586± 9.11 b

449± 7.14 b

CaCl2 47.55± 8.59 49.14 ± 4.09 d

3.58 ± 0.02 c

467± 1.45 d

437± 2.34 c

Cd(NO3)2 83.98 81.40 ± 3.53 b

3.58± 0.04 c

597± 2.08 b

443± 3.76 bc

CuSO4 90.21± 2.25 92.85± 2.19

a 3.71± 0.06

bc 388± 3.53

e 496± 10.71

a

FeSO4 99.1± 3.92 98.80 ± 1.29 a

3.73± 0.03 b

688± 6.67 a

377 ± 16.1 e

ZnSO4 62.73± 4.45 59.92± 4.06

c 4.03± 0.09

a 464 ± 4.05

d 324± 3.93

d * Medium, II; pH, 3.5 ; temperature (30±20C) ; Wheat Bran, 1.0%; MGM(60%), 0.05% ; Mediator, H2O2 (1mM).


192

0

100

200

300

400

500

600

700

800


Metal Ion (1mM)

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

En

zym

e A

ctiv

ity

(IU

/mL

)


Fig. 4.30a Effect of different metal ions on decolorization of Soar Golden Yellow R byPleurotus otreatus and lignolytic enzymes production in 24 hours

y = 6.6837x + 118.74

R2 = 0.262

y = 5.4886x + 100.48

R2 = 0.1622

100

300

500

700

900

1100

1300

1500

40 50 60 70 80 90 100

Decolorization (%)

Enzy

me

acti

vity

(IU

/mL)


r (MnP) = 0.512*r (Lac) = 0.335

Fig.4.30b: Relationship between dye decolorization and enzyme activities with different metal ions


193

Table 4.30).The sign of slope (Fig. 4.30b) indicated that increase in decoloriztion

was correlated with the enzyme activities.The value of R2 indicated that dye

decolorization was due to 26 % variation in MnP and 16% in laccase activities was due to

decolorization. R2 for MnP (0.26) was higher than R2 for laccase (0.16) clearly showing

that dcolorization may best be achieved through MnP production followed by laccase.

The synergistic action of MnP, laccas and Fe+3 reducing activity from wood

decaying fungi for degradation of Azure B (a non phenolic compound) has been reported

(Arnates et al; 2007). Iron is an essential element for microbial growth. It exists in nature

in the insoluble form (Fe+3) which is not readily available for assimilation. To make

soluble Iron (III) many organism have evolved efficient high-affinity acquisition system

(Fekete etal; 1989). The reactivity and limited solubility of most metal ions require

constant chelation once they are taken up into the cell.The metal ios bound by chelators

(organic acids) contribute to metal detoxification by buffering cytosolic metal

concentration (Stewphan and Clemens; 2001).

Presence of Fe in MnP makes role of H2O2 crucial. The reactive species involved

in this reaction was found to be OH hydroxyl free radical, the powerful oxidizing agent in

biological syetem. The OH free radical is so highly reactive that it can react with

whatever organic molecule like lignin or xenobiotic pollutants to produce secondary less

reactive radicals. OH radicals are produced by the oxidation of Fe(II) by H2O2 through a

popular chemical reaction known as Fenton reaction (Aken and Agathos 2001).

Effect of varying concentrations of dye

Effect of varying concentrations of the dye was investigated.This indicated the

maximum uptake of the dye. Dye decolorization was variable due to different dye

concentration level. Decolorization was 99.10, 80.02, 46.43, 16.16 and 11.87 % inflasks

receiving 0.01, 0.05, 0.1, 0.15 and 0.2 % dye solutions respectively(as shown in the Table

4.31). Change in pH was 3.98, 3.85, 3.82, 3.87 and 3.95. MnP activities were 687, 571,

459, 310 and 222 IU/mL while laccase activit profile was 376, 365, 324, 278 and 216

IU/mL inflasks having receiving 0.01, 0.05, 0.1, 0.15 and 0.2 % dye concentrations

respectively (Fig.4.31a). Analysis of variance of the data (Appendex-1, Table 4.31)

shows that our results are significant as (p<0.01) as shown in the table 4.31b.The sign of

slope indicated that increase in decoloriztion was correlated with enzyme activities. The


194

Table 4.31 Decolorization of Solar Golden Yellow RSN by Pleurotus ostreatus and its lignolytic enzyme profile using

varying concentrations of the dye under optimum conditions*

Different Dye Conc. (%)


pH


1 2 MnP Lac

0.01 99.1±1.02 98.76±0.68 a

3.98 ±0.01 a

687±11.72 a

376±4.33 a

0.05 80.02±0.52 79.43±0.97 b

3.85±0.03 ab

571±6.13 a

365±1.20 a

0.1 46.25±3.02 46.51±1.51 c

3.82±0.01 b

459±10.38 c

324±3.93 b

0.15 16.16±11.87 15.90±1.47 d

3.87±0.02 ab

310±2.34 d

278 ±2.73 c

0.2 11.87±0.90 11.10±0.84 e

3.95±0.01 a

222±2.91 e

216±3.53 d

* Medium, II; pH, 3.5 ; temperature (30±20C) ; Wheat Bran, 1.0%; MGM (60%), 0.05%; Mediator, H2O2 (1mM); Metal Ion, FeSO4 (1mM)


195

0

20

40

60

80

100

120

0.01 0.05 0.1 0.15 0.2

Dye concentration (%)

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

700

800

900

1000

En

zym

e A

ctiv

ity

(IU

//mL

)


Fig. 4.31a Effect of varying concentrations of the dye on decolorization of Solar Golden Yellow R by P.ostreatus and lignolytic enzymes production in 24 hours

y = 8.4915x + 257.34

R2 = 0.9973

y = 2.7901x + 206.35

R2 = 0.99

100

300

500

700

900

1100

1300

0 20 40 60 80 100

Decolorization (%)

En

zym

e ac

tivi

ty (

IU/m

L)


r (MnP) = 0.999**r (Lac) = 0.994**

Fig.4.31b Relationship between decolorization and enzyme activities at varying concentrations of Solar Golden Yellow R


196

Table 4.32 Adsorption of Solar Golden Yellow R by fungal mycelia at optimum temperature and pH*

Different Dye Concentrations

(%)

Adsorption (%Mean ± S.E) Incubation Time (Days) Final

pH

1 2

0.01 0.03±0.00

0.007±0.00

e 4.08±0.02

a

0.05 0.47±0.04

0.034±0.01

d 4.02±0.01

a

0.1 0.57±0.05

0.046±0.02

c 3.83 ±0.01

b

0.15 0.70±0.03

0.059±0.03

b 3.73±0.03

c

0.2 1.87±0.23

0.69±0.16

a 3.59±0.04

d *pH, 3.5 ; temperature (30±20C)

0

0.5

1

1.5

2

2.5

3

0.01 0.05 0.1 0.15 0.2


Ad

sorp

tio

n (

%)

Fig.4.32a Dye adsorption on fungal mycelia of Solar Golden Yellow R with varying concentrations of the dye


197

value of R2 indicated that decolorization was due to 99 % variation in MnP and laccase

activities. Two parallel slopes (Fig. 4.31b) indicated that R2 for MnP as wel as laccase is

same i.e.0.99 which clearly shows that dcolorization may best be achieved through

laccase as wel as MnP equally and simultaneously each other.

Fungal culture growth may be largely affected in presence of dye concentration at

toxic level. This factor also influences the dye decolorizing ability of fungi. Moreover,

class of dye which illustrates its structure leaves strong influence in deciciding the extent

of dye removal extent. Pazarlioglu et al.(2005) reported the decolorization of Direct

Blue15 (azodye) by crude culture filtrate of P.chrysosporium decreased from 100% to

60% at initial concentration of 15 mg/L and 120 mg/L respectively.In our research work

it was. Golden Yellow RSN by Pleurotus otreatus and its lignolyitic enzymes activities

found that fungus successfully tolerated dye concentration up to 0.01g/100 mL of Direct

dye Soar Golden Yellow R which indicates greater potential of P.ostratus IBL as Solar

compared to previous studies illustrated above. However, increase in dye concentration

from optimum level caused toxicity and subsequently enzyme inhibition resulting in

lower dye degradation rate as wel as prolongation in incubation period. So dye

concentration and decolorization efficiency of fingal strain are inversely propotional to

Effect of dye adsorption on fungal mycelia

During dye degradation adsorption takes place also but level of adsorption

depends on the nature of dye and fungal strain potential capability. Dye adsorption was

0.03, 0.47, 0.57, 0.70 and 1.87% in flasks recieving 0.01, 0.05, 0.1, 0.15 and 0.2% dye

solution after 24 hours of incubation. Drift in pH was 4.08, 4.02, 3.83, 3.73 and 3.59

(Table 4.32). Analysis of variance of the data (Appendex-1, Table 4.32) shows that our

results are significant (p<0.01). DMR test reveals thattreatment means of dye adsorption

show significant diffeence.

In a previous study of Pazarlioglu et al. 2005, it has been reported that during

biodecolorization of Chrysophenine and Direct Red 26, a rapid adsorption on the fungal

mycelial was observed in autovlaved cultures. Adsorption was 2, 17, and 24% in

Tatrazine, Direct Red23, and Direct Blue 15 respectively. Our indeginous strain showed

more decolorization capability than adsorption affinity since there was very low % of dye

adsorption on fungal mycelia.


198

During biodegradation process, biosorption might also play role in decolorization

of dye by living fingal biomass. For dead cells, the mechanism is called biosorption,

which occur due to physiochemical changes like adsorption, deposition, and

ionexchange. As for as adsorption is concerned it is not yet clear that wheather external

bonding only or externeal binding plus internal interactions take place (Bonnarme and

Jeffries 1990). Complex chemistry of textile dyes and therefore their interactions with the

microorganism depend on structure of a particular dye and the specific chemistry of the

fungal biomass (Polman et al., 1998). In addition, decolorization of RBBR by P.

ostreatus showed that color rmoval from the cultivation medium was due to dye

transformation rather than adsorption of the dye on the fungal mycelium. Moreover,

changes in absorption spectra indicate that decolorization is not a single step process

instead involves complex mechanism (Palmieri et al., 2005). Adsorption % decreased as

the time of incubation increased.

4.4 DECOLORIZATIO N OF VAT DYES

4.4.1 Screening of white rot fungi on Vat dyes Decolorization.

Five WRF strains were used for decolourization of four vat dyes including

Cibanon Red 2B-MD, Cibanon G. Yellow PK-MD, Cibanon Blue GF-MD and

Indanthren Direct Black RBS Colloisol and the results of 10 days decolourization trial

have been presented in Table 4.33 Different fungi showed different pattern of

decolorization on different dyes. P. ostreatus showed maximum decolorization (71.56%)

of Indanthren Direct Black RBS Colloisol followed by 65.79% decolorization of Cibanon

G. Yellow PK-MD in 10-days while no significant decolorization of other vat dyes was

observed. G. lucidum showed maximum decolorization (89.48%) of Cibanon Blue GF-

MD in 9-days followed by 88.33% decolorization of Cibanon Red 2B-MD and 52.34%

decolorization of Cibanon G. Yellow PK-MD in 7 and 9 days, respectively while no

decolorization of Indanthren Direct Black RBS Colloisol was observed even in 10-days.

S. commune showed very poor decolourization efficiency on all the vat dyes. C.

versicolor showed very good efficiency for all the vat dyes; best efficiency (92.12%) was

onserved for Cibanon Blue GF-MD only in 6-days followed by 76.49% decolorization of

Cibanon Red 2B-MD, 88.19% decolorization of Cibanon G. Yellow PK-MD and 88.23%

decolorization of Indanthren Direct Black RBS Colloisol in 8,9 and 10 days, respectively.


199

Table 4.33 Decolorization of Vat dyestuffs by different white rot fungi in time course study

Incubation Time (Days)

WRF Cultures

Decolorization(%)

Cibanon Red 2B-MD Cibanon Golden Yellow PK- MD

Cibanon Blue GFJ-MD

Indanthren Direct Black RBS Collisol

1

P.O. 16.25 0.82 22.56 0.22 10.00 0.00 29.9 1..32 G.L. 4.49 0.69 6.12 0.60 11.3 0.46 0.00 0.00 S.C. 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C.V. 31.1 0.55 24.31 0.60 34.0 0.05 18.19 0.30 P.C 36.8 0.25 25.0 0.03 59.1 0.19 19.0 0.14

2

P.O. 21.1 0.65 38.1 0.24 12.00 0.00 30.9 0.18 G.L. 21.2 0.27 10.8 0.10 27.4 0.39 0.00 0.00 S.C. 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C.V. 46.0 0.2 34.24 0.33 44.1 0.06 47.11 0.23 P.C 45.7 0.45 26.1 0.05 61.5 0.45 32.6 0.62

3

P.O. 26.8 0.05 41.9 0.40 14.47 0.00 35.9 0.55 G.L. 21.9 0.30 16.8 0.42 31.9 0.60 0.00 0.00 S.C. 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C.V. 52.13 0.38 46.57 0.12 57.0 0.08 58.19. 0.08

P.C 56.1 0.63 28.6 0.12 63.8 0.30 47.0 0.05

4

P.O. 27.9 0.7 57.9 0.20 19.00 0.00 43.8 0.30 G.L. 30.9 0.09 21.6 0.40 39.2 0.37 0.00 0.00 S.C. 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C.V 63.14 0.00 51.36 0.50 70.41 0.11 61.45 0.45

P.C 60.6 0.39 31.5 0.32 70.9 0.25 47.4 0.17

5

P.O. 28.0 0.36 63.1 0.03 21.00 0.00 53.4 0.25 G.L. 50.8 0.28 40.1 0.43 61.3 0.45 0.00 0.00 S.C. 0.00 0.00 0.00 0.00 0.00 0.00 11.2 0.40 C.V 69.25 1.25 60.75 0.65 85.42 0.95 67.69 0.65

P.C 64.0 0.21 42.2 0.15 71.7 0.31 48.8 0.14

6

P.O. 29.1 0.60 64.0 0.86 26.31 0.00 60.0 0.34 G.L. 79.1 0.50 45.2 0.25 81.7 0.25 0.00 0.00 S.C. 21.7 0.46 0.00 0.00 26.3 0.45 11.9 0.07 C.V 74.5 0.50 69.17 0.00 92.32 0.27 71.7 0.00

P.C 65.6 0.14 60.5 0.25 74.0 0.20 49.6 0.09

7

P.O. 30.0 0.00 64.2 0.25 28.65 0.00 70.3 0.40 G.L. 88.3 0.60 51.2 0.04 88.8 0.12 0.00 0.00 S.C. 24.9 0.20 0.00 0.00 28.55 0.42 14.1 0.53 CV 76.5 0.00 78.8 1 0.00 91.31 0.00 81.20 0.00 P.C 68.9 0.14 73.5 0.29 85.3 0.30 53.6 0.02

8

P.O. 31.9 0.01 64.1 0.10 29.20 0.00 71.0 0.04 G.L. 78.3 0.03 52.7 0.55 88.8 0.01 0.00 0.00 S.C. 24.9 0.06 0.00 0.00 30.1 0.15 15.5 0.41 CV 76.49 0.00 81.29 0.00 91.13 0.00 83.19 0.00

P.C 69.0 0.05 73.6 0.29 85.3 0.00 53.8 0.15

9

P.O. 32.4 0.24 65.0 0.03 30.00 0.00 71.2 0.00 G.L. 88.5 0.05 52.3 0.10 89.4 0.39 0.00 0.00 S.C. 25.2 0.02 0.00 0.00 30.9 0.07 16.9 0.03 CV 76.49 0.00 87.12 0.00 91.2 0.00 83.22 0.00

P.C 70.7 0.02 73.7 0.03 86.6 0.00 54.1 0.10

10

P.O. 33.5 0.18 65.7 0.10 32.00 0.00 71.5 0.05 G.L. 88.7 0.02 52.7 0.13 89.5 0.40 0.00 0.00 S.C. 25.5 0.05 0.00 0.00 32.67 0.25 17.5 0.49 CV 74.0 0.00 88.1 9 0.00 91.1 4 0.00 83.23` 0.00 P.C 70.9 0.03 74.7 0.15 86.9 0.08 54.5 0.00


200

P. chrysosporium also showed good performance on Cibanone dyes causing maximum

decolorization (86.98%) of Cibanon Blue GF-MD, followed by 74.75% decolorization of

Cibanon G. Yellow PK-MD, 70.92% decolorization of Cibanon Red 2B-MD. It however,

poorly decolorized (54.59%) Indanthren Direct Black RBS Colloisol in 10-days.The net

result of decolourization performance of different WRF cultures on Vat dyes is given

Table 4.33.

Results of initial time course study showed that different strains of WRF have

variable decolorization potential for different dyes. Also different decolourization

patterns for individual dyes were observed for the four white rot fungi cultures.

Decolorization of some dyes took longer time for some cultures as compared to others,

and in some cases it was incomplete even at the end of 10 days study period. However,

the time taken by our selected local isolates showing maximum decolorization on

different dyes compares favourably with reports on other white rot fungi which required

7-20 days for 90% decolorization of a diverse range of synthetic dyes (Kargi et al. 2000;

Kirby et al. 2000; Boer et al. 2004).

All the five locally isolated strains of WRF degraded all the dyes more than 50%.

Pleurotus ostreatus IBL-02, Phanerochaete chrysosporium IBL-03 and Ganoderma

lucidum IBL-05 decolorized different dyes upto and more than 70%, Schyzophyllum

commune IBL-06 successfully degraded all the dyes mostly upto 60% within 10 days.

While Coriolus versicolor IBL-04 degraded all the other dyes upto 45-50% but it showed

maximum decolorization (upto 90%) on Vat dyes only within 3-days. The variation in

colour removal or extent of dye degradation depends on the dye complexity, nitrogen

availability in the medium and the ligninolytic activity of the culture i.e., strains of WRF

used (Glenn and Gold 1983).

Sanghi et al. (2006) revealed that C. versicolor could decolorize dyes almost up to

90%. Levin et al. (2006) investigated the potential of 26 WRF fungal cultures and noted

excellent performance by C.versicolor.

Previous studies on WRF and their ability to decolorize dyes as wel as their

enzyme activity profile indicate strong variation in their decolorizing ability on individual

level (Chander Arora, 2004; Eichlerova et al. 2006; Chander and Arora, 2007). The

complex LMEs profile of different WRF strains involved in dye degradation and pattern

of its expression may also vary with the chemical and structural characteristics of the

dyes being degraded (Nagai et al. 2002; Boer et al. 2004; Mazmanci and Unayayar


201

2005). However, the relative rates of decolorization for different groups of dyes can not

be easily explained. Degradation of a dye involves aromatic ring cleavage, which is

dependant on the identity of ring substituents (Sparado et al. 1992; Chander et al. 2004).

Asgher et al; 2006 also found different patterns of decolorization by different dyes.

However, overall complexity of structure is not the sole criteion of a particular dye

degradation (Toh et al; 2003; Maas and Chaudhri 2005). Structurally similar dyes may

still be differently degraded by white rot fungi. Physiological differences among WRF

and varying levels of LMEs produces in response to different dyes present in the medium

may account for differences in their decoloration abilities (Yesilada et al. 2003; Chander

et al. 2004).

4.4.2 P rocess optimisation

Effect of media composition

The effect of four media was investigated by using the Coriolus versicolor for

decolorization of Cibanon Blue GFJ-MD. After 6 days of incubation it was found that the

dye decolorization was 46.51, 92.06, 18.06 and 10.46 % in medium I, II, III and IV

respectively. Change in pH from optimum level (4.5) was noted as 7.07, 7.09, 7.78 and

7.41 in medium I, II, III and IV respectively (Table 4.34). Enzyme activities of LiP were

54, 67, 16 and 10 IU/mL. MnP and laccase activities profile on 6th day of incubation was

35, 76, 16,19 IU/mL and 185, 326, 106 and 110 IU/mL in four media medium I, II, III

and IV respectively (Fig.4.34a).

Analysis of variance of the data (Appendex-1 Table 4.34) shows that our results

are significant (p < 0.01). DMR test revealed that treatment means of activities with

different media compositions shows significant difference (p < 0.01). The sign of slope

indicated that increase in decoloriztion was correlated positively with thelaccase

activities.The value of R2 indicated that dye color removal was due to 80% variation in

laccase activities. The slope in the figure (4.34b) indicated that R2 for laccase (0.80)

clearly indicating that dcolorization may best be achieved through laccase synthesis.

Laccasewas the major enzyme in dye degradation process.


202

Tabel 4.34 Decolorization* of Cibanone Blue GFJ-Mdby Coriolus versicolr and its lignolytic enzymes profile using different


Med

ium

Decolorization (%Mean ±S.E) Incubation Time (Days)


on 6h Day

1 2 3 4 5 6 7 pH LiP MnP Lac

M-I 16.76±1.05 17.77±1.69 25.93±1.61 29.08±1.16 39.61±1.41 46.51±1.85 51.46 ±1.13

b 6.07±0.10

c 54±1.45

b 35±1.86

b 1 85±3.39

b

M-II 32.86±1.84 45.75±1.27 54.99±1.21 70.76±1.70 85.89±0.64 92.06±1.32 91.46±1.45

a 6.09±0.31

c 67±5.63

a 76 ±3.76

a 326±4.73

a

M-III 14.72±1.15 18.55±1.89 24.28±1.31 28.40±1.34 26.68±1.10 18.06±1.91 18.06±1.12

c 7.78 ±0.01

a 16±2.36

c 16±3.56

c 106±4.74

c

M-IV 5.80±1.65 6.96±1.85 8.26±1.51 11.58±1.54 16.88±1.97 10.46±1.04 10.46±1.87

c 7.41±0.02

b 1 0±4.11

d 19 ±4.56

c 110±2.89

c



203

0102030405060708090

100

M-1 M-2 M-3 M-4Medium

Dec

olo

riza

tio

n (

%)

050100150200250300350400450500

En

zym

e A

ctiv

ity

(IU

/mL

)

Decolorization%LiPMnPLac

Fig.4.34a: Effect of varying medium compositions on decolorization of Cibanon Blue GFJ-MD by Coriolus versicolor and lignolytic enyme production in 6 days

y = 2.4435x + 232.34

R2 = 0.8054

100

150

200

250

300

350

400

450

500

40 50 60 70 80 90 100

Decolorization (%)

Enzy

me

activi

ty (IU

/mL)

r (Lac) = 0.897**

Fig.4.34b: Relationship between dye decolorization and enzyme activities with different media


204

The medium I commonly known as Kirk’s basal nutrient medium contained

costly components like Veratryl alcohol and Tween-80 (surfactant). Medium II is

modified form of the Kirk’basal medium (lacking both Veratryl alcohol and Tween-80).

Medium III and IV differed mainly from M-I and M-II due to the presence of urea.Our

indeginous strain of C.versicolor strain showed better result in medium II than Kirk’s

basal medium and the media containing urea. Dye decolorization and enzyme profile

were impressive in II medium. Laccase role in dye decolorization has been proved by

ample evidences reported by the researchers. Mayer and Richard 2002 reported partial

decolorization of two azodyes (orange G and amaranth) and complete decolorization of

two triphenylmethane dyes (bromophenol blue and malachite green) was obtained

cultures of Pycnoporus sanguineus producing laccase as the sole phenoloxidase.Th

fungus was grown in submerged liquid cultures. Moreover it was found tha there was

direct correlation between enzyme production and dye decolorization. Our fungal strain

produced laccase in liquid state culture and decolorized the dye in same fashion as

reported above.

Effect of Additional carbon

i) Effect of different carbon sources

Carbon sources can induce laccase activity regulation consequently expression

can differ greatly mong species (Mansur etal.1997). Optimisation of different carbon

sources was carried out using glucose, glycerol, lactose wheat straw and wheat bran as

energy sources at 30oC. After 3 days of incubation decolorization was 92.16, 96.81,

57.27, 60.77 and 55.81% in glucose, glycerol, lactose, wheat straw and wheat bran

respectively (Table 4.35). Controlled flasks showed maximum dye degradation 71.51%

on 3rd day. Changes in pH levels noted were 4.33, 4.23, 5.24, 5.27 and 4.48 in glucose,

glycerol, lactose, wheat straw and wheat bran respectivelyEnzyme activities increased to

much an extent in the presence of carbon sources.The laccase activity profile was 396,

430, 202, 212 and198 IU/mL in glucose, glycerol, lactose, wheat straw and wheat bran

respectively. Glycerol showed maximum decolorization % among all sources (the

Fig.4.35a).

Analysis of variance of the data (Appendex-1, Table 4.35) shows that our results

are significant (p < 0.01). DMR test revealed that treatment means activities with

different carbon sources sharing same letters are not significant difference (p < 0.01).


205

Table 4.35 Decolorization of Cibanon blue GFJ-MD by C.versicolor and its lignolytic enzyme profile carbon sources carbon

sources under optimum conditions*

Carbon sources (1%)


Final pH

Enzyme Activity (IU/mL) on 3rd day

1 2 3 4 Lac

0 31.96±1.11 46.05±1.42 71.51±1.29 69.19±1.58 c

4.45±0.05 b

327±14.12 c

Glucose 58.62±1.89 66.97±2.89 92.16±3.48 91.88±2.99 b

4.33±0.04 c

396 ±8.90 b

Glycerol 71.26±1.02 84.89±1.60 96.81±4.14 96.41±26.25 a

4.23 ±0.04 c

430 ±430 a

Lactose 59.22±1.91 42.51±2.95 57.27±4.12 55.83±3.41 e

5.24±0.01 a

202±3.18 d

Wheat straw 59.22±2.89 64.97±2.65 60.77±3.19 59.16±5.78 d

5.27±0.04 d

212±3.18 d

Wheat Bran 49.19±2.13 58.96±2.60 55.81±4.56 21.26±2.56 e

4.48±0.05 d

198±4.62 e

* Medium II; pH, 4.5 ; temperature (30±20C).


206

0

20

40

60

80

100

120

0 Glucose Glycerol Lactose Wheatstraw

WheatBran

Carbon source (1%)

Dec

olo

riza

tio

n (

%0

050

100150

200250300

350400

450500

En

zym

e A

ctiv

ity

(IU

/mL

)

Decolorization%Lac

Fig. 4.35a: Effect of different carbon sources on decolorization of Cibanon Blue

GFJ-MD and its lignolytic enyme production by Coriolus versicolor in 3 days

y = 10.958x + 140.79

R2 = 0.8564

100

300

500

700

900

1100

1300

20 30 40 50 60 70 80 90

Decolorization (%)

Enzy

me

acti

vity

(IU

/mL)

r (Lac) = 0.926**

Fig.4.35b: Relationship between dye decolorization and enzyme activities with different carbon sources


207

The sign of slope (Fig.4.35b) indicated that dye decolorization was positively

correlated with the enzyme activities.The value of R2 indicated dye decolorization was

due to 85% variation in laccase activities. R2 for laccase (0.85) clearly indicates that

dcolorization may best be achieved through laccase synthesis. Laccase is the major

enzyme in dye degradation process.

Effect of varying concentrations of glycerol

After the selection of glycerol as the best carbon source its varying concentrations

ranging from 0.1% to 2.0% were used with a difference of 0.5% for the decolorization of

Cibanon Blue GFJ-MD. Maximum dye decolorization was experienced in triplicate set of

flasks recieving 1.0% glycerol. Decolorization was 89.9, 92.24, 97.71, 68.40 and 49.51 %

in 0.1, 0.5, 1.0, 1.5 and 2.0% respectively on 3rd day of incubation. pH change from

optimum level (4) was showing trend towords basic medium. Observed pH changes were

5.73, 5.19, 5.74, 5.99 and 4.61 in medium containing 0.1, 0.5, 1.0, 1.5, and 2.0 %

glycerol respectively (Table.4.36). Laccas activities determined on 3rd day of incubation

were 364, 371, 455, 391, and 325 IU/mL (Fig.4.36a)

Analysis of variance of the data shows that our results are significants (p < 0.01,

Appendex-1, Table 4.36). DMR test shows that treatment means of activities with

varying concentrations of glycerol shows significant differenc (p < 0.01).The sign of

slope (Fig. 4.36b) indicated that increase in decoloriztion was correlated with the enzyme

activities.The value of R2 indicated that dye decolorization was due to 97 % variation in

laccase activities and it may best be achieved through laccase synthesis. Laccase is the

major enzyme involved dye degradation process.

Although glucose can serve as a best carbon and enrgy source and accelerate the

decolorization of the dyes (Kim et al;1996). In our case, glucose showed better

performance but lesser than glycerol. The study of Revanker and Lele (2007) revealed

that poor decolorization of dyes was observed in presence of Glycerol as a carbon source.

Since there is no evidence of glycerol use as a carbon source in literature so comparison

can be made specifically. However, glycerol being polyhydroxy alcohol might trigger

laccase induction like Veratryl alcohol in case of LiP which is aromatic in nature. So we

can say that our strain depended much on glycerol and showed strong relationship

between the glycerol contents and dye decolorization. So glycerol was selected as a best


208

Table 4.36 Decolorization of Cibanon blue GFJ-MD by C. versicolor and its lignolytic enzyme profile using varying varying

concentrations of glycerol under optimimum conditions*


of Glycerol (%)

Decolorization (%Mean ±S.E) Incubation Time (Days) Final

pH


on 3rd Day

1 2 3 4 Lac

0.1 58.35±2.83 66.57±2.69 89.9±1.46 90.38±2.54 a

5.73±0.02 a

364±18.0 c

0.5 65.7±4.81 77.92±2.93 92.24±2.30 92.03±2.80 ab

5.19±0.02 c

371 ±10.98 c

1 70.61±1.16 86.89±1.73 96.71±1.25 96.41±1.22 b

5.16±0.07 c

455 ±18.30 a

1.5 55.08±3.15 63.81±2.04 68.4±0.95 60.01±0.62 c

4.74 ±0.01 a

391±8.46 b

2 43.24±4.16 46.52±5.63 49.51±4.32 56.22±2.36 d

4.61±0.05 b

325±4.56 d

* Medium II ; pH, 4 ; temperature (30±20C)


209

0

20

40

60

80

100

120

0.1 0.5 1 1.5 2

Glycerol Concentration (%)

Dec

olo

riza

tio

n (

%)

050100150200250300350400450500

En

zym

e A

ctiv

ity

(IU

/mL

)

Decolorization%Lac

Fig.4.36a Effect of different carbon sources on decolorization of Cibanon Blue GFJ-MD by Coriolus versicolor and its lignolytic enyme production in 3 days

y = 15.933x - 302.01

R2 = 0.9732

100

300

500

700

900

1100

1300

35 45 55 65 75 85 95 105

Decolorization (%)

Enzy

me

activi

ty (IU

/mL)

r (Lac) = 0.986**

Fig. 4.36b: Relationship between dye decolorization and enzyme activities with varying concentrations of glycerol


210

carbon source and used in further optimaztion steps. Lactose was poor decolorizer as

compared to glucose and glycerol but better one than agro waste carbon sources like

wheat straw and wheat bran which are rich soure of cellulose.Wheat straw and wheat

bran exhibited more adsorption than dye degradation in this case.

In a subsequent experiment, effect of varying concentrations of Glycerol was also

investigated to choose its best concentration level. Results (Fig.4.36) showed that dye

decolorization increased with glycerol addition and peaked with 1.0% (same as in

previous experiment) as additional carbon supplement after 3 days of incubation..

Maximum laccase activity (455 IU/mL) was also noted in the culture supernatant from

the most decolorized medium.

There was also a point to ponder that maximum dye decolorization (96.71%)

occurred with in 3 days of incubation, rather than in 5 or 6 days as in case of previous

experiments suggesting tremendous stimulation of fungal growth and dye decolorization

by 1% glycerol with out any nitrogen source. Revankar and Lele (2007) also reported

maximum decolorization (96%) of recalcitrant dyes in 3 days by Ganoderma sp. after 8h in

optimized medium containing 2% starch and 0.125 % yeast extract. However, our results

are compareable with recent study of Asgher et al; 2008; in which the C. versicolor strain

showed better performance on Cibanon Blue GFJ-MD with 1% starch. Additional carbon

sources have also been utilised to enhance ligninase formation by WRF for achieving

maximum decolorization of a spectrum of dyes (Robinson et al., 2001; Selvam et al.,

2006; Sanghi et al., 2006; Hander and Arora, 2007).

Effect of Additional Nitrogen Sources

i) Effect of Different nitrogen sources

Nitrogen like carbon is as an essential element for most of the white rot fungi as

for rest of the the microorganisms. But its role as an activating or inhibiting agent

depends upon the nature of the fungal starin, medium for its growth and nature of

substrate.To check the role of nitrogen in dye decolorizing capability of C.versicolor

different kinds of nitrogen sources mainly organic ones were supplied. Dye

decolorization was strongly inhibited in presence of all nitrogen sources. Decolorization

was 40.04, 18.84, 14.47, 11.64 and 26.31% after 72 hours of incubation in Ammonium


211

Table 4.37 Decolorization of Cibanon blue GFJ-MD by C.versicolor and its lignolytic enzyme profile using different

nitrogen sources under optimisation conditions*

Nitrogen source (0.1%)


pH


on 3rd Day

1 2 3 4 Lac

0 58.55±1.26 66.41±1.23 88.88±1.49 90.01±1.90 a

4.73±10.06 d

407±15..41 a

Ammonium Oxalate 16.44±1.44 23.13±4.67 40.04±3.50 16.05±2.50 b

5.29 ±0.01 c

150±15.73 b

Corn Steep Liquor (CSL)

22.63±1.24 25.53±0.81 18.84±6.34 10.23±10.52 c

5.75±0.02 a

89±7.45 c

30% Maize Gluten Meal

24.06±1.36 19.84±1.28 14.47±1.01 11.25±0.23 c

5.67±0.02 ab

47 ±8.91 d

60% Maize Gluten Meal

27.78±2.06 16.7±1.11 11.64±0.63 6 09±0.23 d

4.75±0.45 a 33 ±1.30 e

Yeast Extract 16±0.04 22.51±1.37 26.31±0.90 17.48±0.88 b

5.61±0.04 b

92 ±1.77 c

* Medium II ; pH, 4 ; temperature (30±20C) ; Glycerole, 1%


212

0

50

100

150

200

250

300

350

400

450

0 A mmoniumOxalate

Corn SteepLiquor



Yeast Extract

Nitrogen Source (%)

Dec

olo

riza

tio

n (

%)

0

50

100

150

200

250

300

350

400

450

En

zym

e A

ctiv

ity

(IU

/mL

)

Decolorization%Lac

Fig.4.37a Effect of different nitrogen sources on decolorization of Cibanon Blue GFJ-MD by Coriolus versicolor and its lignolytic enyme production in 3 days

y = 9.4691x + 46.945

R2 = 0.3735

0

20

40

60

80

100

120

140

160

180

0 2 4 6 8 10 12 14

Decolorization (%)

Enzy

me

activi

ty (IU

/mL)

r (Lac) = 0.611*

Fig. 4.37a Relationship between dye decolorization and enzyme activities with different nitrogen sources.


213

oxalate, CSL, MGM 30%, MGM 60% and Yeast Extract respectively. Contolled medium

decolorized dye up to 88.88% with 407 IU/mL of laccase activities after 3 days. This

showed poor role of nitrogen source in the degradation of Cibanon Blue GFJ-MD. Drift

in pH was 4.29, 4.75, 4.67, 4.75, 4.61 in Ammonium oxalate, CSL, MGM 30% and

MGM 60% respectively (Table 4.37). Laccase activities were 150, 89, 47, 33 and 92

IU/mL in Ammonium oxalate, CSL, MGM 30%, MGM 60% and Yeast Extract

respectively. All nitrogen sources inhibitded laccase production and depressing level of

activities were observed in each case (Fig.4.37a).


are significant (p < 0.01). DMR test revealed that treatmeant means OF activities with

different nitrogen sources showed significant difference (Table 4.37). The sign of slope


activities.The value of R2 indicated that dye decolorization was caused by 37% variation

in laccase activities. R2 for laccase (0.37) clearly showed that dcolorization may best be

achieved through laccase synthesis. Laccase plays active role involved dye degradation

process.

WRF show variable growth response in nitrogen deficient and nitrogen sufficient

media and it also depends on the overall composition of the basal nutrient medium

(Robbinson et al., 2003). Previous study show that Nitrogen enriched media have been

found to cause better decolorization by Ganoderma lucidum with higher lignolytic

activities as compared to nitrogen deficient media (Kaal et al., 1995; D’Souza et al.,

1999; Revankar and Lele, 2007). However, addition of nitrogen source beyond optimum

level leads to poor dye decolorization (Radha et al., 2005). Ramsay et al. (2006), in

Amaranthy dye decolorization by T. vesicolor, used the nitrogen free Kirk’s medium with

1g/L glucose to achieve best performance by the fungal strain. In our research, the

alkaline Kirk,s basal medium already had 0.22% ammonium tartarate as nitrogen source.

(Kamei et al., 2006) also experienced better dye removal rates in nitrogen limited

medium.Induction of LME’s occurs during secondary metabolism, usually because of

nutrientb depletion especially nutrient nitrogen (Leung and Pointing, 2002).

The effect of nitrogen on laccase activity have remained a controversial issue and

its role differs from organism to organism (Breen and Singleton 1997). So opinion about

the definite role of nitrogen still needs to be discussed. All the nitrogen sources inhibited


214

enzyme induction and subsequently dye degradation. However, extent of inhibition was

quite dufferent in each source.MGM 30%, MGM 60% and yeast extract showed complete

inhibition while little actvation was found in case of ammoniumoxalate, and CSL.

Laccase activities were also either zero or negligible in all sources as compared to

nitroegen free medium.Lower laccase activites in nitrogen supplemented medium

confirmed the inhibition caused by N sources.Variable growth response of white rot fungi

in nitrogen rich and nitrogen sufficient medium depends upon the composition of basal

nutrient medium (Robinson et al., 2003). Nitrogen enriched media was found to give

better color removal by Ganoderma lucidum with higher lignolytic activities as compared

to nitrogen deficient media (Kaal et al., 1995; D’Souza et al., 1999). However, presence

of nitrogen beyond optimum level causes fast decline in dye decolorization (Radha et al.,

2005). Ramsay et al. (2006) used the nitrogen free Kirk’s medium; Revankar and Lele,

2007) with 1g/L glucose to achieve best Amaranthy dye decolorization by T. vesicolor. In

our case the alkaline Kirk,s basal medium already contained 0.22% ammonium tartarate

as nitrogen source (Kamei et al., 2006) also observed higher dye removal rates in

nitrogen limited medium.

Decrease in lignolytic activity due to nitrogen might be due to glutamate

metabolism and functions partly during RNA synthesis. On the otherhand, the stimulation

of lignolytic activity under nitrogen limited condition is caused by an increase of

intracellular c-AMP level, suggesting deeper modification in fungus physiology which

may characterize the transition between primary and secondary metabolism (Buswel and

odier 1987).

Effect of low molecular mass mediators

Dye decolorization level was increased by using various kinds of low molecular

mass mediators which catalyses decolorization. The redox mediators first reported by

Bourbonnais and Paice (1990) permits laccase to oxidize wide range of non–phenolic

compounds. Redox mediators are the compounds that enhance the rate of reaction by

taking up electron from the biological oxidation system to the electron accepting azo

dyes. Low molecular mass, diffusible mediators provide high redox potential.to attack

dye (Bourbainnais et al., 1995; Xu, 1996; Reyes et al., 1999; Call and Mucke, 1997;

Johannes and Ajcherczyk, 2000). The mediating capibilities of these compounds (ABTS,


215

H2O2, Tween-80, MnSO4 and Veratryl Alcohol) were tested by nmonitoring the

decolorizatioon of Cibanon Blue GFJ-MD for further process of optimization.

Decolorization noted was 98.91, 80.75, 93.76, 88.27 and 88.49 % after 48 hours

of incubation in the flasks containing ABTS, H2O2, Tween-80, MnSO4 and Veratryl

respectively (Table 4.38). Decolorization in controlled flasks was 86.61%. Drift in pH

was 6.07, 6.85, 6.79, 5.17 and 5.01. Laccase activities calculated after 48 hours were 512,

368, 378, 345 and 286 IU/mL ABTS, H2O2, Tween-80, MnSO4 and Veratryl Alcohol

respectively (Fig.4.38a).


are significants (p < 0.01). DMR test revealed that treatment means of activities with

different mediators shows significant difference (Table 4.38).

The sign of slope (Fig.4.38b) indicated that increase in decoloriztion was correlated

with the enzyme activities. The value of R2 indicated that dye decolorization was due to

39% variation in laccase activities. R2 for laccase (0.39) clearly shows that laccase

synthesis is positively correlated with the dye dcolorization. A mediator like ABTS acts

like a co-substrate which act as a diffusible lignin degrading agent. The prevalence of

mediator was supposed on basis of fact that purified laccase could not react directly with

intact fibrous cell wall. For this purpose another component was a prerequisite which

could play the role of oxidizing agents secreted by fungi (Been and Singleton, 1999).

In a studiy carried out by Romero et al. (2006) it was shown that decolorization of

Grey Lanaset could be obtained by fungus degradation or by adding laccase mediators to

enzymatic degradation. However, mediators usually available in the market are not useful

for pilot scale dye decolorization due to their mutagenic as wel as carsenogenic

character.In addition to this problem some mediators cause residual coloration of the

waste water. On such grounds for sustainable process for textile dye decolorization

fungal bioreactor process should be preferred.


216

Table-4.38 Decolorization of Cibanon blue GFJ-MD by C.versicolor and its lignolytic enzyme profile using using low

molecular mass mediators under optimimum conditions*

Mediators (1mM)



after 48 Hours

1 2 3 pH Laccase

0 57.92±1.23 86.61±1.34 89.96± 1.65 bc

4.78±0.06 c

409±14.56 b

ABTS 84.81±1.49 98.91±1.84 98.41±1.13 a

6.07±0.07 a

512 ±6.57 a

H2O2 72.56±2.09 80.75±2.27 87.9 ±2.04 c

6.85±0.01 a

368±18.10 c

Tween-80 76.41±2.56 93.76±3.15 90.56 ±0.75 b

6.79± 0.03 a

378±15.36 c

MnSO4 82.35±1.79 88.27±2.39 67.99±0.97 d

5.17±0.03 b

345±12.25 d

Veratryl Alchol 70.14±0.98 88.49±5.39 91.66±4.69 bc

5.01±0.05 b

286 ±5.05 e

* Medium II ; pH, 4 ; temperature (30±20C) ; Glycerole, 1%


217

0102030405060708090

100

0 ABTS H2O2 Tween-80 MnSO4 VeratrylaaAlcohol

Mediator (1mM)

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

700

800

En

zym

e A

ctiv

ity

(IU

/mL

)

Decolorization%

Lac

Fig.4.38a Effect of low molecular mass mediators on decolorization of Cibanon Blue GFJ-MD by Coriolus versicolor and its lignolytic enyme production in 2days

y = 12.217x - 99.535

R2 = 0.9402

100

300

500

700

900

1100

1300

20 30 40 50 60 70 80 90 100

Decolorization (%)

Enzy

me

acti

vity

(IU

/mL)

r (Lac) = 0.970**

Fig. 4.38b: Relationship between dye decolorization and enzyme activities with different nitrogen sources


218

Lower redox potential of laccase as compared to the non phenolic compound

makes it unit for their oxidation. However, it was observed that in the presence of small

molecule capable to act as electron transfer mediators laccase were also able to oxidise

nonphenolic compounds (Couto and Herrera, 2006).

Effect of different metal ions

Metal ions are considered to be an important group of enzymatic activity

modulators or enhancers and leave a positive impact on enzymatic activity and treatment

efficiency (Kim and Nicell; 2006). These can directly interact with extracellular enzymes

of white rot fungi. However, to investigate a physiological response, heavy metals must

be taken up by the fungus. The uptake of metal ions from liquid environment is the most

simple situation (Baldrian, P.2003). Due to the frequency of these metal ions in dye

dased waste waters and their potential capibilty to modulate enzyme activities, it is

necessary to investigate the their impact on laccase based treatment (Kim and Nicell

2006). Different metal ions play important role in fungal physiology. So metal ions

normally affecting microganisms include copper, manganese, calcium, zinc, cadmium

and iron were used in the form of their respective salts. As and Hg are found to be toxic

in most of the cases so these were avoided. To investigate the potential of C.versicolor

for dye color removal in presence of copper ions, triplicate flasks containing1mM of

CaCl2, CdNO3, CuSO4, FeSO4 and ZnSO4 were used and dye decolorization was 45.62,

87.82, 99.77, 60.77 and 65.04 % after 24 hours of incubation. Drift in after metal salts

addition was 5.70, 5.74, 5.27, 5.01, 4.87 and 5.16 (Table.4.39). Laccase activities

followed were 358, 568, 609, 427 and 490 IU/mL in CaCl2, Cd(NO3)2, FeSO4, MnSO4,

and ZnSO4 respectively (Fig. 4.39a). Controlled flasks decolorized dye up to 98.92% with

laccase activity as 482 IU/mL in 24 hours of incubation.


are significant (p < 0.01). The sign of slope (Fig. 4.39b) indicated that increase in

decoloriztion was corellated with the enzyme activities. R2 indicated that dye colo

removal was due to 50 % variation in laccase activities.R2 for laccase (0.50) clearly

shows that dcolorization was completely dependent on laccase synthesis.


219

Table.4.39 Decolorization of Cibanon blue GFJ-MD by C.versicolor and its lignolytic enzyme profile under optimum

conditions*

Metal Ions (1mM)


pH


1 2 Laccase

0 84.01±1.45 98.92±1.59 a

4.98±0.02 b

482± 14.25 c

CaCl2 45.62±6.03 45.36 ±3.04 e

5.70±0.05 a

358±9.22 e

Cd(NO3)2 87.82±2.93 84.88± 3.55 b

5.74±0.07 a

568±15.61 a

CuSO4 99.77±0.75 96.78 ±1.15 a

5.27±0.06 b

609±11.12 a

FeSO4 60.77±1.85 76.65±2.32 c

5.01±0.04 bc

427±15.74 d

ZnSO4 65.04±2.95 57.19 ±1.63 d

5.16±0.02 b

490±9.40 c

* Medium II ; pH, 4 ; temperature (30±20C) ; Glycerole, 1% ; Mediator, ABTS (1m M)


220

0

20

40

60

80

100

120


Metal Ion (1mM)

Dec

olo

riza

tio

n (

%)

0

100

200

300

400

500

600

700

800

900

1000

En

zym

e A

ctiv

ity

(IU

/mL

)

Decolorization%Lac

Fig.4.39a Effect of metal ions on decolorization of Cibanon Blue GFJ-MD by Coriolus versicolor and its lignolytic enyme production in 2 days

y = 13.101x - 159.38

R2 = 0.4986

100

300

500

700

900

1100

1300

1500

20 30 40 50 60 70 80 90 100

Decolorization (%)

Enzy

me

activi

ty (IU

/mL)

r (Lac) = 0.706*

Fig.4.39: Relationship between decolorization and enzyme activities with different metal ions


221

The addition of metal ions showed variable trend. The order of their efficiency in dye

degradation was as CuSO4 > Cd(NO3)2 > ZnSO4 > FeSO4 > CaCl2. Copper showed

maximum dye dcolorization and laccase activities. Our results supports the data in

literature where copper has been reported as strong laccase inducer in white rot fungi

(Collin and Dobsin 1997).The indegnous strain of C.versicolor worked wel in presence of

1mM CuSO4. Recent research by Tichanowicz etal; (2006) has proved that at this

concentration the laccase was 20% more active than in the absence of ccopper. However,

higher concentration of copper is toxic to microbial cell (Labbe and Thiele1997). When

the copper was increased to 3mM, complete inhibiton was noted in majority of the white

rot fungi (Guillen and Machuca, 2008).

Effect of varying concentrations of the dye

After optimization of different parameters, varying concentrations of the dye were

applied to investigate maximum fungal potential for dye decolorization or toxicity

tolerance exhibited by the fungal strain. Different concentrations applied included 0.01,

0.05, 0.1, 0.15, and 0.2%.The C.versicolor showed maximum decolorization (98.63%) in

medium recieving 0.01% dye concentration. There was gradual decline in decolorization

% with the increase in the dye concentration. So 98.88, 81.60, 42.80, 16.12 and 8.04 %

dye was degraded in media having 0.05, 0.10 0.15, 0.20 % dye respectively (Table 4.40).

pH change was 5.29, 5.40, 5.45, 5.58, 5.68 in flasks having 0.1, 0.5, 0.10, 0.15 and 0.2 %

respectively in culture supernatant having 0.01% dye. Laccase activities declined as the

dye concentration level increased. Since maximum decolorization was observed in 0.01%

dye solution so laccase showed its activity at peak level i.e.595 IU/ mL while110 IU/mL

lacase activity was found in 0.20 % dye solution which was minimum among the

Fig.4.40a. Due to toxicity and complex structure of the dye WRF show variable potential

for dye degradation. At low level of concentration level, fungus degraded wel.

Analysis of variance of the data showed that our results were significants (p <

0.01, Table 4.40). The sign of slope indicated that increase in decoloriztion was

correlated with the enzyme activities.The value of R2 indicated that dye decolorization

was due to 91% variation in laccase activitiy was due to decolorization. R2 for laccase

(0.91) clearly shows that dcolorization may mainly dependent on laccase synthesis.


222

Table 4.40 Decolorization of Cibanon blue GFJ-MD by C.versicolor and its lignolytic enzyme profile under optimum

conditions*




after 24 Hours 1 2 pH

0.01 99.12±0.20 98.88±0.63 a

5.29±0.02 b

595±13.44 a

0.05 0.67±1.10 81.60 ±1.49 a

5.40±0.02 b

480±13.54 b

0.01 44.43±2.05 42.80±3.23 b

5.45±0.03 b

345±12.22 c

0.15 16.16±1.82 16.12±2.30 c

5.58±0.02 a

223±14.26 d

0.2 8.11±2.83 8.04±2.09 d

5.68±0.03 a

110±15.42 e

* Medium II ; pH, 4 ; temperature (30±20C) ; Glucose, 1% ; Mediator, ABTS (1m M); Metal Ion, CuSO4 (1mM)


223

0

20

40

60

80

100

120

0.01 0.05 0.1 0.15 0.2


Dec

olo

riza

tio

n (

%)

01002003004005006007008009001000

En

zym

e A

ctiv

ity

(IU

/mL

)

Decolorization%Lac

Fig.4.40a. Effect of varying concentrations of dye on the decolorization of Cibanon Blue GFJ-MD and lignolytic enzymes production by C.versicolor

y = -86.494x + 1510.3

R2 = 0.9179

0

200

400

600

800

1000

1200

1400

1600

0 5 10 15 20

Decolorization (%)

Enzy

me

acti

vity

(IU

/mL)

r (Lac) = -0.272**

Fig. 4.40b: Relationship between dye decolorization and enzyme activities with varying concentrations of the dye


224

Maximum dye decolorization and negligblel dye adsorption was noted in meduium

receiving lowest dye concentration (0.01%). Howevr, dye decolorization decreased

gradually with increase in dye concentration. No considerable dye adsorption took place

on fungal mycelia. While with higher dye concentration, there was decline in dye

degradation and adsorption was slightly visble which disappered as the ligninases

enzymes increased.with the formation of fungal biomass.

Variable extent of dye decolorization (%) of toxic compounds was entirely due to

different chemical structures of toxic compounds like dyes tolerated by different WRF

cultures. In different previous studies (Kapdan et al., 2000; Nilsson et al., 2005; Rigas

and Dritsa, 2006; Eichlerova et al., 2006; Hai et al., 2006; Kariminiaae-Hamedaani et al.,

2007) different concentrations of a variety of dyes were used and lower concentrations

ranging from 50–500 mg/l were recorded to be best decolorized. It has been already

reported in previous study (Levin et al., 2004; Wang and Yu, 1998) that C. versicolor F.

antarcticuswas were able to decolorize in an hour 28, 30, 43, 88 and 98% of Xylidine (24

mg/l), Poly R-478 (75 mg/l) proving as a potential candidate for decolorizing a spectrum

of dyes, Malachite green (6 mg/l) and Indigo carmine (23 mg/l), produced by a 10 days

old fungal mycelium.

Effect of dye adsorption

Dye adsorption was investigated by applying different oncentrations. Despite

excellent potential of our starin, there was dye adsorption by its fungal mycelia.

Adsorption was directly propotional to the varying dye concentration. However, extent of

adsorption varied with repect to initial dye concentration. As the dye concentration

increased adsorption extent also increasd. Adsorption was 0.016, 0.024,0.14, 0.17 and

0.23 % in 0.01, 0.05, 0.10, 0.15 and 0.20 % respectively (Fig.4.41). Change in pH was

5.13, 5.04, 5.0, 5.0, 5.04 in 0.01 %, 0.05 %, 0.10 %, 0.15 %, and 0.20 % dye solutions

(Table 4.41).Analysis of variance of the data (Appendex-1 Table 4.41) showed that our

results were significant (p <0.01). DMR test showed that the means of tretmante values

were significant (p <0.01, Table 4.41).Dye adsorption and decolorization were inversely

proportional.Medium receiving higher dye concentration showed more adsorption fungal

mycelial pallets while with lower dye concentration there was negligible dye adsorption.

Increase in dye adsorption might be due to increase in toxicicity and enzymes inhibition.


225

Table. 4.41 Adsorption of Dye on fungal mycelia

Dye Concentration

(%)

Adsorption (% Mean± S.E) Incubation Time(Days)

Final pH

1 2

0.01 0.016±5.13 0.003±0.00

d 5.13±0.11

0.05 0.024±5.24 0.01±0.00

d 5.24±0.18

0.1 0.14±5.39 0.05±0.01

c 5.39±0.05

0.15 0.17±5.55 0.09±0.02

b 5.55±0.02

0.2 0.23±5.62 0.15±0.07a 5.62±0.01

0

0.5

1

0.01 0.05 0.1 0.15 0.2

Dye Concentration(%)

Ad

sorp

tio

no

n (

%)

Fig. 4.41 Adsorption of Cibanon Blue GFJ-MD on C.versicolor mycelia

226

Conclusion 1. WRF locally isolated cultures have best potential to decolorize textile dyes and dye

based effluents. Their capabilities can be enhanced by varying optimization

conditions keeping in view the kind and concentration of dyes.

2. The local WRF cultures worked well in pH range of 3.5 to 5 and at optimum

temperature from 30-35 0C.

3. All fungal cultures produced major ligninases enzymes (LiP, MnP, and laccase) but

their activities varied from each others. MnP and laccase were major enzymes

produced by C.versicolor in the reactive dye decolorization while Lip, MnP and

laccase were competitors in disperse dye degradation. P. ostreatus produced MnP and

laccase as major enzymes but role of MnP remained dominat. C.versicolor strain

synthesized laccase as the major enzyme which degraded vat dye.

4. Agrobased substrate used as a carbon source were effective in Foron Turquoise, and

Solar Golden Yellow R while glucose and glycerol were efficient in Remazole

Brilliant Yellow 3-GL and Vat dye decolorization.

5. Dye decolorization was enhaced in the presence of nitrogen souces while strong

inhibition was observed in vat dye using C.versicolor.

227

Recommendations 1. Mutant or genetically modified strains of White Rot Fungi should be exploited to

make sure rapid growth and hyper production of ligninases enzymes and dye

degradation.

2. Co-culturing is a good alternative for treating industrial effluents and the mixture of

dyes falling down in the outlets. There are many strains which work conducively in

the range of same optimum conditions.

3. Immobilied ligninases enzymes produced by WRF might be a good alternative. It will

reduce the cost of treatment technology as well as save plenty of time. For this

purpose three or four colums carrying suitable solid supports can be arranged in series

in such a manner that each column should contain one enzyme at a time and effluents

should pass through different columns

4. More research is needed to identify the new strains showing great potential for

cleaning up the textile dyes effluents sites containing complex mixtures.

5. Since all bioremediation projects require the use of the specific microorganisms under

the environmental conditions so prior to the implementation of a project, a

multidisciplinary approach to illustrating the hydrogeologic, geologic chemical, and

microbiological characteristics should be undertaken.

6. Despite limitations of bioremediation it is an ecofriendly technology. But it should be

coupled with other technology for making it attractive for industrialists.

7. Textile units and dyes manufacturing plants situated in the Faisalabad city must be

shifted away from the residential areas.

8. Make sure the NOC from EPA before the installation of new units.

9. Each dye manufacturing unit and textile factory must appoint a “Bioremedial

Technologist” to monitor the whole process on site.

228

Literature Cited

Abadulla, E., Tzanko, T., Costa, S., Robra, K.., Cavaco-Paulo A. and Gubitz. G. M. (2000).

Decolorization and Detoxification of Textile Dyes with a Laccase from Trametes hirsuta.

Applied and Environ. Microbiol. 66, 3357-3362.

Adosinda, M., Martins, M., Isabel, C., Ferreira, C., Isabel, M. and Santos, M. (2001).

Biodegradation of bioaccessible textile azo dyes by Phanerochaete chrysosporium. J

Biotechnol 89, 91-8.

Adosinda, M., Martins, M., Nelson, L., Armando, J. D. and Silvestre, M., Jo~ao Q. (2003).

Comparative studies of fungal degradation of single or mixed bioaccessible reactive azo

dyes. Chemosphere. 52, 967-973.

Ahmadi, M., Vahabzadeh F., Bonakdarpour, B, Mehranian, M, and Mofarrah, E. (2006).

Phenolic removal in olive oil mill wastewater using loofah-immobilized Phanerochaete

chrysosporium. World J. Microbiol. Biotechnol. 22, 119–127.

Ahmadi, M., Vahabzadeh, F., Bonakdarpour, B., Mofarrah, E. and Mehranian, M. (2005).

Application of the central composite design and response surface methodology to the

advanced treatment of olive oil processing wastewater usingFenton’s peroxidation. J.

Hazard. Mater. 123, 187-195.

Aken, B. V. and Agathos, S. N. (2001). Biodegradation of Nitro-Substituted Explosives by

White Rot Fungi:AmECHANISTIC Approach. Adv. in Appl. Microbiol. 48, 1-77.

Aksu, Z., Kilic, N. K., Ertugrul, S. and Donmez, G. (2007b). Inhibitory effects of chromium (VI)

and Remazol Black B on chromium (VI) and dyestuff removals by Trametes versicolor.

Enzyme Microb. Technol. 40, 1167-74.

229

Alessandra, Z. D. S., Neto, J. M. C., Tavares, C. R. G. and Costa, S. M. G. D. (2004). Screening

of filamentous fungi for the decolourization of a commercial reactive dye. Basic Microb.

44, 288-295.

Alfred, M. M. and Richard, C. S. (2002). Review : Laccase: new functions for an old enzyme.

Phytochem. 60, 551-565.

Aranda, E., Sampedro, I., Ocampo, J. A. and Garcı´a-Romera, I. (2006). Phenolic removal of

olive-mill dry residues by laccase activity of white-rot fungi and its impact on tomato

plant growth. Int. Biodet. Biodeg. 58, 176-179.

Asgher, M., Asad, M. J., Bhatti, H. N. and Legge, R. L. (2007). Hyperactivation and

thermostabilization of Phanerochaete chrysosporium lignin peroxidase by

immobilization inxerogels. World J. Microbiol. Biotechnol. 23, 525-531.

Asgher, M., Kausar, S., Bhatti, H. N., Shah, S. A. H. and Ali, M. (2008). Optimization of

medium for decolorization of Solar golden yellow R direct textile dye by Schizophyllum

commune IBL-06. Int. Biodet. Biodeg. 61, 189-193.

Asgher, M., Kausar, S., Bhatti, H. N., Shah, S. A. H. and Ali, M. (2008). Optimization of

medium for decolorization of Solar golden yellow R direct textile dye by Schizophyllum

commune IBL-06. Int. Biodet. Biodeg. 61, 189-193.

Asgher, M., Shah, S. A. H., Ali, M. and Legge, R. L. (2006). Decolorization of some reactive

textile dyes by white rot fungi isolated in Pakistan. World J. Microbiol.Bioiotechnol. 22,

89-93.

Asther, M., Capdevila., and Corrien, G, (1988). Control of liqnin pesesidase production by

phanerochaete Chrysosperium, INA-12 by temperature shifting Appl. Environ Microbiol.

Atagana, H.I., Ejechi, B. O. and Ayilumo, A. M. (1999). Fungi associated with degradation of

wastes from rubber processing industry. Environ. Monit. Assess. 55, 401-408.

Azmi, W., Sani, R. K. and Banerjee, U. C. (1998). Biodegradation of triphenylmethane dyes.

Enzyme Microbiol. Technol. 22,185-191.

230

Baborova, P., Moder, M., Baldrian, P., Cajthamlova, K., and Cajthaml, T. (2006). Purification of

a new manganese peroxidase of the white-rot fungus Irpex lacteus and degradation of

polycyclic aromatic hydrocarbons by the enzyme. Res. Microbiol. 157, 248–253

Balan, D. S. L., Monteiro, R. T. R. (2001). Decolorization of textile indigo dye by ligninolytic

fungi. J. Biotechnol. 89, 141–145.

Baldrian, P. (2003). Interactions of heavy metals with white-rot fungi. Enzyme Microb. Technol.

32, 78-91.

Baldrian, P. and Gabriel, J. (1997). Effect of heavy metals on the growth of selected wood-

rotting basidiomycetes. Folia Microbiol. 42, 521-3.

Baldrian, P. and Gabriel, J. (2002). Copper and cadmium increase laccase activity in Pleurotus

ostreatus. FEMS Microbiol. Lett. 206, 69-74.

Banat, I. M., Nigam, P., Singh, D. and Marchant, R. (1996). Microbial decolorization of textile-

dye-containing effluents: a review. Bioresour. Technol. 58, 217-227.

Barr, D. P. and Aust, S. D. (1994) .Mechanisms white rot fungi use to degrade pollutants.

Environ. Sci. Technol. 28, 78–87.

Bioremediation of textile azo dyes by Trichophyton rubrum LSK-27.World J. Microbiol. &

Biotechnol. 22, 1027-1031. biotreatment of new wastewater: a review. Afr. J. Biotechnol.

2, 620–635.

Bl!anquez, P., Casas, N., Font, X., Gabarrell, X., Sarr M. and Caminal, T. G. (2004).

VicentMechanism of textile metal dye biotransformation by Trametes versicolor. Water

Research. 38, 2166-2172.

Bllanquez, P., Blanquez, M. and Sarra`T. V. (2008). Development of a continuous process to

adapt the textile wastewater treatment by fungi to industrial conditions Process Biochem.

43, 1-7.

231

Boer, C. G., Obici, L., de Souza, C. G. M. and Peralta, R. M. (2004). Decolorization of synthetic

dyes by solid state cultures of Lentinula (Lentinus) edodes producing manganese

peroxidase as the main ligninolyticenzyme. Biores. Technol. 94, 107-112.

Bonnarme, P. and Jeffries, T. W. (1990). Appl. Environ. Microbiol. 56, 210.

Boominathan, K. and Reddy, C. A. (1992). Fungal degradation of lignin: biotechnological

applications. In Handbook of applied mycology. 4, 763-782.

Bourbonnais, R., Paice, M. G., Reid, I. D., Lanthier, P. and Yaguchi, M. (1995). Lignin

oxidation by laccase isozymes from Trametes versicolor and role of the mediator 2,20-

azino-bis(ethylbenzothiazoline-6-sulfonic acid) in Kraft lignin depolimerization. Appl.

Environ. Microbiol. 61, 1876-1880.

Bourbbonnais , R. and Paice, M. G. (1990). Oxidation of non-phenolic substrates. An expended role for laccase in lignin degradation. FEBS. Lett. 267, 99-102.

Bumpus,J.A., (1989).Biodegradation of polycyclic aromatic hydrocarbons by Phanerochaete

chrysosporium. Appl. Environ. Microbiol. 55, 154-158. Brandi,p., Aleesandro., D. A., Carlo, G., Patrizia, G., and Ana, S. N. P. (2006). In serach for

practical advantages from the immobilisation of an enzyme:the case of laccase. (2006). J. Molec. Cat.B. Enzymatic. 41, 61-69.

Bredberg, K., Andersson, B.E., Landfors, E. and Holst, O. (2002). Microbial. of waste rubber

material by wood-rotting fungi. Biores. Technol. 83, 21–224.

Bredberg, K., Persson, J., Christiansson, M., Stenberg, B. and Holst, O. (2001). Anaerobic

desulfurization of ground rubber with the thermophilic archaeon Pyrococcus furiosus-a

new method for rubber recycling. Appl. Microbiol. Biotechnol. 55, 43–48.

Breen, A. and Singleton,. F. L. (1999). Fungi in lignocellulose breakdown and biopulping. Curr.

Opin. Biotechnol. 10, 252-258.

Brown, A., (1985). Review of lignin in biomass. J.Appl. Biochem. 7, 371-387.

Buswell, J. A. and Odier, E. (1987). Lignin biodegradation. Crit Rev. Biotechnol. 6, 1-60.

Cadimaliev, D.A., Revin, V. V., Atykyan, N. A. and Samuilov, V. D. (2005). Extracellular

oxidases of the lignin-degrading fungus Panus tigrinus. Biochem. 70, 703-707.

232

Call, H. P. and Muecke, I. (1997). J. Biotechnol. 53, 163.

Camarero, Sarkar, S., Ruiz-Duenas, F. J., Martinez, M. J. and Martinez, A. T. (1999).

J.Biolo.Chem. 274,10324-103330.

Campos, R., Kandelbauer, A., Robra, K. H., Cavaco-Paulo, A. and Gubitz, G. M. (2001). Indigo

degradation with purified laccases from Trametes hirsuta and Sclerotium rolfsii. J

Biotechnol. 89, 131- 9.

Chagas, E. P. and Durrant, L. R. (2001) Decolorization of azo dyes by Phanerochaete

chrysosporium and Pleurotus sajorcaju. Enzyme Microbiol. Technol. 29, 473-77.

Chairattanamanokrn, P., Imai, T., Kondo, R., Ukita, M. And Presertasan, P. (2006). Screening

thermotolerant fungi for decolorisation of waste wters.Applied Biochem Biotechnol. 128,

195-204.

Chander, M. and Arora, D. S. (2007). Evaluation of some white-rot fungi for their potential to

decolourise industrial dyes. Dyes and Pigments 72, 192-198.

Chander, M., Arora D. S. and Bath. H. K. (2004). Biodecolourisation of some industrial dyes by

white rot fungi. J. of Industrial Microbiol. and Biotechnol. 31, 94-97.

Chang, H.C., and Bumpus, J.A., (2001). Inhibition of lignin peroxidasemediated oxidation

activity by ethylenediamine tetraaceticacid and N-N-N0-N0-tetramethylenediamine.

Proc. Natl. Sci .Counc. Rep. China Part B Life Sci. 25, 26–33.

Cheng, X-B., Jia, R, Lip, S., Zhu, Q., Tu, S-Q., Tang, W-Z;(2007).Studies on the properties and

co-immobilization of manganese peroxidase. Chin. J. Biotechnol. 23, 90–96.

Chivukula, M. and Renganathan, V. (1995). Phenolic azo dye oxidation by laccase from

Pyricularia oryzae. Appl. Environ. Microbiol. 61, 4374-7.

Cho, N. S., Jarosa-Wilkolazka, A., Luterek, J., Cho, H. Y., Ohga, S. And Leonowicz, A. (2006).

Effect of fungal laccase and low molecular weight mediators on decolorization of Direct

Blue dye. J. Fac. Agric. Kyushu Univ. 51, 219-225.

233

Christian, V. And Vyas, B. R. M. (2005). Enzymatic decolorization of sulfonphthalein dyes.

Enzyme Microbiol. Technol. 36, 333-7.

Christiansson, M., Stenberg, B., Wallenberg, L. R. and Holst, O. (1998). Reduction of surface

sulfur upon microbial devulcanization of rubber materials. Biotechnol Lett 20, 637–642.

Chung, K. T., and Stevens, S. E. (19930. Decolorization of azo dyes environmental Cing, S., and

D. Asma, E. A. and O. Yeslida. (2003) Different approaches to improving the textile dye

degradation capacity of Trametes versicolor. 48, 639-642.

Cing, S., Asma, D., Apophan, E. and Yeslida, O. (2003). Department of Biology, Science and

Art Faculty, Inonu University, 44069. Malatya, Turkey. Folia Microb. (Praha). 48, 639-

642.

Ciullini, I., Tilli, S., Scozzafava, A. and Briganti, F. (2008). Fungal laccase, cellobiose

dehydrogenase, and chemical mediators: combined actions for the decolorization of

different classes of textile dyes. Biores. Technol. 99, 7003-7010.

Cloete, T. .E. and Celliers, L. (1999). Removal of Aroclor 1254 by the white rot fungus Coriolus

versicolor in the presence of different concentrations of Mn(IV) oxide. International

Biodeterioration and Biodegradation. 44, 243-253.

Collins, P. J. and Dobson, A. D. W. (1997). Regulation of laccase gene transcription in Trametes

versicolor. Appl. Environ. Microbiol. 63, 3444-50.

Couto, S. R. and Sanroman, M. A. (2005). Coconut flesh: a novel raw material for laccase

production by Trametes hirsuta under solid-state conditions. Application to Lissamine

Green B decolourization. J Food Eng. 71, 208-13.

Couto, S. R. and Toca-Herrera, J. L. (2007). Laccase production at reactor scale by filamentous

fungi. Biotechnol. Adv. 25, 558-69.

Couto, S. R., Rivela, I., Munoz, M. R. and Sanroman, A. (2000) Stimulation of ligninolytic

enzyme production and the ability to decolorize Poly R-478 in semisolid-state cultures of

Phanerochaete chrysosporium. Biores. Technol. 74, 159-164.

234

Couto, S. R., Rosales, E. and Sanroman, M. A. (2006). Decolorisation of synthetic dyes by

Trametes hirsute in expended-bedreactors. Chemosphere. 62, 1558-1563.

Crchcley, J.h. (1994). Removal of dye waste color from sewage effluents. The use of a

full scale ozone plant. Water, Sci. Tech.30, 275-284.

Cripps, C., Bumpus, J. A. and Aust, S. D. (1990). Biodegradation of azo and heterocyclic dyes

by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 56, 1114–1118.

Cullen, D. and Kersten, P. (1992). Fungal enzymes for lignocellulose degradation. In: Kinghorn

J.R. Turner G. Glasgow (eds) Applied molecular genetics of filamentous fungi. Blackie

Academic and Professional, Chapman & Hall, pp 100–l31.

D,Souza, D. T., Ashutosh, K. V., Merill, M. and Chandralata, R. (2006). Effect of nutrient

nitrogen on laccase production, its isoenzyme patterns and effluent decolorisation,by the

fungus NIOCC=2A isolated from mangoove wood. Indian journal Of marine sciences.35,

364-372.

D’Souza, D. T., Rakesh, T., Awdhesh, Kumar, S. And Chandralata, R. (2006). Enhanced

Production of Laccase by a marine fungus during treatment of colored effluents and

synthetic dyes.Enzyme and Microb. Technol. 38, 504-511.

D’Souza, T. M., Merritt, C. S. and Reddy, C. A. (1999). Lignin-modifying enzymes of the white

rot basidiomycete Ganoderma lucidum. Appl. Environ. Microbiol. 65, 307-313.

D’Annibale, A., Quaratino, D., Federici, F. and Fenice, M. (2006). Effect of agitation and

aeration on the reduction of pollutant load of olive mill wastewater by the white-rot

fungus Panus tigrinus. Biochem. Eng. J. 29, 243-249.

de Jong, E., Field, J. A. De Bont, J. A. M. (1994). Aryl alcohols in the physiology of ligninolytic

fungi. FEMS Microbiol. Rev. 13, 153-88.

Dhouib, A., Ellouz, M., Aloui, F. and Sayadi, S. (2006). Effect of bioaugmentation of activated

sludge on olive mill wastewater detoxification with white-rot fungi. Lett. Appl.

Microbiol. 42, 405-411.

235

Dittmer, J. K., Patel, N. J., Dhawale, S. W. and Dhawale, S. S. (1997). Production of multiple

laccase isoforms by Phanerochaete chrysosporium grown under nutrient sufficiency.

FEMS Microbiol Lett. 149, 65-70.

Dodson, P. J. Evans, P. J. and Palmer, J. M. (1987). Production and properties of an extracellular

peroxidase from Coriolus versicolor which catalyses Cα-C β cleavage in a lignin model

compound..FEMS microbial. Lett. 42, 17-22.

Driessel, B. V. and Christov, L. (2001). Decolorization of bleach plant ffluent by mucoralean and

white-rot fungi in a rotating biological contactor reactor. J Biosci. Bioeng. 92, 271-276.

Eaton, R. A. and Hale, M. D. C. (1993) Wood: decay, pests and protection. Chapman and Hall,

London.

Echlerova, I., Homolka, L., Laisa, L. And Nerud, F. (2005) Orange G, AND Remazole Brilliant

Ble R decolorisation of by white rot fungi Dichomitus squalens, Ishnoderma rosinum,

and Pleurotus calyptratus. Chemosphere. 60, 398-404.

Eichlerova, I., Homolka, L. and Nerud, F. (2006). Evaluation of synthetic dye decolorization

capacity in Ischnoderma resinosum. Journal of Industrial Microbiology

(33), 759–766.

Echlerova, I., Ladislav, H. and Frantisek, N. (2006). Synthetic dye decolorisation capacity of

white rot fungus Dichmitus Squalens. Biores. Technol. 97, 2153-2159.

Eggert, C., Temp, U. and Eriksson, K. E. L. (1996) The ligninolytic system of the white rot

fungus Pycnoporus cinnabarinus: purification and characterization of the laccase. Appl.

Environ. Microbiol. 62, 1151-1158.

Eichlerová, I; Ladislav, H, Ludmila, L. and František, N. (2005). Orange G and Remazol

Brilliant Blue R decolorization by white rot fungi Dichomitus squalens, Ischnoderma

resinosum and Pleurotus calyptratus. Chemo. 60, 398-404.

Erkurt, E. A., Unyayar, A. and Kumbur, H. (2007). Decolorization of synthetic dyes by white rot

fungi,involving laccase enzyme in the process. Process Biochem. 42, 1429-35.

236

Fang, H., Wenrong, Hu. and Yuezhong, Li. (2004). Biodegradation mechanisms and kinetics of

azo dye 4BSby a microbial consortium. Chemosphere. 57, 293-301.

Farnet, A. M., Criquet, S., Cigna, M., Gil, G. and Ferré, E. (2004). Purification of a laccase from

Marasmius quercophilus induced with ferulic acid: reactivity towards natural and

xenobiotic aromatic compounds. Enzyme Microb. Technol. 34, 549-554.

Farnet, A. M., Tagger, S. and Lepetit, J. (1999). Effects of copper and aromatic inducers on the

laccases of the white-rot fungus Marasmius quercophilus. C R L Acad Sci Ser III, Sci La

Vie, Life. 322, 499-503.

Feijoo, G., Dosoretz, C. and Lema, J. M. (1994).Production of lignin peroxidase from

Phanerochaete chrysosporium in packed bed bioreactors with recycling. Biotechnol.

Tech. 8, 365-8.

Fewson, C. A. (1988). Biodegradation of xenobiotics nd other persistant compounds:the causes

of recalcitrantance.Trends Biotechnol. 6, 148-153.

Field, J. A., De Long, E., Feijo, C. G. and De Bont, J. A. M.. (1993). Screening for lignolytic

fungi applicable to the biodegradation of xenobiotics. Trends Biotechnol. 11, 44-9.

Font, X., Caminal, G., Gabarrell, X. And Vicent. T. (2006). Treatment of toxic industrial

wastewater in fluidized and fixed-bed batch reactors with Trametes versicolor: influence

of immobilization. Environ. Technol. 27, 845-854.

Frijters, C. T., Vos, H. Scheffer, G. and Mulder, R. (2006). Decolorizing and detoxifying textile wastewater,

containing both soluble and insoluble dyes in a full scale combined anaerobic/aerobic system.

Water. Res., 40, 1249-1257.

Fu, Y. and Viraraghavan, T. (2001). Fungal decolorization of dye wastewater: a review. Biores.

Technol. 79, 251-262.

Gabriel, J., Kofronova, O., Rychlovsky, P. and Krenzelok, M. (1996). Accumulation and effect

of cadmium in the wood-rotting basidiomycete Daedalea quercina. Bull. Environ.

Contam. Toxicol. 57, 383-390.

237

Gadd, G. M. (2001). Fungi in Bioremediation. Cambridge University Press.

Galhaup, C., Wagner, H., Hinterstoisser, B. and Haltrich, D. (2002). Increased production of

laccase by the wood degrading basidiomycetes Tramete pubescens. Enzyme and Microb.

Technol. 30, 529-536.

Gao, D. W., Wen, X. H. and Qian. Y.2006. Decolourization of reactive brilliant red K-2BP by

white rot fungus under sterile and non-sterile conditions. Journal of Environmental

Sciences, 18, 428-432.

Garcia, I. G., Pena. P. R. J., Venceslada, J. L. B., Martin, A. M., Santos, M. A. M. and Gomez, E.

R. (2004). Removal of phenol compounds from olive mill wastewater using

Phanerochaete chrysosporium,Aspergillus niger, Aspergillus terreus and Geotrichum

candidum. Proc. Biochem. 35, 751-758.

Gavril, M. and Hodson, P.V. (2007). Chemical evidence for the mechanism of the

biodecoloration of Amaranth by Trametes versicolor. World J. Microbiol. Biotechnol. 23,

103-124.

Ge, Y., Yan, L. and Qinge, K. (2004). Effect of environment factors on dye decolourization by P.

sordida ATCC90872 in a aerated reactor. Process. Biochem. 39, 1401-5.

Georgiou, D., Hatiras, J. And Aivasidis, A. (2005). Microbial immobilization in a twostage

fixed-bed-reactor pilot plant for on-site anaerobic decolorization of textile wastewater,


Gianfreda, L., Xu, F. and Bollag, J. M. (1999). Laccases: a useful group of oxidoreductive

enzymes. Bioremediat J. 3, 1-26.

Gill, P. K.,. Arora, D. S. and Chander, M. (2002). Biodecolourization of azo and

triphenylmethane dyes by Dichomitus squalens and Phlebia spp. Journal of Industrial

Microbiol. & Biotechno. 28, 201-203.

238

Glenn, J. K. and Gold, M. H. (1983). Decolorization of several polymeric dyes by the lignin-

degrading basidiomycete Phanerochaete chrysosporium. Appl. Environ. Microbiol. 45,

1741-1747.

Glenn, J. K. and Gold, M. H. (1985). Purification and characterization of an extracellular Mn(II)-

dependent peroxidase from the lignin degrading basidiomycete Phanerochaete

chrysosporium. Arch. Biochem. Biophys. 242, 329-341.

Gold, M.H and Alic, M. (1993). Molecular biology of the lignindegrading basidiomycete

Phanerochaete chrysosporium.Microbiol. Rev. 57, 605–622.

Gonza´lez, T., Terron, M. C., Yague, S., Junca, H., Carbajo, J. M., Zapico, E. J., Silva, R.,

Arana-Cuenca, A., Tellez, A. and Gonzalez, A. E. (2007). Melanoidin-containing

wastewaters induce selective laccase gene expression in the white rot fungu.

Grabski,, A.C, Grimek, H. J. Burgess P.R. (1998). Immobilisation of manganese peroxidase

from lentinula aedodes, nd its biocatalytic generation of MnIII-chelate as a chemical

oxidant of chlorophenols. Biotechnol. Bioeng. 60, 204 -15

Guille´n , Y. and Angela, M. (2008). The effect of copper on the growth of wood-rotting fungi

and a blue-stain fungus. World J. of Microbiol. Biotechnol. 24, 31–37.

Gulnaz,O., Asyenur, K. and Sadik, D. (2006). The use of dried activated sludge for adsorption of reactive dye. J. Hazard.Mat. B. 134, 190-196.

Ha, H. C., Honda, Y., Watanabe, T. and Kuwahara, M. (2001). Production of manganese

peroxidase by pellet culture of the lignindegrading basidiomycete, Pleurotus ostreatus.

Appl. Microbiol. Biotechnol. 55, 704-11.

Hafiz, I., Muhammad, A. and Haq, N. B. (). Optimization of Cibacron Turquoise P-GR

Decolorization by Ganoderma lucidum IBL-05.

Hai, F. I., Fukushi, K. and Yamamoto, K.(2003). Treatment of textile wastewater: Membrane

bioreactor with special dye-degrading microorganism. Proc. Asian Waterqual, Bangkok,

Thailand.

Hai, F. I., Yamamoto, K. and Fukushi, K. (2006). Development of a submerged membrane fungi

reactor for textile wastewater treatment. Desalination. 192, 315-322.

239

Hakala, T. K., Hilde´n, K., Maijala ,P., Olsson, C. and Hatakka, A. (2006). Differential

regulation of manganese peroxidases and characterization of two variable MnP encoding

genes in the white-rot fungus Physisporinus rivulosus. Appl. Microbiol. Biotechnol. 73,

839–849.

Hakala, T. K., Lundell, T., Galkin, S., Maijala, P., Kalkkinen, S. and Hatakka, A. (2005).

Manganese peroxidases, laccase and oxalic acid from the selective white-rot fungus

Physisporinus rivulosus grown on spruce wood chips. Enzyme Microb. Technol. 36,

461–468.

Hamedani, H. K., Akihiko, S. and Mikio, S. (2007). Decolorization of synthetic dyes by a newly

manganese peroxidase-producing white rot fungus. Dyes and Pigments.72, 157-162.

Harvey, P. J., Schoemaker, H. E. and Palmer, J. M. (1986). Veratryl alcohol as a mediator and

the role of radical cations in lignin biodegradation by Phanerochaete chrysosporium.

FEBS Lett. 195, 242-246.

Hatakka A (2001) Biodegradation of lignin. In: Steinbu¨chel A,Hofrichter M (eds) Biopolymers.

Lignin, humic substances, and coal, vol 1. Wiley-VCH, Weinheim, pp 129–180.

Hatakka, A. (1994). Lignin modifying enzymes from selected white-rot . . fungi: production and

role in lignin degradation. FEMS Microbiol 13, 125-135.

Hatvani, N. and Mecs, I. (2002). Effect of nutrient composition on dye decolorisation and

extracellular enzyme production by Lentinus edodes on solid medium. Enzyme

Microbiol. Technol. 30, 381-386.

Heinfling, A., Martinez, M. J., Martinez, A. T., Bergbauer, M. and Szewzyk, U. (1998).

Transformation of industrial dyes by manganese peroxidases from Bjerkandera adusta

and Pleurotus eryngii in a manganese-independent reaction. Appl. Environ. Microbiol.

64, 2788–2793.

Heinfling, A., Bergbauer, M. and Szewzyk, U. (1997). Biodegradation of azo and phthalocyanine

dyes by Trametes versicolor and Bjerkandera adusta. Appl. Environ. Microbiol. 48, 261-

266.

240

Higuchi, T. (1993). Biodegradation mechanism of lignin by white-rot basidiomycetes.

Biolechnol, 30, 1-8.

Hirai, H., Sugiura, M., Kawai, S. and Nishida, T. (2005). Characteristics of novel lignin

peroxidases produced by white-rot fungus Phanerochaete sordida YK-624. FEMS

Microbiol. Lett. 246, 19-24.

Horfrichter, M., Scheibner, k., Scheeengab, I., Fritsche, W. (1998).Enzymatic combination of

aromatic and aliphatic compounds by manganese peoxidase from Nematoloma forwardii

Appl. Environ.Microbiol. 64, 399-404.

Hofrichter, M. (2002). Review: lignin conversion by manganese peroxidase (MnP). Enzyme

Microb. Technol. 30, 454-566.

Honda,Y. and Watanabe, T. (2006). Exclusive overexpression and structure-function analysis of

a versatile peroxidase from white-rot fungus, Pleurotus ostreatus. Sustainable

Humanosphere 2, 2–6.

Hublik.G.,F. Schinner 2000. Characteriztion and immobilization of laccase from. Pleurotus.

Ostereatus andits use for the continuous elimination of phenolic pollutants. Enzyme and

Microbial Technalogy.27:330-336.

Hughes, M. N. and Poole, R. K. (l991). Metal speciation and microbial growth—the hard (and

soft) facts. J Gen Microbiol. 137, 725-34.

Jia, J., Yang, J., Liao, J.and Wang, Z. (1999). Treatment of dyeing waste water, with AFC electrodes. Water, Rees. 22, 881-884.

Johansson, T. and Nyman, P. (1992). Isozymes of lignin peroxidase and manganese (II) from the

wghite rot basidiomycetes Trametes versicolor -- peroxidase. Isolation of enzyme forms

and and characterization of physical and catalytic properties. Arch. Biochem. Biophys.

300, 49-56.

Jolivalt, C., Neuville, L., Boyer, F. D., Kerhoas, L. and Mougin, C. (2006). Identification and

formation pathway of laccase-mediated oxidation products formed from

hydroxyphenylureas. Journal of Agricultural and Food Chem. 54, 5046-5054.

241

Junghanns, C.,Gudrun, K. and Deitmar, S. (2008). Potential of Aquatic fungi derived from

diverse freshwater environment to decolourise synthetic azo and anthraquinone dyes.

Biores. Technol. 99,125-1235.

Kaal, E. E. J., Field, J. E. and Joyce, T. W. (1995). Increasing ligninolytic enzyme activation in

several white rot basidiomycetes by nitrogen sufficient media. Biores. Technol. 53, 133-

139.

Kalmis, E. and Sargin, S. (2004). Cultivation of two Pleurotus species on wheat straw substrates

containing olive mill wastewater. Int. Biodet. Biodeg. 53, 43–47.

Kamei, I., Kogura, R. and Kondo, R. (2006). Metabolism of 4,4/-dichlorobiphenyl by white-rot

fungi Phanerochaete chrysosporium and Phanerochaete sp. MZ142. Appl. Microbiol.

and Biotechnol. 72, 566-575.

Kamitsuji, H., Honda, Y., Watanabe, T. and Kuwahara, M. (2004a).Production and induction of

manganese peroxidase isozymes in a white rot fungus Pleurotus ostreatus.

Appl.Microbiol. Biotechnol. 65, 287-294.

Kamitsuji, H., Honda, Y., Watanabe, T. and Kuwahara, M. (2005a). Direct oxidation of

polymeric substrates by multifunctional manganese peroxidase isozyme from Pleurotus

ostreatus without redox mediators. Biochem. J. 386, 387-393.

Kandelbauer, A., Maute, O., Kessler, R. W., Erlacher, A. and Gubitz, G. M. (2004). Study of dye

decolorization in an immobilized laccase enzyme-reactor using online spectroscopy.

Biotechnol. Bioeng. 87, 552-63.

Kapdan, I. K. and Kargi, F. (2002). Biological decolorization of textile dyestuff containing waste

water by Coriolus versicolor in a rotating biological contractor. Enzyme and Microb.

Technol. 30, 195-199.

Kapdan, I. K., Kargi, F., McMullan, G. and Marchant, R. (2000). Comparison of white rotfungi

cultures for decolorization of textile dyestuffs. Bioprocess Eng. 22, 347-351.

Kapdan, I.K., Fikret, K., 2002. Simultaneous biodegradation and adsorption of textile dyestuff in

an activated sludge unit. Process Biochemistry 37, 973-981.

242

Kapich, A. N., Prior, B. A., Botha, A., galkin, S. L. T. and Hattaka, A. (2004). Effects of

lignocellulose containing substrate on production of lignolytic peroxidases in submerged

cultures of Phanerochaete Chrysosporium ME-446. Enzyme and Microb. Technol.

34,187-195.

Kariminiaae-Hamedaani, H.R., Sakurai, A. and Sakakibara, M. ( 2007). Decolorization of

synthetic dyes by a new manganese peroxidase-producing white rot fungus.Dyes and

Pigments. 72, 157–162.

Karahanian, E., Corsini, G., Lobos, S. and Vicuna, R. (1998). Structure and expression of a

laccase gene from the ligninolytic basidiomycete Ceriporiopsis subvermispora. Biochim

Biophys Acta. 1443, 65-74.

Kaushik, P. and Anushree, M.(2009). Fungal dye decolourization: Recent advances and future

potential. Environ. Intern. 35, 127–141.

Kim, S. J. and Shoda, M. (1999). Decolorization of molasses and a dye by a newly isolated strain

of the fungus Geotrichum andidum Dec. 1. Biotechnol Bioeng. 62, 114-119.

Kim, Y-J. and James A. N. (2006). Impact of reaction conditions on the catalysed conversion od

bisphenol A. b Biores.Technol. 97, 1431-1442.

Kirby, N., Marchant, R. and McMullan, G. (2000). Decolourisation of synthetic textile dyes by

Phlebia tremelflosa. FEMS Microbiol. Letters. 188, 93-96.

Kirk, T. K. and Farrell, R. L. (1987). Enzymatic ‘‘combustion’’: the microbial degradation of

lignin. Ann. Rev. Microbiol. 41, 465-505.

Knap, J. S; Zhang, F. and Tapely, N. (1997). Decolorization of Orange II y a wood rotting

fungus. J. Chem. Technol. and Biotechnol. 69, 289-296.

Knapp, J. S., Newby, P. S. and Reece, L. P. (1995). Decolorization of dyes by wood-rotting

basidiomycete fungi. Enzyme Microbiol. Technol. 17, 664-668.

Harazono, K., and Kazunori, N. (2005). Decolorization of mixtures of different reactive textile

dyes by the white-rot basidiomycete Phanerochaete sordida and inhibitory effect of

polyvinyl alcohol Chemosphere, 59, 63-68.

243

Harazono, K., Yoshio, W. and Kazunori, N. (2003). Decolorization of mixtures of different

reactive textile dyes by the white-rot basidiomycete hanerochaete sordida and inhibitory

effect of polyvinyl alcoholChemosphere. 59, 63-68.

Kokol, V; Aleˇs, D. Ivana, E; Petr, B; Frantiˇsek, N. (2007). Decolorization of textile dyes by

whole cultures of Ischnoderma resinosumand by purified laccase and Mn-peroxidase

Enzyme and Microbial Technology. 40, 1673–1677.

Koray, S., Yesiladalý, G., Pekin1, H., Bermek1, I., Arslan-Alaton, D., Orhon. and Tamerler, C.

(2006). Bioremediation of textile azo dyes by Trichophyton rubrum LSK-27. World. J.

Microbiol and Biotechnol. 22, 1027-1031.

Koroleva, O. V., Gravilova, V. P., Stepanova, E. V., Lebedeva, V. I., Sverdlova, N. I.,

Landesman, E. O., Yavmetdinov, I. S. and Yaropolov, A. I. 2002. Production of lignin

modifying enzymes by co-cultivated white-rot fungi Cerrena maxima and Coriolus

hirsutus and characterization of laccase from Cerrena maxima. Enzyme Microb. Technol.

30, 573-580.

Kotterman, M. J. J., Wasseveld, R. A. and Field, J. A. (1996). Hydrogen peroxide production as

a limiting factor in xenobiotic compound oxidation by nitrogen-sufficient cultures of

Bjerkandera sp. strain BOS55 overproducing peroxidases. Appl. Environ. Microbiol. 62,

880-5.

Kumar, K., Devi, S. S., Krishnamurthi, K., Gampawar, S., Mishra, N., Pandya, G. H. and

Chakrabarti, T. (2006). Decolorisation, biodegradation and detoxification of benzidine

based azo dye. Biores. Technol. 97, 407-413.

Labbé, S. and Thiele, D. J. (1997). Pipes and wiring:the regulation of copper uptake and

distribution in yeast. Trends Microbiol., 7, 500-505.

Lacina, C., Gourene, G. and Agathos, S. (2003)., Utilization of fungi for biotreatment of raw

wastewaters. Afric. J. Biotechnol. 2, 620-630

244

Lan, J., Wang, F., Huang, X. R., Li, Y. Z., Qu, Y. B. and Gao, P. J. (2006).Studies on the

hydrogen peroxide regulated veratryl alcohol mediated oxidation of Pyrogallol red

catalyzed by lignin peroxidase. Acta. Chimi. Sin. 64, 463-468.

Lau, C. M. and Subramaniam, A. (1991). Recovery and applications of waste solids from natural

rubber latex. In: Proceedings of rubber growers conf., Kuala Lumpur, Malaysia, pp 24-

26.

Leung, Pui-Chi and Stephen B.P. (2002). Effect of different carbon and nitrogen sources on Poly

R declorization by white- rot fungi.Mycol.Res. 106, 86-92.

Levin, L., Forchiassin, F. and Papinutti, L. (2002). Effect of copper on the ligninolytic activity of

Trametes trogii. Int Biodeterior Biodegrad. 49, 60.

Levin, L., Forchiassin, F. F. and Viale, A. (2005). Ligninolytic enzyme production and dye

decolorization by Trametes trogii:application of the Plackett–Burman experimental

design to evaluate nutritional requirements. Proc. Biochem. 40, 381-1387.

Li, K., Xu, F. and Eriksson, K. E. L. (1999). Comparison of fungal laccases and redox mediators

in oxidation of a nonphenolic lignin model compound. Appl. Environ. Microbiol. 65,

2654-2660.

Libra, J. A., Borchert, M. and Banit, S. (2003). Competition strategies for the decolourisation

atextile reactive dye with the white rot fungi Trametes versicolor under non-sterile

conditions.Biotechnol. Bioeng. 82, 736-743.

Liu, A., Huang, X., Song, S., Wang, D., Lu, X., Qu. Y. and Gao. P. (2003). Kinetics of the H2O2-

dependent ligninase-catalyzed oxidation of veratryl alcohol in the presence of cationic

surfactant studied by spectrophotometric technique. Spectrochim.Acta Part A Mol.

Biomol. Spectrosc. 59, 2547-2551.

Liu, H. S., Mead, J. L. and Stacer, R. G. (2000). Environmental effects of recycled rubber in

light-fill applications. Rubber Chem. Technol. 73, 551-564.

Lopez, M. J., Guisado, G., Vargas-Garcıa, M. C., Suarez-Estrella, F. and Moreno, J. (2006).

Decolorization of industrial dyes by ligninolytic microorganisms isolated from

composting environment.Enzyme Microb. Technol. 40, 42-5.

245

Lorenzo, M., Diego, M. M. And Angeles, S. (2006). Effect of heavy metals on the production of

several laccase isoenzymes by Trametes versicolor and on their ability to decolorize

dyes.Chemosphere. 63, 912-917.

Lu, L., Zhao, M. and Wang, Y. (2007). Immobilization of laccase by alginate-chitosan

microcapsules and its use in dy decolorization.World J. Microbiol. Biotechnol. 23,159–

166.

Lu, R., Shen, X. L. and Xia, L. M. (2005). Studies on laccase production by Coriolus versicolor

and enzymatic decoloration of dye.

Lucas, M., Mertens, Corbisier, A. M. and Vanhulle., S. (2008). Synthetic Dye

decolourization by white -rot fungi: Development of original microtitre plate

method and screening.42, 97-106.

Majcherczk, A., Johannese, C., Huttermann, A., (1998). Oxidation of polycyclic aromatic

hydrocarbons(PAH) by laccase of Trametes versicolor. Enzyme Microbiol.Technol. 22 ,

335-341.

Ma¨kela,¨ M.R., Hilde´n, K. S; Hakala, T. K., Hatakka, A. and Lundell, T.K. (2006). Expression

and molecular properties of a new laccase of the white rot fungus Phlebia radiata grown

on wood. Curr. Genet. 50:323–333.

Ma´ximo, C., Amorim, M. T. P. and Costa-Ferreira, M. (2003) Biotransformation of industrial

reactive azo dyes by Geotrichum sp. Enzyme Microb. Technol. 32, 145-151.

Maas, R. and Chaudhari, S. (2005). Adsorption and biological decolorization of azo dye Reactive

Red-2 in semicontinuous anaerobic reactors. Proc. Biochem. 40, 699-705.

Makela, M. R., Galkin, S., Hatakka, A. and Lundell, T. K. (2005). Production of organic acids

and oxalate decarboxylase in lignin-degrading white rot fungi. Enzyme Microb.Technol.

30, 542-549.

Mandal, T. K., Baldrian, P., Gabriel, J., Nerud, F. and Zadrazil, F. 1998. Effect of mercury on the

growth of wood-rotting basidiomycetes Pleurotus ostreatus, Pycnoporus cinnabarinus

and Serpula lacrymans. Chemosphere. 36, 435-440.

246

Mansur, M., Suarez, T, Fernandez-Larrea, J. B., Brizuela, M. A. And Gonzalez, A. E. (1997).

Identification of a laccase gene family in the new lignin-degrading basidiomycete CECT

20197. Appl. Environ. Microbiol. 63, 2637-2646.

Martin, R., Jumino, A., Dubief, C., Rosenbaum, G. and Audousset, M. P. (1994).Oreal, Patent

FR2694018.

Martınez, A. T. (2002). Molecular biology and structure-function of lignin-degrading heme

peroxidases. Enzyme Microb.Technol. 30, 425- 44.

Martins, M. A., Ferreira, I. C., Santos, I. M., Queiroz, M. .J. and Lima, N. (2001).Biodegradation

of bioaccessible textile azo dyes by Phanerochaete chrysosporium. J. Biotechnol. 89, 91-

98.

Mazmanci, M. A. and Unyayar, A. (2005). Decolourisation of Reactive Black 5 by Funalia trogii

immobilised on Luffa cylindrica sponge. Process. Biochem. 40, 337-42.

McKay, G. (1979). Waste colour removal from textile effluents. American Dyes Report, 68, 29-36.

McMullan, G., Meehan, C., Conneely, A., Kirby, N. and Robinsonigam, P. (2001). Mini-review:

microbial decolorisation and degradation of textile dyes. Appl. Microbiol. Biotechnol. 56,

81- 87.

Meilgo, I; Lopez, C., Moreira, M.T.,Ferjoo,G., and Lema, J.M., (2003).Oxidative degradation

of azodyes by manganese peroxidase under optimized conditions. Biotechnol.Prog.19,

325-331

ster, T. And Field, J. A. (1998). Characterization of a novel manganese peroxidase-lignin per-

oxidase hybrid isozyme produced by Bjerkandera species strain BOS55 in the absence of

manganese. J. Biol. Chem. 273, 15412-15417.

Michniewicz, A., Ullrich, R., Ledakowicz, S. and Hofrichter, M. (2006). The white-rot fungus

Cerrena unicolor strain 137 produces two laccase isoforms with different

physicochemical and catalytic properties. Appl. Microbiol. Biotechnol. 69, 682-688.

microorganisms and helminthes. Environ. Toxicol. Chem.12, 2121–2132.

247

Minussi, R. C., Pastore, G. M. and Duran, N. (2007). Laccase induction in fungi and laccase/N–

OH mediator systems applied in paper mill effluent. Biores. Technol. 98, 158-164.

Minussi, R. C., Pastore, G.M. and Dura´n N. (2007). Laccase induction in fungi and laccase/N–

OH mediator systems applied in paper mill effluent. Biores. Technol. 98,158–164.

Mohorcic, M., Simona, T., Vera, G. and Jozzzefa, F. (2006). Fungal and enzymatic

Decolourization of artificial textile dye baths. Chemosphere. 63, 1709-1717.

Moreira, P. R., Bouillenne, F., Almeida-Vara, E., Xavier, Malcata, F. Frere, J. M. and Duarte, J.

C. (2006). Purification, kinetics and spectral characterisation of a new versatile

peroxidase from a Bjerkandera sp. isolate. Enzyme Microb. Technol. 38, 28-33.

Murugesan, K. and Kalaichelvan, P. T. (2003). Synthetic dye decolourization by white rot fungi.

Indian J. Exp. Biol. 41, 1076-1087.

Murugesan, K., Arulmani, M., Nam, I. H., Kim, Y. M., Chang, Y. S. and Kalaichelvan, P. T.

(2006). Purification and characterization of laccase produced by a white rot fungus

Pleurotus sajorcaju under submerged culture condition and its potential decolorization of

azo dyes. Appl.Microbiol. Biotechnol. 72, 939-946.

Murugesan, K., Dhamija, A., Nam, I. H., Kim, Y. M. and Chang, Y. S. (2007). Decolourization

of reactive black 5 by laccase: optimization by response surface methodology. Dyes

Pigm. 75, 176-84.

Nagai, M., Sato, T., Watanabe, H., Saito, K., Kawata, M. and Enei, H. (2002). Purification and

characterization of an extracellular laccase from the edible mushroom Lentinula edodes,

and decolorization of chemically different dyes. Appl. Microbiol. and Biotechnol. 60,

327-335.

Nagai, M., Sato, T., Watanabe, H., Saito, K., Kawata, M. and Enei, H. (2002). Purification of

synthetic dyes by solid state cultures of Lentinula (Lentinus) edodes producing

manganese peroxidase as the main ligninolytic enzyme. Biores. Technol. 94, 107-112.

Nie, G., Reading, N. S. and Aust, S. D. (1999). Relative stability of recombinant versus native

peroxidases from Phanerochaete chrysosporium. Arch Biochem Biophys. 365, 328-334.

248

Nigam, P., Armour, G., Singh, D. and Marchant, R. (2000). Physical removal of textile dye from

effluents and solid state fermentation of dye-absorbed agricultural residues. Bioresource

Technol. 72, 219-226.

Nilsson, I., Miller, A., Mattiasson, B., Rubindamayugi, M. S.T. and Welander, U. (2005).

Decolorization of synthetic and real textile wastewater by the use of whiterot fungi.


Noroozi, B., Sorial, G. A., Bahrami, H. and Arami, M. (2007). Equilibrium and kinetic

adsorption study of a cationic dye by a natural adsorbent Silkworm pupa. Journal of

Hazardous Material B, 139, 167-174.

Novotny, C., Svobodova, K., Kasinatii, A. and Erbanova, P. (2004). Biodegradation of synthetic

dyes by Irpex lacteus under various growth). conditions. lnternat. Biodeter. Biodegrad.

54, 215-223.

O’Neill, C., Lopez, A., Esteves, S., Hawkes, F. R., Hawkes, D. L. and Wilcox, S. (2000). Azo-

dye degradation in an anaerobic-aerobic treatment system operating on simulated textile

effluent. Appl Microbiol Biotechnol. 53, 249-254.

of activated sludge on olive mill wastewater detoxification with white-rot fungi. Lett. Appl.

Microbiol. 42, 405–411.

Ollika, P., Ollika, P., Alhonmaki, K., Leppanin, V.M., Glumoff, T., Raijola, T., and Souminen , I. (1993). Decolorization of azo, triphenyl methane, hetrocyclic and polymeric dyes. byligninperoxidase,isoenzymesformfromPhanerochaete.Chryysoporium.Appl.Environ.Microbiol.59, 4010-4016.

Ollika, P., Alhonmaki, K., Leppanin, V.M., Glumoff, T., Raijola, T., and Souminen, I. (1993). Decolorization of azo, triphenyl methane, hetrocyclic and polymeric dyes. byligninperoxidase,isoenzymesformfromPhanerochaete.Chryysoporium.Appl.Environ.Microbiol.59, 4010-4016.

Orth, A. B., Denny, M. and Tien, M. (1993). Overproduction of lignin degrading enzymes byan

isolate of Phanerochaete chrysosporium. Appl. Environ. Microbiol. 57, 2591-2596.

249

Ozsoy, H. D., Unyayar, A. and Mazmanci, M. A. (2005). Decolourisation of reactive textile dyes

Drimarene Blue X3LR and Remazol. Brilliant Blue R by Funalia trogii ATCC 200800.

Biodegradation. 16, 195-204.

Padgonik, H., Grgic, I. and Predih, A. (1999). Decolourisation rate of dyes using lignin

peroxidase of Phanerochaete chrysosporium. Chemosphere. 38, 1353-9.

Palmieri, G., Giardina, P., Bianco, C., Fontanella, B. and Sannia, G. (2000). Copper induction of

laccase isoenzymes in the ligninolytic fungus Pleurotus ostreatus. Appl. Environ.

Microbiol. 66, 920-924.

Palmieri, G. , Giovanna C., Vincenza, F., A., Giovanni S. and Paola G. (2003). ) Atypical laccase

isoenzymes from copper supplemented Pleurotus ostreatus cultures Enzyme Microbio.

Technolo.33, 220–230.

Palmieri, G., Cennamo, G. and Sannia, G. (2005). Remazole Brilliant Blue R decolourisationby the fungus

Pleurotus ostreatus and its oxidative enzymatic system. Enzyme and Microb. Technol. 36, 17-24.

Parshetti, G. K., Kalme, S. D., Gomare, S. S. and Govindwar, S. P. (2007). Biodegradation of

Reactive blue-25 by Aspergillus ochraceus NCIM-1146. Biores. Technol. 98, 3638-3642.

Pasti-Grigsby, M. B., Paszczynski, A., Gosczynski, S., Crawford, D. L. and Crawford, R. L.

(1992). Influence of aromatic substitution patterns on azo dye degradability by

Streptomyces sp. and Phanerochaete chrysosporium. Appl. Environ. .Microbiol. 58,

3605- 13.

Paszeznski, A., Pasti-Grigsby, M. B., Goszezynski, S., Crawford, R. L. And Crawford, D. L.

(1992). Mineralisation of sulfonated azodyes and sulfanilic acid by Phanerochaete

Chrysosporium and Streptomyces chromofucus.Appl. Environ. Microbiol. 58, 3598-3604.

Paszeznski, A., Pasti-Grigsby, M. B., Goszezynski, S., Crawford, D. L. and Crawford, R.L.

(1991). New Approaches to degradation of recalcitrant azodyes by Streptomyces and

Phanerochaete Chrysosporium.

250

Pazarlioglu, K., Raziye, O. and Urek, F. E. (2005). Biodecolourization of Direct Blue15 by

immobilized Phanerochaete Chrysosporium. Process. Biochem. 40, 1923-1929.

Perie, F. H. and Gold, M. H. (1991). Manganese regulation of manganese peroxidase expression

and lignin degradation by the white-rot fungus Dichom#us squalens. Appl.Environ.

Microbiol. 57, 2240-2245.

Pirie, F. H. and Gold, M. H. (1991). Manganese regulation of manganese peroxidase expression

and lignin degradation by the white-rot fungus Dichomitus squalens. Appl. Environ.

Microbiol. 57, 2240-5.

Pogni, R., Teutloff, C., Lendzian, F. and Basosi, R. (2007). Tryptophan radicals as reaction

intermediates in versatile peroxidases:multifrequency EPR, ENDOR and density

functional theory studies. Appl. Magn. Reson. 31, 509-526.

Pointing, S.B. (2001). Feasibility of bioremediation by white-rot fungiS. B. Pointing. Appl.

Microbiol. Biotechnol. 57,:20–33.

Pointing, S. B. and Vrijmoed, L. L. P. (2000). Decolorization of azo and triphenylmethane dyes

by Pycnoporus sanguineus producing laccase as the sole phenoloxidase. World J

Microbiol. Biotechnol. 16, 317- 8.

Polman, A. and Brekenridge, C. R. (1996). Biomass-mediated binding and recovery of textile

dyes from waste e.uents. Tex. Chem. Colour. 28, 31-35.

Poots V,J; Kay, M. and Heakt, J.(1976). The removal of Acid Dye From Effluents using natural

adsorbants---II, Water Resour. 10, 1067-1070.

Quaratino, D., Federici, F., Petruccioli, M., Fenice, M. and D0Annibale, A. (2007). Production,

purification and partial characterization of a novel laccase from the white-rot fungus

Panus tigrinus CBS. 577-79. Antonie Van Leeuwenhoek Int. J.Genet. Mol. Microbiol. 91,

57-69.

Radha, K. V., Regupathi, I., Arunagiri, A. and Murugesan, T. (2005). Decolorization studies of

synthetic dyes using Phanerochaete chrysosporium and their kinetics. Process Biochem.

40, 3337-3345.

251

Raghukumar, C. (2002). Bioremediation of colored pollutants by terrestrial versus facultative

marine fungi. In: Hyde KD (ed) Fungi in marine environments. Fungal Diversity

Research Series, 7, 317-344.

Ramos-Cormenzana, A., Monteoliva-Sanchez, M. and Lopez, M. J.(1995). Bioremediation of

alpechin. Intl. Biodet. Biodeg. 35, 249–268.

Ramsay, J. A. and Chris, G. (2004). Decoloration of a Carpet Dye Effluent Using Trametes

VersicolorBiotechnol. Lett. 26, 197-201.

Ramsay, J., Shin, M., Wong, S. and Goode, C. (2006). Amaranth decoloration by Trametes

versicolor in a rotating biological contacting reactor. Journal of Industrial Microbiol. &

Biotechno. 33, 791-795.

Reddy, C. A. (1995). The potential for white-rot fungi in the treatment of pollutants. Curr. Opin.

Biotechnol. 6, 320-328.

Renganathan, S. (2006). Accumulation of Acid Orange7, Acid Red 18 and Reactive Black5 by

growing Schizophyllum commune. Bioresource Technolo. 97, 2189-2193.

Revankar, M. S. and Lele, S. S. (2007). Synthetic dye decolorization by white rot fungus,

Ganoderma sp. WR-1. Biores. Technol. 98, 775-780.

Reyes, P., Pickard, M. A. and Vazquez-Duhalt, R. (1999). Hydroxybenzotriazole increases the

range of textile dyes decolorized by immobilized laccase. Biotechnol Lett 21, 875-80.

Rigas, F. and Dritsa, V. (2006). Decolorization of a polymeric dye by selected fungal

strains in liquid cultures. Enzyme and Microbial Technology. 39, 120–124.

Robinson. (2003). Production of lignin peroxidase, manganese peroxidase and laccases by WRF

in nitrogen deficient medium. Enzyme and Microb. Technol. 30, 142-147.

Robinson, T., McMullan, G., Marchant, R. and Nigam, P. (2001a). Remediation of dyes in

textile effluent: a critical review on current treatment technologies with a proposed

alternative. Bioresour. Technol. 77, 247-55.

252

Rodakiewicz-Nowak, J., Jarosz-Wilkołazka, A. and Luterek, J. (2006). Catalytic activity of

versatile peroxidase from Bjerkandera fumosa in aqueous solutions of water-miscible

organic solvents. Appl. Catal. A Gen. 308, 56-61.

Rodriguez, E, Pickard, M.A. and Vazquez-Duhalt, R. . (1999) Industrial dye decolorization by

laccases from ligninolytic fungi. Curr. Microbiol. 38, 27–32.

Rogalski, J., Szczodrak, J. and Janusz, G. (2006). Manganese peroxidase production in

submerged cultures by free immobilized mycelia of Nemtoloma froowardii. 97, 469-476.

Romero, S., Paqui, B., Gloria, C., Xavier, F., Montserrat, S., Xavier, G., Teresa, V. (2006).

Different approaches to improving the textile dye degradation capacity of Trametes

versicolor. Biochemical Eng. Journal 31, 42-47.

Ruiz-Duenas, F. J., Morales, M., Perez-Boada, M., Choinowski, T., Martınez, M. J., Piontek, K.

and Martınez, A. T. (2007). Manganese oxidation site in Pleurotus eryngii versatile

peroxidase: a site-directed mutagenesis, kinetic, and crystallographic study. Biochem. 46,

66-77.

Ryan, S., Schnitzhufer, W., Tzanov, T., Cavaco-Paulo, A. and Gubitz, G.M. (2003). An acid-

stable laccase from Sclerotium rolfsii with potential for wool dye decolourization.

Enzyme Microb. Technol. 33, 766–74.

Salas, C., Lobos, S., Larrain, J., Salas, L., Cullen, D. and Vicuna, R. (1995). Properties of laccase

isoenzymes produced by the basidiomycete Ceriporiopsis subvermispora. Biotechnol.

Appl. Biochem. 21, 323-33.

Salony, S. M. and Bisaria, V. S. (2006). Production and characterization of laccase from Cyathus

bulleri and its use in decolorization of recalcitrant textile dyes. Appl. Microbiol.

Biotechnol. 71, 646-653.

Salvam, K., Swaminathan, K. and Chae, K. S. (2003). Decolourization of azodyes and a dye

industry effluent by a white rot fungus Thelephora sp. Biores. Tehnol. 88, 115-119.

Sanghi, R., Dixit A.and Guha, S. (2006). Sequential batch culture studies for the decolorisation

of reactive dye by Coriolus versicolor. Biores. Technol. 3, 396-400.

253

Sanjust, E., Pompei, R., Resciggno, A., Augusto, R. and Ballero, M. (1991). Olive milling

wastewater as medium for growth of four Pleurotus species. Appl. Biochem. Biotechnol.

31, 223-235.

Sayer, J. and Gadd, G. M. (1997). Solubilization and transformation of insoluble inorganic metal

compounds to insoluble metal oxalates by Aspergillus niger. Mycol. Res. 106, 653-61.

Scheibner, K., Hofrichter, M. and Fritsche, W. (1997). Mineralization of 2-amino-4,6-

dinitrotoluene by manganese peroxidase of the white-rot fungus Nematoloma frowardii.

Biotechnol. Lett.19, 835–9.

Schliephake, K. and Lonergan, T. (1996). Laccase variation during dye decolorization in a 200 L

packed bed bioreactor. Biotechnol. Lett. 18, 881-886.

Schlosser, D., Grey, R. and Fritsche, W. (1997). Patterns of lignolytic enzymes in Trametes

versicolor: distribution of extra- and intracellular enzyme activities during cultivation on

glucose, wheat straw and beech wood. Appl. Microbiol. Biotechnol. 47, 412-8.

Selvam, K., Swaminathan, K., Myung Hoon Song, M. H. and Chae, K. (2002). Biological

treatment of a pulp and paper industry effluent by Fomes lividus and Trametes versicolor.

World J. Microbiol. Biotechnol. 18, 523–526.

Selvam, K., Swaminathan, K., Rasappan, K., Rajendran, R. and Pattabhi, S. (2006).

Decolourization and dechlorination of a pulp and paper industry effluent by Thelephora

sp. Ecol. Environ. Conserv. 12, 223-226.

Seshadri, S., Bishop, P. L. and Agha, A. M. (1994) Anerobic /aerobic treatment of selected

azodyes in wastewater.Wastwe Mnage.15, 127-137.

Shah, V., Nerud, F. (2002). Lignin degrading system of white-rot fungi and its exploitation for

dye decolorization. Can. J. Microbiol. 48, 457–870.

Shaul, G. M., Holdsworth, T. J., Dempsey, C. R. and Dostal, K. A. (1991). Fate of water soluble

azo dyes in the activated sludge process. Chemosphere. 22, 107-9.

254

Shin, K. S., Oh, I. K. and Kim, C. J. (1997). Production and purification of Remazol brilliant

blue R decolorizing peroxidase from the culture filtrate of Pleurotus ostreatus. Appl.

Environ. Microbiol. 63, 1744-1748

Shin. (2004). The Role of Enzymes Produced by White-Rot Fungus Irpex lacteus in the

Decolorization of the Textile Industry Effluent. The Journal of Microbiol. 2, 37-41.

Shrivastava, R., Christian, V. and Vyas, B. R. M. (2005). Enzymatic decolorization of

sulfonphthalein dyes. Enzyme. Microb.Technol. 36, 333-337

Silva, C. M .M., Itmar, S.M.and Pablo,R.O. (2005). Lignolytic Enzymes Production by

Ganoderma lucidum spp. Enzyme Micr1ob. Technol.37, 324-329.

Silvia, R., Paqui, B., Gl`oria, C., Xavier, F., Montserrat, S., Xavier, G. and Teresa, V. (2006).

Different approaches to improving the textile dye degradation capacity of Trametes

versicolor Biochemical Eng. Journal 31, 42–47.

Singh, P; Rashmi, S., Anjali, P. and Leela. (2007). Decolorization and partial egradation of

monoazo dyes in sequential fixed-film anaerobic batch reactor (SFABR) Biores.Technol.

98, 2053-2056.

Soares, G. M. B., Amorim, M. T. P. and Costa-Ferreira, M. (2001). Use of laccase together

Soden, D. M. and Dobson, A. D. W. (2001). Differential regulation of laccase gene expression in

Pleurotus sajor-caju. Microbiol. 147, 1755-63.

Spadaro, J. T., Gold, M. H. and Ranganathan, V. (1992). Degradation of azo dyes by the lignin-

degrading fungus Phanerochaete chrysosporium. Appl. and Environ. Microbiol. 58,

2397-2401.

Steel, R. G. d. and J. H. Torrie. (1992). Principles and Procedures of statistics. 2nd Ed. Mc

Grawhill Book Company, Inc. New York.

Sumathi, S. and Manju, B. S. (2000). Uptake of reactive textile dyes by Aspergillus foetidus

Enzyme and Microb. Technol. 27, 347-355.

255

Sundaramoorthy, K., K, Kishi, M.H. Gold and T.L., Poulos, 1994: The crystal structure of

manganese Perosidase from Phanerochaete chrysosporium. Biol. Chem. 269:32759-

32767.

Supaka, N., Juntagjin, L., Damrobglerd, S., Delia, M. L., Strehaiano, S. (2004).Microbial

decolorization of reactive azo dyes in a sequential anaerobic/aerobic reactor system.

Chem. Eng. J. 99,1 46-169.

Susla, M., Novotny, C. and Svobodova, K. (2007). The implication of Dichomitus squalens

laccase isoenzymes in dye decolorization by immobilized fungal cultures. Biores.

Technol. 2109-15.

Swamy, J. and Ramsay, A. (1999). The evaluation of white rot fungi in the decoloration of

textile dyes. Enz. Microb. Tech. 24, 130-7.

Swamy, J. and Ramsay, J. (1999b). Effects of glucose and NH4+ concentrations on sequential

dye decoloration by Trametes versicolor.Enzyme. Microb. Technol. 25, 278-284.

Tekere, M., Zvauya, R. Maswaka, A.Y. and Read J. S.(2001). Growth, dye degradation and

ligninolytic activity studies on Zimbabwean white rot fungi. 28, 420-426.

Teunissen, P. J. M. and Field, J. A. (1998). 2-Chloro-1,4-dimethoxybenzene as a mediator of

lignin peroxidase catalyzed oxidations. FEBS Lett. 439, 219-223.

Thurston, C. F. (1994). The structure and function of fungal laccase. Microbiol. 140, 19-26.

Tien, M. and Kirk, T. K. (1988). Lignin peroxidase of Phanerochaete chrysosporium. Methods

Enzymol. 161, 238-248.

Tinoco, R., Verdin, J. and Vazquez-Duhalt, R. (2007). Role of oxidizing mediators and

tryptophan 172 in the decoloration of industrial dyes by the versatile peroxidase from

Bjerkandera adusta. J Mol. Catal. B Enzyme. 46, 1-7.

Toh, Y. C., Yen, J. J. L., Obbard J. P. and Ting, Y. P. (2003). Decolourization of azo dye by

white rot fungi isolated in Singapore. Enzyme and Microb. Technol. 33, 569-575.

256

Trevor, M., D’Souza, Carlos, S. M. and Adinarayana, C. R. (1999). Lignin-Modifying Enzymes

of the White Rot Basidiomycete Ganoderma lucidum. Appl. And Environ. Microbiol.

5307–5313.

Trovaslet, M, Enaund, E., Guiavarc’h Y., Corbisier, A.M. and Vanhulle, S. (2007).

Potential of Picnoporus Sanguineus laccase in bioremediation of waste water and

kinetic activation in the presence of anthraquinone acid dyes. Enzyme Microb.

Technol.41, 68-376.

Tsioulpas, A., Dimou, D., Iconomou, D. and Aggelis, G. (2002). Phenolic removal in olive oil

mill wastewater by strains of Pleurotus spp. in respect to their phenol oxidase (laccase)

activity. Biores. Technol. 84, 251-257.

Tsukihara, T., Honda, Y., Watanabe, T. and Watanabe, T. (2006). Molecular breeding of white

rot fungus Pleurotus ostreatus by homologous expression of its versatile peroxidase

MnP2. Appl. Microbiol. Biotechnol. 71, 114-120.

Tuomela, M., Oivanen, P. and Hatakka, A. (2002). Degradation of synthetic 14C-lignin by

various white-rot fungi in soil. Soil Biol. and Biochem. 34, 1613-1620.

Tychanowicz, G.K; Daniela F. de S; Cristina, G. M; Souza, M. K. K. and Rosane, M. P.

(2006).Copper Improves the Production of Laccase by the White-Rot Fungus Pleurotus

pulmonarius in Solid State Fermentation Brazilian Archives of BIO. &Technol.5, 699-

704

U¨rek, R.O., and Pazarlioglu N.K. (2004). Purification and partial characterization of manganese

peroxidase from immobilizedPhanerochaete chrysosporium. Proc Biochem 39, 2061–

2068.

Ullrich, R., Le, M. H., Nguyen, L. D. and Hofrichter, M. (2005). Laccase from the medicinal

mushroom Agaricus blazei: production,purification and characterization. Appl.

Microbiol. Biotechnol. 67, 357-363.

257

Unyayar, A., Mazmanci, M. A., Atacag, H., Erkurt, E. A. and Coral, G. (2005). A Drimaren Blue

X3LR dye decolorizing enzyme from Funalia trogii;one step isolation and identification.


Urek, R. O. and Pazarlioglu, N. K. (2005). Production and stimulation of manganese peroxidase

by immobilized Phanerochaete chrysosporium. Proc. Biochem. 40, 83-87.

Vahabzadeh, F., Mehranian, M. and Saatari, A. R. (2004). Color removal ability of

Phanerochaete chrysosporium in relation to lignin peroxidase and manganese peroxidase

produced in molasses wastewater. World J Microbiol.Biotechnol. 20, 859-864.

Vaidya, A. A. and Datye, K. V. (1982). Environmental pollution during chemical processing of

synthetic fibres. Colourage. 14, 3-10.

Van, A, B., Hofrichter, M., Scheibner, K., Hatakka, A. I., Naveau, H., and Agathos, S.N.

(1999a). Transformation and mineralization of 2,4,6-trinitrotoluene (TNT) by anganese

peroxidase from the white-rot basidiomycete Phlebia radiata. Biodeg. 10, 83– 91.

Vandevivere, P.C., Bianchi, R., Verstraete, W. (1998). Treatment and reuse of wastewater from

the textile wet-processing industry: review of emerging technologies. J. Chem. Technol.

Biotechnol. 72, 289–302.

Vanhulle, S., Enaud, E., Troavaslet, M., Billottet, L., Kneeipe, L., Jiwan, J. L. H., Corbisier, A.

M. and Mrachand-Brynaert, J. (2008). Coupling occurs beforebreakdown

ringbiotransformation of Acid Blue62 by white rot fungi. Chemosphere.70, 1097-1107.

Vanhulle, S., Enaud, E., Trovaslet, M., Billottet, L., Kneipe, L. and Jiwan, J. L. H. (2008).

Coupling occurs before breakdown during biotransformation of Acid Blue 62 by white

rot fungi. Chemosphere. 70, 1097-107.

Verma, P. and Madamwar, D. (2002). Production of lignolytic enzymes for dye decolorization

by co-cultivation of white rot fungi Pleurotus ostreatus and Phanerochaete

chrysosporium under solid state fermentation. Appl. Biochem. Biotechnol. 102-103, 109-

118

258

Verma, P. and Madamwar, D. (2005). Decolorization of azo dyes using basidiomycete strain

PV002. World J. Microbiol. Biotechnol., 21, 481-485.

Vyas, B. R. and Molitoris, H. P. (1995). Involvement of an extracellular H2O2-dependent

ligninolytic activity of the white rot fungus Pleurotus ostreatus in the decolorization of

Remazol brilliant blue R. Appl. Environ. Microbiol. 61, 3919-3927.

Wang, Y. and Yu, J. (1998). Adsorption and degradation of synthetic dyes on the mycelium of

Trametes versicolor. Water Sci. Technol. 38, 233-238.

Wariishi, H., Akileswaran, L. and Gold, M. H (1988). Manganese peroxidase from the

basidiomycete Phanerochaete chrysosporium spectral characterization of the oxidized

states and the catalytic cycle, Biochem. 27, 5365-5370.

Wariishi, H., Valli, K., Renganathan, V. and Gold, M. H. (1989). Thiol-mediated oxidation of

nonphenolic lignin model compounds by manganese peroxidase of Phanerochaete

chrysosporium. J Biol. Chem. 264, 14185-91.

Watanabe, T., Shirai, N., Okada, H., Honda, Y. And Kuwahara, M. (2001). Production and

chemiluminescent free radical reactions of glyoxal in lipid peroxidation of linoleic acid

by the ligninolytic enzyme,manganese peroxidase, Eur. J. Biochem. 268, 6114-6122.

Wells, a., Teira, M. and Eve, T.,(2006). Green Oxidation with laccase mediator Systems.

Biochem. Soci. Trans. 34, 304-308.

Wesenberg, Irene, K. and Spiros, N. A. (2003). White-rot fungi and their enzymes for the

treatment of industrial dye effluents. Biotechnol. Advances. 22, 161-187.

Willmott, N., Guthrie, J. and Nelson, G. (1998). The biotechnology approach to colour removal

from textile effluent. J. Soc. Dyers. Colour. 114, 38-41.

with redox mediators to decolorization Remazol Brilliant Blue R. J Biotechnol. 123–9.

Wong, Y., and Yu. J. (1999) .Laccase Caetalysed decolorization of dyes.Water Res. 33, 3512-

3520.

259

Wu, J., Xiao, Y. Z. and Wang, Y. P. (2002). Treatment of pulp mill wastewaters by white-rot

fungi. J. Biol. 19, 17-19.

Wu, F., Ozaki, H., Terashima, Y., Imuda, t, and Ohkouchi., T. (1996). Activities of lignolytic

enzymes of the white rof fungus phanesodiaete Chrysosporium and its recalcitrant

substances degoadibility. Water sic teclinol. 34,69-78

Yang, J. S., Yuan, H. L. and Chen, W. X. (2004). Studies on extracellular enzymes of lignin

degrading fungus Penicillium sp. P6.China Environ Sci. 24, 24-27.

Yaropolov, A. I., Skorobogat’ko, 0. V., Vartanov, S. S. and Varfolomeyev, S. D. (1994)

Lactase: properties, catalytic mechanism, and applicability. Appl. Biochem. Biotechnol.,

49, 257-275.

Yesilada, O., Fiskin, K., Yesilada, E. (1999). The use of white rot fungus Funalia trogii

(Malatya) for the decolorization and phenol removal from olive mill wastewater.

Environ. Technol.16, 95–100.

Yesilada, O., Asma, D. and Cing, S. (2003). Decolorization of textile dyes by fungal pellets.

Process Biochem. 38, 933-8.

Yesilada, O., Cing, S. and Asma, D. (2002). Decolourisation of the textile dye Astrazon Red

FBL by Funalia trogii pellets. Biores. Technol. 81, 155-157.

Young, L. And Yu, J. (1997). Ligninase-catalyzed decolorization of synthetic dyes.Water Res.

31, 1187-93.

Youngs, H. L., Sundaramoorthy, M. and Gold, M. H. (2000). Effects of cadmium on manganese

peroxidase—competitive inhibition of Mn-II oxidation and thermal stabilization of the

enzyme. J. Biochem. 267, 1761-9.-------

Yu, G., Wen, X., Li, R. and Qian, Y. (2006). In vitro degradation of a reactive azo dye by crude

ligninolytic enzymes from nonimmersed liquid culture of Phanerochaete chrysosporium.

Proc. Biochem. 41, 1987-1993.

Zervakis, G., Yiatras, P. and Balis, C. (1996). Edible mushrooms from olive oil mill waste. Int.

Biodet. Biodeg. 38, 237-243.

260

Zhang, F. M., J. S. Knapp and K. N. Tapley. 1999. Decolorisation of cotton bleaching effluent

with white rot fungus. Water Research, 33, 919-928.

Zhang, F., Knapp., Tapley, KN., 1999. Development of bioreactor systems for

decolourization of Orange II., using white rot fungus. Enzyme Microb.Technol.

24, 48-53.

Zhao, X. and I. R. Hardin. 2007. HPLC and spectrophotometric analysis of biodegradation of azo

dyes by Pleurotus ostreatus. Dyes and Pigments, 73, 322-325.

Zhao, J., Koker, T. H. and Janse, B. J. H. (1996). Comparative studies of lignin peroxidases and

manganese-dependent peroxidases produced by selected white rot fungi in solid media.

FEMS Microbiol. Lett. 145, 393-399.

Zhao, X., Hardin, I. R. and Hwang, H. M. (2006). Biodegradation of a model azo disperse dye by

the white rot fungus Pleurotus ostreatus. Int. Biodet. Biodeg. 57, 1-6.

Zhao, X., Hardin, I.R. (2007). HPLC and spectrophotometric analysis of biodegradation of azo

dyes by Pleurotus ostreatus. Dyes Pigm. 73, 322–325.

Zhao, X., Yiping, Lu, Y. and Hardin, I. (2005). Determination of biodegradation products from

sulfonated dyes by Pleurotus ostreatus using capillary electrophoresis coupled with mass

spectrometry. Biotechnol. Lett. 27, 69-72.

Zilly, A., de Souza, C. G. M., Barbosa-Tessmann, I. P. and Peralta, R. M. (2002). Decolorization

of industrial dyes by a Brazilian strain of Pleurotus pulmonarius producing laccase as the

sole phenol-oxidizing enzyme. Folia Microbiol. 47, 315– 319.

Zouari-Mechichi, H., Mechichi, T., Dhouib, A., Sayadi, S., Martınez, A. T. and Martınez, M. J.

(2006). Laccase purification and characterization from Trametes trogii isolated in

Tunisia: decolorization of textile dyes by the purified enz me. Enz. Microb. Technol. 39,

141-148.

261

Appendices

APPENDEX-1 Table 4.2: Analysis of variance of the data on decolorization of Remazole

Brilliant Yellow 3-GL by C. versicolor and production of lignolytic enzymes with different media compositions

Source of variation

d.f Mean squares

Decolorization (%)

Final pH "Enzyme Activity (IU/mL)"

MnP Lac Between

Within

3

8

7296.91**

1.71

4.784**

0.042

19255.6**

74.2

24646.1**

71.3

** = Highly significant (P<0.01) Table 4.3: Analysis of variance of the data on decolorization of Remazole

Brilliant Yellow 3-GL by C. versicolor and production of lignolytic enzymes with varying Ph levels

Source of variation

d.f Mean squares

Decolorization (%)


MnP Lac Between

Within

4

10

1446.7**

13.1

3.8267**

0.0466

3184.0**

360.0

4910.0**

316.0


Brilliant Yellow 3-GL by C. versicolor and production of lignolytic enzymes with varying temperatures

Source of variation

d.f Mean squares

Decolorization (%)


MnP Lac Between

Within

4

10

1111.9**

10.8

0.7287**

0.0552

1244.0**

150.0

4326.5**

56.7

** = Highly significant (P<0.01)

Appendices

262

Table 4.5: Analysis of variance of the data on decolorization of Remazole Brilliant Yellow 3-GL by C. versicolor and production of lignolytic enzymes with different carbon sources

Source of variation

d.f Mean squares

Decolorization (%)


MnP Lac Between

Within

4

10

5626.69**

2.99

7.7179**

0.0056

1493.0NS

472.0

1576.0**

886.0

Table 4.6: Analysis of variance of the data on decolorization of Remazole

Brilliant Yellow 3-GL by C. versicolor and production of lignolytic enzymes with varying concentrations of wheat bran

Source of variation

d.f Mean squares

Decolorization (%)


MnP Lac Between

Within

4

10

5869.4**

106.5

0.1791**

0.0084

30093.0**

380.0

32857.0**

211.0


Brilliant Yellow 3-GL by C. versicolor and production of lignolytic enzymes with different nitrogen sources

Source of variation

d.f Mean squares

Decolorization (%)


MnP Lac Between

Within

4

10

1760.2**

27.9

0.23961**

0.00313

2950.0NS

1115.0

113806.0**

1309.0

MGM = Maize gluten meal, CSL = Corn steep liquor NS = Non-significant (P>0.05); ** = Highly significant (P<0.01) Table 4.8: Analysis of variance of the data on decolorization of Remazole

Brilliant Yellow 3-GL by C.versicolor and production of lignolytic enzymes with varying concentrations of CSL

Source of variation

d.f Mean squares

Decolorization (%)


MnP Lac Between

Within

4

10

4777.0**

617.3

1.37918**

0.00457

13565.0**

560.0

143827.0**

1471.0


Appendices

263

Table 4.9: Analysis of variance of the data on decolorization of Remazole Brilliant Yellow 3-GL by C.versicolor and production of lignolytic enzymes with low molecular mass

Source of variation

d.f Mean squares

Decolorization (%)


MnP Lac Between

Within

3

8

1103.6**

13.5

0.01750NS

0.00809

46978.0**

751.0

294980.0**

1088.0


Brilliant Yellow 3-GL by C. versicolor and production of lignolytic enzymes with different metal ions

Source of variation

d.f Mean squares

Decolorization (%)


MnP Lac Between

Within

5

12

864.9

15.6

0.05773**

0.00244

19780.0**

240.0

107765.0**

913.0


Brilliant Yellow 3-GL by C.versicolor and production of lignolytic enzymes with different dye concentrations

Source of variation

d.f Mean squares

Decolorization (%)


MnP Lac Between

Within

4

10

4651.88

9.04

0.264NS

0.281

10422.0**

153.0

376103.0**

641.0

** = Highly significant (P<0.01) Table 4.12: Analysis of variance of the data on adsorption of Remazole Brilliant Yellow 3-GL by C. versicolor and production of lignolytic enzymes on Fungal mycelia

Source of variation

d.f Mean squares

Adsorption (%)

Final pH

Between

Within

4

10

0.1219**

0.0158

0.0249NS

0.0665


Appendices

264

Table 4.14: Analysis of variance of the data on decolorization of Foron Turquise SBLN-200 by G. lucidum and production of lignolytic enzymes with different media compositions

Source of variation

d.f

Mean squares

Decolori-zation (%)


Lip MnP Lac

Between

Within

3

8

1692.1**

15.2

10.006**

0.032

92073.0**

57.0

37840.9**

62.1

32345.0**

61.6


Table 4.15: Analysis of variance of the data on decolorization of Foron Turquise

SBLN-200 by G. lucidum and production of lignolytic enzymes with different carbon sources

Source of variation

d.f

Mean squares

Decolori-zation (%)


Lip MnP Lac

Between

Within

4

10

2378.1**

10.9

0.8787**

0.0068

124376.8**

225.3

64308.3**

110.1

10687.2**

77.0



SBLN-200 by G. lucidum and production of lignolytic enzymes with varying

concentrations of wheat bran

Source of

variation

d.f

Mean squares

Decolori-zation (%)


Lip MnP Lac

Between

Within

4

10

439.35**

18.94

0.6414**

0.0184

55993.3**

322.0

13457.6**

43.3

2805.9**

85.1


Appendices

265


SBLN-200 by G. lucidum and production of lignolytic enzymes with different

nitrogen sources

Source of variation

d.f

Mean squares

Decolori-zation (%)


Lip MnP Lac

Between

Within

4

10

1965.6

29.5

0.9765**

0.0023

85940.1**

261.0

29670.1**

310.3

8350.1**

169.4



SBLN-200 by G. lucidum and production of lignolytic enzymes with varying concentrations of Maize Glutein Meal 60%

Source of variation

d.f Mean squares

Decolori-zation (%)


Lip MnP Lac Between

Within

4

10

2608.9

10.4

0.0259**

0.0011

131640.4**

190.3

63091.7**

313.1

1165.1**

76.9


Table 4.19 Analysis of variance of the data on decolorization of Foron Turquise

SBLN-200 by and G. lucidum production of lignolytic enzymes with low molecular mass mediators

Source of variation

d.f Mean squares

Decolori-zation (%)


Lip MnP Lac Between

Within

5

12

1167.2

20.4

0.1371**

0.0042

11648.0**

22.1

4016.4**

67.2

65255.3**

78.0


Appendices

266


SBLN-200 by G. lucidum and production of lignolytic enzymes with different metal ions

Source of variation

d.f

Mean squares

Decolori-zation (%)


Lip MnP Lac

Between

Within

5

12

1982.4**

18.2

0.2501**

0.0404

25411.0**

57.1

15334.6**

911.3

35047.5**

53.3



SBLN-200 by G. lucidum and production of lignolytic enzymes with different dye

concentrations

Source of variation

d.f

Mean squares

Decolori-

zation (%) Final pH

"Enzyme Activity (IU/mL)"

Lip MnP Lac

Between

Within

4

10

5190.2**

13.1

0.0706*

0.0184

261131.3**

81.1

87410.5**

515.3

43144.1**

25.3


Table 4.22 Analysis of variance of the data on adsorption of Foron Turquise SBLN-200 by G. lucidum and production of lignolytic enzymes

Source of variation

d.f Mean squares

Adsorption (%) Final pH

Between

Within

4

10

5283.4**

7.9

1.3334**

0.0693


Appendices

267

Table 4.24: Analysis of variance of the data on decolorization of Solar Golden Yellow R by P. ostreatus and production of lignolytic enzymes with different media compositions

Source of

variation d.f

Mean squares

Decolorization

(%) Final pH


MnP Lac

Between

Within

3

8

1339.1**

21.3

0.2166NS

0.0801

10438.8**

80.1

5034.8**

81.5


Table 4.25 Analysis of variance of the data on decolorization of Solar Golden Yellow R by P. ostreatus and production of lignolytic enzymes with different carbon sources

Source of variation

d.f

Mean squares

Decolorization

(%) Final pH


MnP Lac

Between

Within

4

10

1614.0**

23.7

0.2569**

0.0321

20786.2**

32.1

39638.6**

88.9


Table 4.26 Analysis of variance of the data on decolorization of Solar Golden

Yellow R by P. ostreatus and production of lignolytic enzymes with varying concentration of wheat bran

Source of variation

d.f

Mean squares

Decolorization (%)


MnP Lac

Between

Within

4

10

1696.8**

18.0

0.13926**

0.00121

93069.0**

244.0

12423.4**

80.0


Appendices

268


Yellow R by P. ostreatus and production of lignolytic enzymes with different nitrogen sources

Source of variation

d.f

Mean squares

Decolorization (%)


MnP Lac

Between

Within

4

10

1850.1**

40.3

1.3588**

0.0104

67782.2**

790.3

14681.8**

55.5

MGM = Maize gluten meal, CSL = Corn steep liquor NS = Non-significant (P>0.05); ** = Highly significant (P<0.01) Table 4.28 Analysis of variance of the data on decolorization of Solar Golden

Yellow R by P. ostreatus and production of lignolytic enzymes with varying concentrations of Maize Glutein Meal 60%

Source of variation

d.f

Mean squares

Decolorization (%)


MnP Lac

Between

Within

4

10

2266.6

23.0

0.11957**

0.00074

65376.5**

931.2

14045.6**

75.3



Yellow R by P. ostreatus and production of lignolytic enzymes with low molecular

mass mediators

Source of variation

d.f

Mean squares

Decolorization (%)


MnP Lac

Between

Within

5

12

917.24**

8.58

1.6088**

0.0083

173228.0**

679.0

31525.3**

434.1


Appendices

269


Yellow R by P. ostreatus and production of lignolytic enzymes with different metal ions

Source of variation

d.f

Mean squares

Decolorization (%)


MnP Lac

Between

Within

5

12

2026.8**

35.1

0.08644**

0.00687

359862.0**

57.3

267789.2**

83.3



Yellow R by P. ostreatus and production of lignolytic enzymes with different different dye concentrations

Source of variation

d.f

Mean squares

Decolorization (%)


MnP Lac

Between

Within

4

10

4616.67**

3.92

0.02212**

0.00149

335947.2**

97.4

37490.1**

22.1


Table 4.32 Analysis of variance of the data on adsorption of Solar Golden Yellow

R on mycelia of P. ostreatus

Source of variation

d.f

Mean squares

Adsorption (%)

Final pH

Between

Within

4

10

0.7729**

0.0372

0.12497**

0.00092


Appendices

270

Table 4.34 Analysis of variance of the data on decolorization of Cibanon Blue

GFJ-MD by C. versicolor and production of lignolytic enzymes with different media compositions

Source of variation

d.f

Mean squares

Decolorization (%)

Final pH "Enzyme Activity

(IU/mL)" Lac

Between

Within

4

10

1120.5**

14.0

0.92015**

0.00356

299697.23**

687.12



GFJ-MD by C. versicolor and production of lignolytic enzymes with different carbon sources

Source of variation

d.f

Mean squares

Decolorization (%)


(IU/mL)" Lac

Between

Within

4

10

3437.86**

4.78

0.54062**

0.00359

437183.1**

344.0



GFJ-MD by C. versicolor and production of lignolytic enzymes with varying concentrations of glycerol

Source of variation d.f Mean squares

Decolorization (%)


Lac Between

Within

3

8

1432.99**

2.62

0.3355*

0.0775

10546.0**

48.3


Appendices

271

Table 4.37 Analysis of variance of the data on decolorization of Cibanon Blue GFJ-MD by C. versicolor and production of lignolytic enzymes with different nitrogen sources

Source of variation

d.f

Mean squares

Decolorization (%)


(IU/mL)" Lac

Between

Within

4

10

61.31**

1.22

0.10951**

0.00205

15057.6**

157.3



GFJ-MD by C. versicolor and production of lignolytic enzymes with low molecular

mass mediators

Source of variation

d.f

Mean squares

Decolorization (%)


(IU/mL)" Lac

Between

Within

5

12

93.3**

16.4

2.15987**

0.00428

525054.1**

4545.3



GFJ-MD by C. versicolor and production of lignolytic enzymes with different metal ions

Source of variation

d.f

Mean squares

Decolorization (%)


(IU/mL)" Lac

Between

Within

5

12

1368.3**

17.6

0.38434**

0.00760

484445.0**

483.3


Appendices

272

Table 4.40 Analysis of variance of the data on decolorization of Cibanon Blue GFJ-MD by C. versicolor and production of lignolytic enzymes with different dye concentrations

Source of variation

d.f

Mean squares

Decolorization (%)


(IU/mL)" Lac

Between

Within

4

10

3592.1**

13.6

0.00132NS

0.00179

555976.7**

1780.1


Table 4.41 Analysis of variance of the data on adsorption of Cibanon Blue GFJ-

MD by C. versicolor

Source of variation

d.f Mean squares

Adsorption (%) Final pH

Between

Within

4

10

372.92**

4.97

0.00892NS

0.00949


Syed Agha Hassan - Higher Education...

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