Comparative study of various decolorization processes for ...prr.hec.gov.pk/jspui/bitstream/123456789/2126/2/1621S.pdf · Comparative study of various decolorization processes for

Comparative study of various decolorization processes for treatment of synthetic dyes

By

Shumaila Kiran

M.Phil. Chemistry (UAF)

A Thesis submitted in partial fulfillment of the

requirements for the degree of

Doctor of Philosophy In

Chemistry

Department of Chemistry & Biochemistry, Faculty of Sciences,

University of Agriculture,

Faisalabad, Pakistan.

2012

IN THE NAME OF ALLAH,

THE MOST MERCIFUL AND THE MOST GRACIOUS

ACKNOWLEDGEMENT

All the praises are attributed to the sole creator of the entire whole universe the “ALMIGHTY ALLAH” who bestowed on me power of vision to unravel the mysterious of universe enabling me to make this material contribution to already existing of knowledge. In invoke Allah’s blessings and peace for Hazrat Muhammad (peace be upon him). The most perfect, whose moral and spiritual teaching enlightened my hearts and mind. I would like to express my heartiest and sincerest gratitude to my worthy supervisor Dr. Shaukat Ali, Assistant Professor, Department of Chemistry & Biochemistry, University of Agriculture, Faisalabad for his inspiring guidance, constructive criticism, keen personal interest, invaluable suggestions and close supervision of this manuscript. The completion of this research work would have been a dream for me without his consistent motivation and appreciation. I will ever acknowledge his dextrous guidence, never ending moral support and enlightened supervision. I tender my sincerest thanks to Dr. Farooq Anwar, Assistant Professor, Department of Chemistry & Biochemistry, University of Agriculture, Faisalabad and Dr. Muhammad Ashghar, Professor, Department of Chemistry & Biochemistry, University of Agriculture, Faisalabad , member of my supervisory committee for their co-operation, skillful suggestions, amicable attitude and guidance in analytical work. I want to extend my special thanks to Dr. Munir Ahmed Sheikh, Professor, Department of Chemistry & Biochemistry, University of Agriculture, Faisalabad for his cooperation and providing me technical guidance for carrying out accomplishment of my Ph.D degree. I want to extend my special thanks to Dr. Sofia Nosheen, Assistant Professor, Department of Chemistry & Biochemistry, University of Agriculture, Faisalabad for her cooperation, amiable attitude and providing me sincerel guidance throughout my Ph.D studies. I tender my heartiest and sincerest thanks to Dr. Ijaz Hussain Siddiqi for his help, encouragement, prayers and extreme care during the educational careerer.

This is a great opportunity to pay my special thanks to my friends, Shamyla Nawazish, Naila Mukhtar, Gulnaz Afzal, Nosheen Mujtaba & Maryam Aslam for their Prayers, help and companionship. I am also thankful to Hafiz Muhammad Nasir for his consistent help during lab work.

No Acknowledge could ever adequately express my obligation and indebtedness to my affectionate father Allah Ditta and kind mother Razia Bano and all my beloved teachers who taught even a single word to me during my educational career whose supermoral training and unique impressive life style enabled me to build my personality. This is all because of extreme care and prayers of my parents, brothers, sister and sincere friends whose hands are always raised for successful life. May Allah grant the above cited personalities with long and happiest life, peace of mind, prosperity, honour and greater heights…….. Aamin

Shumaila Kiran

DEDICATION

DEDICATED

TO

That golden part of my life which I have spent with

My Affectionate Parents

Whose hands are always raised for my well-being

even at this moment of time

My Brothers & Sister

Who are the world for me Whose love encouraged me

At every step

& To every one who teach me a single word

during my academic career

Declaration

I hereby declare that the contents of the thesis, “Comparative study of various decolorization

processes for treatment of synthetic dyes” are product of my own research and no part has

been copied form any published source (except the references, standard equations/formulae/

protocols etc). I further declare that this work has not been submitted for award of any other

diploma/degree. The University may take action if the information provided is found inaccurate

at any stage. (In case of any default the scholar will be proceeded against as per HEC plagiarism

policy).

Shumaila Kiran

Regd. No. 2002-ag-2582

To, The Controller of Examination, University of Agriculture, Faisalabad.

“We the supervisory committee certify that the contents and form of thesis submitted by Miss

Shumaila Kiran, Reg. No. 2002-ag-2582 have been found satisfactory and recommended that it

be processed for evaluation, by the external examiner(s) for the award of degree”

SUPERVISORY COMMITTEE

CHAIRMAN : ________________________________ (Dr. Shaukat Ali)

MEMBER : _________________________________ (Dr. Farooq Anwar)

MEMBER : _________________________________ (Prof. Dr. Muhammad Asgher)

CONTENTS

Chapter No.

TITLE Page No.

Acknowledgments

Table of contents

List of Tables

List of figures

Abstract

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 6

3 MATERIALS AND METHODS 26

4 RESULTS & DISCUSSIONS 44

5 SUMMARY 193

LITERATURE CITED 195

TABLE OF CONTENTS

No. Title Page

1 Introduction

2 Review of literature

2.1 Textile industry

2.1.1 Flow chart showing process carried in the textile industry

2.1.2 Schematic representation of operations and the main pollutants from

each step

2.2 Dyeing

2.3 Azo dyes

2.3 Synthesis of azo dyes

2.3.2 Reactive azo dyes

2.4 Conventional methods for the treatment of wastewater containing dyes

2.4.1 Physical methods

2.4.1.1 Adsorption

2.4.1.2 Coagulation/Flucculation

2.4.1.3 Membrane filtration

2.4.1.4 Ultra filtration

2.4.1.5 Reverse osmosis

2.4.1.6 Nanofiltration

2.4.2 Chemical methods

2.4.2.1 Photo catalytic decolorization and oxidation of synthetic dyes

2.4.2.2 Photo catalysis and oxidation with Hydrogen peroxide

2.4.2.3 Ozonation

2.4.2.4 Sodium hypochlorite

2.2 Table showing Advantages and Disadvantages of treatment technologies

for the treatment of dyes wastewaters

2.5 Microbial decomposition of synthetic dyes

2.5.1 Mixed cultures (microorganism consortiums)

2.5.2 Anaerobic decolorization of synthetic dyes

1

6

6

7

8

8

9

10

11

12

14

15

15

15

15

15

15

15

16

16

16

16

16

17

30

18

18

18

2.5.3 Aaerobic decolorization of synthetic dyes

2.6 White rot fungi and their enzymes

3 MATERIALS AND METHODS

3.1 PART ONE: Optimization of conditions for decolorization of Reactive dye

222

3.1.1 Photo-Fenton Treatment Of Reactive dye 222

3.1.1.1 Azo dyes

3.1.1.1a Apparatus

3.1.1.1b Chemicals

3.1.1.1c Instruments

3.1.1.1d Preparation of solutions

i. Preparation of dye solution

ii. Preparation of H2O2 (1 × 10 -2 M) solution

iii. Preparation of FeSO4.7H2O (3.5 × 10 -5 M) solution

3.1.1.2 Optimization of light intensity

3.1.1.3 Optimization of pH

3.1.1.4 Optimization of H2O2

3.1.1.5 Optimization of FeSO4

3.1.1.6 Optimization of temperature

3.1.1.7 Optimization of NaCl

3.1.1.8 Optimization of Na2SO4

3.1.2 Biodecolorization of Reactive dye 222 by white rot fungi

3.1.2.1a Apparatus

3.1.2.1b Chemicals

3.1.2.2 Preparation of inoculum

3.1.2.3 Preparation of biodecolorizable mixture

3.1.2.4 Optimization of biodecolorization process

3.1.2.4a Effect of carbon additives

3.1.2.4b Effect of nitrogen additives

3.1.2.4c Effect of low molecular mass mediators

18

19

20

26

26

26

26

26

26

27

27

27

27

28

28

28

28

28

28

28

29

29

29

30

30

30

31

31

32

33

3.1.2.4d Effect of metal ions

3.1.2.5 Enzyme assays

3.1.2.5a Lignin peroxidase assay

3.1.2.5b Manganese peroxidase assay

3.1.2.5c Laccase assay

3.1.3 Sequential Treatments

3.1.3.1 Photo-Fenton treatment followed by biological treatment

3.1.3.2 Biological treatment followed by Photo-Fenton treatment

3.2 PART TWO: Decolorization of synthetic and Industrial effluents

3.2.1 Photo-Fenton Treatment

3.2.2 Biodecolorization Method




3.3 Evaluation of Treated dye Solutions through quality assurance parameters

Preparation of Solutions for quality assurance parameters

i. Digestion solution

ii. Catalyst solution

iii. Standard Solution of Potassium hydrogen phthalate

iv. 2N potassium dichromate solution (K2Cr2O7)

v. Standard solution of glucose (organic carbon)

vi. Standard solution of phenol

vii. 0.5N NH4OH

viii. Solution of Potassium ferricyanide (K3Fe (CN)6)

ix. Sloution of 4-aminoantipyrine

3.3.1 Biological Oxygen Demend (BOD)

3.3.2 Chemical Oxygen Demand (COD)

3.3.3 Total organic carbons (TOC)

3.3.4 Determination of phenols by the 4-aminoantipyrine

3.3.5 Total suspended sollids (TSS)

3.4 UV-Visible and FTIR analysis

33

34

34

34

35

35

35

36

36

37

37

38

38

38

39

38

39

39

39

39

39

39

39

39

40

40

40

40

41

41

42

3.5 Toxicity test

3.6 Statistical analysis of data

RESULTS AND DISCUSSION


4.1.1 Photo-Fenton,s Treatment




4.1.1.4 Optimization of Temperature



4.1.1.7 UV-Visible and FTIR analysis of Reactive dye 222 after Photo-Fenton

treatment

4.1.2 Biological treatment

4.1.2.1 Screening of white rot fungi for Reactive dye 222

4.1.2.2 Effect of dye concentration

4.1.2.3 Effect of pH

4.1.2.4 Effect of Inoculum size

4.1.2.5 Effect of temperature

4.1.2.6 Effect of additional carbon sources

4.1.2.7 Effect of additional nitrogen sources

4.1.2.8 Effect of mediators

4.12.9 Effect of metal ions

4.1.2.10 UV-Visible and FTIR analysis of Reactive dye 222 by Photo-Fenton

treatment followed by biological (P. ostreatus IBL-02) treatment

4.1.2.11 UV-Visible and FTIR analysis of Reactive dye 222 by biological

(P. chrysosporium IBL-03) treatment



4.1.3.1.1 UV-Visible and FTIR analysis of Reactive dye 222 by Photo-Fenton


42

43

44

44

44

47

49

54

56

58

62

62

65

71

77

82

88

95

102

108

115

118

121

121

123

126

4.1.3.1.2 UV-Visible and FTIR analysis of Reactive dye 222 by photo-Fenton

treatment followed by biological (P. chrysosporium IBL_03) treatment


4.1.3.2.1 UV-Visible and FTIR analysis of Reactive dye 222 by biological

(P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

4.1.3.2.2 UV-Visible and FTIR analysis of Reactive dye 222 by biological

(P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment



4.2.1.1 UV-Visible analysis of one synthetic and three industrial effluents by

Photo-Fenton treatment



biological treatment

4.2.3 Sequential treatments


4.2.3.1.1 UV-Visible analysis of one synthetic and three industrial effluents by





4.3 Evaluation of treated samples through Quality assurance parameters

4.3.1 Biological oxygen demand (BOD)

4.3.2 Chemical oxygen demand (COD)

4.3.3 Total organic carbon (TOC)

4.3.4 Total Suspended Solids (TSS)

4.3.5 Phenolic contents

4.4 Haemolytic Activity (Toxicity Assay)

4.5 Economic analysis of the data

5 SUMMARY

6 LITERATURE CITED

128

131

134

137

137

138

143

148

156

156

161

169

171

180

181

182

185

186

189

191

193

195

197

LIST OF FIGURES Fig Title Page 2.1.1 Flow chart showing the process carried in textile industry 7 2.1.2 Schematic of operations involved in textile cotton industry and the main

pollutants from each step 8

2.3.1 Synthesis of azo dyes 11 2.6.1 Desulphonation after ring cleavage 22 2.6.2 Proposed mechanism for reduction of azo dye 23

4.1.1.1. Effect of pH on the decolorization of Reactive dye 222 by Photo-Fenton treatment

46

4.1.1.2 Effect of H2O2 on the decolorization of Reactive dye 222 by Photo-Fenton treatment

48

4.1.1.3 Effect of FeSO4 on the decolorization of Reactive dye 222 by Photo-Fenton treatment

51

4.1.1.4 Effect of temperature on the decolorization of Reactive dye 222 by Photo-Fenton treatment

53

4.1.1.5 Effect of NaCl on the decolorization of Reactive dye 222 by Photo-Fenton treatment

57

4.1.1.6 Effect of concentrations of Na2SO4 on the decolorization of Reactive dye 222 by Photo-Fenton treatment

59

4.1.1.7a UV-Visible spectrum of reactive dye 222 before Photo-Fenton treatment 60 4.1.1.7b UV-Visible spectrum of reactive dye 222 after Photo-Fenton treatment 60 4.1.1.7c FTIR spectrum of reactive dye 222 before Photo-Fenton treatment 61 4.1.1.7d FTIR spectrum of reactive dye 222 after Photo-Fenton treatment 61 4.1.2.1a Decolorization of Reactive dye 222 by five white rot fungi 64 4.1.2.1b Comparision of ligninolytic enzymes produced by five white rot fungi

during decolorization of Reactive dye 222

64

4.1.2.2a Effect of dye concentrations on decolorization of Reactive dye 222 by P. ostreatus IBL-02

67

4.1.2.2b Effect of dye concentrations on decolorization of Reactive dye 222 by P. chrysosporium IBL-03

67

4.1.2.2c Correlation between decolorization activities of ligninolytic enzymes (U/mL) for effect of dye level by P. ostreatus IBL-02

69

4.1.2.2d Correlation between decolorization activities of ligninolytic enzymes (U/mL) for effect of dye level by P. chrysosporium IBL-03

70

4.1.2.3a Effect of pH on decolorization of Reactive dye 222 by P. ostreatus IBL-02 72 4.1.2.3b Effect of pH on decolorization of Reactive dye 222 by P. chrysosporium 73

IBL-03 4.1.2.3c Correlation between decolorization and activities of ligninolytic enzymes

(U/mL) for effect of pH by P. ostreatus IBL-02 75

4.1.2.3d Correlation between decolorization activities of ligninolytic enzymes (U/mL) for effect of pH by P.chrysosporium IBL-03

75

4.1.2.4a 4a Effect of Inoculum size on decolorization (%) of Reactive dye 222 by P.ostreatus IBL-02

78

4.1.2.4b Effect of Inoculum size on decolorization (%) of Reactive dye 222 by P. chrysosporium IBL-03

79

4.1.2.4c Correlation between Decolorization and activities of ligninolytic enzymes (U/mL) for effect of Inoculum size by P. ostreatus IBL-02

81

4.1.2.4d Correlation between decolorization and activities of ligninolytic enzymes (U/mL) for effect of Inoculum size by P. chrysosporium IBL-03

81

4.1.2.5a Effect of temperature on decolorization of Reactive dye 222 by Pleurotus ostreatus IBL-02

84

4.1.2.5b Effect of temperature on decolorization of Reactive dye 222 by P. chrysosporium IBL-03

84

4.1.2.5c Correlation between decolorization and activities of ligninolytic enzymes (U/mL)) for effect of temperature by P. ostreatus IBL-02

86

4.1.2.5d Correlation between decolorization and activities of ligninolytic enzymes (U/mL) for effect of temperature by P. chrysosporium IBL-03

87

4.1.2.6a Effect of additional carbon sources on decolorization of Reactive dye 222 by P. ostreatus IBL-02

90

4.1.2.6b Comparison of ligninolytic enzymes produced by P. ostreatus IBL-02 during decolorization of Reactive dye 222

90

4.1.2.6c Effect of additional carbon sources on decolorization of Reactive dye 222 by P. chrysosporium IBL-03

91

4.1.2.6d Comparison of ligninolytic enzymes produced by P. chrysosporium IBL-03 during decolorization of Reactive dye 222

91

4.1.2.6e Correlation between decolorization and activities of ligninolytic enzymes (U/mL) for effect of additional carbon sources by P .ostreatus IBL-02

93

4.1.2.6f Correlation between decolorization and activities of ligninolytic enzymes (U/mL) for effect of additional carbon sources by P. chrysosporium IBL-03

94

4.1.2.7a Effect of additional nitrogen sources on decolorization of Reactive dye 222 by P. ostreatus IBL-02

97


97

4.1.2.7c Effect of additional nitrogen sources on decolorization of Reactive dye 222 by P. chrysosporium IBL-03

98

4.1.2.7d Comparison of ligninolytic enzymes produced P. chrysosporium IBL-03 during decolorization of Reactive dye 222.

98

4.1.2.7e Correlation between decolorization and activities of ligninolytic enzymes (U/mL)) for effect of additional nitrogen sources by P. ostreatus IBL-02

100

4.1.2.7f Correlation between decolorization and activities of ligninolytic enzymes (U/mL)) for effect of additional nitrogen sources by P. chrysosporium IBL-03

101

4.1.2.8a Effect of mediators on decolorization of Reactive dye 222 by P. ostreatus IBL-02

103


104

4.1.2.8c Effect of mediators on decolorization of Reactive dye 222 by P.chrysosporium IBL-03

104


105

4.1.2.8e Correlation between decolorization and activities of ligninolytic enzymes (U/mL) for effect of mediators by P. ostreatus IBL-02

107

4.1,2,8f Correlation between decolorization and activities of ligninolytic enzymes (U/mL) for effect of mediators by P.chrysosporium IBL-03

107

4.1.2.9a Effect of metal ions on decolorization of Reactive dye 222 by P. ostreatus IBL-02

110


110

4.1.2.9c Effect of metal ions on decolorization of Reactive dye 222 by P. chrysosporium IBL-03

111


111

4.1.2.9e Correlation between decolorization and activities of ligninolytic enzymes (U/mL) for effect of metal ions by P. ostreatus IBL-02

113

4.1.2.9f Correlation between decolorization and activities of ligninolytic enzymes (U/mL) for effect of metal ions by P. chrysosporium IBL-03

114

4.1.2.10a UV-Visible spectrum of reactive dye 222 before biological (P. ostreatus IBL-02) treatment

116

4.1.2.10b UV-Visible spectrum of reactive dye 222 after biological (P. ostreatus IBL-02) treatment

116

4.1.2.10c FTIR spectrum of reactive dye 222 before biological (P. ostreatus IBL-02) treatment

117

4.1.2.10d FTIR spectrum of reactive dye 222 after biological (P. ostreatus IBL-02) treatment

117

4.1.2.11a UV-Visible spectrum of reactive dye 222 before biological (P. chrysosporium IBL-03) treatment

119

4.1.2.11b UV-Visible spectrum of reactive dye 222 after biological (P. chrysosporium IBL-03) treatment

119

4.1.2.11c FTIR spectrum of reactive dye 222 before biological (P. chrysosporium IBL-03) treatment

120

4.1.2.11d FTIR spectrum of reactive dye 222 before biological (P. chrysosporium IBL-03) treatment

120

4.1.3.1a Effect of Photo-Fenton treatment followed by biotreatment (P. ostreatus IBL-02) on decolorization of Reactive dye 222

121

4.1.3.1b Effect of Photo-Fenton treatment followed by biotreatment

P. chrysosporium IBL-03) on decolorization of Reactive dye 222

122

4.1.3.1.1a UV-Visible spectrum of reactive dye 222 before Photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment

124

4.1.3.1.1b UV-Visible spectrum of reactive dye 222 after Photo-Fenton treatment followed by biological (P.ostreatus IBL-02)treatment

124

4.1.3.1.1.c

FTIR spectrum of reactive dye 222 before Photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment

125

4.1.3.1.1d

FTIR spectrum of reactive dye 222 after photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment

125

4.1.3.1.2a UV-Visible spectrum of reactive dye 222 before Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

127

4.1.3.1.2b -Visible spectrum of reactive dye 222 after Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

127

4.1.3.1.2c FTIR spectrum of of reactive dye 222 before Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

127

4.1.3.1.2d FTIR spectrum of of reactive dye 222 after Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

128

4.1.3.2a Effect of biotreatment (P. ostreatus IBL-02) followed by Photo-Fenton treatment on decolorization of Reactive dye 222

129

4.1.3.2b Effect of biotreatment (P. chrysosporium IBL-03) followed by Photo-Fenton treatment on decolorization of Reactive dye 222

130

4.1.3.2.1a UV-Visible spectrum of reactive dye 222 before biological (P. ostreatus) treatment followed by Photo-Fenton treatment

132

4.1.3.2.1b UV-Visible spectrum of reactive dye 222 after biological (P. ostreatus) treatment followed by Photo-Fenton treatment

132

4.1.3.2.1c FTIR spectrum of reactive dye 222 before biological (P. ostreatus treatment followed by Photo-Fenton treatment

133

4.1.3.2.1d FTIR spectrum of reactive dye 222 after biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

133

4.1.3.2.2a UV-Visible spectrum of reactive dye 222 before biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

135

4.1.3.2.2b UV-Visible spectrum of reactive dye 222 after biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

135

4.1.3.2.2c FTIR spectrum of reactive dye 222 before biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

136

4.1.3.2.2d FTIR spectrum of reactive dye 222 after biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

136

4.2.1 Effect of Photo-Fenton treatment on decolorization of synthetic and industrial effluents

139

4.2.1.1a UV-Visible spectrum of synthetic effluent before Photo-Fenton treatment 139

4.2.1.1b 1b UV-Visible spectrum of synthetic effluent after Photo-Fenton treatment 140

4.2.1.1c UV-Visible spectrum of industrial effluent 1 before Photo-Fenton treatment 140

4.2.1.1d UV-Visible spectrum of industrial effluent 1 after Photo-Fenton treatment 141

4.2.1.1e UV-Visible spectrum of industrial effluent 2 before Photo-Fenton treatment 141

4.2.1.1f UV-Visible spectrum of industrial effluent 2 after Photo-Fenton treatment 142

4.2.1.1g UV-Visible spectrum of industrial effluent 3 before Photo-Fenton treatment 142

4.2.1.1h UV-Visible spectrum of industrial effluent 3 after Photo-Fenton treatment 143 4.2.2a Effect of biological treatment (P. ostreatus IBL-02) on decolorization of

synthetic effluent

144

4.2.2b Effect of biological treatment (P. chrysosporium IBL-03) on decolorization of synthetic effluent

144

4.2.2c Effect of biological treatment (P. ostreatus IBL-02) on decolorization of Industrial effluent 1

145

4.2.2d Effect of biological treatment (P. chrysosporium IBL-03) on decolorization of Industrial effluent 1

145

4.2.2e Effect of biological treatment (P. ostreatus IBL-02) on decolorization of Industrial effluent 2

146

4.2.2f Effect of biological treatment (P. chrysosporium IBL-03) on decolorization of Industrial effluent 2

146

4.2.2g Effect of biological treatment (P. ostreatus IBL-02) on decolorization of Industrial effluent 3

147

4.2.2h Effect of biological treatment (P. chrysosporium IBL-03) on decolorization of Industrial effluent 3

148

4.2.2.1a UV-Visible spectrum of synthetic effluent before biological (P. ostreatus IBL-02) treatment

149

4.2.2.1b UV-Visible spectrum of synthetic effluent after biological (P. ostreatus IBL-02) treatment

149

4.2.2.1c UV-Visible spectrum of synthetic effluent before biological (P. chrysosporium IBL-03) treatment

150

4.2.2.1d UV-Visible spectrum of synthetic effluent after biological (P. chrysosporium IBL-03) treatment

150

4.2.2.1e UV-Visible spectrum of industrial effluent 1 before biological (P. ostreatus IBL-02) treatment

151

4.2.2.1f UV-Visible spectrum of industrial effluent 1 after biological (P. ostreatus IBL-02) treatment

151

4.2.2.1g UV-Visible spectrum of industrial effluent 1 before biological (P. chrysosporium IBL-03) treatment

152

4.2.2.1h 4.2.2.1h UV-Visible spectrum of industrial effluent 1 after biological (P. chrysosporium IBL-03) treatment

152

4.2.2.1i UV-Visible spectrum of industrial effluent 2 before biological (P. ostreatus IBL-02) treatment

153

4.2.2.1j UV-Visible spectrum of industrial effluent 2 after biological (P. ostreatus IBL-02) treatment

153

4.2.2.1k UV-Visible spectrum of industrial effluent 2 before biological (P. chrysosporium IBL-03) treatment

154

4.2.2.1 l UV-Visible spectrum of industrial effluent 2 after biological (P. chrysosporium IBL-03) treatment

154

4.2.2.1m UV-Visible spectrum of industrial effluent 3 before biological (P. ostreatus IBL-02) treatment

155

4.2.2.1n UV-Visible spectrum of industrial effluent 3 before biological (P. chrysosporium IBL-03) treatment

155

4.2.2.1o UV-Visible spectrum of industrial effluent 3 after biological (P. chrysosporium IBL-03)treatment

156

4.2.3.1a Effect of biotreatment (P. ostreatus IBL-02) followed by Photo-Fenton treatment on decolorization of synthetic effluent

157

4.2.3.1b Effect of biotreatment (P. chrysosporium IBL-03) followed by Photo-Fenton treatment on decolorization of synthetic effluent

157

4.2.3.1c Effect of biotreatment (P. ostreatus IBL-02) followed by Photo-Fenton treatment on decolorization of industrial effluent 1

158

4.2.3.1d Effect of biotreatment (P. chrysosporium IBL-03) followed by Photo-Fenton treatment on decolorization of industrial effluent 1

158

4.2.3.1e Effect of biotreatment (P. ostreatus IBL-02) followed by Photo-Fenton treatment on decolorization of industrial effluent 2

159

4.2.3.1f Effect of biotreatment (P. chrysosporium IBL-03) followed by Photo-Fenton treatment on decolorization of industrial effluent 2

159

4.2.3.1g Effect of biotreatment (P. ostreatus IBL-02) followed by Photo-Fenton treatment on decolorization of industrial effluent 3

160

4.2.3.1h Effect of biotreatment (P. chrysosporium IBL-03) followed by Photo-Fenton treatment on decolorization of industrial effluent 3

160

4.2.3.1.1a UV-Visible spectrum of synthetic effluent before Photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment

162

4.2.3.1.1b UV-Visible spectrum of synthetic effluent after Photo-Fenton treatment followed by biological (P. ostreatus IBL-02)) treatment

162

4.2.3.1.1c UV-Visible spectrum of synthetic effluent before Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

162

4.2.3.1.1d UV-Visible spectrum of synthetic effluent after Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

163

4.2.3.1.1e UV-Visible spectrum of industrial effluent 1 before Photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment

163

4.2.3.1.1f UV-Visible spectrum of industrial effluent 1 after Photo-Fenton treatment 164

followed by biological (P. ostreatus) treatment 4.2.3.1.1g UV-Visible spectrum of industrial effluent 2 before Photo-Fenton treatment

followed by biological (P. chrysosporium IBL-03) treatment 164

4.2.3.1.1h UV-Visible spectrum of industrial effluent 2 after Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

165

4.2.3.1.1i UV-Visible spectrum of industrial effluent 2 after Photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment

165

4.2.3.1.1j UV-Visible spectrum of industrial effluent 2 before Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

166

4.2.3.1.1k UV-Visible spectrum of industrial effluent 2 after Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

166

4.2.3.1.1l UV-Visible spectrum of industrial effluent 3 before Photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment

167

4.2.3.1.1m

UV-Visible spectrum of industrial effluent 3 after Photo-Fenton treatment followed by biological (P.s ostreatus IBL-02) treatment

167

4.2.3.1.1n UV-Visible spectrum of industrial effluent 3 before Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

168

4.2.3.1.1o UV-Visible spectrum of industrial effluent 3 after Photo-Fenton treatment followed by biological P. chrysosporium IBL-03) treatment

168

4.2.3.2a Effect of biotreatment (P. ostreatus IBL-02) followed by Photo-Fenton treatment on decolorization of one synthetic and three industrial effluents

169

4.2.3.2b 4.23.2a Effect of biotreatment (P. chrysosporium IBL-03) followed by Photo-Fenton treatment on decolorization of one synthetic and three industrial effluents

170

4.2.3.2.1a UV-Visible spectrum of synthetic effluent before biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

171

4.2.3.2.1b UV-Visible spectrum of synthetic effluent after biological (P. ostreatus IBL-02) treatment followed by photo-Fenton treatment

172

4.2.3.2.1c UV-Visible spectrum of synthetic effluent before biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

172

4.2.3.2.1d UV-Visible spectrum of synthetic effluent after biological P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

173

4.2.3.2.1e UV-Visible spectrum of industrial effluent 1 before biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

173

4.2.3.2.1f UV-Visible spectrum of industrial effluent 1 after biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

174

4.2.3.2.1g UV-Visible spectrum of industrial effluent 1 before biological 174


4.2.3.2.1h UV-Visible spectrum of industrial effluent 1 after biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

175

4.2.3.2.1i UV-Visible spectrum of industrial effluent 2 before biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

175

4.2.3.2.1j UV-Visible spectrum of industrial effluent 2 after biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

176

4.2.3.2.1k UV-Visible spectrum of industrial effluent 2 before biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton

176

4.2.3.2.1l UV-Visible spectrum of industrial effluent 2 after biological


177

4.2.3.2.1m

UV-Visible spectrum of industrial effluent 3 before biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

177

4.2.3.2.1n UV-Visible spectrum of industrial effluent 3 after biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

178

4.2.3.2.1o UV-Visible spectrum of industrial effluent 3 before biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

178

4.2.3.2.1p UV-Visible spectrum of industrial effluent 3 after biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

179

4.4a Toxicity assay of treated Reactive dye 222 by Photo-Fenton, biological and sequential treatment

192

4.4b Toxicity assay of treated synthetic and industrial effluents by Photo-Fenton, biological and sequential treatment

192

LIST OF TABLES Table Title Page

2.1 Advantages and Disadvantages of Treatment technologies for Dye removal from Industrial Wastewaters

17

3.1 Composition of Kirk’s basal nutrient medium for white rot fungi 31 4.1.1.1a Decolorization of Reactive dye 222 by Photo-Fenton with varying

pH 45

4.1.1.1b Analysis of variance on decolorization of Reactive dye 222 by Photo-Fenton with varying pH

46

4.1.1.2a Decolorization of Reactive dye 222 by Photo-Fenton with varying H2O2

48

4.1.1.2b Analysis of variance on decolorization of Reactive dye 222 by Photo-Fenton with varying H2O2

49

4.1.1.3a Decolorization of Reactive dye 222 by Photo-Fenton with varying FeSO4

50

4.1.1.3b Analysis of variance on decolorization of Reactive dye 222 by Photo-Fenton with varying FeSO4

51

4.1.1.4a Decolorization of Reactive dye 222 by Photo-Fenton with varying temperature (˚C)

52

4.1.1.4b Analysis of variance on decolorization of Reactive dye 222 by Photo-Fenton with varying temperature

53

4.1.1.5a Decolorization of Reactive dye 222 by Photo-Fenton with varying concentrations of NaCl

54

4.1.1.5b Analysis of variance on decolorization of Reactive dye 222 by Photo-Fenton with varying concentrations of NaCl

55

4.1.1.6a Decolorization of Reactive dye 222 by Photo-Fenton with varying concentrations of Na2SO4

57

4.1.1.6b Analysis of variance on decolorization of Reactive dye 222 by Photo-Fenton with varying concentrations of Na2SO4

58

4.1.2.1 Screening of WRF cultures for decolorization of Reactive dye 222 61 4.1.2.2a Decolorization of Reactive dye 222 by P. ostreatus IBL-02 with

concentrations of Reactive dye 222 66

4.1.2.2b Decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with different concentrations of Reactive dye 222

66

4.1.2.2c Analysis of variance on percent decolorization of Reactive dye 222 by P. ostreatus IBL-02 with different levels of Reactive dye 222.

68

4.1.2.2d Analysis of variance on percent decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with different levels of Reactive dye 222

69

4.1.2.3a Decolorization of Reactive dye 222 by P. ostreatus IBL-02 with varying pH value

71

4.1.2.3b Decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with varying pH values

72

4.1.2.3c Analysis of variance on percent decolorization of Reactive dye 222 by P. ostreatus IBL-02 with varying pH

74

4.1.2.3d Analysis of variance on percent decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with varying pH

74

4.1.2.4a Decolorization of Reactive dye 222 by P. ostreatus IBL-02 with varying Inoculum size

77

4.1.2.4b Decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with varying Inoculum size

78

4.1.2.4c Analysis of variance on percent decolorization of Reactive dye 222 by P. ostreatus IBL-02 with varying Inoculum size

80

4.1.2.4d Analysis of variance on percent decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with varying Inoculum size

80

4.1.2.5a Decolorization of Reactive dye 222 by P. ostreatus IBL-02 with varying temperature

83

4.1.2.5b Decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with varying temperature

83

4.1.2.5c Analysis of variance of the data on percent decolorization of Reactive dye 222 by by P. ostreatus IBL-02 with varying temperature

85

4.1.2.5d Analysis of variance on percent decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with varying temperature

86

4.1.2.6a Decolorization of Reactive dye 222 by P. ostreatus IBL-02 with varying Carbon sources

88

4.1.2.6b Decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with varying carbon sources

89

4.1.2.6c Analysis of variance on decolorization of Reactive dye 222 by by P. ostreatus IBL-02 with varying carbon sources

92

4.1.2.6d Analysis of variance on decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with varying carbon sources

93

4.1.2.7a Decolorization of Reactive dye 222 by by P. ostreatus IBL-02 with varying nitrogen sources

96

4.1.2.7b Decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with varying nitrogen sources

96

4.1.2.7c Analysis of variance on decolorization of Reactive dye 222 by P. ostreatus IBL-02 with varying additional nitrogen sources

99

4.1.2.7d Analysis of variance on decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with varying additional nitrogen sources

100

4.1.2.8a Decolorization of Reactive dye 222 by by P. ostreatus IBL-02 with different mediators

102

4.1.2.8b Decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with different mediators

103

4.1.2.8c Analysis of variance on decolorization of Reactive dye 222 by by P. ostreatus IBL-02 with different mediators

106

4.1.2.8d Analysis of variance on decolorization of Reactive dye 222 by P.

chrysosporium IBL-02 with different mediators 106

4.1.2.9a Decolorization of Reactive dye 222 by by P. ostreatus IBL-02 with different metal ions

109

4.1.2.9b Decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with different metal ions

109

4.1.2.9c Analysis of variance of the data on decolorization of Reactive dye 222 by P. ostreatus IBL-02 with different metal ions

112

4.1.2.9d Analysis of variance on decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with different metal ions

113

4.3 Characterization and permissible limits of wastewater 180 4.3.1 Comparison of water quality parameter (BOD) by different

treatment methods 182

4.3.2 Comparison of water quality parameter (COD) by different treatment methods

184

4.3.3 Comparison of water quality parameter (TOC) by different treatments methods

186

4.3.4 Comparison of water quality parameter (TSS) by different treatment methods

188

4.3.5 Comparison of water quality parameter (Phenolics) by different treatment methods

190

4.5 Economic cost per functional unit of the different inputs for considered treatments

193

ABSTRACT

Azo dyes constitute the largest and most diverse group of dyes used in commercial

applications. These dyes are carcinogenic and mutagenic in nature, as well as also create an

aesthetic problem, so these must be removed before their disposal into water bodies. Various

methods are in use for the decolorization of azo dyes. Every method has its own shortcomings

and drawbacks. In this study, a comparative study was conducted to get the maximum

decolorization of synthetic azo dyes and industrial effluents. Photo-Fenton’s process, biological

and sequential methods were utilized for the decolorization of synthetic and real wastewater

having azo dyes. In biological method, experiments was performed with five locally isolated

indigenous white rot species, for the selection of two white rot fungal cultures based on their

maximal decolorization potential. Different fermentation conditions (dye level, pH, inoculums

size, temperature, mediators and metal ions) and nutritional factors (carbon and nitrogen

sources) were optimized to enhance the efficiency of white rot fungal cultures for dye

decolorization. In Photo-Fenton,s treatment method, the optimization of different experimental

parameters (pH, FeSO4, H2O2, temperature and effects of salts) was done to get maximum

decolorization (90%) of dye under study. Sequential methods were also studied) to investigate

their effectiveness in the present study. The effectiveness of all treatment technologies was

evaluated by water quality assurance parameters such as COD, BOD, TOC, TSS, phenolic

contents ant toxicity assey, by following the standard methods of treatments. All treatments

under study showed a good potential towards decolorization as well as mineralization, however,

Sequential treatments showed best potential towards decolorization (up to 97%) as well as

mineralization (up to 90%) of synthetic azo dyes. The uv-visible and FTIR spectral studies have

shown decolorization as well as mineralization of dyes under study. An economic analysis has

shown as the cost in the chemical treatment (Photo-Fenton treatment is considered mainly due to

the chemicals, thus at lower doses (it is applied as a pre-treatment step), operating cost of the

treatment can be saved. It was also found, that as the sequential carried out at lower dose of

chemicals, so sludge production was almost negligible and the dye wastewater after sequential

treatments fall within the safer limits, hence dispose off such treated water not be hazardous.

Thus the overall treatment chain of Photo-Fenton oxidation followed by aerobic biological

treatment could be quite effective and economical option for the treatment recalcitrant

compounds like azo dyes in pilot plant scale.

1

CHAPTER 1

INTRODUCTION

Textile industries are found in most of the countries and their progress depends on the

progress of textile sector. These industries have shown a significant increase in the use of

synthetic complex organic dyes as the coloring material. The annual world production of textiles

is about 30 million tones requiring 700,000 tonnes of different dyes (Zollinger, 1987) which is

responsible for considerable environmental pollution. Dyes include a broad spectrum of different

chemical structures, primarily based on substituted aromatic and heterocyclic groups such as

aromatic amine which is a suspected carcinogen. A large number of dyes are azo compounds (-

N=N-), which are linked by an azo linkage (McCurdy at al., 1992).

The azo dyes have great structural variations and hence, offer a great variety of colors.

Azo dyes are classified into various classes including reactive dyes being the most important of

them. Reactive dyes form a covalent bond with fabric on which they are applied (Kirkothmer,

1979). The release of azo dyes not only causes a negative aesthetic effect but also some dyes are

toxic or even carcinogenic. Color is the first contaminant to be recognized in wastewater and has

to be removed before discharging into water bodies. The presence of very small amounts of dyes

in water (less than 1 ppm for some dyes) is highly visible and affects the aesthetic merit, water

transparency and gas solubility in lakes, rivers and in other water bodies. The removal of color

from wastewaters is often more important than the removal of the soluble colorless organic

substances (McCurdy et al., 1992).

The wastewater characteristics from a dye house are highly variable from day to day,

depending on the type of dye, the type of fabric and the concentration of the agents added. The

discharge of dye house wastewater into the environment damages the quality of the receiving

streams and is toxic to aquatic life (Meyer, 1981). Therefore, treatment of industrial effluent

having azo dyes is deemed necessary befor their discharge into wastewater bodies. As the

characteristics of dye wastewater are very variable, many different physical, chemical and

biological treatment methods are in use for its treatment; which one is effective treatment depend

upon the type of dye wastewater (McCurdy et al., 1992).

2

Various Physical methods are numerous including anion exchange resins (Karcher et al.,

2002), flotation (Lin and Lin, 1993), electro flotation (Ogfitveren & Koparal, 1994),

electrochemical destruction (Ulker & Savas, 1994), adsorption and the use of activated carbon

(Pala et al., 2002), etc. One major drawback with these methods is that they are non-destructive

and unable to completely remove the recalcitrant azo dyes and/or their organic metabolites,

generating a significant amount of sludge that may cause secondary pollution problems (Zhang

et al., 2004). Therefore, new and different type of pollution is faced and further treatments are

required.

Chemical oxidation methods include a family of processes that may be appropriate for

treating organic pollutants called as advanced oxidation processes (AOPs). The research and

development in advanced oxidation technologies (AOTs) for water remediation have made great

progress in recant years (Zhang et al., 2004; Shu & Chang, 2005; Shu, 2006), especially the

H2O2 based processes (Ksibs, 2006). Many systems are classified under this wide definition of

AOTs. These include Fenton, Photo-Fenton, Photocatalysis, ozonation, chlorination, electro-

chemical oxidation, coagulation, precipitation etc. They are based on generation of highly potent

oxidants such as hydroxyl radical (.OH), a powerful non-selective chemical oxidant that is a

reactive intermediate, which has a strong potential and acts very effectively with most organic

pollutants. Hydroxyl radical (OH.) attacks organic molecules by abstracting hydrogen atom. The

high reactivity of radicals in driving oxidation processes is suitable for achieving the complete

abatement and often mineralization of the pollutants (Malato et al., 2002).

Chlorine has been reported to be a good dye oxidizing agent and has been applied at low

operating costs. However the potential of chlorine to form undesirable compounds with nitrogen

containing components of the wastewater has limited the acceptability of this method (EPRI,

1996). Ozone is one of the most effective means of decolorizing the wastewater, with no residual

or sludge formation (Ince and Gonene, 1997) and no toxic metabolites (Gahr and Oppermann,

1994). The disadvantage of ozonation is its short half-life (typically 20min) demanding

continuous application (Xu and Lebrun, 1999), high cost and aldehyde formation prevents its

wide acceptance. Coagulation cause near-complete decolorization and water reuse is possible.

However sludge disposal still remains a problem (Kim et al., 2004).

Most of the AOTs use a combination of strong oxidants e.g. O3 and H2O2, with catalysts,

e.g. transition metal ions or photo catalysts, and irradiation, e.g. ultraviolet, visible etc. They are

3

based on generation of highly potent oxidants such as hydroxyl radical (OH.), a powerful non-

selective chemical oxidant, that is a reactive intermediate, which has a strong potential and acts

very effectively with most organic pollutants. This hydroxyl radical attacks organic molecules by

abstracting hydrogen atom (Huston et al., 1999).The high reactivity of radicals in driving

oxidation processes is suitable for achieving the complete abatement and often mineralization of

the pollutants (Malato et al., 2002). The nonspecific reactivity of hydroxyl radicals make them

potentially useful for degradation of a wide range of organic compounds (Yaser et al., 2006).

Fenton reagent and its modification have also received great attention as means for

decolorization of synthetic dyes (Neamtu et al., 2004). Light exposure time has an incremental

effect on color removal (Hanzon & Vigilia, 1999).

Fenton’s reagent is very effective for the oxidation of pollutants present in wastewater

(Pak and Chang, 1999). Advantages of this process include color and toxicity reduction

(Robinson et al., 2001). The decolorization and mineralization of dyes using solar light assisted

Fenton and Photo-Fenton reaction is great promising for good color removal (Torrades et al.,

2004). Although Photo-Fenton is quite effective but is economically less feasible as they require

more energy and cost of chemicals.

A number of previous studies have shown that microbial or enzymatic decolorization of

azo dyes is cost-effective and eco-friendly alternative to chemical treatment methods that might

decrease water consumption rate as compared to physio-chemical treatment methods (Rai et al.,

2005). In recent years, a number of studies have employed microorganisms which are able to

bio-decolorize dyes in wastewaters. A wide variety of microorganisms capable of decolorizing a

wide range of dyes include bacteria, fungi and yeasts. The decolorization of dye containing

wastewater by white rot fungi is a very effective, robust, economical and eco-friendly way

(Sanghi et el., 2006).

White rot fungi have been widely studied in regards to xenobiotic degradation. White rot

fungi are capable of degrading dioxins, polychlorinated biphenyls (PCBs) and other chloro-

organics (Reddy, 1995). White rot fungi having potential of decolorizing dyes include

Phanerochaete chrysosporoum, Pleurotus ostreatus, Pleurotus florida and Coriolus versicolor

(Knapp & Newby, 1999), Tramates versicolor (Wong & Yu, 1999; Libra et al., 2003) and

Funalia trogii (Yesilada et al., 1995). The major mechanism is biodecolorization because they

can produce the lignin modifying enzymes. Laccase, manganese peroxidase (MnP) and lignin

4

peroxidase (LiP) to mineralize synthetic dyes (Raghukumar et al., 1996; Fu & Viraraghavan,

2001). The relative contribution of LiP, MnP and laccase to the decolorization of dyes may be

different for each fungus (Pasti-Grigsby at al., 1992).

Long time duration can be required for the disappearance of the active dye molecule and

its complete mineralization during biodecolorization. There is a still lack of information

concerning the different steps of the mineralization and the formed by-products. Therefore, the

integration of two processes, photocatalysis and biological treatment can be helpful, in order to

reduce the costs (Guilard et al., 2003).

Many studies have reported the use of combination methods for the removal of dyes and

colorants from wastewaters (Kim et al., 2004; Neamtu et al., 2004) and have been to be the ideal

solution for bioremediation of wastewaters. Sequential treatments have been proposed to treat

the wastewater generated by the textile industry containing azo dyes and other dyes as well,

including UV or Photo-Fenton followed by biological treatment. Mineralization of azo dyes was

also examined in a two stage sequential Fenton’s oxidation followed by aerobic biological

treatment (Guilard et al., 2003). Potential advantages of the strategy of combining chemical and

biological processes to treat contaminants in wastewater have also previously been suggested

(Pasti-Grigsby at al., 1992). Advance oxidation processes are used as a pre-treatment step for the

enhancement of the biodegradability of wastewater comprising recalcitrant compounds i.e.

textile waste, can potentially be justified if the intermediate products formed during the reaction

can readily be degraded by microbial consortia in a biological treatment plant Fu &

Viraraghavan, 2001).

The present project was focused on investigating the feasibility of Photo-Fenton reaction,

biological aerobic treatment and sequential treatment for decolorization/degradation of azo dyes

and effluents. The target dye for this research project was chosen from a class of reactive azo

dyes being extensively used and was known to be problematic in terms of both treatibility and

toxicity (Lars and Mallika, 1997). The experimental work was focused on the following

objectives.

Aims and Objectives

1. To optimize the different physico-chemical parameters by both chemical and biological

methods, for the decolorization as well as de-toxification of azo dyes.

5

2. To study the potential of sequential treatment technologies for the detoxification of dye and

effluents at optimized conditions for degradation of selected dyes.

3. To develop an economical and industrial wastewater treatment technology for decolorization

and mineralization of dyes.

4. Spectral analysis of decolorization and dgradation of reactive dyes and effluents by different

treatment technologies.

6

CHAPTER 2

REVIEW OF LITERATURE

A number of synthetic dyes are being used for textile dyeing, printing and in leather

industries. The structural variation of dyes derives from the use of different chromophoric groups

e.g. azo, anthraquinone, triarylmethane and phthalocyanine groups (Heinfling et al., 1998,

Bandyopadhay and Chattopadhay, 2007). The azo group is found in almost all groups of textile

dyes (e.g. disperse, acid, basic and reactive dyes) and it plays an important role in dyestuffs

chemistry. In Pakistan, textile industries spread over large scale which discharges large quantity

of their effluents directly in to water bodies such as rivers, streams and soils. The presence of

dyes in industrial wastewaters is of great concern because loss of dye in textile wastewaters

produces potentially hazardous products, which cause toxicity as well as aesthetic problems, so

the treatment of textile waste water is of great concern (Bandyopadhay and Chattopadhay, 2007).

In this chapter, main focus will be paid to explain the problems created by the presence of azo

dyes in textile wastewater. Various processes used in the textile sector will be highlighted in

order to present a comprehensive view of the problems being faced by the discharge of textile

effluents into the wastewater bodies. Effective treatment methodology for the treatment of textile

wastewater having azo dyes will also be explained. Every process has its own limitations in

terms of costs, environmental impact, sludge production and potential toxic byproducts.

2.1 Textile Industry

As in Pakistan, textile industries are wide spread, so in order to make effluent problems

more understandable being faced by the textile industry, it seems to be deem necessary to have a

clear image of processes which result in effluent production. Fig. 2.1

As textile industry used azo dyes in dyeing and printing which is the main focus of this project,

so it will be described in somewhat detail here.

7

Fig.2.1 Flow chart showing the process carried in textile industry

8

Fig. 2.2. Schematic operations involved in textile cotton industry and main

pollutants from each step

9

2.2 Dyeing

The dyes which are most commonly used for dyeing purpose include direct dyes, reactive

dyes, vat and sulphur dyes. The most important are the reactive ones due to their extra ordinary

properties than other dyes. Different dyes use different dyeing procedures depending on their

nature; however water is used in all of them mostly used as a solvent to facilitate the dyeing

process (Carliell, 1993). Dyeing processes vary greatly in the amount and type of wastes

produced. Exhaust dyeing, continuous dyeing and batch dyeing are mostly used. The continuous

and batch dyeing generally use much lower quantities of chemicals and water than exhaust

method (Carliell, 1993). Due to the continuous nature of the process, the waste streams are easily

segregated for heat recovery and other management strategies. The volume of dye effluent

emerging from dyeing process depends on the volume of azo dye solution used to dye a kilogram

of fabric. The dye treated by batch process, when it is discharged in to the waste water bodies

contains 80-90% of the unreacted dyes and auxiliary chemicals giving rise to the concentrated,

intense color to the effluent (Buckley, 1992).

Presence of color and its causative compounds has been undesirable in water used for

either industrial need. A dye house effluent contains dye in the range of 0.6-0.8gL-1, Hence color

and dye removal, has recently become an area of major scientific concern as indicated by a

number of research reports (Gahr et al., 1994).

It is necessary to review the chemical composition and binding of the dye molecules with fabric,

in order to understand the mechanism of degradation of dyes which are hazardous in nature.

The annual world production of textiles is about 30 million tones requiring 700,000

tonnes of different dyes (Zollinger, 1987) which is responsible for considerable environmental

pollution. Dyes include a broad spectrum of different chemical structures, primarily based on

substituted aromatic and heterocyclic groups such as aromatic amine which is a suspected

carcinogen, phenyl and naphthyls, the only thing in common is their ability to absorb light in the

visible region. A number of dyes are azo compounds

(-N=N-), which are linked by an azo group (McCurdy et al., 1992).

The azo dyes, the largest class of dyes, have great structural variations and hence, offer a

great variety of colors. The biggest users of these dyes are the textile, paper, pulp, tannery and

Kraft bleaching industries which are the most potential contributors as far as color pollution is

concerned (Zhang et al., 2003).

10

2.3 Azo Dyes

This class accounts for 60-70% of all dyes.Azo compounds having the functional group

R-N=N-R', in which R and R' can be either aryl or alkyl. According to IUPAC system, azo

compounds are “Derivatives of diazine (diimide), HN=NH, in which both hydrogens are

substituted by hydrocarbyl groups, e.g. PhN=NPh azobenzene or diphenyldiazene.”

(Encyclopedia)

The more stable derivatives contain two aryl groups. The -N=N- group is called an azo

group. Due to involvement of п-delocalization, aryl azo compounds have different colors,

especially reds, oranges, and yellows. Therefore, they are used as dyes , and are commonly

known as azo dyes, example of which are Disperse orange 1 and Yellow azo dye. They have

excellent coloring properties as well as others. The lightfastness depends on the properties of the

organic azo compound and on the way they attach with fiber. Some azo dyes are non-toxic,

although some have been found to be mutagenic

(“Compendium of chemical Terminology”, 2nd ed.).

Yellow azo dye Chrysoidine dye

Basic red 29 Acid orange 7

11

2.3.1 Synthesis of azo dyes

Overview of azo dye synthesis is given below:

Stage 1- Diazotization

It involves a primary aromatic amine, called the diazo component which is treated with

nitrous acid (generated in situ from the reaction of NaNO2 & HCl) in low temperature conditions

to form an unstable diazonium salt.

Stage 2- Azo coupling

The diazonium salt reacts with a coupling reagent (for example a phenol or an aromatic

amine) to form the stable azo dye.

Different dyes have different substituents or functional groups at ortho, meta or para

positions, have a great variety of structurally variable dyes are used in the industries (McCurdy,

1991).

2.3.2 Reactive azo dyes

There are different types of reactive azo dyes being the most important of them. The

present study will focus on reactive azo dyes. Reactive dyes are categorized by functional group.

Majority of the reactive azo dyes (up to 90%) have azo linkage as chromophoric group (Edward,

2000).

The fibre-reactive dyes contain the reactive groups, which react with cellulose in mild alkali

solution. ICI launched a number of dyes based on this chemistry, called the procion dyes. This

new range was superior than vat and direct dyes, having excellent wash fastness and a broad

12

range of brilliant colors. Procion dyes can be applied in batches, or in continuous manner.

The general structural representation of a fiber-reactive dye is shown below:

The bridging group links the chromogen and the fiber-reactive group. The bridging group

is an amino, -NH-, group. The fiber-reactive group is the only part of the molecule which reacts

with the fiber. The different types of fibers-reactive group will be discussed below. A cellulose

polymer has hydroxyl functional groups, which reactive dyes utilize as nucleophiles. Under

alkaline conditions, the cellulose-OH groups are forced to remove proton to give cellulose-O-

groups. These can then attack electron-deficient regions of the fibre-reactive group, and perform

either aromatic nucleophilic substitution or nucleophilic addition to alkenes.

Reactive dyes have a functional group by which it actually forms a covalent bond with

the fiber molecule. Once the bond is formed, the dye molecule becomes an actual part of it

(Zollinger, 1991). Fiber reactive dyes can be used as acid dyes as well on protein fibers,

including wool and silk. In general, the greater the number of polar groups that are present in the

fiber, the easier the fabric is to dye (www.pburch.net).

2-Naphthyl orange

In a reactive dye a chromophore ontains a sunbstituent that is activated and allowed to

react directly to the surface of the substrate.

13

R = Chromophore

Cell = Cellulose

Reactive dyeing is the important method for the coloration of cellulosic fibres. Reactive dyes can

be applied on wool and nylon (www.indiamart.com/supremodyestuff).

A number of researchers have shown that almost 10-15% of dye is lost during its

application. In most cases, up to 90% of azo dyes go in to the waste as unreacted form. Color has

to be removed before discharge in to the water bodies. The presence of very small amount of azo

dyes in water (less than 1ppm for some dyes) is highly visible and effects water transparency

indicating water pollution. So, the removal of color is the major area of concern for

researchers (McCurdy et al., 1992).

This study directly involves the major emphasis on the study of metabolites

generate when azo dyes are subjected to subsequent different degradation treatments. Azo dyes

having nitro groups (one or more) are found to be mutagenic in nature. Moreover, some of the

azo dyes can lead to the production of toxic compounds upon degradation. The toxic compounds

which may produce are benzidine and its derivatives, amino benzene derivatives, amino naphthol

etc. (Rosenkranz and Kalpmen, 1989).

14

Benzidine

Moreover, industrial effluents having heavy metals can make a complex with azo dyes,

which render them underground when proceed by a treatment method. The use of salts is also

common to push the fiber reaction dye from solution to become attached on fabric. By which

electrolyte (salt) concentration and hence, conductivity of textile effluents increase to a

considerable extent which create toxicity problems. A dye house effluent typically contains 0.6‐

0.8 gl‐1 dye. Hence color removal has recently become an area of major scientific interest as

indicated by a number of research reports. Waste water treatment using physical, chemical and

biological or sequential methods are well known for color removal.

2.4 Conventional methods for treatment of wastewater containing dyes

As discharge standards are becoming more stringent, the development of eco-

technological strategies for minimizing concentration of dyes and their conversion into less toxic

or even harmless products in wastewater are deem necessary. The following methods are

generally in use for the removal of color from wastewaters.

2.4.1 Physical methods

2.4.1.1 Adsorption

Among physical methods adsorption is very effective in the treatment of industrial

effluent. Different types of adsorbents are known and are in use. Activated carbon is commonly

used adsorbent which is effective in removing organic compounds from the industrial effluent.

After use, it must be regenerated or disposed off. Other adsorbents include silica gel, cinder ash

15

and various clays. Bioadsorbants are also in use which is naturally occurring polymers which are

biodegradable or which act as ion-exchangers. Cellulose bioadsorbents are used effectively to

remove color in effluents (Gahr et al., 1994).

2.4.1.2 Coagulation/ Flocculation

This is a effective method for purification of water. Inorganic (alum, lime and iron salts)

and organic (polymers) coagulants are used to treat dye wastewater for color removal, either

alone or in combination with one another. Alum is more effective one in color removal from

textile effluents containing other dyes as well (Gahr et al., 1994).

2.4.1.3 Membrane Process

Membrane technologies have the potential either to remove the dyestuff or allow reuse of

the auxiliaries used for dyeing. This method has the potential to clarify, concentrate and most

importantly to separate dye from effluent (Mishra and Tripathy, 1993; Xu and Lebrun, 1999).

(i) Ultra filtration: Ultra filtration has advantages such as the dyes recovery and water or the

reusing of them. Membrane transport properties are affected by membrane thickness. The

process of dye separation speeds up by increasing the time of solvent evaporation and the

temperature of the solution decrease (Xu and Lebrun, 1999).

(ii) Reverse osmosis: It is sometimes referred to as hyper filtration, is a process in which water

is made to flow forcefully through a semi permeable membrane. It is suitable for removing ions

from dye bath effluents (Marcucci, et al. 2001).

(iii) Nanofiltration: Nanofiltration is a process of separation whose performance characteristics

lies between reverse osmosis and ultra filtration. Stoyko and Pencho (2003) worked out on the

purification of water having reactive dye, using nanofiltration,. They demonstrated dye retention

of 85 – 90 %, showed satisfactory for the reuse of the water.

2.4.2 Chemical methods

2.4.2.1 Photo catalytic decolourisation and oxidation of synthetic dyes

Commercial dyes are seemed to resist photo degradation, so the optimized photo catalytic

conditions for the decolourisation of azo dyes requires considerable attention. The use of

UV/H2O2 is a effective mean of removal of dyes which result in the production of OH. Free

radical which is responsible for the degradation of dyes. Due to the environmental interest, the

16

efficiency of a number of catalysts and irradiation conditions are well known for the

decolourisation of various synthetic dyes (Young et al., 1997).

2.4.2.2 Photocatalysis and Oxidation with Hydrogen Peroxide

Hydrogen peroxide is applied frequently to the decolourisation of industrial effluent

having synthetic dyes. Hydrogen peroxide can decolorize industrial effluent in the presence of Fe

(II) sulphate, very effectively (Kuo, 1992). The results showed that the method could be applied

successfully for the decolorisation of a number of dyes but it ineffective for vat and disperse dyes

(Yang et al., 1998).

2.4.2.3 Ozonation

Ozonation has found its application in the decolorisation of synthetic dyes (Tang and An,

1995). Dye effluents react differently with ozone depending on their composition. A report given

by Nansheng et al., (1997), it was founded that ozone effectively decolorizes azo dyes in

wastewater having dyes. Moreover, it was found that the rate of photo degradation is dependent

on the chemical structure of the dye (Nansheng et al., 1997).

2.4.2.4 Sodium Hypochlorite (NaOCl)

The dye having amino group can successively undergo degradation by this method. The

Cl-1 generated from NaOCl cause chemical destruction of this group, which ultimately lead to the

azo bond cleavage (Bannat et al., 1996).

17

Advantages and Disadvantages OF Treatment technologies for Dye removal

from Industrial Wastewaters

Methods Advantages Disadvantages

Activated carbon Effective for removal of a

variety of dyes

Ineffective for disperse and

vat dyes

Silica gel Effective for removal of

basic dyes

Least effective on

commercial scale

Membrane filtration Effective for all dyes High pressures, expensive

Ion exchange Regeneration of adsorbent Ineffective against disperse

dyes

Electro kinetic coagulation Economical viable sludge production disposal

constraints

Fenton’s reagent Potential for removal of

soluble and insoluble dyes

Formation of byproducts

Photo-Fenton’s reagent A very good Potential for

removal of dyes

May produce toxic products

Ozonation Applied in gaseous Short half life, continuous

supply is necessary

Photochemical No sludge production Production of toxic

byproducts

NaOCl Effective for cleavage of

azo linkage

Formation of byproducts

Electrochemical destruction Products are non toxic High cost of electricity

Biodegradation Cost effective, effective for

removal of all types of dyes

Slow process, effective at

optimal conditions

In view of these disadvantages, removal by dyes by biological materials has gained momentum

from 1990’s.

18

2.5 Microbiological decomposition of synthetic dyes

The use of microorganisms for the biodegradation of synthetic dyes is an effective

method the use of microorganisms for treatment of industrial effluents having dyes offers

considerable advantages. It is relatively inexpensive, low cost and eco-friendly. The end products

of complete mineralization are less toxic or even non-toxic, as reviewed by Stolz (2001).

Unfortunately, the some dyes are chemically stable and hence, resistant to microbial attack. The

isolation of new microbial strains or the use of existing ones for the degradation of dyes can

increase the efficiency of microbiological degradation of dyes in the coming future.

2.5.1 Mixed cultures (microorganism consortiums)

The use of microbial cultures offers great advantages over the use of pure cultures in the

decolorization of synthetic dyes. The individual microbial strains may attack the dye molecule at

different sites or may use end products produced by another strain for further continuation of

decomposition. However, it should be take into account that the composition of mixed cultures

might be changed during the decomposition process, that may interferes with the control of other

technologies using mixed ones. The efficiency of decomposition largely depends on the chemical

nature of synthetic dye and hence, on the biodegradation capability of the mixed cultures. The

advantages and disadvantages of the use of mixed cultures for the decomposition of dyes have

been previously established by Banat et al. (1996). Optimum conditions for the microbial

decomposition of dyes show marked variation both in individual as well as mixed

anaerobic/aerobic processes. It has been found that the efficacy of aerobic process was inferior

over anaerobic decolorization process.

2.5.2 Anaerobic decolorization of synthetic dyes

The efficiency of applications anaerobic technology for the degradation of a number of

dyes has been well demonstrated (Delee et al., 1998). Experiments showed that reduction by

sulphide is somewhat responsible for the conversions of Acid Orange 7 an aerobically.

Mathematical discussion of the experimental results cleared out that autocatalysis played an

effective role where 1-amino-2-naphthol causes the chemical reduction of azo bond. (Zee van der

19

et al., 2000). The use of starch as a supplement enhanced the dye removal rate of industrial

wastewater (Chinkewitvanich et al., 2000).

Many experimenters concluded from their findings that microorganisms could

successively reduce azo dyes, anaerobically, and finally causing cleavage of aromatic ring

(Meyer, 1981; Ganesh, 1992).The reduction of azo bond cause a remarkable reduction of color

of dye house effluent. However, some researchers have reported the resistance of many aromatic

rings to reduction carried by microbes. It is considered that reduction of dye involve various

mechanisms like enzymatic (Baldrian, 2004), non-enzymatic (Julia et al., 2008), intracellular

(Shah & Nerud, 2002), extra cellular (Carliell et al., 1995) or a combination of more than one

mechanisms. Moreover, adsorption of dye on the mycelia of fungi may also take place (Asad et

al., 2007).

Disperse Blue 79 was reduced in anoxic sediment–water system, N, Ndisubstituted 1, 4

azobenzene and 3-bromo-6-nitro-1, 2-diaminobenzene were found to be the end products of

decomposition (Weber and Adams, 1995). Great variations were observed among the

degradation rates of different azo dyes (Baughman and Weber, 1994). The reactive azo dye

Reactive Red 141 was decomposed under anaerobic conditions. The azo bonds were cleaved by

the microbes which result in the liberation of 2-aminonaphtalene-1,5 disulfonic acid (Carliell et

al., 1995).

It was further demonstrated that use of redox mediators enhances the rate of decomposition of

azo dyes (Zee van der et al., 2001). The presence of salts (nitrate and sulfate) greatly effect the

decomposition rate of the azo dye Reactive Red 141 an aerobically. The results indicated that

nitrate delays decomposition process while sulfate not affect the biodegradation process

considerably (Carliell et al., 1998).

2.5.3 Aaerobic decomposition of synthetic dyes

Literature study showed anaerobic reduction of azo dyes to be more satisfactory than

aerobic one; the toxic products (carcinogenic aromatic amines) are degraded by an aerobic

process into less toxic or even harmless products. A number of technologies have been

20

developed for anaerobic/aerobic treatment of dye effluents. Decolorization rates were 20%, 72%,

and 78% for Acid Yellow 17, Basic Blue 3, and Basic Red 2, respectively (Ana et al., 2002).

This combined method has been employed for the decomposition anthraquinine based

reactive dyes (Panswad and Luangdilok, 2000) and the results obtained showed the dependence

of the decolorization rate on the molecular structure of the reactive. Dye effluents were also

treated using a sequential system. Results showed that, the Basic Red was removed very

efficiently an aerobically; however, no removal of the Acid Yellow 17 occurred. (Basibuyuk and

Forster, 1997; FitzGerald and Bishop, 1995).

Anaerobic/aerobic treatment is suitable for the cleavage of the azo bond in various azo

dyes (Seshadri et al., 1994). The azo bond of Acid Orange 8, Acid Orange 10 and Acid Red 14

was cleaved only under anaerobic conditions (Jiang and Bishop, 1994). The efficiency of the

removal of reactive diazo Remazol Black B dye by aerobic/anoxic s has been studied. The results

showed that prolonged anoxic and anaerobic period enhanced decolorization (Panswad et al.,

2001). The azo dye Procion Red H-E7B has been decolorized in a combined anaerobic–aerobic

process (O’Neill et al., 1999) and the positive effect of existence of carbohydrate at higher

concentration on the decolorization has been proved (O’Neill et al., 2000).

2.6 White-rot fungi and their enzymes

White-rot fungi produce a wide variety of extra cellular enzymes (laccase, lignin

peroxidase, Manganese peroxidase) that are responsible for the decomposition of highly lignin,

hemicelluloses, cellulose, etc. Because of their high biodegradation capacity, they are of

considerable interest, and their use in the decolorization process of industrial wastewaters has

been extensively investigated (Young and Yu, 1997). A number of reports have been reviewed

on decolorization of wastewaters by fungi (Fu and Viraraghavan, 2001). The role of enzymes in

microorganisms suitable for the decomposition of dyes has been extensively investigated.

Studies has been focused to the separation, isolation and testing of enzymes. Exact knowledge of

the enzymatic processes involved in the decomposition of dyes isof great concern.

Lignin peroxidase isoenzymes were isolated from P. chrysosporium and purified by

chromatofocusing. The activity of isoenzymes towards decolorization of a number of dyes was

21

compared with that of a crude enzyme preparation. Optimum pH requirements were different in

each case. The results indicated the marked differences among the decomposition capacity of

crude enzyme preparation and purified, although the structural variations of dyes exerted slight

influence (Ollikka et al., 1993). The degradation rate was highly dependent on the pH (Bhunia et

al., 2001). Another experimental finding showed that the enzymes of white rot fungi showed

different potential towards the degradation of different dyes. Veratryl alcohol considerably

enhanced the decomposition rate (Heinfling et al., 1998). Similar findings proved that pure

laccase was unable to degrade Remazol Brilliant Blue R but the degradation rate was enhanced

considerably inthe presence of a mediator (violuric acid) (Ollikka et al., 1993).

Tosyl group Tosylic acid Sodium benzenesulphonic acid

The important characteristics of sulphonic acid (or its salts) containing azo compounds is

their high solubility in water and hence, are present in water bodies. Textile and pharmaceutical

industries are the major consumers of sulphonated azo compounds, so are responsible mainly for

release of such compounds to waste water stream. Desulphonation or mineralization of these

compounds in environment results in the release of sulphur containing groups. During

desulphonation, first step involves the formation of HSO3-1, which in the next step undergoes

oxidation converting in to SO4-2. The desulphonation may occur before, after or during the

aromatic ring opening of sulphonated aromatic compounds.

22

Desulphonation after ring cleavage (Junker et al., 1994)

A number of reports heve shown that degradation of sulphonated aromatic compounds

(dyes) occurs via the remival of sulphonic acid group from the compound. Aerobic degradation

of naphthalene sulphonic acids by Phanerochaete chrysosporium was reported (Spadaro et al.,

1992; Cripps et al., 1993; Paszczunski and Crawford, 1990). After desulphonation, the

naphthalene compound was mineralized to carboxylic acids, CO2, H2O and energy. Different

microbial species are susceptible dofferently to azo dyes (Spadaro et al., 1992). Biodegradation

of reactive azo dyes present in the textile waste water system is a complicated one due to

versatility in the structure of dyes present. A number of other environmental factors can also

effect biodegradation of dyes. These include type of dye, chemical structure, and nature of

substituents, type of microorganisms, cell permitivity, pH of water and concentration of dye and

other additives. Thus color removal is not a simple process, but it’s a complicated one (Xu et al.,

2006).

Literature study revealed the role of enzymates of microbes in the biodegradation of dyes.

Ollikka et al. (1993) studied the oxidation of Congo Red at pH 4.0 in the presence of peroxidase.

In the report given by Cripps et al. (1993), the enzymatic oxidation of Congo Red by crude lignin

23

peroxidase was occurred at pH 4.5. Efficiency of the treatment system is dependent on pH of

waste water or dye solution used. A number of research reports show a relationship between pH

and biological treatment technologies for dyes removal. The optimum pH of process varied

depending upon both on type microorganisms and structure of dyes under study (Chen et al.,

2003).

Proposed mechanism for reduction of azo dye

Cell permeability is another very important factor not related with the structure of dye but

having a major role in dye decolorization mechanism and extent. From reports, it was sugested

that dye redyced after its adsorption or chemical reactions madiated by living cells has been

studied extensively using various types of fungi. Chemical structure of azo dyes has a prominent

effect on the biodegradation rate (Asad et al., 2007). Depending on the position of azo group and

their numbers, different dyes degrade differently, a literature have shown that an increase in the

number of azo groups on dye molecule would laed to decrease in the rate of degradation of dye

24

as it would require more time to reduce more azo groups. Poly azo dyes showed very low

linkages (Brown and Laboureur, 1983).

Fiber reactive azo dyes have side groups responsible for solubilization, along with

reactive groups which are nucleophilic in nature. The degradation of dye is dependent on the

type, chemical nature, number and positions of various substituents in the dye molecule (Ganesh,

1992). The production of toxic dye metabolites from dye molecule is said to cause a further

supression in dye degradation. Textile waste water has high concentration of salt, dispersing and

solubilizing agents which poses adverse negative effects on biodegradation (Carliell, 1994).

Reports have shown a decrease in degradation rates of dyes due to the production and

accumulation of toxic products in the growth culture. It is well cited in the literature that growth

and efficiency of microorganisms is effected badly by production of toxic metabolites which is

very important factor to be considered in effluent treatment having azo dyes (Wurhmann et al.,

1980). Carliell et al. (1995) performed a series of experiments on reactive dyes and concluded

that at concentrations of dyes greater than 100mgL-1, growth of microorganisms was inhibited.

Effect of dye concentration (which act as a substrate for enzymes) on rate of color removal by

various fungal strains have been studied by a number of researchers (Carliell et al., 1995; Ivana

et al., 2010; Kalyani et al., 2008), their findings confirmed earlier investigations that at higher

concentration of dyes, cell growth might be inhibited leading to a decreased rate of

decolorization.

Julia et al. (2008) performed a series of experiments and concluded that if focus was

given on the combination of chemical methods (Photo-Fenton more frequently) with aerobic

biological treatment in order to avoid the drawbacks of each. A minimum oxidation performed as

a pre-treatment to just increase the biodegradability and generate a new effluent more amenable

to biodegradation and results in least toxic metabolites production (Sarria et al., 2003; Farre et

al., 2006). Application of Photo-Fenton as a post-treatment, a complete decolorization and up to

80% mineralization was accomplished in the combined oxidation system (Julia et al., 2008).

25

CONCLUSIONS

From the discussion presented in this chapter, the following conclusions has been drawm:

Chemical method like Photo-Fenton being relatively cost effective, less time consuming

and easy to handle can be employed for remival of dyes.

White rot fungi ( a versatile group) can be successively employed for removal of dyes.

The enzymes of white rot fungi can effectivly reduce the azo linkage, thus causing the

destruction of chromophore of dye depending on the type and structure of dye used. The

resulting metabolites may be more or less toxic than dye itself.

Sulphonation of azo dyes increase their water solubility and hence, increase their

recalcitrance in bith anaerobic and aerobic environments, although some aerobic

microorganisms cause desulphonation thus allows the mineralization of sulphonated

aromatic amines under aerobic conditions.

A sequential treatment of chemical method (Photo-Fenton) followed by biological

treatment can be successively employed for almost complete decolorization and

maximum mineralization of aromatic azo dyes.

Above critical review explains attempts for treatment of waste water bodies having dyes.

Biological and chemical approaches are quite atractive and can be easily applied on industrial

sector. Major economy of Pakistan is dependent on development of textile sector. Hence, it is

a crucial need of time to develop an analytical approach which may help to textile and other

related sectors utilizing dyes, in the effective treatment of their wastes having dyes which is

hazardous to the environment if released to the environment without any effective treatment

technology. Keeping in view of all requirements present work was planned. It is an attempt

to use of chemical, biological and sequential technologies; the most suitable methods for dye

decolorization (as clear from above discussion). The results obtained would be used to

develop a comprehensive analytical approach on industrial sector for investigation of

decolorization of reactive azo dyes and hence, to minimize the hazardous effects caused by

the disposal of dyes in waste water system.

26

CHAPTER 3

MATERIALS AND METHODS

The present project was designed to develop an economical and environment friendly

process for degradation of dyes. The research work was perormed in the textile ccemistry and

Industrial biotechnology laboratories, Department of Chemistry and Biochemistry, University of

Agriculture, Faisalabad. In first part of the study, the optimization of reaction conditions was

done o using Reactive dye 222. The optimized conditions were then applied on the industrial and

synthetic effluents to evaluate their bioremediation potential under preoptimized conditions.

3.1. PART ONE: Optimization of conditions for decolorization of Reactive

dye 222

The decolorization of Reactive dye 222 was studied. The experimental work was divided

into the following three categories that have been described under the following headings




3.1.1 Photo-Fenton Treatment Of Reactive dye 222

3.1.1.1 Azo dyes

3.1.1.1a Apparatus

The following apparatus was used during the experimental research work:

Volumetric flask

Pipette

Measuring cylinders

Beakers

Funnels

Mechanical stirrer

27

Thermometer

Light Lamp (radiation source)

Hot plate with magnetic stirrer

Weighing Balance

COD vials (Lovibond)

Falcon tube (for toxicity test)

3.1.1.1b Chemicals

Chemicals used during whole experimental work enlisted here as under:

Reactive dye 222

Hydrogen peroxide (1×10 -2 M)

Ferrous sulphate (3.5×10 -5 M)

Sodium sulphate

Sodium chloride

Distilled water

Sulphuric acid

Sodium hydroxide

3.1.1.1c Instruments

Instruments were used enlisted here as under:

pH meter

Luxmeter

Weighing balance

UV/Visible Spectrophotometer

Fourier transform infrared spectrohotometer (FTIR)

3.1.1.1d Preparation of solutions

The solutions used in the photo-Fenton treatment were prepared as follows:

i. Preparation of dye solution

The 1% stock solution of Reactive dye 222 was prepared in distilled water. From this

stock solution, a number of 0.001, 0.002, 0.003, 0.004 and 0.005% dilutions were prepared.

28

ii. Preparation of H2O2 (1 × 10 -2 M) solution

For preparation of solution, according to standard calculations, 0.1ml of commercially

available H2O2 (37%) was taken in measuring flask and diluted it upto 100ml with distilled

water.

iii. Preparation of FeSO4.7H2O (3.5 × 10 -5 M) solution

For preparation of solution, according to standard calculations, 0.35g of FeSO4.7H2O was

taken in measuring flask and dissolved in 100ml of distilled water.

3.1.1.2 Optimization of light intensity

70ml of each solution (reactive dye 222, H2O2 1×10 -2 M and FeSO4 3.5×10 -5 M) was

taken in a reaction vessel. Initial pH of the sample was carefully adjusted to 3.5 by pH meter

using 0.5M H2SO4/1M NaOH.The sample was stirred on hot plate with magnetic stirrer under

radiations at 500 Lux and the reaction was let running for one hour at 40˚C.

Sample of solution was taken for absorbance testing after each 10 minutes at λmax of 615 nm. The

remaining three experiments were performed at temperatures 1000Lux, 1500 Lux and 2000 Lux.

The further optimization of parameters (pH, H2O2, FeSO4, temperature, NaCl and Na2SO4) was

carried out with the optimized level of light intensity.



taken in a reaction vessel. The initial pH of the sample was carefully adjusted to 2 by pH meter

using 0.5MH2SO4/1M NaOH.The sample was stirred on hot plate with magnetic stirrer under

radiations at 1500 Lux and the reaction was let running for one hour at 40˚C. Sample of solution

was taken for absorbance testing after each 10 minutes at λmax of 615 nm. The remaining seven

experiments were done with pH 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 and 6.0. The further optimization of

parameters (H2O2, FeSO4, temperature, NaCl and Na2SO4) was carried out with the optimized

level of pH.



taken in a reaction vessel. The initial pH of the sample was carefully adjusted to 3.5 by pH meter

29

using 0.5M H2SO4/1M NaOH.. The sample was placed on hot plate with magnetic stirrer under



remaining four experiments were done with H2O2 5×10 -3 M, 1×10 -2 M, 2×10 -2 M and 4×10-2 M.

The further optimization of parameters (FeSO4, temperature, NaCl and Na2SO4) was carried out

with the optimized level of H2O2.




using 0.5M H2SO4/1M NaOH. The sample was stirred on hot plate with magnetic stirrer under



remaining three experiments were done with FeSO4 of 1.5×10 -5 M, 2.5×10 -5 M, 3.5×10 -5 M and

4.5×10 -5 M. The further optimization of parameters (temperature, NaCl and Na2SO4) was carried

out with the optimized level of FeSO4.

3.1.1.6 Optimization of temperature






remaining three experiments were done at temperatures 40˚C, 50˚C and 60˚C. The further

optimization of parameters (temperature, NaCl and Na2SO4) was carried out with the optimized

level of temperature.







remaining three experiments were done in the presence of different levels of NaCl 6g/L, 9g/L

30

and 12g/L. The further optimization of parameters (NaCl and Na2SO4) was carried out with the

optimized level of NaCl.




using 0.5M H2SO4/1M NaOH.The sample was stirred on hot plate with magnetic stirrer under



remaining three experiments were done in the presence of different levels of Na2SO4 6g/L, 9g/L

and 12g/L.


3.1.2.1a Apparatus

The following apparatus was used during the experimental research work:

Volumetric flask

Pipette

Measuring cylinders

Beakers

Funnels

Autoclave

Shaker

Oven

Leminar air flow

Centrifuge machine

3.1.2.1b Chemicals

Chemicals used during whole experimental work enlisted here as under:

Reactive dye 222

31

Composition of Kirk’s basal nutrient medium for white rot fungi

Sr. No. Ingredients Quantity (g/L)

1 Potato extract 250

2 Glucose 20

3 Potato dextrose agar 15

4 Ammonium tararate 0.22

5 KH2PO4 0.21

6 MgSO4.7H2O 0.05

7 CaCl2.2H2O 0.01

8 Thiamine 0.001

9 10% Tween 80 10 mL

10 100mM veratryl alcohol 1 mL

11 **Chloramphenicol 1 cc

12 ***Trace element solution 10 mL

*pH 4.5; temperature, 30°C

**Chloramphenicol is used to resist bacterial growth.

***The trace element solution contained (g/L): CuSo4, 0.08; H2MoO4, 0.05;

MnSO4.4H2O, 0.07; ZnSO4.7H2O, 0.043; Fe2 (SO4)3, 0.05.

Pure cultures of five indigenous white rot fungi S. commune IBL-01 (SC) , P. ostreatus

IBL-02 (PO) ,P. chrysosporium IBL-03 (PC), T. versicolor IBL-04 (TV) and G. lucidum IBL-05

(GL) were obtained from Industrial Biotechnology Laboratory and grown on potato dextrose

agar (PDA) medium slants for 6-7 days at pH 4.5 and 30°C. The slants were preserved at 4°C in

refrigerator.

32

3.1.2.2 Preparation of inoculum

The flasks containing Kirk’s medium (whose composition mentioned earlier) for

individual fungi were adjusted at pH 4.5 with 1.0 M NaOH/1.0 M HCl and autoclaved at 121°C

for 15 minutes. The flasks were inoculated with loopful fungal spores from individual slant

cultures and placed in incubator (120 rpm) under shaking at 30oC for the days to get a

homogenous spore suspension. The number of spores in the inoculum was counted by using

heamocytometer to get 10-7- 10-8 spores/mL. Fresh inoculums were prepared for each experiment

(Asghar et al., 2006).

3.1.2.3 Preparation of biodecolorizable mixture

The Reactive dye 222 solutions in 500ml Erlenmeyer flasks along with 200ml Kirk’s

medium were treated with each microbial strain. After adjusting pH at 4.5, the flasks were

autoclaved for 15 minutes at 121°C. After cooling to room temperature, the triplicate dye

solution containing flasks were inoculated with 5ml homogenous spore inocula of respective

fungi and were incubated at 32 oC for a period of 10 days. The uninoculated flasks (having

Kirk’s medium and dye) were also incubated as control experiments. For each fungal strain,

triplicate flasks were used. Aliquots (1ml) were taken periodically from flasks after 24 hours to

measure the extent of decolorization of the dye under study. Supernants obtained after

centrifugation (1200rpm for 10 min) was run though spectrometer (Model T60U) to check

absorbance at λmax of 615nm. The uninoculated medium having no dye was used as a blank. The

results obtained are recorded the average of three replicates of various intervals.

Based on the screening experiment, the two white rot fungi P. ostreatus IBL-02 (PO) and

P. chrysosporium IBL-03 (PC) showing best biodecolorization results were selected for

development/optimization of the biodecolorization process.

3.1.2.4 Optimization of biodecolorization process

Different environmental and growth conditions effect the microbial growth, enzyme

production and enzyme activities involved in dyestuff biodecolorization/biodegradation. As the

literature survey showed the dependence of fungal enzymes on certain growth conditions for

their maximum degradation efficiency. Most important of growth conditions are pH, incubation

temperature, inoculum’s size and effect of various amendments (Ganesh et al., 1999). The

process of biological degradation was optimized for selected two white rot fungi best suited for

degradation of Reactive dye 222 on the basis of screening experiment. Degradation experiments

33

were run under various growth conditions by varying one at a time while keeping others

constant.

. Dye concentration (0.01-0.05g/l)

. pH (3.5-5.5)

. Incubation Temperature (25-40oC)

. Inoculum’s size (1-5ml/100ml)

All experiments were carried out in the same way as described earlier. Decolorization

(%) was monitored in the same way as described above. All experiments were performed in

triplicate.

The activity of fungi can be increased by using various additional parameters like carbon,

nitrogen, mediators and metal ions. Study was done to find out the effect of these additional

parameters on the decolorization (%) of reactive dye 222 by P. ostreatus IBL-02 (PO) and P.

chrysosporium IBL-03 (PC).

3.1.2.4a Effect of carbon additives

After optimization of experimental parameters, effect of various carbon sources other

than Reactive dye 222, were used to determine their effects on growth and extent of dye

degrading ability of two screened white rot fungi i.e. P. ostreatus IBL-02 (PO) and P.

chrysosporium IBL-03 (PC). Experiments were done by the protocol followed earlier.

a. Glucose (2g/L)

b. Starch (2g/L)

c. Glycerol (2g/L)

d. Rice bran (2g/L)

e. Wheat bran (2g/L)

3.1.2.4b Effect of nitrogen additives

After optimization of experimental parameters, effect of various nitrogen sources other

than Reactive dye 222, were used to determine their effects on growth and extent of dye

degrading ability of two screened white rot fungi i.e. P. ostreatus IBL-02 (PO) and P.

chrysosporium IBL-03 (PC). Experiments were done by the protocol followed earlier.

a. Corn steep liquor (0.22g/L)

b. Yeast extracts (0.22g/L)

c. Maize gluten 30% (0.22g/L)

34

d. Maize gluten 60% (0.22g/L)

e. Ammonium oxalate (0.22g/L)

3.1.2.4c Effect of low molecular mass mediators

Mediators have a profound effect on microbial growth, ligninolytic enzymes which

decolorize different dyes. Low molecular mass mediators enhance enzyme induction in white rot

fungi. The present study involves biodegradation of reactive dye 222 as a major motive, so effect

of various mediators on the activity of P. ostreatus IBL-02 (PO) and P. chrysosporium IBL-03

(PC) was taken in to consideration.

a. Veretryl Alcohol

b. MnSO4

c. Glycerol

d. Ethanol

e. 2, 2’-Azinobis (3-ethylbenzathiazoline)-6-sulphonic acid

f. Oxalic acid

g. H2O2

The effect of different mediators on decolorization of reactive dye 222 was studied by the

following the same protocol as outlined above.

3.1.2.4d Effect of metal ions

Metal ions play the role of cofactors and enhance accumulative effect of various additives

particularly low molecular mass mediators. In the report given by Nies (1999), it was shown that

metal ions have a profound potential to interact with enzymes of microorganisms directly

involved in biodegradation. To study effect of various metal ions on degradation of Reactive dye

222, various metal ion sources were used given below:

a. Cd (NO3)2

b. CaCl2

c. ZnSO4

d. FeSO4

e. CuSO4

The effect of different metal ions on decolorization of Reactive dye 222 was studied by

the following the same protocol as outlined earlier.

35

3.1.2.5 Enzyme assays

Three ligninolytic enzymes Laccase (Lac), Lignin peroxidase (LiP) and Manganese

peroxidase (MnP) were studied at the end of each experiment.

3.1.2.5a Lignin peroxidase assay

Lignin peroxidase (LiP) was assayed by the method of Tien and Kirk (1983), mixture

contained 1 mL of veratryl alcohol (1 mM) and 1 mL of tartarate buffer (1 mM) of pH 3 and 100

µL of culture supernatant. 500 µL of H2O2 (0.2 mM ) was added as an oxidizing agent. The

oxidation rate of veratryl alcohol to veratraldehyde was determined at 310 nm (molar extinction

co-efficient = 9300 M-1cm-1). One unit of LiP activity was defined as the amount of enzyme

catalyzing the formation of 1μmol of veratryaldehyde per minute.

Enzyme activity (U/mL) = A/9300 × 107

3.1.2.5b Manganese peroxidase assay

Manganese peroxidase (MnP) was determined by the method of Wariishi assay (1992),

mixture contained 1 mL of MnSO4 (1 mM ) and 1 mL of sodium malonate (50 mM ) buffer of

pH 4.5 and 100 µL of culture supernatant. 500 µL of H2O2 (0.1 mM) was added as an oxidizing

agent. The oxidation rate was monitored at 270nm (molar extinction co-efficient = 11590 M-1cm-

1). One unit of MnP activity was defined as an amount catalyzing the production of 1μmol of

product per ml per minute.


3.1.2.5c Laccase assay

Laccase (Lac) activity was measured following the assay given by Wolfendon and

Wilson (1982). Mixture contained 1 mL of ABTS (1 mM ) and 1 mL of malonate buffer (50

mM) of pH 4.5 and 100 µL of culture supernatant. 500 µL of H2O2 (0.2 mM) was added as an

oxidizing agent. Laccase activity was determined by monitoring the oxidation of 2, 2-azinibis (3-

ethylbenzothiazoline-6-sulfonic Acid) (ABTS) at 436nm (molar extinction co-efficient =

36000M-1cm-1). One unit of laccase activity was defined as the amount of enzyme that oxidizes

1μmol ABTS in 1 minute.


36


Sequential treatment study was performed to analyze the efficiency of treatment methods

either they are more effective when they are treated alone or in combination with each other.

Sequential treatment was done via two different ways:


This sequential treatment was done at pre-optimized conditions. For this purpose, the dye

solution was treated by Photo-Fenton method at optimized conditions (pH 3.5, H2O2 1×10 -2 M

FeSO4 3.5×10 -5 M and temperature 50°C). The Photo-Fenton treated dye solution was subjected

to biodecolorization by two white rot fungi, P. ostreatus (PO) and P. chrysosporium (PC) at the

pre-optimized growth conditions (pH 4.5, temperature 30 oC, inoculum size 4mL, rice bran 1.5g,

MnSO4 1mM), separately by following the same protocol as discussed earlier. The percent

decolorization was measured after every 24hrs for a period up to 72hrs. Samples having all

nutrients except fungi were taken as abiotic controls. The ligninolytic enzymes LiP, MnP and

Lac were studied at the end of each experiment by following the protocols as described earlier.


It was done at pre-optimized conditions. The biologically treated dye solutions by both

PO and PC at optimized growth conditions (pH 4.5, temperature 30°C, inoculum size 4mL, rice

bran 1.5g, MnSO4 1mM). Samples having all nutrients except fungi were taken as abiotic

controls. The ligninolytic enzymes LiP, MnP and Lac were studied at the end of each experiment

by following the protocols as described earlier.The biologically treated dye solutions were

subjected to Photo-Fenton treatment at the pre-optimized conditions (pH 3.5, H2O2 1×10 -2 M

FeSO4 3.5×10 -5 M and temperature 50 oC), by following the same method as described earlier.

The percent decolorization was measured after every 10 minutes up to 1hr.


In the part two, decolorization of one synthetic effluent (having a mixture of three

reactive dyes) and three indusrial effluents effluent was studied

37

Preparation of Synthetic effluent

A synthetic textile wastewater was prepared in the laboratory by mixing hydrolysed

reactive dyes (Reactive black (139 mg/l); Reactive Yellow (150 mg/l); Reactive Red (1215

mg/l)), hydrolysed starch (13.9 mg/l), Na2SO4 (27.8 mg/l) and Na2HPO4 (27.8 mg/l) in de-

ionised water. Then it was placed on hot plate on magnetic stirrer at 80°C for 1.5 h after

adjustment at pH 12.


20 ml of wastewater was taken and diluted up to 100 ml with distilled water in volumetric

flask and adjusting the pH 3.5 (optimized pH) using 0.5M H2SO4 and 1M NaOH. After this

process 80 mg of FeSO4.7H2O was dissolved in reaction mixture. The reaction mixture was

stirred on magnetic stir bar under radiations of 1500 Lux and the reaction was let running for one

hr at 50°C. Then hydrogen peroxide was added and started the stirring. Reaction media was

stirred for 1 hour at 50°C. Sample of reaction mixture was taken for absorbance measurement

after each 10 minutes. The solution having FeSO4 and H2O2 solution (without effluent) was used

as standard (Locus and Peres et al., 2006).

3.2.2 Biodecolorization Method

The triplicate flasks having reaction medium (90ml of effluent and 10mL of Kirk’s basal

medium) were adjusted at pH 3.0 and sterilized (121°C) in an autoclave for 15 min. The flasks

were inoculated with 5 ml inocula of strains of P. ostreatus IBL-02 and P. chrysosporium IBL-

03 separately, in laminar air flow aseptically. The inoculated flasks were incubated for 6 days at

300C in shaking cultures at 120 rpm. Aliquots (1ml) were taken periodically from flasks after 24

hours up to the period of 72 hours to measure the extent of decolorization of the dye under study

(Asghar et al., 2006). Supernatants obtained after centrifugation (1200rpm for 10 min) was run

though spectrophotometer (Model T60U) to check absorbance at λmax of 615nm. The

uninoculated medium having no effluent was used as a blank. The results obtained are recorded

the average of three replicates of various intervals.

38


Sequential treatment study was done to evaluate the efficiency of treatment methods


Sequential treatment was done via two different ways.


This sequential treatment was done at pre-optimized conditions. For this purpose, the

each effluent was treated by Photo-Fenton method at optimized conditions (pH 3.5, H2O2 1×10 -2

M FeSO4 3.5×10 -5 M and temperature 50°C). The Photo-Fenton treated effluent was subjected to

biodecolorization by two white rot fungi, P. ostreatus IBL-02 (PO) and P. chrysosporium IBL-

03 (PC) at the pre-optimized growth conditions (pH 4.5, temperature 30°C, inoculum size 4mL,

rice bran 1.5g, MnSO4 1mM), separately by following the same protocol as discussed earlier.

The percent decolorization was measured after every 24hrs for a period up to 72hrs. Samples

having all nutrients except fungi were taken as abiotic controls. The ligninolytic enzymes LiP,

MnP and Lac were studied at the end of each experiment by following the protocols as described

earlier.


This sequential treatment was done at pre-optimized conditions. The biologically treated

effluents by both PO and PC at optimized growth conditions (pH 4.5, temperature 30°C,

inoculum size 4mL, rice bran 1.5g, MnSO4 1mM). Samples having all nutrients except fungi

were taken as abiotic controls. The ligninolytic enzymes LiP, MnP and Lac were studied at the

end of each experiment by following the protocols as described earlier.The biologically treated

effluents were subjected to Photo-Fenton treatment at the pre-optimized conditions (pH 3.5,

H2O2 1×10 -2 M FeSO4 3.5×10 -5 M and temperature 50°C), by following the same method as

described earlier. The percent decolorization was measured after every 10 minutes up to 1hr.

Decolorization assay

The dcolorization (%) efficiency of the parameters was assessed by using absorbance of uv-

visible spectrophotometer. The following formula was used to calculate the decolorization

efficiency of the process:

Decolorization (%) =I – F/I X 100

39

Where

I = Initial absorbance

F = Absorbance of decolorized

All decolorization experiments were performed in triplicate.

3.3 Evaluation of Treated dye Solutions through quality assurance

parameters

Preparation of Solutions for quality assurance parameters

i. Digestion solution

Digestion solution was prepared by adding 2.6g K2Cr2O7, 8.33g HgSO4 in 42ml H2SO4

(98% concentrated) and then diluting it to 250ml with deionized water.

ii. Catalyst solution

5.06 g Ag2SO4 was dissolve in 500 ml concentrated H2SO4 and placed it for 48 h to

ensure that the Ag2SO4 dissolved completely.

iii. Standard Solution of Potassium hydrogen phthalate

A standard solution of Potassium hydrogen phthalate (KHP) was prepared from a stock

solution (1g KHP/1L) of 425ppm concentration in deionized water. 425 ppm solution of KHP

gives 500 mg/L COD.

iv. 2N potassium dichromate solution (K2Cr2O7)

It was prepared by dissolving 98.06 gram per litter.

v. Standard solution of glucose (organic carbon)

250 ppm solution of glucose was prepared by dilution method from a stock solution of

1000ppm solution. 25ml of stock solution was taken and diluted to 100ml in volumetric flask.

vi. Standard solution of phenol

0.5g phenol was dissolved in 500ml distilled water to prepare stock solution of phenol

and further required dilutions were made according to standard dilution method.

vii. 0.5N NH4OH

Dilute 35ml of fresh concentrated NH4OH (30%) to 1L with distilled water.

viii. Potassium ferricyanide (K3Fe (CN)6)

8gram potassium ferricyanide dissolved in 100ml distilled water.

40

ix. 4-aminoantipyrine

2 grams of 4-aminoantipyrine was dissolved in 100ml distilled water.

3.3.1 Biological Oxygen Demand (BOD)

In this test, the test samples along with specified microorganisms in airtight bottle of the

specified size were incubated for a period of 5 days. Dissolved oxygen (DO) was measured

initially and after incubation, and BOD was calculated from the difference between initial and

final values. All oxygen uptakes occurred after this measurement was included in the BOD

measurement (Greenberg et al., 1985).

3.3.2 Chemical Oxygen Demand (COD)

In this test, 3.5ml catalyst solution and 1.5ml digestion solution was added in digestion

vials. Then 2.5ml sample solution was added in to the digestion vial. A blank sample was

prepared by adding all reagents (e.g. acid and oxidizing agent) to a volume of deionized water.

The digestion vials were placed in the oven for 2 hours at 150°C. After the digestion vials were

cooled, results were then read directly on a spectrophotometer at λmax 600nm. The absorbance of

the blank sample was subtracted from the absorbance of the original sample to ensure a true

measurement of organic matter.

The oxidizable matter was calculated in terms of oxygen equivalent (Greenberg et al., 1985).

Calculations

The COD was calculated by using the following formula

COD = standard factor × absorbance

Standard factor was calculated from the standard by using the formula given below:

Standard Factor = concentration of standard/ absorbance of standard

3.3.3 Total organic carbons (TOC)

Total organic carbon (TOC) values indicate the concentration all organic matter present

in a solution after a treatment method. 1ml of 2N K2Cr2O7 solution and 1.6ml H2SO4 (98%) was

added in digestion vials. Then 4ml of sample solution was added in to the digestion vials. The

solution in digestion vials having 1ml of 2N K2Cr2O7 solution and 1.6ml H2SO4 (98%) served as

blank. The digestion vials were placed in the oven for 1.5 hours at 110°C. After the digestion

vials were cooled, results were then read directly on a spectrophotometer at λmax 590nm.

41

The absorbance of the blank sample was subtracted from the absorbance of the original sample

to ensure a true measurement of organic matter. The oxidizable matter was calculated in terms of

oxygen equivalent (Greenberg et al., 1985).

Calculations

The TOC was calculated by using the following formula

TOC = standard factor × absorbance

Standard factor was calculated from the standard by using the formula given below:

Standard Factor = concentration of standard/ absorbance of standard

3.3.4 Determination of phenols by the 4-aminoantipyrine

0.5mg of Reactive dye 222 was added to 100ml distilled water in a 250ml beaker.

Distilled water was used as blank and a series of 100ml phenol standards (0.1-0.5mg). Sample,

blank and standards were treated as follow:

2.5ml of 0.5N NH4OH solution was added to dye solution and immediately pH was

adjusted to 7.9 ± 0.1 with phosphate buffer. 1.0ml of 4-Aminoantipyrine solution was added in to

it, mixed well. Then, 1.0 ml of K3Fe (CN) 6 solution was added and mixed well. Blank and

standard solutions were treated by the way as dye solution was treated (Greenberg et al., 1985).

After 15 min, blank and standards were run through spectrometer at 500 nm to make a

calibration curve. The absorbance of dye sample was also noted at 500 nm.

Calculation

Dye sample phenol content from photometric readings were determined by using the

following formula:

Phenol in mg/L = A/B × 1000

Where:

A = mg phenol in sample, from calibration curve and

B = mL original sample

3.3.5 Total suspended sollids (TSS)

A sample was filtered through a weighed whattmann filter paper No.42 and the residue

obtained on the filter paper was dried to a constant weight at 103 to 105°C for 45 minutes. The

increase in weight of the filter paper represented the total suspended solids (Greenberg et al.,

1985).

42

Calculation

To calculate total suspended solids, the following formula was used

Total suspended solids in mg/L = (A-B) × 1000/ sample volume in ml

Where:

A = weight of filter paper + dried residue (mg)

B = weight of filter paper (mg)

3.4 UV-Visible and FTIR analysis

Decolorization was monitored by UV-Vis spectrophotometeric analysis, whereas

degradation analysis was monitored by FTIR. For this purpose, 100ml of sample (after

decolorization) was taken, centrifuged at 10,000 rpm for 20 minute and extraction of metabolites

was carried out from supernatant using equal volume of ethyl acetate. The extracts were dried

over anhydrous Na2SO4 and evaporated to dryness in rotary evaporator. The crystals obtained

were dissolved in small volume of analytical grade methanol and usd for analysis. During UV-

Vis spectral amalysis, changes in absorption spectrum in the decolorized medium (400-800 nm)

were recorded in comparision with the results from the untreated samples. The FTIR analysis

(model no. Tensor 27, Bruker optics) was done in the done mid IR region of (1000-4000 cm-1)

with 16 scan speed and was recorded in comparision with the results from the untreated dye

samples.

3.5 Toxicity test

Heamolytic activity was checked by using the Powell et al. (2000) method. Three ml of

freshly obtained heparinzed human blood was gently mixed, poured into a sterile 15 ml falcon

tube and centrifuged for 5 min at 850g. The supernatant was poured off and viscous pellets were

washed three times with 5 ml of chilled (4°C) sterile isotonic Phosphate-buffered saline (PBS)

solution , adjusted to pH~7.4, to stabilize the pH, mixed it almost for half an hour at room

temperature (25–30°C).The washed cells were suspended in the 20ml chilled, saline PBS buffer.

Erythrocytes were counted on haemacytometer (Fisher ultra plane Neubauer ruling). The blood

cell suspension was maintained on wet ice and diluted with sterile (PBS),the cell count was

7.068 x108 cell per ml for each assay. 20µl of plant extract in five different solvents was taken in

2ml apen doff tubes. For each assay, 0.1% Triton X-100 was taken as a positive control, 100% of

blood lysis and Phosphate buffer saline (PBS) was taken for each assay as a negative control,

background (0% lysis).In each 2ml apen doff tube already contained 20µl sample, added 180µl

43

diluted blood cell suspension and mixed it with the help of pipette tip. Tubes were incubated for

35 min at 37°C and agitated after 10 min. immediately after incubation; the tubes were placed in

ice for 5 min and centrifuged for 5 min at 1310g. After centrifugation, 100µl supernatant was

taken from the tubes, and diluted with 900 µl chilled PBS. All tubes were maintained on wet ice

after dilution. The 200 µl sample was taken into 96 well plates, and three replicates were taken in

well plate having one positive control (100% of blood lyses) and one negative control (0% of

blood lyses). After this, absorbance at 576nm was taken at µ quantification. Triton-X 100 (0.1

%) was chosen as positive control for 100% lyses and PBS buffer as Negative control for 0%

lyses. The experiment was done in triplicate and mean ±S.E. was calculated. The % hemolysis

formula was calculated by the formula given below:

Data observed was expressed in % of Hb release by comparing with 100% haemolysis of

the same number of cells using (0.1% Triton X-100).

3.6 Statistical analysis of data

All treatments were run in triplicate and all analysis was performed in triplicate. The data

collected were analyzed by computing standard errors and analysis of variance (Steel et al.,

1997).

44

CHAPTER 4

RESULTS AND DISCUSSION


222

In part one, the study was done with reactive dye 222 to optimize different physico-

chemical parameters of different treatment methods to assess maximum decolorization (%) of

dye under study.


The present investigation was carried out to analyse the color intensity of Reactive dye

222 in the solution after the application of Photo-Fenton processes. Concentration of dye, doses

of H2O2, doses of iron (II) salt, pH, temperature and time for decolorization are the variables

that´s why their effect on the color strength is described here. The main process variables

affecting the rate of Fenton's reaction are dye concentration, pH, the molar concentrations of the

oxidant (H2O2) and catalyst (Fe2+), temperature and reaction time.


As indicated in eq. 1, the amount of HO. radical generated by the Photo-Fenton reaction is

affected by the pH. The HO. radical can be efficiently formed especially in acidic conditions Kuo

(1992). To find out the optimum pH for maximum conversion of the aqueous dye solution,

experiments were conducted at its varying levels from 2-6, by keeping other factors (H2O2 1×10-

2 M , FeSO4 3.5 ×10-2 M, pH 3.5, temperature 40°C and dye 0.04%) costant (Table 4.1.1.1a).

Fe2+ + H2O2 → •OH + OH− + Fe3+ (1) •OH + RH → H2O + R • (2)

R •+ Fe3+ → R+ + Fe2+ (3)

R+ + H2O → ROH + H+ (4)

Fig. 4.10 represents the color removal of RB 222 at different pH. The rate of photo-

bleaching of dye under study increases with increasing pH upto 3.5. It is evident from the figure

45

that extent of decolorization decreases with further increase in pH. At pH 3.5, 95.18% color

removal was observed (Fig. 4.1.1.1).

Table 4.1.1.1a Decolorization of Reactive dye 222 by Photo-Fenton with varying pH

pH Decolorization (%)

2 23.995E

2.5 31.11D

3.0 70.463B

3.5 79.06A

4.0 44.943C

4.5 44.13C

5.0 25.505E

5.5 21.63E

6.0 24.836E

LSD = 6.160

*: H2O2, 1× 10-2 M; FeSO4, 3.5 × 10-5 M; Temperature, 50°C; time, 50 min.

Means sharing similar letters in column are statistically non significant (P≤0.05)

46

0

20

40

60

80

100

120

2 2.5 3 3.5 4 4.5 5 5.5 6

pH

Dec

olor

izat

ion

(%

)

Fig.4.1.1.1 Effect of pH on the decolorization of Reactive dye 222 by Photo-Fenton treatment

The statistical analysis of the data by ANOVA under CRD showed a significant

difference in dye decolorization under varying pH (Table 4.1.1.1b). Comparison of means by

LSD test also showed significant (P≤0.05) difference netween dye decolorization at varying pH.

Table 4.1.1.1b Analysis of variance of the data on decolorization of Reactive dye 222 by Photo-Fenton with varying pH

Source of variance

df SS MSS F

Block 5 16170.822 3234.164 67.198***

Treatment 8 43569.013 5446.127 113.156***

Error 94 4524.171 48.130

Total 107 63905.411

(P≤0.05)

47

Malik and Saha (2003) reported pH 3 for the decolorization of dyes. Meric et al. (2004)

showed that more than 99% of color removal was possible in the pH range of 3-3.5. Tang and

Chen (1996) reported similar results sfor decolorization of C.I. RR 120 using H2O2/Fe+2 salts.

The low activity detected at high pH has been reported in the literature (Lee et al., 2003) and

might be due to the formation and precipitation of Fe (OH)3 (Bhattachaya, 1992). Color removal

of synthetic dyes in wastewater by Photo-Fenton’s treatment was achieved at pH range 3.0-4.0

(Dil et al., 2000). Our results correspond to those from literature.


The selection of an optimum concentration for the decolorization of Reactive dye 222 by

Photo-Fenton’s reagent is important from practical point of view (due to the cost of H2O2). To

optimize the dose of H2O2, different levels of H2O2 1× 10-3 to 4× 10-2 M were selected, while

keeping the concentrations of other parameters (FeSO4 3.5 ×10-2 M, pH 3.5, temperature

40°C and dye 0.04%) costant (Table 4.1.1.2a). The effect of H2O2 concentration on the

decolorization of Reactive dye 222 is shown in Fig. 4.1.1.2a It is evident that rate of photo-

bleaching of dye under study increases with increasing H2O2 concentrations up to 1×10-2 M. It

can be explained on the basis that at higher concentration of H2O2, more hydroxyl radicals are

produced which decolorize more dye molecules. Further increase in H2O2 caused a decline in

color removal of dye under study. The color removal observed at optimum level of H2O2 (1×10-2

M) was 95.23%.

Table 4.1.1.2a Decolorization of Reactive dye 222 by Photo-Fenton with varying H2O2

concentrations

H2O2 Decolorization (%)

1× 10-3 M 23.003D

5× 10-3 M 31.351C

1× 10-2 M 79.033A

2× 10-2 M 44.403B

4× 10-2 M 41.218B

LSD = 3.55

48



0

20

40

60

80

100

120

0.001 0.005 0.01 0.02 0.04

H2O2 (M)

Dec

olor

izat

ion

(%)

Fig.4.1.1.2 Effect of H2O2 concentrations on the decolorization of Reactive dye 222 by



difference in dye decolorization under varying under varying concentrations of H2O2.

Comparison of means by LSD test also showed significant (P≤0.05) difference except between

2× 10-2 and 4× 10-2 M concentrations of H2O2 (Table 4.1.1.2b).

49

Table 4.1.1.2b Analysis of variance of the data on decolorization of Reactive dye 222 by

Photo-Fenton with varying H2O2 concentrations

Source of variance

df SS MSS F

Block 5 10697.391 2139.478 113.806***

Treatment 4 22030.798 5507.699 292.972***

Error 50 939.969 18.799

Total 59 33668.158

(P≤0.05)

Dil et al. (2000) have reported the existence of the optimum H2O2 concentration 1×10-2 M

for the decolorization of methylene blue by Photo-Fenton’s reagent. The color removal

efficiency (decrease in absorbance at 510 nm) was increased with increasing the dosage of H2O2.

As the dosage of H2O2 increased from 1×10-3 M to 1×10-2 M color removal reached 20.70-

88.72%. When increasing the H2O2 dosage from 1×10-2 to 4×10-2 M, color removal increased

slightly from 88.72-92.41. According to Bergendahl (2004), decolorization rate of organic

compounds was decreased as a result of scavenging effect. Fernandez et al. (1999) reported that

decline in color at higher concentration of H2O2 might be due to the scavenging effect of HO.

radicals at higher concentration which can be expressed by the eqs. given below:

HO. + H2O2 → H2O + HO2

.

HO2. + HO

. → H2O + O2


Dye decolorization efficiency of Photo-Fenton process is influenced by the concentration

of Fe+2 ions which catalyze hydrogen peroxide decomposition resulting in HO. radical

production and consequently the degradation of organic molecule. According to the literature,

increasing ferrous salt concentration, degradation of organic compound also increases, to certain

level where further addition of iron becomes inefficient (Rathi et al., 2003). The effect of Fe+2

concentration on color removal of reactive dye 222 was examined by changing the Fe+2

50

concentration between 0.5 × 10-5 to 4.5 × 10-5 M, while keeping the concentrations of

H2O2, pH and dye concentration costant (H2O2 1×10-2 M, pH 3.5, temperature 40°C and

dye 0.04%) (Table 4.1.1.3a). As Fe2+ doses were increased from 0.5 × 10-5 to 3.5 × 10-5

M, the removal % increased from 14.39% to 95.28% (Fig. 4.1.1.3). The concentration

higher than 3.5 × 10-5 M results in decrease in decolorization (%) of dye under study.

Table 4.1.1.3a Decolorization of Reactive dye 222 by Photo-Fenton with varying

FeSO4 concentrations

FeSO4 Decolorization (%)

0.5 × 10-5 M 23.003D

1.5 × 10-5 M 61.448B

2.5 × 10-5 M 64.085B

3.5 × 10-5 M 79.247A

4.5 × 10-5 M 30.338C

LSD = 5.998



51

0

20

40

60

80

100

120

0.5×10-5 1.5×10-5 2.5×10-5 3.5×10-5 4.5×10-5

FeSO4 (M)

Dec

olor

izat

ion

(%

)

Fig.4.1.1.3 Effect of FeSO4 concentrations on the decolorization of Reactive dye 222 by



difference in dye decolorization under varying under varying concentrations of FeSO4.

Comparison of means by LSD test also showed significant (P≤0.05) difference except between .5

× 10-5 and 2.5 × 10-5 M concentrations of FeSO4 (Table 4.1.1.3b).


Photo-Fenton with varying FeSO4

Source of variance

df SS MSS F

Block 5 12760.798 2552.159 47.702***

Treatment 4 38598.201 9649.550 180.356***

Error 50 2675.133 53.5026

Total 59 54034.131

(P≤0.05)

52

Report given by Rathi et al. (2003) showed that higher ferrous doses lead to the

generation of hydroxyl radicals that result in the increased removal of dye. At higher doses, the

dye removal efficiency decreases indicating that H2O2 becomes the limiting factor for further

HO. radical generation. Barbusinski and Majenski (2003) examined that overloading of the Fe+2

salt hinders the degradation efficiency. This might be attributed due to the following reasons: (I)

higher concentration of Fe+2 ion results in turbidity shich hinders the penetration of light (II)

high dosage of iron salt increases the concentration of Fe+2 ions in solution which can also act as

hydroxyl radical scavenger as indicated by the following equation:

Fe+2 + HO. → Fe+3 + OH

-

4.1.1.4 Optimization of Temperature

To study the effect reaction temperature on the decolorization of reactive dye 222, a

series of experiments were conducted at different temperatures ranging from 30 to 60°C, by

keeping other factors (pH 3.5; H2O2, 1× 10-2 M; FeSO4, 3.5 × 10-5 M) constant (Table 4.1.1.4a).

By rising the temperature has a positive effect on the decolorization of reactive dye 222. When

the reaction was increased from 30 to 50°C, the color removal efficiency was increased from

80.12 % to 95.72%. The decolorization efficiency was decreased to 60.2%, as temperature was

raised to 60°C (Fig. 4.1.1.4).


temperature (°C)

Temperature (°C) Decolorization (%)

30 41.218C

40 70.462B

50 79.261A

60 31.12D

LSD = 6.160



53

0

20

40

60

80

100

120

30 40 50 60

Temperature (0C)

Dec

olor

izat

ion

(%

)

Fig.4.1.1.4 Effect of temperature on the decolorization of Reactive dye 222 by Photo-

Fenton treatment


difference in dye decolorization under varying under varying concentrations of FeSO4.

Comparison of means by LSD test also showed significant (P≤0.05) difference at varying

temperature (Table 4.1.1.4b).


Photo-Fenton with varying temperature

Source of variance

df SS MSS F

Block 5 13997.522 2799.504 64.656***

Treatment 3 19041.338 6347.113 146.59***

Error 39 1688.636 43.2984

Total 47 34727.496

(P≤0.05)

54

It can be explained that Photo-Fenton,s could be accelerated by raising temperature which

improves the generation of HO. radical and therefore to enhance the decolorization of reactive

dye 222 up to optimum level. The temperature higher than optimum value resulted in decrease in

decolorization efficiency due to scavenging effect of HO. radical (Panizza and Cerisela, 2009;

Sun et al., 2008).


Huge amounts of salts are used in coloring up variety of dyes on textiles and hence, they

are co-existed with dyes in effluent, which could effect the treatment of wastewater. In the

present study, the effect of the presence of NaCl (0,3,6,9 & 12g/L) on the decolorization of

reactive dye 222 was investigated, by keeping all other factors factors (pH 3.5; H2O2, 1× 10-2 M;

FeSO4, 3.5 × 10-5 M; temperature, 50°C) constant (Table 4.1.1.5a). It is clear from the figure that

the addition of NaCl to the dye solution caused a decrease in decolorization percentage.The

decolorization efficiency within 60min reaction decreased from 95.12% to 44.67%, as a

consequence of increasing the concentration of NaCl (Fig. 4.1.1.5).


concentrations of NaCl concentrations

NaCl

(g/L)

Decolorization (%)

3 87.698A

6 57.649B

9 42.188C

12 33.42D

LSD =3.44



55

0

20

40

60

80

100

120

0 3 6 9

NaCl (g/L)

Dec

olor

izat

ion

(%

)

Fig.4.1.1.5 Effect of NaCl concentrations on the decolorization of Reactive dye 222 by



difference in dye decolorization under varying concentrations of NaCl. Comparison of means by

LSD test also showed significant (P≤0.05) difference under varying concentrations of NaCl

(Table 4.1.1.5b).


Photo-Fenton with varying concentrations of NaCl concentrations

Source of variance

df SS MSS F

Block 5 4387.568 877.514 50.439***

Treatment 3 20469.671 6823.224 392.197***

Error 39 678.5001 17.398

Total 47 25535.74

(P≤0.05)

56

Literature survey showed that the inhibitive effect of chloride ion ( generated from NaCl)

on the decolorization of raeztive azo dyes happened due to the scavenging effect of chloride ion

to HO. radical, and the following chemical reactions might happened (Ashraf et al., 2004):

HO. + Cl

- → ClOH

.-+ OH

-

ClOH.-

+ Fe+2 → Cl- + OH

-

The chloride anion can quench the hydroxyl radicals which further affect the

decolorizatioin as well as degradation rate (Pappolymerou et al., 2007). The presence of

inorganic anions reduces the electrostatic repulsion between dye molecules which leads to

aggregation making it less susceptible to hydroxyl radical attack (Dong et al., 2007;

Muruganandham & Swaminathan, 2004).


All the anions showed relative tendency to quench the hydroxyl radicals thus affecting

the degradation rate. The extent of quenching is found to be strongly dependant on nature of

anions, its concentration and on the oxidant used. Different concentrations of Na2SO4 (3 to

12g/L) were used to study their impact on on decolorization efficiency of reactive dye 222 with

respect to oxidizing agent (H2O2) , by keeping all other factors factors (pH 3.5; H2O2, 1× 10-2 M;

FeSO4, 3.5 × 10-5 M; Temperature, 50˚C) constant (Table 4.1.1.6a). It is clear from the figure that

sulphate ion has had a negative impact on the decolorization of reactive dye 222 by Photo-

Fenton oxidation. The decolorization efficiency within 60min reaction decreased from 95.34% to

44.12%, as a consequence of increasing the concentration of Na2SO4 (Fig. 4.1.1.6).

57


concentrations of Na2SO4 concentrations

Na2SO4

(g/L) Decolorization (%)

3 69.283A

6 46.617B

9 51.416B

12 20.02C

LSD =4.911



0

20

40

60

80

100

120

0 3 6 9

Na2SO4 (g/L)

Dec

olor

izat

ion

(%

)

Fig.4.1.1.6 Effect of concentrations of Na2SO4 on the decolorization of Reactive dye 222 by



difference in dye decolorization under varying concentrations of Na2SO4. Comparison of means

by LSD test also showed significant (P≤0.05) difference under varying concentrations of

Na2SO4 (Table 4.1.1.6b).

58


Photo-Fenton with varying concentrations of Na2SO4 concentrations

Source of variance

df SS MSS F

Block 5 9544.690 1908.938 53.976***

Treatment 3 14928.043 4976.014 140.698***

Error 39 1379.299 35.367

Total 47 25852.033

(P≤0.05)

The sulphate anion can form strong complex with Fe+2/Fe+3 ions thereby reducing

concentration of free iron ions in the solution which are necessary for the photo-fenton

mechanism (Orozco et al., 2008). The sulphate anion can quench the hydroxyl radicals which

further affect the decolorizatioin as well as degradation rate (Pappolymerou et al., 2007;

Muruganandham & Swaminathan, 2004). The presence of inorganic anions reduces the

electrostatic repulsion between dye molecules which leads to aggregation making it less

susceptible to hydroxyl radical attack (Dong et al., 2007).

4.1.1.7 UV-Visible and FTIR analysis of Reactive dye 222 after Photo-Fenton

treatment

To disclose the decolorization and degradation of the reactive dye 222, the analysis of the

transformation of reactive dye 222 by UV-Visible and FTIR spectral studies was done. UV-

Visible spectral analysis of untreated dye sample showed a peak in the visible region

(λmax=615nm) while the dye treated by photo-Fenton treatment indicated decrease in height of

spectral line showing maximum absorption which indicated decolorization and a shift of spectral

line towards the UV-region confirmed the degradation of dye under study (Fig. 4.1.1.7a and

4.1.1.7b)

FTIR analysis was done to characterize the metabolites formed by photo-Fenton

treatment of dye under study. FTIR spectrum of control dye with metabolites extracted after

59

complete decolorization indicated the degradation of the parent dye compound by photo-Fenton

treatment. FTIR spectrum of untreated dye displayed peaks at 3344.59, 2352.21, 1646.3, 619.15

and 613.956 cm-1 for –OH stretch of phenol, N=N stretch and C=C stretching of monosubstituted

benzene ring (Fig. 4.1.1.7c) while the FTIR spectrum of the degradation products showed

appearance of peaks at 3468.74 and 3313.55 cm-1 for stretch free –OH, 2936.64 cm-1 and

2830.22 cm-1 for C-H stretching of alkyl benzene, 2351 cm-1 for =C-H stretch, 1748.29 cm-1 for

O=C stretch, 1451.205 cm-1 for -C-H bending, 610-680 cm-1 along with overtone at 1233.93 cm-1

for -C-H deformation and 1034.39 cm-1 along C-O stretch (4.1.1.7d). Absence of peaks between

3400 to 3380 cm-1 for –NH stretch indicated that no aliphatic and aromatic amines were formed

by photo-Fenton. FTIR spectral analysis indicated that decolorization of dye under study was due

to degradation of dye in to intermediate products. The peak characteristic of azo bond at 1646.3

cm-1 of dye was absent in the decolorized medium, indication degradation of dye due to aromatic

amines as intermediate products which are subjected to oxidation giving rise to simpler

compounds.

60

Fig. 4.1.1.7a UV-Visible spectrum of reactive dye 222 before Photo-Fenton treatment

Fig. 4.1.1.7b UV-Visible spectrum of reactive dye 222 after Photo-Fenton treatment

61

Fig. 4.1.1.7a FTIR spectrum of reactive dye 222 before Photo-Fenton treatment

Fig. 4.1.1.7b FTIR spectrum of reactive dye 222 after Photo-Fenton treatment

62

4.1.2 Biological treatment 4.1.2.1 Screening of white rot fungi for Reactive dye 222

Pure cultures of five white rot fungal strains pure cultures of five indigenous white rot

fungi S. commune IBL-01 (SC), P. ostreatus IBL-02 (PO) , P. chrysosporium IBL-03 (PC), T.

versicolor IBL-04 (TV) and G. lucidum IBL-05 (GL) were initially screened for their growth and

decolorization of Reactive dye 222 in liquid growth medium. Maximum decolorization of dye

under study and fungal growth was recorded on the sixth day of incubation period (Table.

4.1.2.1). The incubation time higher or lower than sixth day showed relatively less growth as

well as decolorization rate of reactive dye 222. The maximum decolorization (%) shown by P.

ostreatus IBL-02 (PO) and P. chrysosporium IBL-03 (PC) were 92.7% and 85.9%, respectively,

while S. commune IBL-01 (SC), T. versicolor IBL-04 (TV) and G. lucidum IBL-05 (GL) showed

decolorization (%), 75.1%, 67.6% and 65.1%, respectively (Fig. 4.1.2.1a). Although all

ligninolytic enzymes were involved in the decolorization of dye under study by all fungal strains,

however Manganese Peroxidase (103.9u/ml) by P. ostreatus IBL-02 (PO) and Lignin peroxidase

(104.5u/ml) by P. chrysosporium IBL-03 (PC) turned out to be the major enzymes produced on

6th day of incubation (Fig. 4.1.2.1b) while less enzyme activities were observed produced by SC,

G.L and C.V during the decolorization experiments. On the basis of decolorization (%) findings,

finally P. ostreatus IBL-02 (PO) and P. chrysosporium IBL-03 (PC) were selected for further

optimization of various physicochemical parameters.

63

Table 4.1.2.1: Screening of WRF cultures for decolorization of Reactive dye 222.

WRF cultures

Decolorization (%) ± S.E Enzyme activity (U/mL) cell dry wt. (g)

Day-2 Day-4 Day-6 Day-8 Day-10 Lac MnP LiP

PO 80.2±0.52 83.2±0.5 92.7±0.52 64.3±0.5 60.8±0.52 25.1±0.55 103.9±0.5 114.5±0.5 0.12

PC 79.7±0.51 82.1±0.5 85.9±0.54 60.1±0.5 56.6±0.5 23.3±0.55 133.3±0.5 157.5±0.5 0.09

SC 70.1±0.52 72.4±0.5 75.1±0.52 58.9±0.5 50.6±0.53 20.7±0.53 117.7±0.5 98.4±0.52 0.06

CV 60.8±0.52 63.8±0.5 67.6±0.51 51.6±0.5 47.3±0.51 20.4±0.5 101.9±0.5 95.2±0.53 0.06

GL 56.5±0.53 61.8±0.5 65.1±0.53 46.1±0.5 40.0±0.51 15.1±0.5 99.7±0.52 92.5±0.5 0.04

64

0102030405060708090

100

2 4 6 8 10

Incubation period (Days)

Dec

olor

izat

ion

(%

)PO PC SC TV GL

Fig. 4.1.2.1a Decolorization of Reactive dye 222 by five white rot fungi

0

20

40

60

80

100

120

PO PC SC TV GL

Fungus strains

En

zym

e ac

tivi

ty (

U/m

L)

Lac MnP LiP

Fig. 4.1.2.1b Comparision of ligninolytic enzymes produced by five white rot fungi during

decolorization of Reactive dye 222

65

The white rot fungus contains enzymes and is therefore able to degrade or mineralize

several organic pollutants, researchers tested the white rot fungi T. versicolor and P. florida for

their dye decolorization ability against reactive dyes Blue CA, Black B 133 and Corazol violet

SR and reported reported that mycelia growth of all the fungi started at the very start day and

decolorization appeared from the third day onwards (Rodriguez et al., 2008). Padmavathy et al.

(2003) employed the similar method of decolorization by P. chrysosporium, T. hirusta and a

native marine fungus. Similar work of screening of white rot fungus and azo dye decolorization

assay was done by Abdullah and Zafar (1999). They carried out the decolorization study by

Pleurotus ostreatus on azo dyes such as Orange G, Congo red and Amido black 102 and dye

industry effluents and documented that decolorization was due to the ligninolytic enzymes.

Cetin and Donmez (2006) reported that P. chrysosporium decolorized 94.9% of reactiver

RR, 91.0% of reactive black RB and 63.6% of remazol blue. Abdullah and Zafar (1999) found

that the presence of LiP and/or MnP in addition to Laccase, increased decolorization of Reactive

blue up to 85%. In a number of previous reports, it has been mentioned that fungal dye

decolorization was due to adsorption (Asad et al., 2007). But appearance of very fade zones in

the decolorization experiments during present study clearly suggest that dye decolorization was

an enzymatic process rather than adsorption of dyes. This finding is also in consistent with

findings of Julia et al. (2006), who reported enzymatic color removal by fungal strains. Previous

literature study, point comes stating that dye decolorization is very much dependant on substrate

specificity of an enzyme azo-reductase both for single or mixed microbial consortia

(Zimmermann et al., 1982).

4.1.2.2 Effect of dye concentration

Effect of different concentrations of dye on its decolorization by P. ostreatus IBL-02

(PO) and P. chrysosporium IBL-03 (PC) were studied and the results have been shown in Table

4.1.2.2a and Table 4.1.2.2b. When dye concentration was increased from 0.01-0.05g/L, the

percent decolorization gradually decreased, with maximum values of 92.7% with PO and 85.9%

with PC obtained at 0.01g/L concentration of dye (Fig. 4.1.2.2a & 4.1.2.2b).

66

Table 4.1.2.2a Decolorization of Reactive dye 222 by P. ostreatus IBL-02 with different

concentrations of Reactive dye 222

Dye level

(g/100mL)

Decolorization (%)

Lac MnP LiP Dry Weight

(g)

0.01 72.198±0.51A 11.2±0.45A 116±0.32A 11.51±0.51 3.54±0.34A

0.02

62.508±0.46B 8.1±0.47B 92±0.31B 8.51±0.46B 2.21±0.31B

0.03 58.332±0.47B 7.4±0.46 B 77±0.30B 7.51±0.47B 2.01±0.33B

0.04

44.835±0.50C 5.01±0.45C 52.1±0.30C 5.32±0.46C 1.49±0.37C

0.05 37.512±0.47D 3.02±0.47D 31.8±0.29D 3.42±0.48D 0.73±0.30D

LSD 1= 5.340 LSD 2= 1.71 LSD3=16.21 LSD4= 1.51 LSD5=0.19

pH 4.5; temperature, 30 oC; inoculum size, 4mL; rice bran, 1.5g; MnSO4; CuSO4, 1mM

Means sharing similar letters in column are statistically non significant (P≤0.05) Table 4.1.2.2b Decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with different

concentrations of Reactive dye 222

Dye level (g/100mL)

Decolorization (%)

Lac (U/mL)

MnP (U/mL) LiP

(U/mL) Dry Weight (g)

0.01 67.983±0.44A 14.9±0.41A 9.1±0.33A 103.3±0.48A 1.55±0.41A

0.02

55.675±0.43B 13.2±0.42B 8.5±0.36B 82.21±0.47B 1.207±0.42B

0.03 52.687±0.41B 12.5±0.46C 7±0.32C 74.53±0.46B 1.021±0.40B

0.04

42.448±0.46C 11.4±0.45C 6.1±0.35D 62.76±0.47C 0.986±0.44C

0.05 34.705±0.42D 9.5±0.45D 5.1±0.31E 53.21±0.46D 0.731±0.42D


pH 4.5; temperature, 30°C; inoculum size, 4mL; rice bran, 1.5g; MnSO4; CuSO4, 1mM


67

0

1020

3040

50

6070

8090

100

0.01 0.02 0.03 0.04 0.05Dye level (g/100ml)

Dec

olor

izat

ion

(%

)

0

20

40

60

80

100

120

140

160

180

En

zym

e ac

tivi

ty (

U/M

L)

decolorization Lac Mnp Lip

Fig. 4.1.2.2a Effect of dye level on activities of ligninolytic enzymes of P. ostreatus IBL-02

and decolorization of Reactive dye 222

0

10

20

30

40

50

60

70

80

0.01 0.02 0.03 0.04 0.05

Dye level (g/100ml)

Dec

olor

izat

ion

( %

)

0

20

40

60

80

100

120

140

160

180

En

zym

e ac

tivi

ty (

U/M

L)


Fig.4.1.2.2b Effect of dye level on activities of ligninolytic enzymes of P. chrysosporium IBL-

03 and decolorization of Reactive dye 222

When a comparison was made among two fungal strains for their decolorization ability,

PO showed a little more potential than PC which is evident from its percent decolorization value.

68

The decolorization ability of fungal strains was as a result of the production of extra-cellular

peroxidases. With 0.01g/L concentration of dye, maximum enzyme activity of 116.54U/mL for

MnP (with PO) and 101.3U/mL for LiP (with PC) was recorded. There was no noticeable

production of laccase by both fungal strains during the decolorization experiments. From these

results, it was observed that MnP (for P.O) and LiP (for P.C) are the key enzymes responsible for

the decolorization process.


difference in dye decolorization and ligninolytic enzymes (MnP, LiP, Laccase) activities under

varying dye levels. Comparison of means by LSD test also showed significant (P≤0.05)

difference except between 0.02 and 0.03g/100mL dye levels (Table 4.1.2.2c and 4.1.2.2d).

Correlation analysis between ligninolytic enzyme (MnP, LiP, laccase) activities and dye

decolorization for P. ostreatus IBL-02 (PO), revealed a positive correlation between ligninolytic

enzyme activities and dye decolorization (Fig. 4.1.2.2c). The higher value of correlation (R2 =

0.907) for MnP showed the major role of this enzyme in the decolorization of dye (Fig. 4.1.2.2c).

With increasing MnP activity, the rate of decolorization increased and vice versa. Similar trend

of correlation was observed between ligninolytic enzymes (MnP, LiP, laccase) activities and dye

decolorization for P. chrysosporium IBL-03 (PC), the higher value of correlation (R2 = 0.907)

showed the major role of LiP in the decolorization of dye by PC (Fig. 4.1.2.2d). With increase in

LiP activity, dye decolorization increased.

Table 4.1.2.2c Analysis of variance of the data on percent decolorization of Reactive dye

222 by P. ostreatus IBL-02 with different levels of Reactive dye 222

Source of variance

df SS MSS F

Block 2 4664.375 2332.188 116.685***

Treatment 4 4634.383 1158.596 57.967***

Error 23 459.701 19.987

Total 29 9758.459

69

Table 4.1.2.2d Analysis of variance of the data on percent decolorization of Reactive dye

222 by P. chrysosporium IBL-03 with different levels of Reactive dye 222

Source of variance

df SS MSS F

Block 2 2880.417 1440.209 52.696***

Treatment 4 3908.047 977.012 35.748***

Error 23 628.605 27.331

Total 29 7417.069

(P≤0.05)

y = 2.4682x - 73.576

R2 = 0.9074

y = 2.7031x - 56.436

R2 = 0.872

y = 1.0525x - 32.769

R2 = 0.9025

0

50

100

150

200

0 20 40 60 80 100

Enzyme activity (u/mL)

Dec

olor

izat

ion

(%

)

Lac Mnp LipLinear (Mnp) Linear (Lip) Linear (Lac)

Fig. 4.1.2.2c Correlation between decolorization and ligninolytic enzymes for effect of dye

level by P. ostreatus IBL-02

70

y = 2.4682x - 73.576

R2 = 0.9074

y = 2.7031x - 56.436

R2 = 0.872

y = 1.0525x - 32.769

R2 = 0.9025

0

50

100

150

200

0 20 40 60 80 100


Dec

olor

izat

ion

(%

)


Fig. 4.1.2.2d Correlation between decolorization and activities of ligninolytic enzymes for

effect of dye level by P. chrysosporium IBL-03

Previous studies have shown that color removal depends on the destruction of the

chromophore. The peroxidases of the fungus needs to attack one molecule of the dye several

times, a lower concentration of the dye facilitates the destruction of dye molecules and the higher

concentration of the dye, slower the rate of dye removal (Lawrence and Jian, 1997; Paul et al.,

1993). The dye under study possesses sulphonic groups, at higher concentration of dye,

sulphonic groups may act as detergents and surfactants which may inhibit the growth of

microorganisms (Wuhrmann et al., 1980).

Faison et al. (1980) reported the relevant factors that influence the decolorization process

are: (1) the structure of the dyes (2) loss of the vital requirements by the organisms and (3) might

require an additional amount of veratryl alcohol apart the production from the microorganism

itself. The decrease in percent decolorization of dye at higher concentration has been reported

earlier by (Kalyani et al., 2008; and Kehra et al., 2005). Xu et al. (2006) reported that higher dye

concentration, the longer the time required removing the color.

71

4.1.2.3 Effect of pH

The decolorization of dye by P. ostreatus IBL-02 (PO) and P. chrysosporium IBL-03

(PC) was carried out at varying pH 3.5-5.5 (Table 4.1.2.3a & 4.1.2.3b). The percent

decolorization went on increasing with the rise in pH of the medium, with the maximum

decolorization, 92.92% by P. ostreatus IBL-02 (PO) (Fig. 2a) and 85.69% by P. chrysosporium

IBL-03 (PC) (Fig. 2b), for the dye studied in the present work was observed at pH 4.5. The major

production of MnP (103.70 U/mL) by Pleurotus ostreatus IBL-02 (Fig. 2a) and LiP (105.38

U/mL) by P. chrysosporium IBL-03 (PC) (Fig. 2b), were found at the optimized pH. From these

results, it was observed that MnP (for PO) and LiP (for PC) are the key enzymes responsible for

the decolorization process.

Table 4.1.2.3a Decolorization of Reactive dye 222 by P. ostreatus IBL-02 with varying pH

pH Decolorization

(%) Laccase

(U/mL) MnP (U/mL)

LiP

(U/mL) dry weight

(g)

3.5 40.58D 4.75±0.42D 30.68±0.71D 6.71±0.61D 0.074±0.21D

4.0 58.47B 6.22±0.43C 69.54±0.70C 10.42±0.60C 0.87±0.23B

4.5 71.112A 9.59±0.40A 103.70±0.7A 18.21±0.62A 1.16±0.31A

5.0 62.748B 8.06±0.44B 81.10±0.75B 13.21±0.67B 0.75±0.30B

5.5 47.328C 6.62±0.46 C 59.81±0.77C 9.32±0.64C 0.098±0.32C


*: pH 4.5; temperature, 30°C; inoculum size, 4mL; rice bran, 1.5g; MnSO4; CuSO4, 1mM


72

Table 4.1.2.3b Decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with varying

pH

pH Decolorization

(%) Laccase

(U/mL) MnP (U/mL)

LiP

(U/mL) dry weight

(g)

3.5 41.637D 11.42±0.59C 10.59±0.54C 44.09±0.61C 0.88±0.21B

4.0 57.35B 13.22±0.56B 12.54±0.54B 65.6±0.62B 0. 83±0.20B

4.5 69.07A 16.50±0.56A 14.04±0.49A 105.3±0.63A 0.96±0.22A

5.0 61.371B 14.33±0.54B 13.86±0.50B 82.36±0.64B 0.72±0.23C

5.5 46.592C 8.28±0.55D 10.10±0.51C 26.77±0.64D 0.343±0.22D




0

10

2030

40

50

60

7080

90

100

3.5 4 4.5 5 5.5

pH

Dec

olor

izat

ion

(%

)

0

20

40

60

80

100

120

En

zym

e ac

tivi

ty (

U/m

L)


Fig. 4.1.2.3a Effect of dye level on activities of ligninolytic enzymes of P. ostreatus IBL-02


73

0

10

20

30

40

50

60

70

80

90

100

3.5 4 4.5 5 5.5

pH

Dec

olor

izat

ion

(%

)

0

20

40

60

80

100

120

En

zym

e ac

tivi

ty (

U/m

L)


Fig. 4.1.2.3b Effect of pH on activities of ligninolytic enzymes of P. chrysosporium IBL-03





difference except between pH 4 and 5 (Fig. 4.1.2.3c and 4.1.2.3d). Correlation analysis between

ligninolytic enzyme (MnP, LiP, laccase) activities and dye decolorization for P. ostreatus IBL-

02 (PO), revealed a positive correlation between ligninolytic enzyme activities and dye

decolorization (Fig. 4.1.2.3c). The higher value of correlation (R2=0.9351) for MnP showed the

major role of this enzyme in the decolorization of dye (Fig. 4.1.2.3c). With increasing MnP

activity, the rate of decolorization increased and vice versa. Similar trend of correlation was

observed between ligninolytic enzymes (MnP, LiP, laccase) activities and dye decolorization for

P. chrysosporium IBL-03 (PC), the higher value of correlation (R2=0.9321) showed the major

role of LiP in the decolorization of dye by P.C (Fig. 4.1.2.3d). With increase in LiP activity, dye

decolorization increased.

74


222 by P. ostreatus IBL-02 with varying pH

Source of variance

df SS MSS F

Block 5 6545.56 1309.11 47.942***

Treatment 4 7115.41 1778.85 65.14***

Error 50 1365.32 27.31

Total 59 15026.28

(P≤0.05)


222 by P. chrysosporium IBL-03 with varying pH

Source of variance

df SS MSS F

Block 5 5968.61 1193.722 46.591***

Treatment 4 5917.75 1479.438 57.742***

Error 50 1281.076 25.622

Total 59 13167.438

(P≤0.05)

75

y = 1.6192x - 45.037R2 = 0.9351

y = 0.319x - 11.277R2 = 0.7388

y = 0.1222x - 1.7092R2 = 0.7433

0

20

40

60

80

100

120

0 20 40 60 80 100


Dec

olor

izat

ion

(%

)


Fig. 4.1.2.3c Correlation between decolorization and activities of ligninolytic enzymes for

effect of pH by P. ostreatus IBL-02

y = 1.9306x - 75.798R2 = 0.8777

y = 0.1764x - 2.7783R2 = 0.797

y = 0.1039x - 1.6403R2 = 0.9329

0

20

40

60

80

100

120

0 20 40 60 80 100


Dce

olor

izat

ion

(%

)

Lac Mnp LipLinear (Mnp) Linear (Lac) Linear (Lip)


effect of pH by P. chrysosporium IBL-03

76

The pH effect on enzymatic decolorization was associated with the pH dependence of

enzyme activity and the dye itself. These observations indicate the optimum pH for both fungal

strains depends on the nature of the pH of medium and reported by Alberto et al. (2000).

According to the Gupta and Tanaka, 1995; Wu et al., 2001, pH is one of the factors influencing

the rate of decolorization of some organic compounds in the decolorization process. As pH was

further raised, there was a decrease in decolorization for both fungal strains used for

biodecolorization of Reactive dye 222. From, the Fig. 4.3a & 4.3b, it is quite clear that P.O

showed little more sensitivity towards change in pH as compared to P.C. Our results are in

agreement as reported by Asad et al. (2007) using halotolerant and halophilic bacteria.

As observed by Dirk et al. (2003) and Ana et al. (2002), the white rot fungi produce

organic acids such as malonate, oxalate during the initial growth period, which later decomposed

by ligninolytic enzymes. The white rot fungus such as P. chrysosporium was biologically active

during this period. Medium pH is expected to affect the ionization state of the functional groups

on the fungal cell walls and the entrapped groups such as carboxylic, phosphate and amino

groups (Yakup et al., 2003). Raghukumar et al. (1996) used three marine fungal strains for color

removal and report pH 4.5 to be the most effective. It is a well established fact that fungus grow

at relatively low pH than bacteria cultures which normally ranges from 4-5.

Ollika et al. (1993) studied lignin peroxidase mediated oxidation of Congo red at pH 4.5.

In the report by Cripps et al. (1993), the attempted enzymatic oxidation of Congo Red by crude

lignin peroxidase was performed at pH 4.5. Although pH 4.5 is within the physiological range

that is near optimum for growth and lignin degradation. Research by Tavares et al. (2006)

showed that very low pH values of the cultures medium are not favorable for ligninolytic

enzymes production by Trametes versicolor. There could be two explanations for this: (1) either

the metabolism of the ligninolytic enzymes synthesis is repressed at very low pH value; or (2)

conformational changes in the enzyme three dimensional structures are promoted at very low pH,

affecting the active site, not allowing biocatalytic reactions. Nanhongo et al. (2002) also fount

that ligninolytic enzymes stability from T. modesta was significantly affected by pH < 4.5.

Another study by Jonsson et al. (1997) showed that pH less than four was detrimental for laccase

production and suggested that a possible explanation for this was laccase’s susceptibility to

acidic proteases.

77

4.1.2.4 Effect of Inoculum size

Inoculum size plays a significant role in enzyme production in liquid state medium. A

lower level of inoculum may not be sufficient to initiate the growth, whereas a higher level may

cause competitive inhibition (Sabu et al., 2005), so the determination of optimum inoculum size

becomes a crucial step in liquid state medium. The effect of fungal inoculum size on %

decolorization, and MnP, LiP and Laccase activities by PO and PC was investigated (Table

4.1.2.4a & 4.1.2.4b). The inoculum size used in the study was varied at 1,2,3,4 and 5mL, keeping

the all other factors constant. As inoculum size was increased, the increase in decolorization (%)

was obtained for both PO and PC. Experimental results on the estimation of optimum

concentration of inoculum size necessary to achieve maximum decolorization, showed that the

maximum decolorization (%) shown by P. ostreatus IBL-02 (PO) and P. chrysosporium IBL-03

(PC) at an inoculum size of 4ml, were 91.19% (Fig. 3a) and 85.42%, respectively (Fig. 3b). The

high activity of MnP (101.32U/mL) by PO (Fig. 3a) and LiP (118.28 U/mL) by PC (Fig. 3b) was

detected, at optimized level of inoculum (4mL).

Table 4.1.2.4a Decolorization of Reactive dye 222 by P. ostreatus IBL-02 with varying

Inoculum size

Inoculum size

(mL) Decolorization

(%) Laccase

(U/mL) MnP (U/mL)

LiP

(U/mL) dry weight

(g)

1 32.25D 11.11±0.61B 48.00±0.39D 9.72±0.51C 0. 85±0.2C

2 38.696C 11.23±0.60B 85.99±0.37B 12.32±0.50B 1. 27±0.2B

3 46.465B 10.22±0.62C 73.86±0.42C 13.22±0.54B 1. 31±0.2B

4 59.358A 14.11±0.60A 101.32±0.52A 15.28±0.49A 1.41±0.3A

5 46.632B 8.89±0.63D 88.99±0.50B 6.23±0.52D 0.66±0.27D




78

Table 4.1.2.4b Decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with varying Inoculum size

Inoculum size

(mL)

Decolorization (%)

Laccase

(U/mL) MnP (U/mL) LiP (U/mL)

dry weight (g)

1 28.254D 11.11±0.61D 10.9±0.51D 81.72±0.51D 0.67±0.3D

2 38.058C 14.03±0.62C 12.15±0.50C 90.32±0.50C 0.72±0.31C

3 44.178B 17.22±0.62B 15.54±0.49B 101.22±0.54B 0.92±0.32B

4 55.835A 14.32±0.63A 18.65±0.52A 118.28±0.49A 1.24±0.3A

5 45.169B 10.89±0.64B 16.49±0.53B 103.23±0.52B 0.90±0.31B




0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5Innoculum size ( ml )

Dec

olor

izat

ion

( %

)

0

20

40

60

80

100

120

En

zym

e ac

tivi

ty (

U/m

L)


Fig. 4.1.2.4a Effect of inoculum size on activities of ligninolytic enzymes of P. ostreatus IBL-

02 and decolorization of Reactive dye 222

79

0

10

20

30

40

50

60

70

80

1 2 3 4 5Innoculum size ( ml )

Dec

olor

izat

ion

( %

)

0

20

40

60

80

100

120

140

En

zym

e ac

tivi

ty (

U/m

L)


Fig.4.1.2.4b Effect of Inoculum size on decolorization (%) of Reactive dye 222 by

P. chrysosporium IBL-03




difference except between at 3 and 5 inoculum size (Table 4.1.2.4c and 4.1.2.4d). Correlation

analysis between ligninolytic enzyme (MnP, LiP, laccase) activities and dye decolorization for P.

ostreatus IBL-02 (PO), revealed a positive correlation between ligninolytic enzyme activities and

dye decolorization. The higher value of correlation (R2 = 0.956) for MnP showed the major role

of this enzyme in the decolorization of dye (Fig. 4.1.4c). With increasing MnP activity, the rate

of decolorization increased and vice versa. Similar trend of correlation was observed between

ligninolytic enzymes (MnP, LiP, laccase) activities and dye decolorization for P. chrysosporium

IBL-03 (PC), the higher value of correlation (R2 = 0.9597) showed the major role of LiP in the

decolorization of dye by P.C (Fig. 4.1.2.4d). With increase in LiP activity, dye decolorization

increased.

80


222 by

P. ostreatus IBL-02 with varying Inoculum size

Source of variance

df SS MSS F

Block 3 7379.921 2459.974 120.425***

Treatment 4 3589.341 897.335 43.928***

Error 32 633.252 20.428

Total 39 11156.568

(P≤0.05)

Table 4.1.2.4d Analysis of variance on percent decolorization of Reactive dye 222 by

P. chrysosporium IBL-03 with varying Inoculum size

Source of variance

df SS MSS F

Block 3 7473.5625 2491.1875 162.8363***

Treatment 4 3281.975 820.4938 53.6315***

Error 32 489.5593 15.2987

Total 39 11245.0969

(P≤0.05)

81

y = 0.847x + 44.53

R2 = 0.907

y = 0.1076x + 8.7822

R2 = 0.956

y = 0.1726x + 3.1411

R2 = 0.6683

0

20

40

60

80

100

120

140

0 10 20 30 40 50 60 70 80 90 100


Dec

olor

izat

ion (%

)

Lac Mnp LipLinear (Lip) Linear (Mnp) Linear (Lac)


effect of Inoculum size by P. ostreatus IBL-02

y = 0.847x + 44.53R2 = 0.9597

y = 0.1076x + 8.7822R2 = 0.8068

y = 0.1726x + 3.1411R2 = 0.6683

0

20

40

60

80

100

120

140

0 10 20 30 40 50 60 70 80 90 100


Dec

olor

izat

ion (%

)



effect of Inoculum size by P. chrysosporium IBL-03

82

The results indicate that production of MnP and LiP is dependant on the fungal inoculum

size as highest activity for both was observed at inoculum size of 4ml. At the higher inoculum

size, a decrease in decolorization (%) and enzyme activities was detected. Our results are in

consistent with the experimental findings of Kashyap et al. (2002). They found that lower

inoculum size requires longer time for the multiplication of cells to increase in sufficient

numbers to use substrate and produce enzymes. An increase in inoculum size would ensure a

rapid proliferation of fungal strains and hence increase in biomass synthesis. After a certain limit,

production of enzymes could decrease due to depletion of nutrients and enhanced biomass,

which ultimately decrease in metabolic activity. Our results showed a direct relationship b/w

enzyme activities and dye decolorization as highest activities of enzymes and dye decolorization

was found at the same inoculum size (4ml). The Baldrian and Snajdr (2006) reported that RBBR

dye was more efficiently degraded than RB5 by the liter-decomposing fungi as well as the role of

ligninolytic enzymes in degradation of dye. Shahvali et al. (2000) also reported that inoculum

size of 4-5ml is sufficient for the decolorization of the textile wastewater, above which no

appreciable change in decolorization (%) was recorded.

4.1.2.5 Effect of temperature

Temperature is a factor of paramount importance for all processes related with microbial

vitality, including the remediation of water and soil. Temperature is an evitable factor due to its

impact on microbial growth and enzyme production. In order to study the variation in

temperature and decoloriaztion, studies were carried out at temperature ranges from 25 to 40°C,

keeping other conditions constant (pH 4.5, dye 0.01g/L, inoculum’s size 4ml/100ml of dye

solution). Results are shown in Fig. 4.1.2.5a & 4.1.2.5b and are discussed here under. The

maximum percent decolorization values shown by P. ostreatus IBL-02 (PO) and P.

chrysosporium IBL-03 (PC) at incubation temperature of 30°C were 92.14% and 85.71%,

respectively (Fig. 4a & 4b). The enzyme profiles revealed the major production of MnP

(102.5U/mL) by PO (Fig. 4a) and LiP (104.32U/mL) by PC (Fig. 4b), at the optimized

incubation temperature (30°C). There was no noticeable production of laccase by both fungal

strains during the decolorization experiments. At higher (>30°C) or lower (>30°C) temperatures

the decolorization activity of the fungal strains gets reduced indicating that either the fungus is

83

not able to produce the peroxidases for decolorization or they get denatured (Baldrian and

Snajdr, 2006).


temperature

Temperature

(0C) Decolorization

(%) Laccase

(U/mL) MnP (U/mL)

LiP

(U/mL) dry weight

(g)

25 42.725C 11.74±0.61C 45.83±0.48C 4.57±0.46C 0.85±0.22C

30 69.538A 13.47±0.58A 106.50±0.48A 8.28±0.51A 1.70±0.21A

35 56.207B 11.70±0.60B 84.72±0.46B 5.49±0.48B 0.98±0.21B

40 47.708C 11.42±0.65C 56.39±0.51C 4.89±0.47C 0.81±0.23C





temperature

Temperature

(0C) Decolorization

(%) Laccase

(U/mL) MnP (U/mL)

LiP

(U/mL)

cell dry weight

(g)

25 43.652B 4.12±0.43B 3.34±0.47B 45.32±0.46B 0.72±0.11B

30 61.85A 7.11±0.44A 6.11±0.46A 114.32±0.46A 0.93±0.10A

35 47.009B 5.81±0.46B 3.81±0.48B 48.92±0.47B 0.74±0.13B

40 38.472C 5.23±0.47C 4.11±0.47C 32.47±0.48C 0.61±0.12C




84

0

10

20

30

40

50

60

70

80

90

100

25 30 35 40

Temperature (oC)

Dec

olor

izat

ion

(%

)

0

20

40

60

80

100

120

En

zym

e ac

tivi

ty (

U/m

L)


Fig.4.1.2.5a Effect of temperature on activities of ligninolytic enzymes of P. ostreatus IBL-02


0

10

20

30

40

50

60

70

80

25 30 35 40

Temperature (oC)

Dec

olor

izat

ion

(%

)

0

20

40

60

80

100

120

En

zym

e ac

tivi

ty (

U/m

L)


Fig. 4.1.2.5b Effect of temperature on activities of ligninolytic enzymes of P. chrysosporium

IBL-03 and decolorization of Reactive dye 222

85




difference except between at 30 and 35°C temperatures (Table 4.1.2.5c and 4.1.2.5d). Correlation

analysis between ligninolytic enzyme (MnP, LiP, laccase) activities and dye decolorization for P.

ostreatus IBL-02 (PO), revealed a positive correlation between ligninolytic enzyme activities and

dye decolorization (Fig. 4.1.2.5c). The higher value of correlation (R2=0.9221) for MnP showed

the major role of this enzyme in the decolorization of dye (Fig. 4.1.2.5c). With increasing MnP

activity, the rate of decolorization increased and vice versa. Similar trend of correlation was

observed between ligninolytic enzymes (MnP, LiP, laccase) activities and dye decolorization for

P. chrysosporium IBL-03 (PC), the higher value of correlation (R2=0.9372)) showed the major

role of LiP in the decolorization of dye by PC (Fig. 4.1.2.5d). With increase in LiP activity, dye

decolorization increased.


222 by P. ostreatus IBL-02 with varying temperature

Source of variance

df SS MSS F

Block 4 10959.032 2739.758 76.871***

Treatment 3 4130.073 1376.691 38.626***

Error 32 1140.517 35.641

Total 39 16229.621

(P≤0.05)

86


222 by P. chrysosporium IBL-03 with varying temperature

Source of variance

df SS MSS F

Block 4 8095.056 2023.765 86.349***

Treatment 3 3022.339 1007.446 42.986***

Error 32 749.979 23.437

Total 39 11867.378

(P≤0.05)

y = 2.8543x - 139.31R2 = 0.772

y = 0.1012x - 2.2986R2 = 0.9221

y = 0.0996x - 1.357R2 = 0.7493

0

20

40

60

80

100

120

0 20 40 60 80 100


Dec

olor

izat

ion

(%

)



effect of temperature by P. ostreatus IBL-02

87

y = 2.8543x - 139.31R2 = 0.9372

y = 0.1012x - 2.2986R2 = 0.7221

y = 0.0996x - 1.357R2 = 0.7493

0

20

40

60

80

100

120

0 20 40 60 80 100


Dec

olor

izat

ion

(%

)Lac Mnp LipLinear (Lip) Linear (Mnp) Linear (Lac)


effect of temperature by P. chrysosporium IBL-03

In microorganisms the environmental temperature directly establishes organismal

temperature, as the microbial cell responds to temperature changes by adaptation via biochemical

or enzymatic mechanisms. A direct relationship exists b/w rate of microbial growth and

enzymatic reactions. In many cases, growth increases with increase in temperature but it

decreases sharply at upper and lower extreme limits of temperature. It has been observed that the

decolorization rate of azo dyes increases up to the optimum temperature, and afterwards, there is

a marginal reduction in the decolorization reduction (Chang et al., 2001). The optimum

temperature for substrate oxidation by white rot fungi in the presence of MnP as a major enzyme

by PO along with other ligninocellulosic enzymes has been consistently reported as 30-35°C. The

optima determined by us are consistent with these reports (Tan et al, 2000; Aksu and Tezer,

2000; Nyanhongo et al, 2002; Masud and Anantharaman, 2006), who explained that fungal

growth was supported in a limited temperature range with dye removal.

Chen et al. (2002) reported that optimum temperature for color removal of red azo dye

was 30°C and 35°C. Mehne et al. (1995) also gave the same explanation for color reduction to be

88

optimum at 35°C. The optimal temperature of decolorization by Coriolus sp. was found to be

30°C in a report (Fu and Vararaghavan, 2001). Knapp et al. (2001) demonstrated that the main

potential advantage of microorganism having high optimal temperature does not lie in more

rapid catalysis but in the fact that in large scale bioreactors removal of excess metabolic heat will

present less of a problem at a higher temperature, cooling costs will be less. A report given by

Zhang et al. (2007) explained that lower decolorization at higher temperatures may be attributed

to the deactivation of the decolorization enzymes and low biomass production. Same reports

were given by other researchers (Chen et al. 2008; Rajamohan and Karthikeyan, 2006). Radha et

al. (2005) reported reduction of decolorization activity of fungus at higher temperatures indicate

either the fungus is not able to produce the peroxidases for decolorization or they get denatured

or loss of cell viability (Chen et al. 2008) so microbial growth is greatly affected by change in

temperature.

4.1.2.6 Effect of additional carbon sources

Carbon is a vital source of energy for microorganisms. Fungi consume and grow on

readily available carbon sources at the initial stages of growth and then produce secondary

metabolites and extracellular enzymes for biodegradation of dyestuff. To find an inexpensive and

more suitable carbon source, different carbon sources (starch, glycerol, glucose, rice bran and

wheat bran) were used in decolorization experiments to study their effect on potential removal of

dye under study (Table 4.1.2.6a and 4.1.2.6b). Concentration of carbon sources was 2g/100mL

(pre-optimized value). The addition of rice bran as additional carbon source in growth media

showed maximum decolorization performance 94.34% (by PO) and 92.11% (by PC), whereas

with starch, glycerol, glucose and wheat bran, the color reductions were 56.68%, 57.46%,

65.83% and 90.21%, respectively (Fig. 4.1.2.6a & 4.1.2.6b). The production of ligninolytic

enzymes were also studied under the same conditions as in the decolorization experiments. The

high activity (125.1U/mL) of MnP (by PO) and high activity (136.81U/mL) of LiP (by PC) was

detected (Fig. 4.1.2.6c and 4.1.2.6d). These results clearly indicated that rice bran showed to be a

most effective source of carbon for bio-decolorization of reactive dye 222 by both fungal strains.

89


Carbon sources

Carbon sources

Decolorization (%)

Laccase

(U/mL) MnP (U/mL)

LiP

(U/mL) dry weight

(g)

Starch 42.235D 13.89±0.48D 73.34±0.50D 14.63±0.47D 0.74±0.51D

Glycerol 45.943C 16.67±0.45C 95.07±0.49C 16.63±0.46C 0.87±0.50C

Glucose 49.011C 17.36±0.48C 92.32±0.53C 18.92±0.48C 0.152±0.51C

Rice bran 77.433A 18.25±0.45A 125.1±0.52A 22.81±0.43A 0.162±0.52A

Wheat bran 71.192B 16.20±0.48 98.24±0.51B 21.62±0.42B 0.78±0.52B


pH 4.5; temperature, 30oC; inoculum size, 4mL; rice bran, 1.5g; MnSO4; CuSO4, 1mM



carbon sources

Carbon sources

Decolorization (%)

Laccase

(U/mL) MnP (U/mL)

LiP

(U/mL) dry weight

(g)

Starch 41.11B 15.89±0.48B 8.34±0.50B 94.93±0.47B 1.54±0.32B

Glycerol 44.776B 16.67±0.45B 8.07±0.49B 94.63±0.46B 1.57±0.31B

Glucose 48.423B 17.36±0.48B 8.32±0.53B 98.92±0.48B 1. 52±0.3B

Rice bran 70.25A 18.25±0.45A 11.1±0.52A 136.81±0.43A 1. 61±0.3A

Wheat bran 65.128A 16.20±0.48A 10.24±0.51A 131.62±0.42A 1.68±0.3A




90

0

10

20

30

40

50

60

70

80

90

100

Starch Glycerol Glucose Rice bran Wheat bran

Carbon sources

Dec

olor

izat

ion

(%

)

Fig. 4.1.2.6a Effect of additional carbon sources on decolorization of Reactive dye 222 by

P. ostreatus IBL-02

0

20

40

60

80

100

120

140


Carbon sources

Enz

yme

acti

vity

(u/

mL

)

Lac Mnp Lip

Fig. 4.1.2.6b Comparison of activities of ligninolytic enzymes produced by P. ostreatus IBL-

02 during decolorization of Reactive dye 222

91

0

10

20

30

40

50

60

70

80

90


Carbon sources

Dec

olor

izat

ion

(%

)

Fig. 4.1.2.6c Effect of additional carbon sources on decolorization of Reactive dye 222 by


0

20

40

60

80

100

120

140

Starch Glycerol Glucose Rice bran Wheat branCarbon sources

En

zym

e ac

tivi

ty (

u/m

L)

Lac Mnp Lip

Fig. 4.1.2.6d Comparison of activities of ligninolytic enzymes produced by

P. chrysosporium IBL-03 during decolorization of Reactive dye 222

92



varying dye levels (Table 4.1.2.6c and 4.1.2.6d). Comparison of means by LSD test also showed

significant (P≤0.05) difference except between with glucose, starch and glycerol as additional

carbon sources. Correlation analysis between ligninolytic enzyme (MnP, LiP, laccase) activities

and dye decolorization for P. ostreatus IBL-02 (PO), revealed a positive correlation between

ligninolytic enzyme activities and dye decolorization ((Fig. 4.1.2.6e). The higher value of

correlation (R2=0.9695) for MnP showed the major role of this enzyme in the decolorization of

dye (Fig. 4.1.2.6e). With increasing MnP activity, the rate of decolorization increased and vice

versa. Similar trend of correlation was observed between ligninolytic enzymes (MnP, LiP,

laccase) activities and dye decolorization for P. chrysosporium IBL-03 (PC), the higher value of

correlation (R2=0.9261) showed the major role of LiP in the decolorization of dye by PC (Fig.

4.1.2.6f). With increase in LiP activity, dye decolorization increased.

Table 4.1.2.6c Analysis of variance of the data on decolorization of Reactive dye 222 by P.

ostreatus IBL-02 with varying carbon sources

Source of variance

df SS MSS F

Block 2 4493.318 2246.659 325.844***

Treatment 4 6137.161 1534.290 222.526***

Error 23 158.583 6.895

Total 29 10789.062

(P≤0.05)

93

Table 4.1.2.6d Analysis of variance of the data on decolorization of Reactive dye 222 by

P. chrysosporium IBL-03 with varying carbon sources

Source of variance

df SS MSS F

Block 2 4694.896 2347.448 58.122***

Treatment 4 4021.224 1005.306 24.891***

Error 23 928.937 40.389

Total 29 9645.057

(P≤0.05)

y = 1.5148x - 19.595R2 = 0.5261

y = 0.0509x + 12.789R2 = 0.5938

y = 0.0378x + 5.8715R2 = 0.9665

0

20

40

60

80

100

120

140

0 20 40 60 80 100


Dec

olor

izat

ion (%

)

Lac Mnp LipLinear (Lip) Linear (Lac) Linear (Mnp)

Fig. 4.1.2.6e Correlation between decolorization and activities of ligninolytic enzymes for

effect of additional carbon sources by P. ostreatus IBL-02

94

y = 1.5148x - 19.595R2 = 0.9261

y = 0.0509x + 12.789R2 = 0.5938

y = 0.0378x + 5.8715R2 = 0.5665

0

20

40

60

80

100

120

140

0 20 40 60 80 100


Dec

olor

izat

ion

(%

)

Lac Mnp LipLinear (Lip) Linear (Lac) Linear (Mnp)

Fig. 4.1.2.6f Correlation between decolorization and activities of ligninolytic enzymes for

effect of additional carbon sources by P. chrysosporium IBL-03

The purpose of supplement of additional carbon sources other than dye itself has two

reasons: First, it promotes the growth and rapid establishment of fungus within provided raw

material (basal nutrient medium). Second, the white rot fungus needs additional easily

matabolizable carbon sources to degrade dye more efficiently (Kaal et al., 1995). Earlier reports

on decolorization studies (Nyanhango et al., 2002; Masud and Anantharaman, 2006) indicated

the necessity of additional carbon sources other than dye itself to enhance the decolorization

process. The effect of organic carbon sources on adaption of the fungus for the production of

ligninolytic enzymes is of importance. Many previous studies have proved that both and the

concentration of carbon sources is powerful nutritional factors regulating ligninolytic enzymes

production by wood rotting basidiomycetes (Galhaup et al., 2002).

Others reported that in start metabolism of additional carbon like rice bran might cause

an increased production of nucleotides leading to increase in decolorization efficiency

(Kumarasamy et al., 2009). Sathiy-moorthi also found effective decolorization by P. florida

supplemented with 2% rice bran as a supplement (Carliell et al., 1995). Variability among the

95

earlier reported studies and forgoing research might be due to difference in microbial

characteristic and enzymes responsible for color removal. It can be concluded that in metabolism

of additional carbon sources glucose or rice bran might caused an increased production of

nucleotides leading to increase in decolorization efficiency (Khehra et al., 2005).

4.1.2.7 Effect of additional nitrogen sources

Since nitrogen is an essential component of fungal metabolism so the effect of different

nitrogen sources (ammonium oxalate, corn steep liquor (CSL), maize glutein meal 60%, maize

meal 30% and yeast extract) was studied on color reduction (Table 4.1.2.7a & 4.1.2.7b). It was

notable that with addition of ammonium oxalate as additional nitrogen source, both PO and PC

showed more color reduction 82.12% and 75.3%, respectively (Fig. 4.1.2.7c and 4.1.2.7d).

Other additional nitrogen sources showed less color reduction with both fungal strains.

Moreover, production of ligninolytic enzymes (MnP & LiP) by both fungal strains was also

reduced in the presence of additional nitrogen sources, as it is evident from Fig. 4.1.2.7c and

4.1.2.7d. The percent decolorization values in the presence of additional nitrogen sources were

less than in the absence of additional nitrogen sources (92.7% and 85.9%, by both PO and PC,

respectively), suggesting that fungal strains are more efficient in utilizing the nitrogen of the dye

present in the medium than added nitrogen, thus enhancing the color removal in the absence of

additional nitrogen in the growth medium.

96


nitrogen sources

Nitrogen sources

Decolorization (%)

Laccase (U/mL)

MnP (U/mL) LiP (U/mL) dry weight

(g) Corn steep

liquor 51.952C 5.97±0.49C 31.93±0.51C 10.7±0.44C 0.71±0.22C

Yeast extract 57.995B 9.03±0.47B 37.10±0.52B 14.8±0.42B 0. 81±0.23B

Maize gluetein 60%

70.618A 14.8±0.46A 50.49±0.54A 18.4±0.41 1.12±0.21A

Ammonium oxalate

72.323A 15.1±0.46A 49.61±0.50A 19.5±0.43 1.19±0.2A

Maize gluetein 30%

72.577A 15.2±0.46A 49.61±0.50A 18.0±0.45 1.11±0.21A




Table 4.1.2.7b Decolorization of Reactive dye 222 by P. chrysosporium IBL-03with varying

nitrogen sources

Nitrogen sources

Decolorization (%)

Laccase (U/mL)

MnP (U/mL) LiP (U/mL) dry weight

(g) Corn steep

liquor 53.238B 5.00±0.52B 3.73±0.42B 30.1±0.52B 0.309±0.24B

Yeast extract 55.913B 7.36±0.52B 4.65±0.41B 33.3±0.53B 0.723±0.23B

Maize gluetein 60%

63.456A 8.4±0.53A 5.99±0.40A 50.3±0.54A 0.893±0.21A

Ammonium oxalate

67.08A 9.0±0.51A 6.77±0.42A 65.7±0.55A 1.117±0.22A

Maize gluetein 30%

64.08A 8.47±0.5A 5.30±0.45A 38.5±0.56A 1.02±0.23A




97

10

20

30

40

50

60

70

80

Corn steepliquor

Yeast extract M.G 60% Amm. Oxalate M.G 30%

Nitrogen sources

Dec

olor

izat

ion

(%

)

Fig. 4.1.2.7a Effect of additional nitrogen sources on decolorization of Reactive dye 222 by

P. ostreatus IBL-02

0

10

20

30

40

50

60

70

80

Corn steep liquor Yeast extract M.G 60% Amm. Oxalate M.G 30%

Nitrogen sources

Enz

yme

acti

vity

(u/

mL

)

Lac Mnp Lip

Fig. 4.1.2.7b Comparison of ligninolytic enzymes produced by P. ostreatus IBL-02 during


98

0

10

20

30

40

50

60

70

80

90

Corn steep liquor Yeast extract M.G 60% Amm. Oxalate M.G 30%

Nitrogen sources

Dec

olor

izat

ion

(%)

Fig. 4.1.2.7c Effect of additional nitrogen sources on decolorization of Reactive dye 222 by


0

10

20

30

40

50

60

70

80

Corn steep liquor Yeast extract M.G 60% Amm. Oxalate M.G 30%Nitrogen sources

En

zym

e ac

tivi

ty (

u/m

L)

Lac Mnp Lip

Fig. 4.1.2.7d Comparison of ligninolytic enzymes produced P. chrysosporium IBL-03 during


99




significant (P≤0.05) difference except between maize yeast extract, glutein 30% and 60% as

additional nitrogen sources. Correlation analysis between ligninolytic enzyme (MnP, LiP,

laccase) activities and dye decolorization for P. ostreatus IBL-02 (PO), revealed a positive

correlation between ligninolytic enzyme activities and dye decolorization (Fig. 4.1.2.7e). The

higher value of correlation (R2 = 0.8479) for MnP showed the major role of this enzyme in the

decolorization of dye (Fig. 4.2.7e). With increasing MnP activity, the rate of decolorization

increased and vice versa. Similar trend of correlation was observed between ligninolytic

enzymes (MnP, LiP, laccase) activities and dye decolorization for P. chrysosporium IBL-03

(PC), the higher value of correlation (R2 = 0.775) showed the major role of LiP in the

decolorization of dye by PC (Fig. 4.1.2.7f). With increase in LiP activity, dye decolorization

increased.


ostreatus IBL-02 with varying additional nitrogen sources

Source of variance

df SS MSS F

Block 2 1682.751 841.375 80.565***

Treatment 4 2171.331 542.8328 51.978***

Error 23 240.199 10.444

Total 29 4094.282

(P≤0.05)

100


P. chrysosporium IBL-03 with varying additional nitrogen sources

Source of variance

df SS MSS F

Block 2 1885.204 942.602 85.039***

Treatment 4 877.922 219.481 19.801***

Error 23 243.854 11.084

Total 29 2900.469

(P≤0.05)

y = 1.2097x - 43.466

R2 = 0.6916

y = 0.1094x - 2.5875

R2 = 0.808

y = 0.1467x - 2.9124

R2 = 0.8479

0

10

20

30

40

50

60

70

0 20 40 60 80 100


Dce

olor

izat

ion

(%

)



effect of additional nitrogen sources by P. ostreatus IBL-02

101

y = 3.3693x - 164.52R2 = 0.3409

y = 0.1108x + 8.2071R2 = 0.775

y = 0.1562x + 0.3665R2 = 0.204

020406080

100120140160180

0 20 40 60 80 100 120


Dec

olor

izat

ion

(%

)


Fig. 4.1.2.7f Correlation between decolorization and activities of ligninolytic enzyme for

effect of additional nitrogen sources by P. chrysosporium IBL-03

Decrease in decolorization potential may be due to the accumulation of simple nitrogen

compound added, acting as a co-metabolic substrate in media. Similar trend has been observed

by others (Xu et al., 2006). These results are also in harmony with those of Zhang et al. (1998),

Tatarko and Bumpus (1998) and Sanghi et al. (2006), who also reported the less efficient

removal of dye with supplemental nitrogen. Literature survey have shown that the addition of

nitrogen sources seemed to be less effective in promoting decolorization, probably due to the

preference of the cells in assimilating the dye as the nitrogen source. It might be due to the

decreased microbial activity in the presence of supplemental nitrogen which causes a shift of pH

more towards acidic side, which decreased the color removal, growth as well as enzymatic

activity of fungal strains (Saratale et al., 2009).

102

4.1.2.8 Effect of mediators

Redox mediators have a profound effect on microbial growth, ligninolytic enzymes

which decolorize different dyes. Different redox mediators like Veretryl Alcohol, MnSO4,

Glycerol, Ethanol, ABTS, Oxalate and H2O2 were added in flasks having dye to evaluate their

effect on decolorization of dye under study (Table 4.1.2.8a and 4.1.2.8b). Maximum

decolorization (93.82% and 91.11%) was achieved in the presence of MnSO4 as a redox

mediator by both PO and PC, respectively (Fig. 4.1.2.8a & 4.1.2.8b).


mediators

Mediators

Decolorization (%)

Lac (U/mL)

MnP (U/mL)

LiP (U/mL)

dry weight (g)

Glycerol 59.367E 8.7±0.50E 36±0.62E 14.15±0.5E 1.376±0.41E

ABTS 65.292D 9.3±0.52D 70±0.64D 16.1±0.56D 1.395±0.41D

Oxalic acid 68.968C 10.6±0.51C 93.5±0.61C 17.1±0.59C 1.446±0.42C

MnSO4

89.417A 15.2±0.54A 150±0.63A 20±0.57A 2.103±0.43A

H2O2

73.788B 11.6±0.49B 105±0.64B 19.1.2±0.56B 1.604±0.44B

V.Alcohol

68.792C 10.2±0.50C 97±0.62C 17.8±0.58C 1.44±0.42C




103

Table 4.1.2.8b Decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with

different mediators

Mediators

Decolorization (%)

Lac (U/mL)

MnP (U/mL)

LiP (U/mL)

dry weight (g)

Ethanol

54.61F 31.6±0.41F 28±0.47F 35.0±0.57F 0.15F

Glycerol 57.827E 40.7±0.44E 45±0.51E 58.1±0.56E 0.34E

ABTS 64.108D 43.5±0.38D 48±0.48D 105±0.56D 0.77D

Oxalic acid 67.533C 47.5±0.44C 54.1±0.50C 110±0.59C 1.480C

MnSO4

88.305A 62.8±0.39A 79±0.48A 149±0.57A

1.901A

H2O2

72.068B 33.5±0.42B 73±0.46B 130±0.56B 1.635B

V.Alcohol

66.572C 48.1±0.43C 65±0.51C 111±0.58C 1.564C




0

10

20

30

40

50

60

70

80

90

100

Ethanol Glycerol ABTS Oxalic acid MnSO4 H2O2 V.AlcoholMediators

Dec

olor

izat

ion

(%

)

Fig. 4.1.2.8a Effect of mediators on decolorization of Reactive dye 222 by P. ostreatus IBL-

02

104

0

20

40

60

80

100

120

140

160

180

Ethanol Glycerol ABTS Oxalic acid MnSO4 H2O2 V.Alcohol

Mediators

En

zym

e ac

tivi

ty (

u/m

L)

Lac Mnp Lip



0

10

20

30

40

50

60

70

80

90


Mediators

Dec

olor

izat

ion

(%

)

Fig. 4.1.2.8c Effect of mediators on decolorization of Reactive dye 222 by P. chrysosporium

IBL-03

105

0

20

40

60

80

100

120

140

160


Mediators

En

zym

e ac

tivi

ty (

u/m

L)

Lac Mnp Lip

Fig. 4.1.2.8d Comparison of ligninolytic enzymes produced by P. chrysosporium IBL-03


Decolorization by MnP of PO occurred to a relatively less degree (92.7%) wit no MnSO4

added to the reaction system, increased quickly (94.55%) with elevated MnSO4 concentration

(1mM), suggesting MnSO4 to be an effective mediator of MnP (150U/mL) produced as a major

ligninolytic enzyme by P. ostreatus IBL-02 (PO). The other mediators like Veretryl Alcohol,

MnSO4, Glycerol, Ethanol, ABTS, Oxalate and H2O2 showed no appreciable increase in

decolorization rate (Fig. 4.1.2.8c). Similarly, decolorization by LiP of PC occurred to a relatively

less degree (85.9%) wit no veratryl alcohol added to the reaction system, increased quickly

(92.11%) with elevated veratryl alcohol concentration (1mM), suggesting veratryl alcohol to be

an effective mediator of Lip (149U/mL) produced as a major ligninolytic enzyme by P.

chrysosporium IBL-03 (PC) ((Fig. 4.1.2.8d).




significant (P≤0.05) difference. Correlation analysis between ligninolytic enzyme (MnP, LiP,

106



higher value of correlation (R2 = 0.833) for MnP showed the major role of this enzyme in the

decolorization of dye (Fig. 4.1.2.8e). With increasing MnP activity, the rate of decolorization



(PC), the higher value of correlation (R2 = 0.9316) showed the major role of LiP in the


increased.


ostreatus IBL-02 with different mediators

Source of variance df SS MSS F

Block 2 229.677 114.839 71.321***

Treatment 6 4352.988 725.498 450.571***

Error 33 53.136 1.6102

Total 41 4635.801

(P≤0.05)


P. chrysosporium IBL-03 with different mediators


Block 2 219.172 109.586 52.766***

Treatment 6 4257.018 709.503 341.628***

Error 33 8.536 2.0768

Total 41 4544.725

(P≤0.05)

107

y = 2.9244x - 120.11R2 = 0.4481

y = 0.2649x - 1.3567R2 = 0.8333

y = 0.1873x - 2.6498R2 = 0.3723

0

2040

6080

100120

140160

180

0 20 40 60 80 100


Dec

olor

izat

ion

(%

)Lac Mnp LipLinear (Lip) Linear (Mnp) Linear (Lac)


effect of mediators by P. ostreatus IBL-02

y = 5.4158x - 372.31R2 = 0.9023

y = 0.3015x - 12.517R2 = 0.5578

y = 0.3194x - 18.06R2 = 0.9316

020406080

100120140160180

0 20 40 60 80 100 120


Dec

olor

izat

ion (%

)


Fig. 4.1.2.8f Correlation between decolorization and activities of ligninolytic enzymes for

effect of mediators by P. chrysosporium IBL-03

108

Enhancement in the anaerobic reduction of azo dyes by Escherichia coli was observed in

the presence of redox mediator, and this is because of the induction in the azoreductases activity

reported earlier (Liu et al., 2009; Rau, et al., 2002). Azo reduction is supposed to occur

through reduction of dye as terminal as acceptor in microbial electron transport chain, so a

number of oxidoreductase enzymes are involved in this process (Lorenco et al., 2000). (Other

researchers also reported color removal from textile wastewater in the redox mediators like

ABTS, MnSO4 and oxalic acid, suggesting the enhancement of decolorization performance of

azo dyes at a concentration of 0.1-0.2mM by Escherichia coli JM 109. Our results are in

consistent with these findings (Liu et al., 2009).

4.12.9 Effect of metal ions

The azo dyes of textile wastewater not enough to act as a sufficient substrate for the

growth of microbial flora and thus complete decolorization to takes place. Therefore, it is

necessary to have an electron donor (usually metal ions) supply to enhance the decolorization

performance (Georgiou et al., 2004). Metal ions play the role of cofactors and enhance

accumulative effect of various additives particularly low molecular mass mediators. Most

commonly used metal ions include CuSO4, FeSO4, ZnSO4, Cd (NO3)2 and CaCl2 (Table 4.1.2.9a

and 4.1.2.9b). The maximum decolorization (%) values shown by P. ostreatus IBL-02 (PO) and

P. chrysosporium IBL-03 (PC) in the presence of CuSO4 as metal ion source were 96.12% and

94.96%, respectively (Fig. 4.1.2.9a & 4.1.2.9b). The enzyme profiles revealed the major

production of MnP (152.5U/mL) by PO (Fig. 4.1.2.9c & 4.1.2.9d) and LiP (154.95U/mL) by PC,

in the presence of CuSO4 as metal ion source (Fig. 4.1.2.9c & 4.1.2.9d).

109


metal ions

Metal ions

Decolorization (%)

Lac (U/mL)

MnP (U/mL)

LiP (U/mL)

dry weight (g)

Cd (NO3)2

70.562E 11.8±0.55E 48.5±0.60E 9.3±0.54E 0.96±0.61E

CaCl2

74.19D 12.5±0.53D 63.5±0.61D 11.95±0.55D 1.01±0.60D

ZnSO4

79.78C 13.4±0.51C 73.5±0.57C 13.32±0.54C 1.139±0.63C

CuSO4

88.515A 18.2±0.5A 152.5±0.57A 17.88±0.55A

1.332±0.62A

FeSO4

83.132B 13.9±0.52B 78±0.58B 10.23±0.58B 1.290±0.61B




Table 4.1.2.9b Decolorization of Reactive dye 222 by P. chrysosporium IBL-03 with different

metal ions

Metal ions

Decolorization (%)

Lac (U/mL)

MnP (U/mL)

LiP (U/mL)

dry weight (g)

Cd (NO3)2 69.23E 8.01±0.43E 9.8±0.60E 98.3±0.54E 0.62±0.33E

CaCl2

72.823D 10±0.43D 11±0.60D 108.3±0.54D 0.76±0.32D

ZnSO4

77.84C 12.78±0.4C 12±0.62C 114.95±0.55C 1.90±0.33C

CuSO4

86.77A 19.2±0.41A 18.5±0.58A 149.32±0.54A 1.43±0.31A

FeSO4

81.54B 17.5±0.41B 16.5±0.61B 126.88±0.55B

1.18±0.31B




110

0

10

20

30

40

50

60

70

80

90

100

Cd (NO3)2 CaCl2 ZnSO4 CuSO4 FeSO4

Metal ions

Dec

olor

izat

ion

(%

)

Fig. 4.1.2.9a Effect of metal ions on decolorization of Reactive dye 222 by P. ostreatus IBL-

02

0

20

40

60

80

100

120

140

160

180


Metal ions

En

zym

e ac

tivi

ty (

u/m

L)

Lac Mnp Lip



111

0

10

20

30

40

50

60

70

80

90


Metal ions

Dec

olor

izat

ion

(%)

Fig. 4.1.2.9c Effect of metal ions on decolorization of Reactive dye 222 by P. chrysosporium

IBL-03

0

20

40

60

80

100

120

140

160

Cd (NO3)2 CaCl2 ZnSO4 CuSO4 FeSO4Metal ions

En

zym

e ac

tivi

ty (

u/m

L)

Lac Mnp Lip

Fig. 4.1.2.9d Comparison of ligninolytic enzymes produced by P. chrysosporium IBL-03


112




significant (P≤0.05) difference. Correlation analysis between ligninolytic enzyme (MnP, LiP,



higher value of correlation (R2=0.9562) for MnP showed the major role of this enzyme in the

decolorization of dye (Fig. 4.1.2.9e). With increasing MnP activity, the rate of decolorization



(PC), the higher value of correlation ((R2=0.8646) showed the major role of LiP in the


increased.

Table 4.1.2.9c Analysis of variance of the data on decolorization of Reactive dye 222 by

P. ostreatus IBL-02 with different metal ions


Block 2 440.233 220.117 122.090***

Treatment 4 1213.669 303.417 168.294***

Error 23 41.467 1.803

Total 29 1695.369

(P≤0.05)

113


P. chrysosporium IBL-03 with different metal ions


Block 2 437.609 218.804 80.279***

Treatment 4 1155.212 288.803 105.961***

Error 23 62.688 2.726

Total 29 1655.509

(P≤0.05)

y = 4.3237x - 291.87R2 = 0.8646

y = 0.1212x + 2.0762R2 = 0.9526

y = 0.3127x - 16.001R2 = 0.6785

0

20

40

60

80

100

120

0 20 40 60 80 100


Dec

olor

izat

ion

(%

)


Fig. 4.1.2.9e Correlation between decolorization and enzyme activity (u/mL) for effect of

metal ions by P. ostreatus IBL-02

114

y = 4.3237x - 291.87R2 = 0.8646

y = 0.1212x + 2.0762R2 = 0.526

y = 0.3127x - 16.001

R2 = 0.6785

0

20

40

60

80

100

120

0 20 40 60 80 100


Dec

olor

izat

ion

(%

)


Fig. 4.1.2.9f Correlation between decolorization and enzyme activity (u/mL) for effect of

metal ions by P. chrysosporium IBL-03

The metal ions are general potent inhibitors of enzyme reactions but they might play a

role of co-factors and enhance the activity of enzymes upto 1mM. However, increasing the metal

ion concentration decreased the reactivity of ligninolytic enzymes. These results are in harmony

with those reported many researchers (Georgiou et al., 2004). It is well reported that addition of

metal ions apparently induces the reduction of azo bonds effectively. In another study,

decolorization of Reactive orange 16 by Bacillus sp. in the presence of CuSO4 as metal ion

source was greatly enhanced upto 97% (Pearce et al., 2003; Van der Zee et al., 2001).

115

4.1.2.10 UV-Visible and FTIR analysis of Reactive dye 222 by Photo-Fenton





(λmax=615nm) while the dye treated by photo-Fenton treatment followed by biological (P.

ostreatus IBL-02) treatment indicated decrease in height of spectral line showing maximum

absorption which indicated decolorization and a shift of spectral line towards the UV-region

confirmed the degradation of dye under study (Fig. 4.1.2.10a and 4.1.2.10b).



complete decolorization indicated the degradation of the parent dye when it was subjected to by

photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment. FTIR spectrum

of untreated dye displayed peaks at 3344.59, 2352.21, 1646.3, 619.15 and 613.956 cm-1 for –OH

stretch of phenol, N=N stretch and C=C stretching of monosubstituted benzene ring (Fig.

4.1.2.10c) while the FTIR spectrum of the degradation products showed appearance of peaks at

2364.64 cm-1 for =C-H stretch, 1562.06 cm-1 for aromatic ring skeleton and 610-690 cm-1 for of

monosubstituted benzene ring (Fig. 4.1.2.10d). Absence of peaks between 3650 to 3590 cm-1

indicated that no formation of phenolic compounds and absence of peaks between 3400 to 3380

cm-1 for –NH stretch indicated that no aliphatic and aromatic amines were formed by photo-

Fenton as a treatment followed by biological (P. ostreatus IBL-02) treatment. FTIR spectral

analysis indicated that decolorization of dye under study was due to degradation of dye in to

intermediate products. The peak characteristic of azo bond at 1646.3 cm-1 of dye was absent in

the decolorized medium, indication degradation of dye due to aromatic amines as intermediate

products which are subjected to oxidation giving rise to simpler compounds.

116

Fig. 4.1.2.10a UV-Visible spectrum of reactive dye 222 before biological (P. ostreatus IBL-

02) treatment

Fig. 4.1.2.10b UV-Visible spectrum of reactive dye 222 after biological biological (P. ostreatus IBL-02) treatment

117

Fig. 4.1.2.10c FTIR spectrum of reactive dye 222 before biological biological (P. ostreatus IBL-02) treatment

Fig. 4.1.2.10d FTIR spectrum of reactive dye 222 after biological (P. ostreatus IBL-02)

treatment

118

4.1.2.11 UV-Visible and FTIR analysis of Reactive dye 222 by biological (P.

chrysosporium IBL-03) treatment




(λmax=615nm) while the dye treated by biological (P. chrysosporium IBL-03) treatment indicated

decrease in height of spectral line showing maximum absorption which indicated decolorization

and a shift of spectral line towards the UV-region confirmed the degradation of dye under study

(Fig. 4.1.2.11a and 4.1.2.11b).



complete decolorization indicated the degradation of the parent dye compound by biological (P.

chrysosporium IBL-03) treatment. FTIR spectrum of untreated dye displayed peaks at 3313.59,

2352.21, 1646.3, 619.15 and 613.956 cm-1 for –OH stretch of phenol, N=N stretch and C=C

stretching of monosubstituted benzene ring (Fig. 4.1.2.11c) while the FTIR spectrum of the

degradation products showed appearance of peaks at 3313.55 cm-1 for –OH stretch, 2945.51 cm-1

and 2834.66 cm-1 for C-H stretching of alkyl benzene, 1460.07 cm-1 for -C-H bending, 1127.51

and 1025.52 cm-1 for C-O stretch and 613.15 cm-1 for monosubstituted benzene ring and (Fig.

4.1.2.11d). Appearance of peak at 3313.55 cm-1 indicated the formation of carboxylated

products. Absence of peaks between 3400 to 3380 cm-1 for –NH stretch indicated that no

aliphatic and aromatic amines were formed by biological (P. chrysosporium IBL-03) treatment.

FTIR spectral analysis indicated that decolorization of dye under study was due to degradation of

dye in to intermediate products. The peak characteristic of azo bond at 1646.3 cm-1 of dye was

absent in the decolorized medium, indication degradation of dye due to aromatic amines as

intermediate products which are subjected to oxidation giving rise to simpler compounds.

119

Fig. 4.21.11a UV-Visible spectrum of reactive dye 222 before biological (P. chrysosporium IBL-03) treatment

Fig. 4.1.2.11b UV-Visible spectrum of reactive dye 222 after biological (P. chrysosporium IBL-03) treatment

120

Fig. 4.1.2.11c FTIR spectrum of reactive dye 222 before biological (P. chrysosporium IBL-03) treatment

Fig. 4.1.2.11d FTIR spectrum of reactive dye 222 before biological (P. chrysosporium IBL-03) treatment

121






In order to find out the biodegradability rate using the Photo-Fenton reaction as a pre-

treatment step, experiments were conducted at the pre-optomized reaction conditions. The

maximum decolorization (%) values shown by P. ostreatus IBL-02 were 97.72% and by P.

chrysosporium IBL-03 were 96.45%, respectively. The enzyme profiles revealed the major

production of MnP (163.23U/mL) by PO and LiP (161U/mL) by PC (Fig. 4.1.3.1a and

4.1..1b)

0

20

40

60

80

100

120

24 hrs 48 hrs 72 hrsTime (hrs)

Dec

olor

izat

ion

(%

)

0

20

40

60

80

100

120

140

160

180

En

zym

e ac

tivi

ty

(U/m

L)


Fig. 4.1.3.1a Effect of Photo-Fenton treatment followed by biotreatment (P. ostreatus IBL-

02) on decolorization of Reactive dye 222

122

0

20

40

60

80

100

120

24 hrs 48 hrs 72 hrs

Time (hrs)

Dec

olor

izat

ion

(%

)

0

20

40

60

80

100

120

140

160

180

En

zym

e ac

tivi

ty (

U/m

L)


Fig. 4.1.3.1b Effect of Photo-Fenton treatment followed by biotreatment (P. chrysosporium

IBL-03) on decolorization of Reactive dye 222

The decolorization efficiency of both fungal strains was increased by combinig both

photo-fenton treatment followed by biotreatment in our experimental findings due to the

combined oxidative effect of both treatments and it is agreed well with available literature

(Brosillon et al., 2008). The application of a combined method i.e. biotreatment and AOPs for

treating dyes as well as effluents, is highly advantageus. This combined approach not only allows

better achievement of decolorization efficiency, but also contributes towards contributes towards

reducing treatment costs (Azbar et al., 2004). Mario et al. (1997) reported the enhancement of

dyes removal efficiency of industrial wastewater treatment by using sequential treatments rather

than biological treatment processes alone. Pagga and Brown (1986) documented that sequential

treatments semed to be efficient due to the complex polyaromatic structure and recalcitrant

nature of dyes; wastewater containing azo dyes are not possible to degrade by means of

biological treatment unit. Though Fenton and photo-Fenton processes are capable for

dearomatozation of dyestuff, the main problem with this treatment is high reagent cost and

123

production of sludge containing high amunt of Fe (III), which needs to be managed by safe

disposal methods, hence there seems a need for further research for finding an alternative

economical treatment method for maximum mineralization of textile azo dyes and textile

effluents (Liou et al., 2003; Fischer and Hahn, 2005; Ahmadi et al., 2005; Fongsatitkul et al.,

2004).







ostreatus IBL-02) treatment indicated decrease in height of spectral line showing maximum


confirmed the degradation of dye under study (Fig. 4.1.3.1.1a and 4.1.3.1.1b).



complete decolorization indicated the degradation of the parent dye when it was subjected to by

photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment. FTIR spectrum

of untreated dye displayed peaks at 3344.59, 2352.21, 1646.3, 619.15 and 613.956 cm-1 for –OH


4.1.3.1.1c) while the FTIR spectrum of the degradation products showed appearance of peaks at

2364.64 cm-1 for =C-H stretch, 1562.06 cm-1 for aromatic ring skeleton and 610-690 cm-1 for of

monosubstituted benzene ring (Fig. 4.1.3.1.1d). Absence of peaks between 3650 to 3590 cm-1

indicated that no formation of phenolic compounds and absence of peaks between 3400 to 3380

cm-1 for –NH stretch indicated that no aliphatic and aromatic amines were formed by photo-

Fenton as a treatment followed by biological (P. ostreatus IBL-02) treatment. FTIR spectral

analysis indicated that decolorization of dye under study was due to degradation of dye in to

intermediate products. The peak characteristic of azo bond at 1646.3 cm-1 of dye was absent in

the decolorized medium, indication degradation of dye due to aromatic amines as intermediate


124

Fig. 4.1.3.1.1a UV-Visible spectrum of reactive dye 222 before Photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment

Fig. 4.1.3.1.1b UV-Visible spectrum of reactive dye 222 after Photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment

125

Fig. 4.1.3.1.1c FTIR spectrum of reactive dye 222 before Photo-Fenton treatment followed

by biological (P. ostreatus IBL-02) treatment

Fig. 4.1.3.1.1d FTIR spectrum of reactive dye 222 after Photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment

126


treatment followed by biological (P. chrysosporium IBL-03) treatment





chrysosporium IBL-03) treatment indicated decreases in height of spectral line showing

maximum absorption which indicated decolorization and a shift of spectral line towards the UV-

region confirmed the degradation of dye under study (Fig. 4.1.3.1.2a and 4.1.3.1.2b).



complete decolorization indicated the degradation of the parent dye compound photo-Fenton

treatment followed by biological (P. chrysosporium IBL-03) treatment. FTIR spectrum of

untreated dye displayed peaks at 3344.59, 2352.21, 1646.3, 619.15 and 613.956 cm-1 for –OH



3326.85 cm-1 for –OH stretch, 2918.91 cm-1 for C-H stretching of alkyl benzene, 2373.51 cm-1

for =C-H stretch, 803.81 and 777.21 cm-1 for -C-H out of plane deformation and 630-680 cm-1

for C=C stretching of monosubstituted benzene ring (Fig. 4.1.3.1.2d). Absence of peaks between

3400 to 3380 cm-1 for –NH stretch indicated that no aliphatic and aromatic amines were formed

by photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment. FTIR

spectral analysis indicated that decolorization of dye under study was due to degradation of dye

in to intermediate products. The peak characteristic of azo bond at 1646.3 cm-1 of dye was absent

in the decolorized medium, indication degradation of dye due to aromatic amines as intermediate


127

Fig. 4.1.3.1.2a UV-Visible spectrum of reactive dye 222 before Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

Fig. 4.1.3.1.2b UV-Visible spectrum of reactive dye 222 after Photo-Fenton treatment

followed by biological (P. chrysosporium IBL-03) treatment

128

Fig. 4.1.3.1.2c FTIR spectrum of of reactive dye 222 before Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

Fig. 4.1.3.1.2d: FTIR spectrum of of reactive dye 222 after Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment


In order to find out the biodegradability rate using the biological treatment followed by

photo-Fenton treatment, experiments were conducted at the pre-optomized reaction conditions.

129

The maximum decolorization (%) values shown by P. ostreatus IBL-02 were 94.01 and by P.

chrysosporium IBL-03 were 89.23% (Fig. 4.1.3.2a & 4.1.3.2b), respectively. The enzyme

profiles revealed the major production of MnP (163.23U/mL) by PO and LiP (161U/mL) by PC.

0

20

40

60

80

100

120

10 20 30 40 50 60

Time (min)

Dec

olor

izat

ion

(%

)

Fig. 4.1.3.2a Effect of biotreatment (P. ostreatus IBL-02) followed by Photo-Fenton

treatment on decolorization of Reactive dye 222

130

0

10

20

30

40

50

60

70

80

90

100

10 20 30 40 50 60

Time (min)

Dec

olor

izat

ion

(%

)

Fig. 4.1.3.2b Effect of biotreatment (P. chrysosporium IBL-03) followed by Photo-Fenton

treatment on decolorization of Reactive dye 222

The application of a combined method i.e. biotreatment and AOPs for treating dyes as

well as effluents, is highly advantageus. This combined approach not only allows better

achievement of decolorization efficiency, but also contributes towards contributes towards

reducing treatment costs (Azbar et al., 2004). Previous researchers have focused on only one

method of treatment, whereas the preferred method for treatment of recalcitrant dyes is to use

AOP (for partial degradation) followed by aerobic biological process (Liou et al., 2003; Fischer

and Hahn, 2005; Ahmadi et al., 2005).

Fongsatitkul et al (2004) reported that chemical treatment prior to biodegradation

delivered the best performance for treating the textile effluent rather than only biological or

chemical treatment. In another study, degradation of two azo dyes (reactive black 5 & reactive

yellow 145) was carried out by means of integrated process involving photocatalysis UV/TiO2

followed by biological treatment and more than 90% degradation was achieved (Liou et al.,

2003).

131

4.1.3.2.1 UV-Visible and FTIR analysis of Reactive dye 222 by biological (P.

ostreatus IBL-02) treatment followed by Photo-Fenton treatment




(λmax=615nm) while the dye treated by biological (P. ostreatus IBL-02) treatment followed by

photo-Fenton treatment indicated decrease in height of spectral line showing maximum





complete decolorization indicated the degradation of the parent dye compound when it was

subjected to by biological (P. ostreatus IBL-02) treatment followed by photo-Fenton treatment.

FTIR spectrum of untreated dye displayed peaks at 3344.59, 2352.21, 1646.3, 619.15 and

613.956 cm-1 for –OH stretch of phenol, N=N stretch and C=C stretching of monosubstituted

benzene ring (Fig. 4.1.3.1.1c) while the FTIR spectrum of the degradation products showed

appearance of peaks at 3295.81 cm-1 for –OH stretch due to the presence of –COOH group,

2941.08 cm-1 and 2825.79 cm-1 for C-H stretching of alkyl benzene, 2351.33 cm-1 for =C-H

stretch, 1739.42 cm-1 for -C-H deformation, 1460.07 cm-1 for -C-H bending, 1251.66 and

1025.52 cm-1 for C-O stretch in –COOH group, 604.28 and 657.49 cm-1 for monosubstituted

benzene ring (4.1.3.2.1d). Absence of peaks between 3400 to 3380 cm-1 for –NH stretches

indicated that no aliphatic and aromatic amines were formed by biological (Pleurotus P.

ostreatus IBL-02) treatment followed by photo-Fenton treatment (Fig.). FTIR spectral analysis

indicated that decolorization of dye under study was due to degradation of dye in to intermediate

products. The peak characteristic of azo bond at 1646.3 cm-1 of dye was absent in the decolorized

medium, indication degradation of dye due to aromatic amines as intermediate products which

are subjected to oxidation giving rise to simpler compounds.

132

Fig. 4.1.3.2.1a UV-Visible spectrum of reactive dye 222 before biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

Fig. 4.1.3.2.1b UV-Visible spectrum of reactive dye 222 after biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

133

Fig. 4.1.3.2.1c: FTIR spectrum of reactive dye 222 before biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

Fig. 4.1.3.2.1d: FTIR spectrum of reactive dye 222 after biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

134

4.1.3.2.2 UV-Visible and FTIR analysis of Reactive dye 222 by biological (P.

chrysosporium IBL-03) treatment followed by Photo-Fenton treatment




(λmax=615nm) while the dye treated by biological (P. chrysosporium IBL-03) treatment followed

by photo-Fenton treatment indicated decrease in height of spectral line showing maximum





complete decolorization indicated the degradation of the parent dye compound biological (P.

chrysosporium IBL-03) treatment followed by photo-Fenton treatment. FTIR spectrum of

untreated dye displayed peaks at 3344.59, 2352.21, 1646.3, 619.15 and 613.956 cm-1 for –OH



3326.85 cm-1 for –OH stretch, 2941..08 cm-1 and 2830.22 cm-1 for C-H stretching of alkyl

benzene, 2364.64 cm-1 for =C-H stretch, 1757.16 cm-1 for -C-H deformation, 1446.77 cm-1 for -

C-H bending, 610-680 cm-1 along with overtone at 1260..368 cm-1 for -C-H deformation and

1021.09 cm-1 along C-O stretch (Fig. 4.1.3.2.2d). Absence of peaks between 3400 to 3380 cm-1

for –NH stretch indicated that no aliphatic and aromatic amines were formed by biological (P.

chrysosporium IBL-03) treatment followed by photo-Fenton treatment. FTIR spectral analysis

indicated that decolorization of dye under study was due to degradation of dye in to intermediate

products. The peak characteristic of azo bond at 1646.3 cm-1 of dye was absent in the decolorized

medium, indication degradation of dye due to aromatic amines as intermediate products which

are subjected to oxidation giving rise to simpler compounds.

135

Fig. 4.1.3.2.2a UV-Visible spectrum of reactive dye 222 before biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

Fig. 4.1.3.2.2b UV-Visible spectrum of reactive dye 222 after biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

136

Fig. 4.1.3.2.2c FTIR spectrum of reactive dye 222 before biological (P. chrysosporium IBL-

03) treatment followed by Photo-Fenton treatment

Fig. 4.1.3.2.2d FTIR spectrum of reactive dye 222 after biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

137


The first series of experiments were carried out in order to determine the optimal

parameters of biological and chemical processes for decolorization and mineralization of reactive

dye 222 in model wastewater. In part two, decolorization of one synthetic effluent (having a

mixture of three reactive dyes) and three indusrial effluents effluent was studied


The experiments were conducted at the optimized experimental conditions (pH 3.5, H2O2

1× 10-2 M, FeSO4 3.5 × 10-5 M;, temperature 50˚C, time 50 minutes) of Photo-Fenton treatment

to find out the decolorization potential of one synthetic and three industrial effluents. All

effluents were decolorized by Photo-Fenton oxidation process but showed less decolorization

(%) potential as were observed for reactive dye 222 solutions by the same treatment. Synthetic

effluent was decolorized (70.12%) more than three industrial effluents (64.12%, 60.92% &

49.99%), respectively (Fig.4.2.1).

0

10

20

30

40

50

60

70

80

1 2 3 4 5 6 7

Time (min)

Dec

olor

izat

ion

(%

)

synthetic effluent industrial effluent 1industrial effluent 2 industrial effluent 3

Fig. 4.2.1 Effect of Photo-Fenton treatment on decolorization of synthetic and industrial

effluents

138

In a study investigating C.I orange 7 (a commonly detected dye in textile wastewater)

removal, Daneshvar et al. (2008) reported that the absence of uv radiations or H2O2 decreased

dye removal efficiency, but their optimized level resulted in 60-70% of dye removal efficiency.

Rezaee et al. (2008) used photo-fenton process for decolorization of textile effluent having

mixture of reactive dyes and found that the effluent was decolorized up to 80% under optimal

H2O2 dosage (2.5mM) and a pH within 3-3.5. Ugurly and Kula (2007) investigated the

decolorization and degradation of phenolic compounds present in textile effluent by photo-fenton

process, almost 75% removal was achieved at the optimized level (H2O2 2mM, pH 3,

temperature 50˚C). These findings highly support our experimental findings.

According to Moraes et al. (2005), photo-fenton process is sufficient for the removal of

dyes present in textile effluents. Malik and Saha (2003) reported that the removal rate is strongly

dependent on the optimal concentration dye, Fe+2 and H2O2. Muruganandham and Swaminathan

(2004) have carried out studies where similar results were obtained; they suggested a pH 3 is the

optimum pH for photo-fenton process.





Visible spectral analysis of each untreated effluents sample showed a peak in the visible region

(λmax=615nm) while the each effluent treated by biological (P. chrysosporium IBL-03) treatment

followed by photo-Fenton treatment indicated decrease in height of spectral line showing


region confirmed the degradation of dye under study (Fig. 4.2.1.1a-4.2.1.1h).

139

Fig. 4.2.1.1a UV-Visible spectrum of synthetic effluent before Photo-Fenton treatment

Fig. 4.2.1.1b UV-Visible spectrum of synthetic effluent after Photo-Fenton treatment

140

Fig. 4.2.1.1c UV-Visible spectrum of industrial effluent 1 before Photo-Fenton treatment

Fig. 4.2.1.1d UV-Visible spectrum of industrial effluent 1 after Photo-Fenton treatment

141

Fig. 4.2.1.1e UV-Visible spectrum of industrial effluent 2 before Photo-Fenton treatment

Fig. 4.2.1.1f UV-Visible spectrum of industrial effluent 2 after Photo-Fenton treatment

142

Fig. 4.2.1.1g UV-Visible spectrum of industrial effluent 3 before Photo-Fenton treatment

Fig. 4.2.1.1h UV-Visible spectrum of industrial effluent 3 after Photo-Fenton treatment

143


The experiments were conducted at the optimized growth conditions for fungal strains (*:

pH 4.5; temperature, 30oC; inoculum size, 4mL; rice bran, 1.5g; MnSO4, 1mM; CuSO4, 1mM)

used in the present study to investigate the decolorization potential of one synthetic and three

industrial effluents by P. ostreatus IBL-02 (PO) and P. chrysosporium IBL-03 (PC).The color

reduction following biological treatment (by both PO & PC) of synthetic and industrial effluents

were similar to those observed when the reactive dye 222 solutions were treated biologically but

the data was less consistent and color reduction was lower (Fig. 4.2.2a-4.2.2h). Synthetic effluent

was decolorized (68.23% & 65.23%) more than three industrial effluents (58.23% &

55.21%.50.23% & 45.11%, 40.45% & 37.23%) by P. ostreatus IBL-02 (PO) and P.

chrysosporium IBL-03 (PC), respectively. While comparing the decolorization potential of all

effluents by both fingal strains, PO exhibited a little more decolorization potential than PC (as it

is evident from their decolorization values shown in given figures.

0

10

20

30

40

50

60

70

80

24 hrs 48 hrs 72 hrsTime (hrs)

Dec

olor

izat

ion

(%

)

0

10

20

30

40

50

60

En

zym

e ac

tivi

ty (

U/m

L)


Fig. 4.2.2a Effect of biological treatment (P. ostreatus IBL-02) on decolorization of synthetic

effluent

144

0

10

20

30

40

50

60

70

80


Time (hrs)

Dec

olor

izat

ion

(%

)

0

10

20

30

40

50

60

En

zym

e ac

tivi

ty (

U/m

L)


Fig. 4.2.2b Effect of biological treatment (P. chrysosporium IBL-03) on decolorization of

synthetic effluent

0

10

20

30

40

50

60

70


Time (hrs)

Dec

olor

izat

ion

(%

)

0

10

20

30

40

50

60

En

zym

e ac

tivi

ty (

U/m

L)


Fig. 4.2.2c Effect of biological treatment (P. ostreatus IBL-02) on decolorization of

Industrial effluent 1

145

0

10

20

30

40

50

60

70


Time (hrs)

Dec

olor

izat

ion

(%

)

0

5

10

15

20

25

30

35

40

45

En

zym

e ac

tivi

ty (

U/m

L)


Fig. 4.2.2d Effect of biological treatment (P. chrysosporium IBL-03) on decolorization of


0

10

20

30

40

50

60


Time (hrs)

Dec

olor

izat

ion

(%

)

0

5

10

15

20

25

30

35

40

45

En

zym

e ac

tivi

ty (

U/m

L)


Fig. 4.2.2e Effect of biological treatment (P. ostreatus IBL-02) on decolorization of


146

05

101520253035404550


Time (hrs)

Dec

olor

izat

ion

(%

)

0

5

10

15

20

25

30

35

40

En

zym

e ac

tivi

ty (

U/m

L)


Fig. 4.2.2f Effect of biological treatment (P. chrysosporium IBL-03) on decolorization of


0

5

10

15

20

25

30

35

40

45


Time (hrs)

Dec

olor

izat

ion

(%

)

0

5

10

15

20

25

30

35

40

En

zym

e ac

tivi

ty (

U/m

L)


Fig. 4.2.2g Effect of biological treatment (P. ostreatus IBL-02) on decolorization of


147

0

5

10

15

20

25

30

35

40

45


Time (hrs)

Dec

olor

izat

ion

(%

)

0

5

10

15

20

25

30

35

40

En

zym

e ac

tivi

ty (

U/m

L)


Fig. 4.2.2h Effect of biological treatment (P. chrysosporium IBL-03) on decolorization of


Some researchers established that at optimized growth conditions and in the presence of

appropriate carbon source, aerobic color remival is mainly is due to the azo dye reduction (Cruz

and Buitron, 2001; Pinheiro et al., 2004).In another study conducted with an industrial effluent at

pre-optimized experimental conditions, color reduction was found to be 60-80% after 15hrs

(Sponza and Isik, 2005). Albuquerque et al. (2005) observed 70% decolorization of 2.5mg/L

acid orange 7 at the pre-optimized reaction conditions after only 10hrs. Aerobic decolorization of

effluent having reactive azo dyes was examined by Mass and Chaudhari (2005), and found that

biodegradation played a major role in color abatement and accounted for more than 70% removal

of color from the effluent. The ligninolytic enzymes of fungi are highly non-specific and can

effectively treat even dilute wastes (Atiken, 1993) enzymatic treatment of industrial effluent has

been considered as potential method. Biodecolorization was found to be more effective in the

real dye bath effluent system rather than the industrial effluents due to the high complex nature

(Murugesan et al., 2007).

148





Visible spectral analysis of untreated effluents sample showed a peak in the visible region


followed by photo-Fenton treatment indicated decrease in height of spectral line showing


region confirmed the degradation of dye under study (Fig. 4.2.2.1a-4.2.2.1o).

Fig. 4.2.2.1a UV-Visible spectrum of synthetic effluent before biological (P. ostreatus IBL-02) treatment

149

Fig. 4.2.2.1b UV-Visible spectrum of synthetic effluent after biological (P. ostreatus IBL-02) treatment

Fig. 4.2.2.1c UV-Visible spectrum of synthetic effluent before biological (P. chrysosporium

IBL-03) treatment

150

Fig. 4.2.2.1d UV-Visible spectrum of synthetic effluent after biological (P. chrysosporium IBL-03) treatment

Fig. 4.2.2.1e UV-Visible spectrum of industrial effluent 1 before biological (P. ostreatus IBL-02) treatment

151

Fig. 4.2.2.1f UV-Visible spectrum of industrial effluent 1 after biological (P. ostreatus IBL-02) treatment

Fig. 4.2.2.1g UV-Visible spectrum of industrial effluent 1 before biological (P. chrysosporium IBL-03) treatment

152

Fig. 4.2.2.1h UV-Visible spectrum of industrial effluent 1 after biological (P. chrysosporium IBL-03) treatment

Fig. 4.2.2.1i UV-Visible spectrum of industrial effluent 2 before biological (P. ostreatus IBL-02) treatment

153

Fig. 4.2.2.1j UV-Visible spectrum of industrial effluent 2 after biological (P. ostreatus IBL-02) treatment

Fig. 4.2.2.1k UV-Visible spectrum of industrial effluent 2 before biological (P. chrysosporium IBL-03) treatment

154

Fig. 4.2.2.1 l UV-Visible spectrum of industrial effluent 2 after biological (P. chrysosporium IBL-03) treatment

Fig. 4.2.2.1m UV-Visible spectrum of industrial effluent 3 before biological (P. ostreatus IBL-02) treatment

155

Fig. 4.2.2.1m UV-Visible spectrum of industrial effluent 3 before biological (P. ostreatus IBL-02) treatment

Fig. 4.2.2.1n UV-Visible spectrum of industrial effluent 3 before biological (P. chrysosporium IBL-03) treatment

156

Fig. 4.2.2.1o UV-Visible spectrum of industrial effluent 3 after biological (P. chrysosporium IBL-03) treatment

4.2.3 Sequential treatments





In order to find out the decolorization rate using the Photo-Fenton reaction as a pre-

treatment step, experiments were conducted at the pre-optomized reaction conditions (optimized

conditions of biological treatment), and the results are presented in Fig. 4.2.3.1a-4.2.3.1h. The

maximum decolorization (%) values shown by synthetic effluent by P. ostreatus IBL-02 and by

P. chrysosporium IBL-03 were 74.43% and 60.42%, respectively. The industrial effluents

decolorized comparatively less than synthetic effluent (as it is evident from figure). The enzyme

profiles revealed the major production of MnP (83.23U/mL) by by P. ostreatus IBL-02 and Lip

(67.23U/mL) by P. chrysosporium IBL-03 during the experiments by synthetic effluents while

less enzyme activities were found during the decolorization experiments with both fungal strains.

157

0

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24 48 72

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olor

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)

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80

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En

zym

e ac

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decolorization Lac MnP LiP


treatment on decolorization of synthetic effluent

0

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24 48 72

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Dec

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Fig. 4.2.3.1b Effect of biotreatment (P. chrysosporium IBL-03) followed by Photo-Fenton

treatment on decolorization of synthetic effluent

158

0

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24 48 72

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olor

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Fig. 4.2.3.1c Effect of biotreatment (P. ostreatus IBL-02) followed by Photo-Fenton

treatment on decolorization of industrial effluent 1

0

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Fig. 4.2.3.1d Effect of biotreatment (P. chrysosporium IBL-03) followed by Photo-Fenton


159

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Fig. 4.2.3.1e Effect of biotreatment (P. ostreatus IBL-02) followed by Photo-Fenton


0

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olor

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05101520253035404550

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Fig. 4.2.3.1f Effect of biotreatment (P. chrysosporium IBL-03) followed by Photo-Fenton


160

0

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Fig. 4.2.3.1g Effect of biotreatment (P. ostreatus IBL-02) followed by Photo-Fenton


0

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Fig. 4.2.3.1h Effect of biotreatment (P. chrysosporium IBL-03) followed by Photo-Fenton


161

The decolorization efficiency of both fungal strains was increased by combinig both

Photo-Fenton treatment followed by biotreatment in our experimental findings due to the

combined oxidative effect of both treatments and it is agreed well with available literature

(Brosillon et al., 2008). Although photo-fenton itself is a highly effective oxidation method, even

then the application of biological treatment has an enhanced effect on the decolorization rate of

azo dyes (Camel and Bermond, 1998; Azbar et al., 2004). Many studied have demonstrated that

AOPs are effectively removing color and partially removing the organic content of dyestuffs, but

further treatment by biological process enhance the dye removal rate and COD reduction (Chen

et al., 1997; Galindo et al., 2001; Thobanoglous et al., 2003).


biological treatment followed by Photo-Fenton treatment



Visible spectral analysis of untreated effluents sample showed a peak in the visible region while

the each effluent treated by biological treatment followed by Photo-Fenton treatment indicated

decrease in height of spectral line showing maximum absorption which indicated decolorization

and a shift of spectral line towards the UV-region confirmed the degradation of dye under study

(Fig. 4.2.3.1.1a-4.2.3.1.1o).

162

Fig. 4.2.3.1.1a UV-Visible spectrum of synthetic effluent before Photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment

Fig. 4.2.3.1.1b UV-Visible spectrum of synthetic effluent after Photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment

163

Fig. 4.2.3.1.1c UV-Visible spectrum of synthetic effluent before Photo-Fenton treatment

followed by biological (P. chrysosporium IBL-03) treatment

Fig. 4.2.3.1.1d UV-Visible spectrum of synthetic effluent after photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

164

Fig. 4.2.3.1.1e UV-Visible spectrum of industrial effluent 1 before Photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment

Fig. 4.2.3.1.1f UV-Visible spectrum of industrial effluent 1 after Photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment

165

Fig. 4.2.3.1.1g UV-Visible spectrum of industrial effluent 2 before Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

Fig. 4.2.3.1.1h UV-Visible spectrum of industrial effluent 2 after Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

166

Fig. 4.2.3.1.1j UV-Visible spectrum of industrial effluent 2 before Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

Fig. 4.2.3.1.1i UV-Visible spectrum of industrial effluent 2 after Photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment

167

Fig. 4.2.3.1.1l UV-Visible spectrum of industrial effluent 3 before Photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment

Fig. 4.2.3.1.1m UV-Visible spectrum of industrial effluent 3 after Photo-Fenton treatment followed by biological (P. ostreatus IBL-02) treatment

168

Fig. 4.2.3.1.1n UV-Visible spectrum of industrial effluent 3 before Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

Fig. 4.2.3.1.1o UV-Visible spectrum of industrial effluent 3 after Photo-Fenton treatment followed by biological (P. chrysosporium IBL-03) treatment

169

4.2.3.2 Biological treatment followed by Photo-Fenton treatment In order to find out the biodegradability rate using the biological (by both fungal strains

under study) as a pre-treatment step, experiments were conducted at the pre-optomized reaction

conditions. The maximum decolorization (%) values shown by synthetic effluent by P. ostreatus

IBL-02 and by by P. chrysosporium IBL-03 were 74.43% and 60.42%, respectively (Fig.

4.2.3.2a and 4.2.3.2b). The industrial effluents decolorized comparatively less than synthetic

effluent (as it is evident from figure).

0102030405060708090

0 10 20 30 40 50 60Time (min)

Dec

olor

izat

ion

(%

)

Greeen effl. Red effl. Blue effl.

Synth. effl.


treatment on decolorization of one synthetic and three industrial effluents

170

0

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60

70

80

0 10 20 30 40 50 60

Time (min)

Dec

olor

izat

ion (%

)Green effl. Red effl. Blue effl.Synth. Effl.

Fig. 4.23.2a Effect of biotreatment (P. chrysosporium IBL-03) followed by Photo-Fenton

treatment on decolorization of one synthetic and three industrial effluents

Potential advantages of the strategy of combining chemical and biological processes to

treat contaminants in wastewater were previously suggested (Scott and Ollis, 1995; Scott and

Follis, 1997). Although photo-fenton itself is a highly effective oxidation method, even then the

application of biological treatment has an enhanced effect on the decolorization rate of azo dyes

(Camel and Bermond, 1998; Azbar et al., 2004). Many studied have demonstrated that AOPs are

effectively removing color and partially removing the organic content of dyestuffs, but further

treatment by biological process enhance the dye removal rate and COD reduction (Chen et al.,

1997; Galindo et al., 2001; Thobanoglous et al., 2003). Potential advantages of the strategy of

combining chemical and biological processes to treat contaminants in wastewater were

previously suggested (Scott and Ollis, 1995; Scott and Follis, 1997).

171





Visible spectral analysis of untreated effluents sample showed a peak in the visible region


followed by Photo-Fenton treatment indicated decrease in height of spectral line showing


region confirmed the degradation of dye under study (Fig. 4.2.3.2.1a-4.2.3.2.1o).

Fig. 4.2.3.2.1a UV-Visible spectrum of synthetic effluent before biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

172

Fig. 4.2.3.2.1b UV-Visible spectrum of synthetic effluent after biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

Fig. 4.2.3.2.1c UV-Visible spectrum of synthetic effluent before biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

173

Fig. 4.2.3.2.1d UV-Visible spectrum of synthetic effluent after biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

Fig. 4.2.3.2.1e UV-Visible spectrum of industrial effluent 1 before biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

174

Fig. 4.2.3.2.1f UV-Visible spectrum of industrial effluent 1 after biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

Fig. 4.2.3.2.1g UV-Visible spectrum of industrial effluent 1 before biological


175

Fig. 4.2.3.2.1h UV-Visible spectrum of industrial effluent 1 after biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

Fig. 4.2.3.2.1i UV-Visible spectrum of industrial effluent 2 before biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

176

Fig. 4.2.3.2.1j UV-Visible spectrum of industrial effluent 2 after biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

Fig. 4.2.3.2.1k UV-Visible spectrum of industrial effluent 2 before biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

177

Fig. 4.2.3.2.1l: UV-Visible spectrum of industrial effluent 2 after biological (P.

chrysosporium IBL-03) treatment followed by Photo-Fenton treatment.

Fig. 4.2.3.2.1m UV-Visible spectrum of industrial effluent 3 before biological (P. ostreatus IBL-02) treatment followed by Photo-Fenton treatment

178

Fig. 4.2.3.2.1n UV-Visible spectrum of industrial effluent 3 after biological (P. ostreatus IBL-2) treatment followed by Photo-Fenton treatment

Fig. 4.2.3.2.1o UV-Visible spectrum of industrial effluent 3 before biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

179

Fig. 4.2.3.2.1p UV-Visible spectrum of industrial effluent 3 after biological (P. chrysosporium IBL-03) treatment followed by Photo-Fenton treatment

180

4.3 Evaluation of treated samples through Quality assurance

parameters

Chemical methods of treatment rely upon the chemical interactions of the contaminants

we wish to remove from water, and the application of chemicals that either aid in the separation

of contaminants from water, or assist in the destruction or neutralization of harmful effects

associated with contaminants. In general, most of the reports on the biotreatment of dyes deal

mainly with decolorization, but few report on the reduction in toxicity or on the biodegradation

products or intermediates of degradation. Driessel and Christov (2001) reported that fungal

treatment with C. versicolor rendered the effluents essentially non-toxic. Same report is also

given by Song and Chang (2004) that fungal treatment may reduce 35% toxicity in effluents.

The textile effluent that is wasted has higher value of water quality parameters (COD, TOC,

TSS, DO, Phenols) than their permissible limits. The characterization of actual as well as

synthetic waste water is shown in table No. 1 along with permissible limits of water quality

parameters.

Table No.4.3 Characterization and permissible limits of wastewater

Water quality

Parameters

Synthetics Actual Permissible Limits

COD (mg/L) 1570 1985 120

TOC(mg/L) 998 1465 5

TSS(mg/L) 770 1205 35

DO(mg/L) 4.02 3.40 *3-6

Phenols(mg/L) ------ ------- 0.5

pH 12 5 – 9

*5–6 ppm Sufficient for most species <3 ppm Stressful to most aquatic species <2 ppm Fatal to most species

181

4.3.1 Biological oxygen demand (BOD)

Biological oxygen demand measures the biodegradable material and helps in the

development of organic by-products (Manahan, 1994). Both untreated and treated samples of dye

and effluents by all treatment methods under study were analyzed to determine the value of BOD

(mg/L).

The results for BOD measurement of reactive dye 222, synthetic effluent and three industrial

effluents by photo-fenton, biological and sequential treatments are represented in table.

Significant decrease in BOD was observed in all treated samples by all treatment methods (Table

4.3.1). However, results showed that BOD dye samples treated by sequential treatment was

lowest (5.23 mg/L) while that in the industrial effluent (79.5mg/L) was highest in comparison

with that of other samples treated by all other treatment methods (Table 4.3.1).

182

Table 4.3.1 Comparison of water quality parameter (BOD) by different treatment methods

The BOD values of all treated samples fell in the permissible limits i.e. 30-30mg/L

(ISI: 2490 1982). Similar conclusions were also drawn by Shah (1999) and Khan and Noor

(2002).

4.3.2 Chemical oxygen demand (COD)

Measurement of the COD was carried out in order to provide information about the

concentration of the organic matter (dyes) present in wastewater after different treatment

(chemical, biological and sequential) technologies used in the present study. Both treated and

untreated samples of dye and effluents were analyzed to determine the value of COD (mg/L).

Reactive dye

222

Synthetic

effluent

Industrial

effluent 1

Industrial

effluent 2

Industrial

effluent 3

BOD (mg/L)

Before After Before After Before After Before After Before After

Photo-

Fenton 55.4 4.13 61.32 12.02 75.34 19.5 83.21 23.5 99.21 25.52

PO 55.4 4.34 61.32 14.55 75.34 20.5 83.21 24.2 99.21 25.81

PC 55.4 4.65 61.32 15.08 75.34 20.55 83.21 24.78 99.21 25.95

Chem/PO 55.4 3.23 61.32 10.54 75.34 16.1 83.21 18.2 99.21 20.2

Chem/PC 55.4 3.44 61.32 10.66 75.34 16.4 83.21 18.7 99.21 20.9

PO/Chem 55.4 3.35 61.32 10.82 75.34 18.2 83.21 19.5 99.21 21.1

PC/Chem 55.4 3.39 61.32 10.78 75.34 18.52 83.21 20.1 99.21 21.8

183

The results for COD measurement of reactive dye 222, synthetic effluent and three industrial

effluents by photo-fenton, biological and sequential treatments are represented in table.

Significant decrease in COD was observed in all treated samples by all treatment methods (Table

4.3.2 ) was lowest (5.03 mg/L) while that in the industrial effluent (79.5mg/L) was highest in

comparison with that of other samples treated by all other treatment methods (Table 4.3.2) which

indicate the partial mineralization of dyes under study). COD value of treated sample fell in

permissible limits i.e. 30 mg/L (ISI: 2490 1982).

184

Table 4.3.2 Comparison of water quality parameter (COD) by different treatment methods

Kuo (1992) reported approximately 80% COD removal via photo-fenton oxidation

process. Malik and saha (2003) documented that 70% COD removal can be achieved by

treatment with Pleurotus ostreatus. Sanghi et al. (2006) investigated that Phanerochaete

chrysosporium (PC) and C. versicolor removed COD up to 75%. Srikanlayanukul et al. (2006)

found that after decolorization, 80% and 67% of color and COD removal were achieved within

48 h, respectively.

Fongsatitkal et al. (2004) found more than 90% COD reduction in chemical treatment

followed by biological treatment sequence for simulated wastewater. According to Gianluca and

Nicola (2001), COD removal of biotreated textile wastewater was dependent on initial COD of

textile wastewater. About 67% and 39% of COD removal was achieved when initial COD was

Reactive dye

222

Synthetic

effluent

Industrial

effluent 1

Industrial

effluent 2

Industrial

effluent 3

COD (mg/L)


Photo-

Fenton 105.4 6.13 115.32 42.02 175.34 69.5 183.21 73.5 199.21 75.5

PO 105.4 7.34 115.32 44.55 175.34 72.5 183.21 78.2 199.21 79.2

PC 105.4 7.65 115.32 45.08 175.34 74.2 183.21 79.1 199.21 79.5

Chem/PO 105.4 5.03 115.32 38.54 175.34 66.1 183.21 69.2 199.21 73.2

Chem/PC 105.4 5.04 115.32 38.66 175.34 67.4 183.21 69.7 199.21 73.9

PO/Chem 105.4 5.15 115.32 40.52 175.34 68..2 183.21 70.5 199.21 75.1

PC/Chem 105.4 5.19 115.32 40.78 175.34 68.52 183.21 70.1 199.21 74.8

185

160 and 203mg/L, respectively. In another study, it was documented that up to 90% COD

removal can be possible with a combined chemical and biological treatment (Kuo, 1992).

4.3.3 Total organic carbon (TOC)

Measurement of the TOC was carried out in order to provide information about the

of concentration of the organic matter (dyes) present in wastewater after different treatment

(chemical, biological and sequential) technologies used in the present study. Both untreated and

treated samples of dye and effluents were analyzed to determine the value of TOC (mg/L). The

decolorization potential was evaluated by reduction in TOC of dye and four effluents treated by

photo-Fenton, biological and sequential treatments. A significant reduction in TOC was achieved

by all treated samples by different treatment methods (Table 4.3.3). However, results showed

that TOC in the dye treated samples by sequential treatment was lowest (5.19mg/L) while that in

the industrial effluent 3 (73.2mg/L) was highest in comparison with that of treated samples by all

other treatment methods (Table 4.3.3) which indicate the partial mineralization of dyes under

study.

186

Table 4.3.3 Comparison of water quality parameter (TOC) by different treatment methods

Shu et al. (2004) studied the degradation of acridine orange using the photo-fenton

reaction. They obtained 75% of mineralization. Lucas and Peres (2006) achieved 66.4% of TOC

removal of reactive black 5 aqueous solutions by the photo-fenton reaction. Neamtu et al. (2003)

obtained 49.32% of TOC remival of reactive yellow 84 and 73.52% reactive red 120

mineralization by biological degradation using P.s ostreatus which are in consistent with our

Reactive dye

222

Synthetic

effluent

Industrial

effluent 1

Industrial

effluent 2

Industrial

effluent 3

TOC (mg/L)


Photo-

Fenton 106.32 41.23 176.34 70.2 185.6 78.1 199.4 81.2 106.32 41.23

PO 105.8 3.97 106.32 43.02 176.34 74.2 185.6 80.2 199.36 84.2

PC 105.8 4.69 106.32 45.52 176.34 74.45 185.6 80.7 199.36 84.6

Chem/PO 105.8 5.85 106.32 38.53 176.34 65.3 185.6 72.5 199.36 78.2

Chem/PC 105.8 5.97 106.32 39.83 176.34 66.48 185.6 73.1 199.36 79.11

PO/Chem 105.8 6.19 106.32 40.21 176.34 67.65 185.6 77.1 199.36 80.1

PC/Chem 105.8 6.31 106.32 40.45 176.34 67.82 185.6 77.51 199.36 80.7

187

experimental findings. Chacon et al. (2006) documented 84% of TOC of acid orange 24 using

photo-fenton degradation followed by biological treatment by white rot fungal cultures. In

another study, the degradation of textile wastewater exhibited 55% of TOC removal by

UV/Fe+2/H2O2 system (Kusic et al., 2006).

4.3.4 Total Suspended Solids (TSS)

In textile waste water total suspended solids (TSS) can include a wide variety of

material like insoluble pigments. High TSS can block light from reaching submerged vegetation

and also cause an increase in surface water temperature, thus dissolved oxygen levels falls

beyond the normal (Mulligan et al., 2009). A significant reduction in TSS was achieved by all

treated samples by different treatment methods (Table 4.3.4). However, results showed that TSS

in the dye treated samples by sequential treatment was lowest (1.1mg/L) while that in the

industrial effluent 3 (24.75mg/L) was highest in comparison with that of treated samples by all

other treatment methods (Table 4.3.4) which indicate the partial mineralization of dyes under

study. However all the values were within the permissible limits compared with the WHO and

US-EPA standards set for drinking water.

188

Table 4.3.4 Comparison of water quality parameter (TSS) by different treatment methods

The highest TSS in industrial effluents as compared to synthetic effluent might be due to

the fact that water used in textile industries for dyeing processes carried away the solid particles

and increase the solid contents in the industrial effluents and found a significant decrease in TSS

could be achieved by using an effective treatment technology. Research done on textile

Reactive dye

222

Synthetic

effluent

Industrial

effluent 1

Industrial

effluent 2

Industrial

effluent 3

TSS (mg/L)


Photo-

Fenton 14.21 1.72 33.21 6.81 67.21 11.6 74.21 15.71 78.24 21.23

PO 14.21 1.82 33.21 8.51 67.21 13.32 74.21 17.62 78.24 24.67

PC 14.21 1.83 33.21 8.83 67.21 13.41 74.21 17.81 78.24 24.75

Chem/PO 14.21 1.1 33.21 5.22 67.21 9.11 74.21 12.6 78.24 16.2

Chem/PC 14.21 1.22 33.21 5.34 67.21 9.23 74.21 12.76 78.24 16.32

PO/Chem 14.21 1.43 33.21 6.21 67.21 10.42 74.21 13.11 78.24 17.11

PC/Chem 14.21 1.47 33.21 6.42 67.21 10.45 74.21 13.31 78.24 17.19

189

wastewaters by sequential treatments (AOP followed biologicak treatment) resulted in 70%

reduction in TSS in comparision with untreated wastewater (Azizullah et al., 2011).

4.3.5 Phenolic contents

Waste waters of many industries constitute a major environmental problem because of

the toxicity of the phenolic compounds present. Phenols can be produced by the oxidation of

benzene or aromatic compounds and same is the case here in biological and Photo-Fenton

treatments. This phenolic toxicity is the only drawback of such treatments. These treatments

need further biological treatments to decline phenolic toxicity. These phenols are produced when

a mixture of hydrogen peroxide and ferrous sulfate, called Fenton’s reagent, used to hydroxylate

aromatic rings, though yields are usually not high (Santos et al., 2001).

Both untreated and treated samples of dye and effluents were analyzed to determine the value of

phenols (mg/L). The mineralization potential was evaluated by reduction in phenolic contents of

dye and four effluents treated by photo-Fenton, biological and sequential treatments. Decrease in

phenolic contents was observed in all treated samples by all treatment methods (Table 4.3.5),

however, phenolic contents in the dye treated samples by sequential treatment was lowest

(5.43mg/L) while that in the industrial effluent (80.65mg/L) was highest in comparison with that

of treated samples by all other treatment methods (Table 4.3.5) which indicate the partial

mineralization of dyes under study. However all the values were within the permissible limits

compared with the WHO and US-EPA standards set for drinking water.

190

Table 4.3.5 Comparison of water quality parameter (Phenolics) by different treatment

methods

Comparision of researches have shown that up to 75% reduction in phenolic contentsby

using photo-Fenton treatment as a pre-treatment step (Hosseini et al., 2007), Suryaman et al.

(2006) 50mg/L phenol and documented that it required 30min of AOP (photocatalytic process)

coupled biological treatment. From the aforementioned researches, it is evident that mineralizing

phenol based on conditions of our findings (Kavitha and Palanivelu, 2004).

Reactive dye

222

Synthetic

effluent

Industrial

effluent 1

Industrial

effluent 2

Industrial

effluent 3

Phenolics (mg/L)


Photo-

Fenton 102.32 5.93 132.32 18.58 173.34 58.42 182.2 65.54 196.26 76.12

PO 102.32 6.14 132.32 20.52 173.34 60.89 182.2 67.42 196.26 80.42

PC 102.32 6.65 132.32 20.87 173.34 60.98 182.2 68.56 196.26 80.65

Chem/PO 102.32 5.43 132.32 15.10 173.34 53.3 182.2 62.1 196.26 71.23

Chem/PC 102.32 5.54 132.32 15.78 173.34 53.94 182.2 62.34 196.26 71.41

PO/Chem 102.32 5.65 132.32 16.33 173.34 55.98 182.2 63.54

196.26 73.24

PC/Chem 102.32 5.69 132.32 16.52 173.34 56.23 182.2 63.76 196.26 73.76

191

4.4 Haemolytic Activity (Toxicity Assay)

The lipid bilayer that constitutes a biological membrane is primarily stabilized by the

hydrophobic force, which compels the hydrophobic tails of phospholipids to join together.

Secondary stabilization is provided by Van der Waals attractive forces, which keep these groups

gathered together in the anhydrous interior of the membrane, and by hydrogen bonds formed

between the polar heads of the phospholipids and water, both within the cell and at its external

surface (Aki and Yamamoto, 1991; Powell et al., 2000). The cytoplasmatic membrane

constitutes the first cell target that suffers damage caused by external denaturing agents such as

heat or ethanol. Heat increases the vibrational energy of chemical groups, favoring rupture of the

non-covalent bonds that stabilize the native structure of the biological complex. Ethanol

specifically decreases the intensity of the hydrophobic force by solvating the apolar groups that

were hidden inside the native structure of the biological complex (Mohan et al, 1992).

The mechanical stability of the erythrocytic membrane is a good indicator of the effect of

various in vitro insults levied on it by various compounds for the screening of cytotoxicity and is

dependent on their physical and structural properties. It is also well known that in infectious

diseases too, hemolysis occurs due to the action of the microbial products and resident parasites

like Plasmodium falciparum (Mohan et al., 1992) on the red blood corpuscles membranes. In

itself the erythrocytic membrane is a dynamic structure that can dictate significant changes in its

interaction, best illustrated with detergents ( Aki and Yamamoto, 1991) and well characterized

drug-induced hemolysis. Hemolysis is best evaluated using an in vitro method, which can show

the effect of increasing concentration and to be sigmoidally related to the logarithm of contact

time, as studied for various surfactants.

The rate of haemolysis is a reflection of the toxicity using different concentration of the

synthetic compound on human erythrocytes. On Y-axis lysis of Human Red Blood was

compared with treated samples on X-axis comparing with positive control having (100%) lysis.

In this study, rate of hemolysis of untreated and treated dye and effluents samples by photo-

fenton, biological and sequential treatments was evaluated to assess the toxicity rate. Triton –x

.1% as a positive control, showed 100% lysis while Phosphate Buffer Saline-PBS, as a negative

control, showed 0% lysis. The results of treated samples were compared with these controls.

From the Fig. 4.4a & 4.4b, it is clear that there was a marked decrease in toxicity of treated dye

and effluent samples by all treatment methods used in the present study.

192

Fig. 4.4a Toxicity assay of treated Reactive dye 222 by Photo-Fenton, biological and

sequential treatments

TOXICITY ASSAY

0.0

20.0

40.0

60.0

80.0

100.0

120.0

sample-1

sample-3

sample-5

sample-7

sample-9

sample-11

sample-13

sample-15

sample-17

sample-19

sample-21

sample-23

sample-25

sample-27

positive

contr

ol

Sample along with Control

Pe

rce

nta

ge

of

RB

Cs

Ly

sis

Fig. 4.4b Toxicity assay of treated synthetic and industrial effluents by Photo-Fenton, biological and sequential treatments

TOXICITY ASSAY

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8

Phosphate buffer sa

line

Average Trito

n X 100

Sample and control

% o

f R

BC

s L

ysis

193

4.5 Economic analysis of the data

The use of all treatment (photo-Fenton, biological and sequential) technologies in the

present study for wastewater treatment could be the development of efficient and low cost

materials to promote sufficient treatment, the adoption of strategies for processes integration, the

targeting of mew classes of pollutants and the commercialization of processes which has been

safer used in the laboratory.

The application of the figure-of-merit proposed by IUPAC was of great help in the

determination of scaling up parameters depending on the wastewater composition and the

application of treatment on wastewater. Almost 70-80% decolorization as well as mineralization

(as it was evident from COD, TOC measurement) was obtained by applying biological treatment

using white rot fungi to treat the synthetic wastewater. Similarly, almost 80-90% decolorization

and 80% mineralization (as it was evident from COD, TOC measurement) was obtained by

photo-Fenton treatment using 3.5 × 10-5 M of FeSO4 and 1 × 10-5 M H2O2 in 50 minutes of

visible irradiation time when synthetic wastewater was used. Although both processes were

effective but they were resulted in formation of phenolic products, which are not eco-friendly. It

was determined when sequential treatments were considered, 95% decolorization and almost

90% mineralization was achieved and a negligible amount of phenolic products were generated

(Julia et al., 2008) making it an effective eco-friendly technology. The actual energy

consumption can be effectively decreased raising reasonable values.

Table 4.5 Economic cost per functional unit of the different inputs for considered

treatments

Cost ($)

Photo-Fenton Biological Sequential

Electricity (KWh) 2.23 × 10-3 --------------- 2.187 × 10-4

H2O2 (rupees/L) 0.05 0.000023 0.0056

FeSO4 (rupees/L) 0.04 --------------- 0.0043

Kirk’s medium (rupees/L) ------------------- 0.008 0.0008

Total 9.2 × 10-3 8 × 10-4 1.9 × 10-6

194

In view of this, considering both environmental and economic data to decide about the

sustainability of the studied wastewater treatments, the best environmental/cost option seems to

be the coupling of artificial light photo-Fenton process and biological treatment. As a final

remark, and taking these results as a starting point, it is predictable that a photo-Fenton process

coupled to a biological treatment would be the best economic and environmental option to

remove reactive dyes from textile effluents. These scaling up parameters should be used in a

pilot plant and the results from this treatment are under collection at this time. Such a

combination reduced the treatment time and optimized the overall economics, since the photo-

detoxification system can be significantly smaller.

195

CHAPTER 5

SUMMARY

A large number of dyes are used by textile sector, paper, cosmetic and leather industries

for dyeing and printing purposes. The discharge of dye house wastewater into the environment

damages the quality of the receiving streams and toxic to aquatic life (Meyer, 1981). A number

of physical and chemical methods are in use for de-toxification of wastewater having dyes. The

restrictive environmental legislation and the ecological problems have resulted in the search of

economically viable wastewater treatment technology. In the present project, Photo-Fenton,

Biological and sequential treatment technologies were applied to treat synthetic and real

wastewater of reactive azo dyes. First, different experimental parameters were optimized with

synthetic wastewater having reactive dye 222. In Photo-Fenton,s treatment methods, the

optimization of different experimental parameters (pH, FeSO4, H2O2, temperature and effects of

salts) were optimized to get maximum decolorization (95%) of dye under study. In biological

methods, experiments was performed with five locally isolated indigenous white rot species, for

the selection of two white rot fungal cultures based on their maximal decolorization potential.

Different fermentation conditions (pH, inoculums size, temperature, mediators and metal ions)

and nutritional factors (carbon and nitrogen sources) were optimized to enhance the efficiency

of white rot fungal cultures for dye decolorization. Then optimized conditions were applied to

treat the synthetic wastewater having a mixture of dyes and three industrials. The rate of

decolorization was determined by the change in color of dye whuch was detected by UV-Visible

spectrophotometer, following the decolorization assay. The effectiveness of all treatment

technologies was evaluated by water quality assurance parameters such as COD, BOD, TOC,

TSS, Phenolic contents by following the standard methods of treatments. Toxicity test was

performed to evaluate the detoxification of all treatments under study. Extent of mineralization

was evaluated by measuring total organic carbon (TOC), chemical oxygen demand (COD)

measurement, reduction in uv-visible spectrum and FTIR spectral studies, by all treatment

technologies under study. Almost 80-85% decolorization as well as 70% mineralization (as it

was evident from COD, TOC measurements) was obtained by applying biological treatment

using white rot fungi to treat the synthetic wastewater. Similarly, almost 90-95% decolorization

196

and 80% mineralization (as it was evident from COD, TOC measurements) was obtained by

photo-Fenton treatment using 3.5 × 10-5 M of FeSO4 and 1 × 10-5 M H2O2 in 50 minutes of

visible irradiation time when synthetic wastewater was used. Although both processes were

effective but they were resulted in formation of phenolic products in lesser amounts, which are

not eco-friendly. It was determined when sequential treatments were considered, 97%

decolorization and almost 90% mineralization was achieved and a negligible amount of phenolic

products was generated, making it an effective eco-friendly technology. The UV-Visible and

FTIR spectral studies have shown decolorization as well as mineralization of dyes under study.

The actual energy consumption can be effectively decreased raising reasonable values. An

economic analysis has shown as the cost in the chemical treatment (photo-fenton treatment is

considered mainly due to the chemicals, thus at lower doses (it si applied as a pre-treatment

step), operating cost of the treatment can be saved. It was also found, that as the sequential

treatments were carried out at lower dose of chemicals, so sludge production was almost

negligible. Thus sludge handling and disposal cost can also be saved. Later the better

mineralization of the dyes (removal of aromatic amines and hydroxylated products) can be

achieved in the aerobic biological treatment system, which is considered to be economical. Thus

the overall treatment chain of photo-Fenton,s oxidation followed by aerobic biological treatment

could be quite effective and economical option for the treatment of recalcitrant compounds like

azo dyes in pilot plant scale.

197

LITERATURE CITED

Abdullah, N. and S.I Zafar. 1999. Lignocellulose biodegradation by white rot basidiomycetes:

overview. Int. J. Mushroom sci. 2: 59-78.

Aksu, Z. and S. Tezer. 2000. Equilibrium and kinetic modeling of biosorption of Remazol Black

B by Rhizopusarrhizus in a batch system: effect of temprarure.Process Biochem. 36: 431-

446.

Alberto, D., R. Isabel, C.R. Susanaand M.S. Angeles. 2000. Design of new rotating drum

bioreactor for lignolytic enzyme production by Phenerochaetechrsosporiumgrown on

inert support. Process Biocem. 37: 549-54.

Albuquerque, M.G.E., A. T. Lopes, M.L. Serralheiro, S.M. Novais and H.M, Pinbeiro. 2005.

Biological sulphate reduction and redox mediator effects, an azo dye decolorization in

anaerobic-aerobic sequencing batch reactors. Enz. Microb. Technol.36: 790-797.

Ana, C., J.P. Peter, A.M.J.J. Cess and H.D. Van. 2002. Fungal peroxidases: Model aspects and

applications. J. Biotechnol. 93: 143-158.

APHA.1985. Standard methods for the examination of water and wastewaters (16thEd.). APHA-

AWWA and WPCF, Washington.

Asad, S., M.A. Amoozegar, A.A. Pourbabaee, M.N. Sarbolouki and S.M.M. Dastgheib. 2007.

Decolorization of textile azo dyes by newly isolated halophilic and halotolerant bacteria.

Bioresource Technol. 98: 2082-2088.

Asghar, M.,M.T. Asad and R.G. Legge.2006. Enhanced lignin peroxidase synthesis by

Phanerochaetechrysosporiumin solid state bioprocessing of lignocellulosic

substrate.World J. Microbiol. Biotechnol. 22: 449-453.

Ashraf, S.S., M.A. Rauf and S. Alhodrami. 2006. Degradation of methyl red using Fenton,s

reagent and the effect of various salts. Dyes Pigments. 69: 74-78.

Azbar, N., T. Tonar and K. Kestioglu. 2004. Composition of various oxidation processes and

chemical treatment methods for COD and color removal from a Polyester and acetate

fibre dyeing effluent. Chemosphere. 55: 25-43.

198

Azizullah, A., P. Richter and D. P. Hader. 2011. Ecotoxicological evaluation of wastewater

samples from Gadoon Amazai Industrial Estate (GAIE), swabi, Pakistan. Int. J. Environ.

Sci. 1: 1-17.

Baldrian, P. 2004. Purification and characterization of laccase from the white rot fungus

Daedallea quercina and decolorization of synthetic dyes by the enzyme. J. Microbial

Biotechnol. 63: 560-563.

Baldrian, P. and J. Snajdr. 2006. Production of ligninolytic enzymes by litter decomposing fungi

and their ability to decolorize synthetic dyes. Enz. Microb. Technol. 39: 1023-1029.

Bannat, I.M., P. Nigam, D. Singh and R. Marchant. 1996. Microbial decolorization of textile dye

containing effluents: a review.Biores. Technol. 58: 217-227.

Bandyopadhay, G and S. Chattopadhay. 2007. Single hidden layer artificial neural network

models versus multiple linear regression in forecasting the time series of total ozone. J.

Environ. Sci. Technol. 4: 141-150.

Barbusinski, K. and J. Majewski. 2003. Degradation of azo dye acid red 18 by Fenton,s reagent

in the presence of iron powder. Pol. J. Environ. Stud. 12: 151-157.

Basibuyuk, M. and C.F. Foster. 1997. The use of sequential anaeribuc/aerobic process for the

biotreatment of simulated dyeing waste water. J. Environ. Technol. 18: 843-848.

Baughman, G.L. and EJ. Waber.1994. Transformation of dyes and related compounds in anoxic

sediment: Kinetics and products. Environ. Sci. Technol. 28: 267-276.

Bhunia, A., S. Durani and P.P. Wangikar. 2001. Horseradish peroxidase catalyzed degradation of

industrially important dyes. J. Biotechnol. Bioeng. 72: 562-567.

Bhattachaya, S.K., L.K. Wang and M.H.S. Wang. 1992. Handbook of industrial wasre treatment,

Vol. 1 Marcel Dekker, New York.

Brown, D. and P. Laboureur. 1983. The degradation of dyestuffs: Part 1.Primary biodegradation

under anaerobic conditions. Chemosphere. 12: 397-404.

Buckley, C.A. 1992. Memberane technology for the treatment of dye house effluents. Water sci.

Technol. 25: 203-209.

199

Tanford, C. 1973. The Hydrophobic Effect: Formation of Micelles in Biological Membranes,

John Wiley, New York.

Camel, V. and A. Bermond. 1998. The use of ozone and associated oxidation processes in

drinking water treament. Water Res. 32: 3208-3222.

Carliell, C.M. 1993. Biological degradation of azo dyes in an aerobic system. Thesis: Master of

science in Engineering, Department of Chemical Engineering, University of Natal

Durban.

Carliell, C.M., S.J. Barclay, C. Shaw, A.D. Wheately and C.A. Buckley. 1998. The effect of salts

used in textile dyeing on microbial decolorization of e reactive azo dye. J. Environ.

Technol. 19: 1133-1137.

Carliell, C.M., S.J. Barclay, N. Nadidoo, C.A. Buckley, D.A. Muuholland and E. Senior. 1995.

Microbial decolorization of a reactive azo dye under anaerobic conditions. Water S.A.

21: 61-69.

Carliell, C.M., S.J. Barclay, N. Nadidoo, C.A. Buckley, D.A. Muuholland and E. Senior. 1994.

Anaerobic decolorization of reactive dyes in a conventional sewage treatment processes.

Water S. A. 22: 341-344.

Cetin, D. and G. Donmez. 2006. Decolorization of reactive dyes by mixed cultures isolated from

textile effluent under anaerobic conditions. J. Enz. Microb. Technol. 38: 926-930.

Chacon, J.M., T. Leal, M. Sanchezm and E.R. Bandala. 2006. Solar photocatalytic degradation

of azo-dyes by pfoto-Fenton process. Dyes Pgments. 69: 189-207.

Chen C.H., C.F. Chang, C.H. Ho, T.L. Tsai and S.M. Liu. 2008. Biodegradation of crystal violet

by a Shewanella sp. NTOUI.Chemoshpere. 72: 1712-1720.

Chen, B.Y., C. Jo-Shu and C. Shan-Yu. 2003. Bacterial species diversity and dye decolorization

of a two species mixed consortium. Environ. Engng. Sci. 20: 337-345.

Chen, J., W.H. Rulkens and H. Brunruing. 1997. Photochemical elimination of phenols and COD

in industrial waste waters. Water Sci. Tech. 35: 231-238.

Chen, M., H. Liu, J. Weng and Y. Wang. 2002. Influence of supplemental nutrition aerobic

decolorization of Acid Red 14 inactivated sludge. Process Biochem. 37:667-678.

200

Chinwekitvanich, S., M. Tuntodvest and T. Panswad. 2000. Anaerobic decolorization of a

reactive dyebath effluent by a two stage UASB with Tapioca as co-substrate. Water

Research. 34:2 223-2232.

Cripps, C., Bumpus, J.A. and Aust, D.A. 1993. Biodagradation of azo and heterocyclic dyes by

Phanerochaetechrysosporium. Appl. Environ. Microbiol., 56: 1114-1118.

Cruz, A. and G. Buitron. 2001. Biodegradation of disperse blue 79 using sequential anaerobic/

aerobicbofertilizers. Water Sci. Technol. 44: 15-166.

Daneshvar, N., S. Abev and F. Hosseinzodeh. 2008. Study of C.I acid orange 7 removal in

contaminated water by photo oxidation processes. Global NEST J. 10: 16-23.

Delee, W., C. O’Neil, F.R. Hawkes and H.W. Pinheiro. 1998. Anaerobic treatment of textile

effluents: a review. J. Chem. Technol. Biotechnol. 73: 323-335.

Dirk, W., K. Irene and N.A. Spiros. 2003. White rot fungi and their enzymes for the treatment of

industrial dye effluents. Biotechnol. Adv. 22: 161-187.

Dong, Y., J. Chen, C. Li and H. Zu. 2007. Decolorization of three azo dyes in water by

photocatalysis of Fe (III)-oxalate complexes/ H2O2 in the presence of inorganic salts.

Dyes Pigments. 73: 261-267.

Driessel, B. V. and L. Christov. 2001. Decolorization of plant effluent by white rot fungi in a

rotary biological contactor reactor. Chemosphere. 92: 271-276.

Edward, J.C. 2000. Investigation of color removal by chemical oxidation for three reactive

textile dyes and spent textile dye wastewatere.Master, s Thesis, Virginia Polytechnic

Institute and State University.p. 56.

EPRI. 1996. The efficiency of color removal technique in textile waste water treatment.

Hydrosuence, EPRI project 3329-01.

Faison, B. D., T. K. Kirk an R. Farrell. 1980. Role of veretryl alcohol in regulating ligninase

activity in Phenerochaete chrsosporium. Appl. Environ. Microbial. 52: 251-254.

Fongsatitkul, P., P. Elefsiniotis, A. Yamasmita and L.N. Yamashita. 2004. Use of sequencing

batch reactors and Fenton’s reagent to treat a wastewater from a textile industry. J.

Biochem. Eng. 21: 213-220.

201

Farre, M.J.X. Domenech and J. Peral. 2006. Assesment of Photo-Fenton and biological treatment

coupling for Diuron and Linuron removal from water. Water Res. 40: 2533-2540.

Fernandez, J., J. Bandara, A. Lopez, P. Buffar and J. Kiwi. 1999. Photo-assisted fenton

degradation of nonbiodegradableazo dye (Orange II) in Fe-free solutions mediated by

citation transfer memberanes. Langmuir. 15: 185-192.

Fischer, A. and C. Hahn. 2005. Biotic and abiotic degradation behaviur of ethylene glycol.

Biores. Technol. 39: 151-162.

FitzGerald, S. and P.L. Bishop. 1995. Two stage anaerobic/aerobic treatment of sulfonatedazo

dyes. J. Environ. Sci. Health Part A, Environ. Sci. Eng. Tox.Hazard. Substance Control

A. 30: 1251-1276.

Fu,Y. and T. Viraraghavan. 2001. Fungal decolorization of dye wastewaters: a review.Biores.

Technol. 79: 251-262.

Gahr, F. and W. Oppermann. 1994. Ozonation- an important technique to comply with new

German laws for textile waste water treatment. Water Sci. Technol. 30: 255-263.

Galhaup, C., H. Wagner, B. Hinterstoisserand D. Haltrich. 2002. Increased production of laccase

by yhe wood degrading basidiomycetesTrametepubescens.Enz. Microb. Technol. 3: 529-

536.

Galindo, C., P. Jacques and A. Kalt. 2001. Photochemical and photocataytic degradation of an

indigoid dye: a case study of acid blue 7 (AB74). J. Photochem. Photobiol.141: 47-56.

Ganesh, R. 1992. Fate of azo dye in sludges.Master, s Thesis, Virginia Polytechnic Institute and

State University. p.193.

Greenberg, A.E., R.L. Trussell and L.S. Clesceri. 1985. Standard methods for the examination of

water and waste water (20th Ed.).

Hanzon, B. and R. Vigilia. 1999. UV disinfection.Wastewater Technol. 2: 24-28.

Heinfling, A., M.J. Martinez, A.T. Martinez, M. Bergbauer and U. Szewzyk. 1998.

Transformation of industrial dyes by manganese peroxidase from Bjerkanderaadusta and

Pleurotuseryngii in a manganese-independent reaction. Appl. Environ. Microb. 64: 2788-

2793.

202

Hosseini , S. N., S. M. Borghei, M. Vossoughi and N. Taghavinia N 2007. Immobilization of

TiO2 on perlite granules for photocatalytic degradation of phenol. Appl. Catal B Environ.

74: 53-62.

Ince, N.H. and D.T. Gonene.1997. Treatibility of a textile azo dye by UV/H2O2. Environ.

Technol. 18:179-185.

Ivana, C., M. Lepretti, S. Martucciello and C. Esposito. 2010. Enzymatic strategies to detoxify

Glutein: Implications fot Celiac disease: Review article. Enzyme Research Article ID.

17435

Jiang, H. and P.L. Bishop. 1994. Aerobic biodegradation of azo dyes in biofilms. Water Science

and technology. 22:525-530.

Jonsson, L., J.M. Saloheimo and A. Penttila. 1997. Laccase from white-rot fungus Trametes

versicolor: cDNA cloning of ICC1 and expression in Pichiapastoris.Curremt Geometrics.

3: 425-430.

Julia, G.M., X. Domenech, A. Jose, G. Hortel, F. Torrades and J. Peral. 2008. The testing of

several biological and chemical coupled treatments for Cibacron Red FN-R azo dye

removal. J. Hazard. Mater. 154: 484-490.

Junker, F., T. Leisinger and A.M. Cook. 1994. 3 Sulphocatechol 2,3dioxygenase and other

dioxygenase (E.Cl 13:11. 2 and E.Cl. 14.12) in degradative pathway of 2 amino benzene

sulphonic acid and 4 toluene sulphonic acids in Alcaligenes sp. Strain 0-1. Microbiology.

140: 1713-1722.

Kalyani, D.C., P.S. Patil, J.P. Jadhav and S.P. Govindwar. 2008. Biodegradation of reactive

textile dye Red BLI by an isolated bacterium Pseudomonas Sp. SUKI.Biores. Technol.

99: 4635-4641.

Kamitsuji, H., Y. Honda, T. Watanabe and M. Kuwahara. 2004. Production and Induction of

manganese peroxidase isozymes in a white rot fungus Pleurotusostreatus. Appl.

Microbial Biotechnol. 70: 497-504.

Karcher, S., A. Kornmuller and M. Jekel. 2002. Anion exchange resins for the removal of

reactive dyes from textile wastewaters. Water Res. 36: 4717-4724.

203

Kashyap, P., A. Sabu, A. Pandey, G. Szakacs, and C.R. soccal. 2002. Extra-cellular L-

glutaminase production by zygosaccharomycesrouxii under solid state fermentation.

Process Biochem. 38: 307-312.

Kavitha V. and K. Palanivelu. 2004. The role of ferrous ion in Fenton andphoto-Fenton

processes for the degradation of phenol. Chemosphere. 55: 1235-1243.

Khehra, M.S., H.S. Saini, D.K. Sharma, B.S. Chadha and S.S. Chimini. 2005. Comparative

studies on potential of consortium and constituent pure bacterial isolates to decolorize azo

dyes. Water Res. 39: 5135-5141.

Kim, T.H., C. Park, J. Yang and S. Kim. 2004. Comparison of disperse and reactive dye

removals by chemical coagulation and Fenton oxidation. J. Hazard. Mat. 112: 95-103.

Kirk-Othmer. 1979. Encyclopedia of chemical technology (3rd Ed.). p. 374-385.

Knapp, J.S., C. Vantoch- wood and F. Zhang. 2001. Use of wood – rotting fungi for the

decolorization of dyes and industrial effluents. P. 242-304. In G. M. Gadd (Ed). Fungi in

bioremediation. Cambridge University Press, UK.

Ksibs, M. 2006. Chemical oxidation with hydrogen peroxide for domestic waste water treatment.

J. Chem. Engg. 119: 161-165.

Kumarasamy, M., Y.M. Kim, J.R. Jeon and Y.K. Chang.2009. Effective of metal ions on dye

decolorization by laccase from Ganodermalucidum. J. Hazard. Mater. 168: 523-529.

Kuo, W.G., 1992. Decolorizing dye wastewater with Fenton’s reagent. Water Res. 26:881-886.

Kusic, H., N. Koprivana, L. Srsan. 2006. Azo dye degradation using Fenton type processes

assisted by UV irradiation: A kinetic study. J. PhotochemPhotobiol.181: 195-202.

Lawerenc, Y. and Y. Jian. 1997. Lignininace-catalyzed decolorization of synthetic dyes. Water

Res. 31: 1187-1193.

Libra, J.A., M. Borchert and S. Banit. 2003. Competition strategies for the decolorization of a

textile reactive dye with white rot fungi Tremates versicolor under non-sterile conditions.

Biotechnol.Bioeng. 82: 736-744.

Lin, S.H. and C.M. Lin. 1993. Treatment of textile waste effluent by ozonation and coagulation.

J. Environ. Engng.120 :437-446.

204

Liou, J.M., M.C. Luv. N. Chen. 2003. Oxidation of explosive by Fenton and Phto-Fenton

process. Water Res. 37: 3172-3179.

Liu, G., J. Wang, M. Zhou, H. Lu and R. Jin. 2009. Acceleration of azo dye decolorization by

using reductase activity of azoreductase and quinine redox mediator. Biores. Technol. 42:

315-321.

Lorenco, J., M. Novais and H.M. Pinheiro. 2000. Reactive textile dye color removal in a

sequencing batch reactor. Water Sci. Technol. 42 Lucas, M.S. and J.A. Peres. 2006.

Kinetics of decolorization of azo dyes Reactive Black 5 by Fenton and photo-Fenton

oxidation. Dyes Pigments. 71: 236-44.

Malato, S.,J. Blanco, A. Vidal and C. Richter.2002. Photo catalysis with solar energy at a pilot-

plant scale.Appl. Catal. B. Environ. 37: 1-15.

Malik, P.K. and S.K. Saha. 2003. Oxidation of direct dyes with hydogen peroxide using ferrous

ion as catalyst. Sep. Purif. Technol. 98: 35-50.

Marcucci, M., G. Nosenzo, G. Capannelli, I. Ciabatti, D. Corrieri and G. Ciardelli. 2001. Color

and COD retention by nano-filtration membrane. Desalination. 138: 78-82.

Mario, A., S. Esplugas and G. Saum. 1997. How and why chemical and biological processes for

wastewater treatment. Water Sci. Technol. 35:321-327.

Mass, R. and S. Chandhari.2 005. Adsorption and biological decolorization of azo dye Reactive

Red 2 in semicontinuous aerobic reactors. Proc. Biohem. 40: 69-705.

Masud, H.S.K. and N. Anantharaman. 2006. Activity enhanement of ligninolytic enzymes of

Trametes versicoler with bagasse powder. Afr. J. Biotechnol. 5: 189-194.

McCurdy, M.W. 1992. Chemical reduction and oxidation combined with biodegradation for the

treatment of a textile dye wastewater. Master’s Thesis, Virginia Polytechnic Institute and

State University.

McCurdy, M.W., G.D. Boardman, D.L. Michelsen and B.M. Woodby. 1992. Chemical reduction

and oxidation combined with biodegradation for the treatment of textile dyes. J. Appl.

Microbial Biotechnology. 56: 81-87.

205

Mehna, A., P. Bajpai and P.K. Bajpai. 1995.Studies on decolorization of effluent from a small

pulp mill utilizing agri residues with Trametes versicoler.Enzyme Microb. Technol. 17:

18-22.

Meric, S., D. Kaptan and T. Olmez. 2004. Color and COD removal from wastewater containing

reactive black 5 using Fenton,s oxidation process. Chemosphere. 54: 435-441.

Meyer, U. 1981. Biodegradation of synthetic organic colorants.In: Microbial degradation of

xenobiotic and recalcitrant compounds.Leisinger, T. et al., (Eds.). Academic Press,

London.

Mishra, G. and M. Tripathy. 1993. A critical review of treatments for decolorization of textile

effluents.Colourage. 48:35-38.

Mohan, K., N.K. Ganguly, M.L. Dubey and R.C.Mahajan. 1992. Oxidative damage of

erythrocytes infected with P. falciparum. An in vitro study. Annals of Hematology.65:

131-134.

Moraes, J.E.F., F.H. Quina, C.A.O. Nascimento, D.N. Silva and O.F. Chiavone. 2004. Treatment

of saline wastewater contamination with hyrdrocarbons by the photo-fentonrocess.

Environ. Sci. Technol. 38: 1183-1187.

Mulligan, N. C., N. Davarpanah, M. Fukue and T. Inoue. 2009. Filtration of contaminated

suspended solids for the treatment of surface water. Chemosphere. 74: 779-786.

Muruganandham, M. and M. Swaminathan. 2004. Decolrization of reactive orange by Fenton

and Photo-Fenton oxidation technology.Dyes and Pigments. 63: 315-321.

Neamtu, M., A. Yediler, I. Siminiceanu and A. Kttrup. 2003. Oxidation of commercial reactive

azo dye aqueous solutions by the photo-Fenton and Fenton-like

processes.J.Photochem.Photobiol.161: 87-93.

Nanhango, G.S.J. Gomes, G. Gubitz, R. Zvauya, J.S. Readand W. Steiner. 2002. Production of

laccase by a newly isolated strain of Trametes modesta. Biores. Technol. 84: 259-263.

Nansheng, D., W. Feng, T. Shizhong and F. Tao. 1997. Photodegradation of dyes in aqueous

solutions containing Fe (111)-hydroxyl complex.Solar photodegradation

Kinetics.Chemosphere. 34: 2725-2735.

206

O, Neil, C., F.R. Hawkes, D.L. Hawkes, N.D. Lourenco, H.M. Pinhiero and W. Delee. 1999.

Color in textile effluents sources, measurements, discharge contents and simulation: a

review. J. Chem. Technol. Biotechnol. 74: 1009-1018.

O, Neil, C., A. Lopez, S. Esteves, F.R. Hawkes, D.L. Hawkes and S. Wilcox. 2000. Azo dye

degradation in an anaerobic-aerobic treatment system operating on simulated textile

effluent. Appl. Microbial Biotechnol. 53: 249-254.

Ogfitveren, U.B. and S. Koparal. 1994. Color removal from textile effluents by electrochemical

destruction. J. Environ. Sci. Health. 29: 1-16.

Ollika, P., L. Alhonmaki, V.M. Leppanen, T. Glumoff, T. Raijola and I. Suominen. 1993. “

Decolorization of azo, triphenylmethane, heterocyclic and polymeric dyes by lignin

peroxidase isoenzymes from Phanerochaete chrysosporium”. Appl. Environ. Microbiol.

59: 4010-4016.

Orozco, S.L., E.R. Bsdala, C.A.A. Bulnes, B. Serra, R.S. Parra and I.H. Perez. 2008. Effect of

iron salt on the color removal of water containing azo dye reactive blue using photo-

assisted Fe (II)/ H2O2 and Fe (III)/ H2O2 system. J. Photochem. Photobiol. 198: 144-

149.

Pak, D. and W. Chang. 1999. Decolorizing dye waste water with low temperature catalytic

oxidation. Water Sci. Technol. 40: 115-121.

Panizza, M. and G. Cerisela.2009. Electro-fenton degradation of synthetic dyes. Water Research.

43: 339-344.

Panswad, T. and W. Luangdilok. 2000. Decolorization of reactive dyes with molecular structure

under different environmental conditions. Water Res. 34: 4177-4184.

Pappolymerou, A.E., D. Fatta and M. Loizidou. 2007. Development and optimization of dark

Fenton oxidation for the treatment of toxic waste water with organic load. J. Hazard.

Mater. 146: 558-563.

Pasti-Grigsby, M.B., A. Paszcczynski, S. Goszczynski, D.L. Crawford and R.L. Crawford. 1992.

Influence of Aromatic substitution patterns on azo dye degradability by Streptomyces sp.

and Phanerochaete chrysosporium. Appl. Environ. Microbiol. 58: 3605-3613.

207

Paszczynski, A. and R. Crawford. 1995. Potential for bioremediation of xenobiotic compounds

by the white rot fungus Phanerochaete chrysosporium. Biotechnol.Prog. 11: 386-379..

Pauli, O., A. Kirsi, L.M. Veli, G. Tumpo, R. Imo and S. Ilari. 1993. Decolorization of azo,

triphenylmethane, hetemocyclic and polymeric dyes by lignin peroxides isoenzymes from

Phenerochaetechrsosporium. Appl. Environ. Microbial. 59: 4014-4016.

Pearce, C.I., Lloyd and J.T. Guthrie. 2003. The removal of color from textile wastewaterusing

whole bacterial cells: a review. Dyes Pigments. 58:179-196.

Perez, M., F. Torrades, X. Domenech and J. Peral. 2002. Photochemical oxidation.textile

effluents by Photo-Fenton reagent. Water Res. 36: 2703-2710.

Pinheiro, H.M., E. Touand and O. Thomas. 2004. Aromatic amines from azo dye reduction:

Status review with emphasis on direct UV spectrophotometric detection in textile

industry wastewaters. Dyes Pigments. 61: 121-139.

Powell, W.A., C.M. Catranis and C.A. Maynard. 2000. Design of self-processing antimicrobial

peptide for plant protection. Lett. Appl. Microbiol. 31: 163-169.

Radha, K.V., J. Regupathi, A. Arungiri and T. Murugesan. 2005. Decolorization studies of

synthetic dyes using Phenerochaete chrysosporium and their kinetics. Proces Biochem.

40: 3337-3345.

Raghukumar, C., D. Chandramohan, F.C. Michel and C.A. Reddy. 1996. Degradation of lignin

and decolorization of paper mill bleach plant effluent (BPE) by marine fungi.

Biotechnol. Lett. 6: 105-106.

Rathi, A., H.K. Rajor and R.K. Sharma. 2003. Photodegradation of direct yellow 12 using

UV/H2O2/Fe+2. J. Hazard. Mater B. 102: 231-241.

Rau, J., H.K. Knackmuss and A. Stolz. 2003. Effects of different quinoid mediators on the

anaerobic reduction of azo dyes by bacteria. Environ. Sci. Technol. 36: 1497.

Reddy, C. A. 1995. The potential for white rot fungi in the treatment of effluents.Curr. Opt.


208

Rezaee, A.M.T. Ghanian, S.J. HashemiamMoussari, A. Khavarin and G. Gharizadeh. 2008.

decolorization of reactive blue 19 dye from textile wastewater by the UV/H2O2 process.

J. Appl. Sci. 8: 1108-122.

Robinson, T., B. Chandran and P. Nigam. 2001. Studies on the production of enzymes by white

rot fungi for the decolorization of textile dyes. J. Enz.Micro. Technol. 29: 575-579.

Rodriguez, E., F.J. Ruiz-Duenas, R. Kooistra, A. Ram, A.T. Martinez and M.J. Martinez. 2008.

Isolation of two laccase genes from the white rot fungus Pleurotus eryngii and

heterologous expression of the PcI3 encode protein. J. Biotechnol., 134: 9-19.

Rosenkranz, H.S. and G. Kalpmen. 1989. Structural basis of mutagenicity of phenylazoanoline

dyes. Mutation Res. 221: 217-234.

Santos, V. L., N. M. Heilbuth and V. R. Linardi. 2001. Degradation of phenols by Trichosporan

sp. Lez cells immobilized in alginate. J. Basic Micribial. 41: 171-178.

Singer, S. J. and G.L. Nicholson. 1972. The fluid mosaic model of the structure of the cell

membranes. Science.175: 720–731.

Sabu, A., A. Pandey, M.J. Daud and G. Szakacs. 2005. Tamarind seed power and palm kernel

cabe: two novel agro esidues for the production of tannase under solid state fermentation

by Aspergillus niger ATCC 16620. J. Bioresource Technology. 96: 1223-1228.

Sami, S. and E. Radhouane 1995. Role of lignin peroxidase and manganese peroxidase from in

the decolorization of Phenerochaete chysosporium olive mill wastewaters.Appl. Environ.

Microbial. 61: 1098-1103.

Sanghi, R., A. Dinit and S. Guha. 2006. Sequential batch culture studies for the decolorization of

reactive dye by Coriolus versicolor. Biores. Biotechnol. 97: 396-400.

Sarria, V., M. Deront, P. Peringer and C. Pulgarin. 2003. Degradation of a biorecalcitrant dye

precursor present in industrial wastewaters by a new integrated iron (photo assisted

biological treatment. Appl. Catal. 40: 231-246.

Scott, J.P. and D.F. Follis. 1995. Integraration of chemical and biological oxidation processes for

water treatment: Review and recommendations. Environ. Prog. 14: 88-103.Scott, J. P.

and D. F. Ollis. Seshadri, S., P.L. Bishop and A.M. Agha. 1994. Anaerobic/aerobic

treatment of selected azo dyes in wastewater. Waste Manag.14: 127-137.

209

Shah, V. and Nerud, F. 2002. Lignin degrading system of white rot fungi and its exploitation for

dye decolorization. Can. J. Microb. 48: 857-870.

Shahvali, M., M.M. Assadi and K. Rostami. 2000. Effects of environmental parameters on

decolorization of textile wastewater using Phenerochaete chrysosporium.

Bioprocess.Enz. 23:7 21-726.

Shu, H. and M. Chang. 2005. Pre-ozonation coupled with UV/H2O2 process for the

decolorization and mineralization of cotton dyeing effluent and synthesized C. I. Direct

22 wastewater. J. Hazard. Mater. 121: 127-133.

Shu, H.Y., M.C. Chang, and H.J. Fan.2004. Decolorization of azo dye acid black 1 by the

UV/H2O2 process and optimization of operating parameters. J. Hazard. Mater. 113: 203-

210.

Shu, H. 2006. Degradation of dye house effluent containing C. I. Direct 199 by process of

ozonation coupled with UV/H2O2 and in a sequence of ozonation with UV/H2O2. J.

Hazard. Mater. 133: 92-98.

Spadaro, J.T., M.H. Gold and V. Renganathan. 1992. Degradation of azo dyes by the lignin

degrading fungus Phanerochaete chrysosporium. Method. Enzymol. 161: 238-249.

Sponza, D.T. and M. Isik. 2005. Toxicity and intermediates of C.I. 84 Red 28 dye through

sequential anaerobic/aerobic treatment. Proc. Biohem. 40: 2735-2744.

Srikanlayankul, M. and W. Kitwechkun. 2006. Decolorization of Orange II by immobilized

thermotolerant white rot fungus Coriolous versicolor RC3 in packed bed bioreactor.

Biotechnology. 7: 280-286.

Steel, R.G., J.H. Torrie and D.A. Dickey. 1997. Principles and procedures of stastics: A

Biochemical Approach (3rd Ed.). McGraw Hill, New York, USA.

Stolz.A. 2001.Basic and applied aspects in the microbial degradation of azo dyes. J. Appl.

Microbial Biotechnol. 56: 69-80.

Stoyko, P.P. and A.S. Pencho. 2003. Color and COD retention by reverse osmosis. .

Desalination. 154:247-252.

210

Sun, J.H., S.P. Sun, M.H. Fan, H.Q. Guo, Y.F. Lee and R.X. Sun. 2008. Oxidative

decomposition of p-nitroaniline in water by solar photo-fenton advanced oxidation

process. J. Hazard. Mater. 153: 187-193.

Suryaman, D., K. Hasegawa and S. Kagaya. 2006. Combined biological and photocatalytic

treatment for the mineralization of phenol in water. Chemosphere, 65: 2502-2506.

Tan. N.C.G., A. Boprger, P. Selenders, A. Svitelskaya, G. Lettirga and J.A. Field. 2000.

Degradation of azo dye mordant yellow 10 in a sequential and bioaugmented aerobic

bioreactor. Water Sci. Technol. 42: 337-344.

Tatarko, M. and Bumpus, J.A. 1998. Biodegradation of Congo red by Phanerochaete

chrysosporium. Water Res. 32: 1731-1717.

Tavares, A.P.M., M.A.Z. Coelho, M.S.M. Agapito, J.A.P. Coutinho and A.M.R.B. Xavier. 2006.

Optimization and modeling of laccase production by Trametes versicolor. Biochem.


Thobanoglous, G., F.L. Burtan and H.D. Stensel. 2003. Wastewater engineering treatment and

reuse. McGraw Hill Companis Inc.

Tien, M. and T.K. Kirk.1983. Lignin degrading enzymes from the basidiomycete Phanerochaete

chrysosporium burds. Science. 221: 661-663.

Torrades, F., J.G. Montano, J. Hortal, X. Domenech and J. Peral. 2004. Decolorization and

mineralization of commercial reactive dyes under solar light assisted Photo-Fenton

conditions. Solar Energy. 77: 573-581.

Ugurly, M. and I. Kula. 2007. Decolorization and removal of some organic compounds from

olive mill wastewater by advanced oxidation processes and lime treatment. Environ. Sci.

Poll. Res. 14: 319-325.

Ulker, B. and K. Savas. 1994. Color removal from textile effluents by electrochemical

destruction. J. Environ. Sci. Health. 29: 1-16.

Vander, F.P., G. Lettinga and J.A. Field. 2001. Azo dye decolourization by anaerobic granular

sludge. Chemosphere. 44: 1169-1176.

211

Wong, Y. and J. Yu. 1999. Laccase-catalyzed decolorization of synthetic dyes. Water Res. 33:

3512-3520.

Weber, E.J. and R.L. Adams. 1995. Chemical and sediment-mediated reduction of the azo dye

disperse blue 79. Environ. Sci. Technol. 29: 1163-1170.

Wolfendon, B.S. and R.L. Wilson. 1982. Radical cations as reference chromogens in studies of

one electron transfer reactions: Pulse radiolysis studies of 2, 2- azinibis-(3-

ethylbenzthiazoline-6-sulfonate). J. Chem. Soci. Perkin Trans. 2: 805-812.

Wuhrmann, K., K. Menscher and T. Kappeler. 1980. Investigation on rate determining factors in

microbial reduction of azo dyes.European J. Appl. Microbiol.Biotechnol. 9: 325-338.

Xu, M., J. Guo, G. Zeng, X. Zheng and G. Sun. 2006.Decolorization of anthraquinone dye

decolrization by Photo-Fnton treatment. Appl. Environ. Microbiol.Biotechnol. 71: 46-

251.

Xu, Y. and R.E. Lebrun. 1999. Treatment of textile dye plant effluent by nano-filtration

membrane. J. Sci. Technol. 34: 2501-2509.

Yakup, A.M., A. Cigdem, E. Aysun, B. Gulay and G. Omer. 2003. Ca-alignate as a support for

Pb (II) and Zn (II) biosorption with immobilized Phenerochaete chrsosporium.

Carbohydr.Polym. 52: 167-174.

Yase, A., A. Nasir and A.A.A. Khan. 2006. Energy requirement of ultraviolet and AOPs for the

post treatment of treated combined industrial effluent. Coloration Technol. 122: 201-206.

Yesilada, O., K. Fiskin and E. Yesilada. 1995. The use of white rot fungus Funaliatrog 11

(Malatya) for the decolorization and phenol removal from olive mill wastewater.

Environ. Technol. 16: 95-100.

Young, L. and J. Yu. 1997. Ligninase-catalyzed decolorization of synthetic dyes. Water

Research. 31: 1187-1193.

Zee van der, F.P., G. Lettinga and J.A. Field.2000. The role of autocatalysis in the mechanism of

an aerobic azo reduction. Water Sci. Technol. 42:301-308.

212

Zhang, F., A. Yediler, X. Liang and A. Kettrup.2004. Effects of dye additives on the ozonation

process and oxidation by-products: A comparative study using hydrolyzed C. I. reactive

red 120.Dyes Pigments. 60: 1-7.

Zhang, F., J.S. Knappand, K.N. Tapley. 1998. Development of bioreactor systems for

decolorizationof Orange II using white rot fungus. Enzyme Microb. Technol. 24: 48-53.

Zhang, X., Y. Lin, K. Yan and H. Wu. 2007. Decolrization of andhraquinane type dye by

Myrodhecium sp. Amer. J. Biores.Bioeng. 104: 104-110.

Zimmermann, T., H.G. Kulla and T. Leisinger. 1982. Properties of purified Orange 11

azoreductase, theenzyme initiating azo dye degradation by Pseudomonas KF46. Eur. J.

Biochem. 129: 197-203.

Zollinger, H. 1991. Color chemistry, synthesis, properties and applications of organic dyes and

pigments. VCH, New York.

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