1.1 The issue of micropollutants . . . . 1

Contents

1 Introduction 1

1.1 The issue of micropollutants . . . . 1

1.2 Laccases: a useful enzyme . . . . 4

1.2.1 What are laccases? . . . . 4

1.2.2 General structure . . . . 5

1.2.3 Mechanism of action . . . . 5

1.2.4 Reactions induced by laccase-catalyzed oxidation . . . . 7

1.3 Laccase-catalyzed degradation of two model micropollutants: a review . . . 11

1.3.1 What is bisphenol A? . . . 11

1.3.2 Identified products of degradation . . . 11

1.3.3 Proposed degradation pathways and mechanisms . . . 12

1.3.4 Models describing the degradation process . . . 12

1.3.5 What is diclofenac? . . . 14

1.3.6 Identified products of degradation . . . 15

1.3.7 Proposed degradation pathways . . . 16

1.3.8 Models describing the degradation process . . . 16

1.4 What happens in mixes? . . . 16

1.5 Objectives and strategy . . . 20

1.5.1 Objectives . . . 20

1.5.2 Strategy . . . 20

2 Degradation of RBBR 23 2.1 Abstract . . . 23

2.2 Keywords . . . 23

2.3 Introduction . . . 23

2.4 Material and methods . . . 25

2.4.1 Material . . . 25

2.4.2 Methods . . . 26

2.5 Calculation . . . 27

2.6 Results and discussion . . . 30

2.6.1 Evolution of the medium absorbance . . . 30

2.6.2 Model validation . . . 30

2.6.3 HPLC analysis . . . 33

2.7 Conclusion . . . 35

2.8 Acknowledgments . . . 36

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CONTENTS

3 Degradation and polymerization of DCF 37

3.1 Abstract . . . 37

3.2 Keywords . . . 37

3.3 Introduction . . . 38

3.4 Materials and methods . . . 39

3.4.1 Materials . . . 39

3.4.2 Methods . . . 39

3.4.3 Calculation . . . 41

3.5 Results and discussion . . . 45

3.5.1 Determination of the kinetic constants . . . 45

3.5.2 Experimental degradation of DCF and IBs formation . . . 45

3.5.3 Features of the model . . . 48

3.5.4 Information for design and scale-up . . . 52

3.6 Conclusion . . . 53

4 Mixes degradation: Sensitivity to concentrations and enzymatic activity 55 4.1 Introduction . . . 55

4.2 Material and Methods . . . 56

4.2.1 Enzyme preparation and chemicals . . . 56

4.2.2 Activity test . . . 56

4.2.3 Enzymatic degradation of BPA and DCF in batch . . . 56

4.2.4 HPLC analyzes . . . 58

4.3 Calculation . . . 59

4.3.1 Compounds alone . . . 59

4.3.2 Degradation of mixes . . . 60

4.4 Results . . . 63

4.4.1 Values of parameters . . . 63

4.4.2 BPA degradation . . . 65

4.4.3 DCF degradation . . . 67

4.4.4 Relation between the degradations of BPA and DCF . . . 69

4.4.5 Implications for treatment design . . . 69

4.5 Conclusion . . . 71

5 Micropollutants degradation: Comparison between two laccase strains 73 5.1 Comparison between the two laccases . . . 73

5.1.1 Catalytic site . . . 74

5.1.2 Binding pocket . . . 76

5.1.3 Tertiary structure . . . 76

5.2 Material and Methods . . . 77

5.2.1 Enzyme preparation and chemicals . . . 77

5.2.2 Activity test . . . 77

5.2.3 Enzymatic degradation of BPA and DCF in batch . . . 77

5.2.4 HPLC analysis . . . 78

5.2.5 Oxygen consumption during BPA reaction with T. versicolor laccases 78 5.2.6 Molecular docking . . . 80

5.2.7 Model . . . 84

5.3 Results of the molecular docking . . . 84

5.3.1 Values obtained . . . 84

5.3.2 Implications . . . 85

5.4 Color change and formation of insoluble byproducts . . . 86

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CONTENTS

5.5 Degradation of Bisphenol A . . . 87

5.5.1 M. thermophila . . . 87

5.5.2 T. versicolor . . . 87

5.5.3 Conclusion . . . 92

5.6 Degradation of Diclofenac . . . 92

5.7 Degradation of mixes . . . 92

5.7.1 BPA removal . . . 92

5.7.2 DCF removal . . . 95

5.7.3 X

as a function of X

. . . 97

5.8 Conclusion . . . 98

6 General conclusions and perspectives 101 6.1 General conclusions . . . 101

6.1.1 Literature review . . . 101

6.1.2 Removal of micropollutants in single-compound solutions . . . 101

6.1.3 Removal of micropollutants in mixes . . . 102

6.1.4 Models . . . 102

6.1.5 Application to wastewater treatment . . . 103

6.2 Perspectives . . . 104

6.2.1 Further study of the model mixes used in this work . . . 104

6.2.2 Laccases production . . . 104

6.2.3 Simultaneous use of multiple laccases strains . . . 105

6.2.4 Immobilization and bioreactors . . . 105

6.2.5 More complex mixes and real WWTP effluents . . . 105

Bibliography 105 Appendices 120 A Supporting information to Chapter 2 121 B Supplementary information to Chapter 2 not included in the article 127 B.1 How to further improve the research presented in Chapter 2 . . . 127

B.2 Limit of detection . . . 127

B.3 Evaluation of R

. . . 127

C Supporting information to Chapter 3 129 D Calibration curves obtained using the Agilent HPLC system 131 E Information added to Chapter 3 after acceptance of the article 133 E.1 Evaluation of R

. . . 133

E.2 Information for design and scale-up – the case of concentration . . . 133

E.3 Fig. 3.1 with error bars displaying the 95% Confidence Interval . . . 134

F Calibration curves and example of chromatograms obtained using the

Hitachi HPLC system 137

G Evaluation of R

for results of Chapter 4 141

G.1 Evaluation of R

. . . 141

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CONTENTS

H Evaluation of R

for results of Chapter 5 143

H.1 Evaluation of R

. . . 143

I Determination of O

consumption 145 I.1 A non-invasive oxygen probe . . . 145

I.2 Determination of the saturation O

concentration . . . 146

I.3 Determination of k

a . . . 147

I.4 Determination of R . . . 147

J Examples of files used in molecular docking 149 J.1 Example of a ligand file: BPA.pdbqt . . . 149

J.2 Example of a configuration file: config-Tv1GYC-BPA.txt . . . 150

J.3 Example of a log file: Tv1GYC-BPA_log.txt . . . 151

J.4 Example of a out file: Tv1GYC-BPA_all.pdbqt . . . 152

K List of publications 157

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