Contents
1 Introduction 13
1.1 Scope, objectives and strategy . . . . 13
1.1.1 The issue of endocrine disruptors . . . . 13
1.1.2 The OXEROM project . . . . 17
1.1.3 Objectives and strategy . . . . 17
1.2 Laccase: a useful friend . . . . 18
1.2.1 What are laccases ? . . . . 18
1.2.2 How do laccases work ? . . . . 19
1.2.3 Laccases and men . . . . 22
2 Methods of water estrogenicity monitoring 25 2.1 Introduction . . . . 25
2.2 Literature review . . . . 25
2.2.1 Bioassays . . . . 26
2.2.2 Analytical methods . . . . 31
2.2.3 Literature review conclusions . . . . 32
2.3 Experimental assessment . . . . 32
2.3.1 Materials and methods . . . . 32
2.3.2 Results . . . . 35
2.4 Discussion . . . . 43
2.4.1 Bioassays . . . . 43
2.4.2 Analytical method . . . . 44
2.5 Conclusions . . . . 45
3 Enzymatic removal of estrogenicity in a batch reactor 47 3.1 Introduction . . . . 47
3.2 Materials and Methods . . . . 48
3.2.1 The choice of BPA . . . . 48
3.2.2 Analysis of laccase activity . . . . 48
3.2.3 Degradation experiments . . . . 50
3.3 Results . . . . 54
3.3.1 BPA and E2 stability . . . . 54
3.3.2 Evolution of estrogenicity . . . . 55
3.3.3 Chromatograms . . . . 57
3.3.4 Evolution of BPA concentration . . . . 59
3.3.5 Correlation between estrogenicity and BPA concentration . . . . 60
3.3.6 Monitoring of BPA degradation byproducts . . . . 61
3.4 Discussion . . . . 62
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Contents
3.4.1 Stability . . . . 63
3.4.2 Degradations . . . . 63
3.5 Conclusions . . . . 67
4 Enzymatic removal of estrogenicity in a continuous packed-bed reactor 69 4.1 Introduction . . . . 69
4.2 Materials and methods . . . . 70
4.2.1 Immobilized laccases . . . . 70
4.2.2 Packed-bed reactor . . . . 71
4.2.3 Characterization of the packed-bed reactor . . . . 72
4.2.4 Estrogenicity removal . . . . 74
4.3 Results . . . . 76
4.3.1 Immobilization and activity . . . . 76
4.3.2 Characterization of the packed-bed reactor . . . . 77
4.3.3 Estrogenicity removal . . . . 82
4.4 Discussion . . . . 86
4.4.1 Immobilization and activity . . . . 86
4.4.2 Characterization of the packed-bed reactor . . . . 86
4.4.3 Estrogenicity removal . . . . 88
4.5 Conclusions . . . . 90
5 Kinetics study 91 5.1 Kinetics in the packed-bed reactor . . . . 91
5.1.1 E2 . . . . 91
5.1.2 BPA . . . . 94
5.2 Kinetics in the batch reactor . . . . 98
5.3 Conclusions . . . . 102
6 Reactor design 103 6.1 Reactor design rules and model . . . . 103
6.2 Comparison with an ozonation process . . . . 105
6.3 Conclusions . . . . 107
7 Conclusions and perspectives 109 7.1 Original objectives . . . . 109
7.2 General conclusions . . . . 109
7.3 Perspectives for future research . . . . 113
Bibliography 126 Appendices 127 A Detailed protocols of bioassays 128 A.1 YES assay . . . . 128
A.2 LYES assay . . . . 130
A.3 MMV-LUC assay . . . . 130
B Coefficient of variation of reproducibility 133
C Fitting curves of bioassays 135
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D Chromatograms 139
E Residence Times Distribution with KCl: calculation of outlet conductivity 141 F Analytic resolution of the 1st-order kinetic model for batch degradations 143
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