Halogenated Toxicity Phenol PDF

Treatment of wastewater containing toxic
halogenated phenolic compounds by immobilized

horseradish peroxidase on a novel support
A thesis submitted in fulfilment of the requirements for

the degree of Doctor of Philosophy
Alon Meizler
B.Sc., M.Sc.
School of Civil, Environmental and Chemical Engineering

College of Science, Engineering and Health
RMIT University, Melbourne
February 2010
Declaration
I certify that except where due acknowledgement has been made, the work is that of the
author alone; the work has not been submitted previously, in whole or in part, to qualify
for any other academic award; the content of the thesis is the result of work which has
been carried out since the official commencement date of the approved research
program; any editorial work, paid or unpaid, carried out by a third party is
acknowledged; and, ethics procedures and guidelines have been followed.
Signed
Name AlonMeizler ....
on //.
Page 1 of 163
Acknowledgements
The author wishes to thank his supervisors, Professor Felicity Roddick and Dr. Nichola
Porter, for their constant guidance, advice and support throughout this research
program. The author gratefully acknowledges the financial support given by RMIT
University.
The author also appreciates the time give by laboratory staff, namely Cameron Crombie,
Philip Francis, Frank Antolasic and Paul Morrison for advice and assistance with
technical instruments, and previous postgraduate students Wee Hong Chin, David Stork
and Yeow Chong Soh for fruitful discussions during the early months of this research.
Geotechnical Services Pty Ltd is acknowledged for conducting the Microtox toxicity
tests on the treated pentabromophenol and pentachlorophenol effluent and the
Ecotoxicology Laboratory at RMIT University for conducting the Microtox tests on
the treated 4-BP effluent and the hydra and cytotoxicity tests on the treated
pentabromophenol and pentachlorophenol effluent.
This research has been a pleasurable experience, improving my academic skill and
laboratory knowledge.
Above all I wish to thank my mom and dad, Ruth and Ben-Zion for their support and
belief in me. You thought me to have patience and understanding and gave me the
foundations on which I stand, hoping to reach higher.
Page 2 of 163
Table of contents
DECLARATION
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
LIST OF FIGURES
LIST OF TABLES
14
SUMMARY
15
LIST OF PUBLICATIONS
18
NOMENCLATURE
19
ABBREVIATIONS
20
CHAPTER 1.
INTRODUCTION
21
CHAPTER 2.
LITERATURE REVIEW
24
2.1
HALOGENATED PHENOLS
2.1.1
Toxicity of halogenated phenols
2.1.2
The application of enzymes to treatment of wastewater containing halogenated phenols
24
24
26
2.2
PEROXIDASES
2.2.1
Horseradish peroxidase
2.2.2
Catalytic cycle of peroxidases
2.2.3
Enzyme immobilization
2.2.4
Covalent binding of enzymes
2.2.5
Disadvantages of immobilized peroxidase
27
28
29
30
31
32
2.3
TITANIUM DIOXIDE
2.3.1
Physical properties of titanium dioxide
2.3.2
Photo-induced hydrophilicity
2.3.3
The photocatalytic mechanism
2.3.4
Mineralization or formation of by-products
2.3.5
Oxidation of halogenated phenols by means of TiO2 photooxidation.
2.3.6
Catalyst lifetime
33
33
34
36
38
39
40
2.4
RESEARCH AIMS
2.4.1
Overall aim
2.4.2
Problems anticipated with the new system
40
40
42
2.5
43
THESIS OUTLINE
CHAPTER 3.
Page 3 of 163
MATERIALS AND METHODS
44
3.1
CHEMICALS
44
3.2
SYNTHESIS OF SUPPORTS
3.2.1
Synthesis of porous glass (PG)
3.2.2
Synthesis of Tglass
3.2.3
Synthesis of casamic
3.2.4
Determination of surface properties of supports
3.2.5
TiO2 coating procedure
3.2.6
Preparation of supports prior to enzyme immobilization
3.2.7
Technique of enzyme immobilization
44
44
44
45
45
45
45
45
3.3
ANALYTICAL METHODS
3.3.1
X-ray spectroscopy techniques
3.3.1.1
X-ray Diffraction (XRD)
3.3.1.2
X-ray Photoemission Spectroscopy (XPS)
3.3.1.3
Environmental Scanning Electron Microscopy (ESEM)
3.3.2
Characterization of products by mass spectrometry
3.3.3
Determination of carbon concentration in solution
3.3.4
HPLC reverse phase chromatography
3.3.4.1
4-Bromophenol analysis
3.3.4.2
Pentabromophenol and pentachlorophenol analysis
3.3.5
Characterization of products by gel permeation chromatography (GPC)
3.3.6
Free and immobilized enzyme assay
3.3.7
Determination of anion concentration in solution
3.3.8
Toxicity tests
3.3.9
Analysis of H2O2
46
46
46
46
46
46
46
47
47
47
48
48
48
49
49
3.4
REACTOR SYSTEMS
3.4.1
Continuously stirred batch reactor
3.4.1.1
Comparison of photocatalytic capacity of Tglass and a range of TiO2-coated supports
3.4.1.2
Determination of pH and temperature range for HRP-Tglass
3.4.1.3
Total adsorption capacity measurements
3.4.1.4
Evaluation of UVB and UVA irradiation for H2O2 formation
3.4.1.5
Evaluation of UVB irradiation on product formation using pyrogallol with free and
immobilized HRP
3.4.2
Plug flow reactor system
3.4.2.1
Evaluation of UVB irradiation for removal of adsorbed polymers
3.4.2.2
Determination of influence of concentration on the system kinetics
3.4.2.3
Determination of influence of flow rate on the system kinetics
3.4.3
Packed bed under constant irradiation
3.4.3.1
H2O2 production
3.4.3.2
4-BP transformation
3.4.3.3
PCP and PBP mixture transformation experiments
3.4.3.4
PCP and PBP(separated and as a mixture) toxicity experiments
49
50
50
50
50
51
51
51
51
52
52
52
52
53
53
53
CHAPTER 4. A COMPARATIVE STUDY OF THE ACTION OF TIO2 IN A

NUMBER OF GLASS SUPPORTS
54
4.1
INTRODUCTION
54
4.2
RESULTS AND DISCUSSION
55
4.2.1
Characteristics of the Tglass support
55
4.2.1.1
The anatase/rutile ratio
55
4.2.1.2
The analysis of the topography of Tglass
57
4.2.1.3
Determination of the photooxidative activity of TiO2 associated with various supports59
4.2.2
Tglass as a support for enzyme immobilization
63
4.3
SUMMARY OF CHAPTER 4
65
CHAPTER 5. DETERMINATION OF THE ROLE OF HRP IMMOBILIZED ON

67
TGLASS WITH AND WITHOUT UV RADIATION
Page 4 of 163
5.1
INTRODUCTION
67
5.2
5.2.1
Analysis of support after enzymatic reaction
5.2.2
Determination of pH and temperature ranges of HRP immobilized on Tglass
5.2.3
Determination of adsorption capacity of immobilized HRP at equilibrium
5.2.4
Generation of hydrogen peroxide by HRP-Tglass under UVB irradiation
5.2.5
The effect of UVB/HRP oxidation on pyrogallol polymerization with and without Tglass
5.2.6
Influence of UVB dose on free and immobilized HRP in transforming pyrogallol
5.2.7
Influence of UV dose on the activity of HRP-Tglass in transforming 4-BP
5.2.8
Plug flow reactor studies
5.2.9
Analysis of products from enzymatic polymerization
5.2.9.1
Analysis of soluble products
5.2.9.2
Analysis of adsorbed products
5.2.10
Removal of adsorbed oligomers
67
67
71
73
75
76
78
80
81
86
86
87
90
5.3
93
CHAPTER 6.
6.1
KINETICS OF HRP IMMOBILIZED ON PG AND TGLASS
INTRODUCTION
95
95
6.2
6.2.1
The influence of substrate concentration and flow rate on HRP-PG
6.2.2
Development of a model for HRP-PG
6.2.3
Validation of the HRP-PG model
6.2.4
The influence of substrate concentration and flow rate on HRP-Tglass
6.2.5
Development of a model for HRP-Tglass
6.2.6
Validation of HRP-Tglass model
6.2.6.1
The effect of concentration
6.2.6.2
The effect of flow rate
6.2.7
Product adsorption capacity of HRP-Tglass
95
95
97
100
101
103
106
106
107
108
6.3
109
CHAPTER 7. KINETICS OF HRP IMMOBILIZED ON TGLASS WITH UV

IRRADIATION
7.1
INTRODUCTION
112
112
7.2
RESULTS
7.2.1
H2O2 production in the packed bed reactor
7.2.2
4-BP transformation in a continuous regime initiated by UV irradiation
7.2.3
Analysis of products from HRP-Tglass system activated by UVB
7.2.4
The effect of amount of HRP-Tglass on the system performance.
7.2.5
Comparison of the HRP-Tglass systems performance
113
113
114
117
118
119
7.3
120
SUMMARY FOR CHAPTER 7
CHAPTER 8. DETOXIFICATION OF HIGHLY HALOGENATED PHENOLIC

COMPOUNDS BY HRP IMMOBILIZED ON TGLASS
121
8.1
INTRODUCTION
121
8.2
RESULTS
121
8.2.1
Treatment of high concentration of PCP and PBP
121
8.2.1.1
Analysis of products from the treatment of PCP and PBP
125
8.2.1.2
Toxicity of treated solutions according to Microtox test
127
8.2.2
Toxicity of treated PCP and PBP solutions in low concentrations according to Microtox and
Hydra tests
127
8.3
Page 5 of 163
131
CHAPTER 9.
CONCLUSIONS
CHAPTER 10.
RECOMMENDATIONS FOR FURTHER WORK
REFERENCES
133
137
138
APPENDIX A. SURFACE AREA AND PORE SIZE MEASUREMENT FOR

SUPPORTS.
153
APPENDIX B. DETERMINATION OF 4-BROMOPHENOL USING HPLC-UV
155
APPENDIX C. DETERMINATION OF PBP AND PCP USING HPLC-UV
157
APPENDIX D. ION CONDUCTIVITY CHROMATOGRAPHY AND STANDARD

CALIBRATION CURVES
159
APPENDIX E. DETERMINATION OF TOXICITY USING HYDRA
HEXACTINELLA.
161
APPENDIX F. DETERMINATION OF 4-BP ADSORPTION ON SUPPORTS IN

THE ABSENCE OF PEROXIDASE REACTION USING TOC.
162
Page 6 of 163
List of Figures
Figure 2-1. Catalytic cycle of a heme peroxidase. ..........................................................29
Figure 2-2. Crystal structures of titanium dioxide formation as (a) rutile (b) anatase....33
Figure 2-3. Photo-induced super hydrophilicity. ............................................................35
Figure 2-4. The mechanism of photo-induced super hydrophilicity...............................35
Figure 2-5. Diagram of photoconductivity model.. ........................................................37
Figure 2-6. Schematic representation of breakdown mechanism of halogenated phenol
under UV radiation in presence of TiO2.. .......................................................................39
Figure 2-7. Schematic representation of the reaction sequence for HRP polymerization
and TiO2 support approach.............................................................................................42
Figure 3-1. Schematic representation of the HRP-Tglass packed bed under UV radiation.
.........................................................................................................................................52
Figure 4-1. The photooxidation products of 4-BP. .........................................................54
Figure 4-2. XRD spectrum of the Tglass particles (anatase rutile)...........................56
Figure 4-3. ESEM image of Tglass (A) and PG (B)..........................................................58
Figure 4-4. XPS analysis of Tglass surface .......................................................................59
Figure 4-5. pH, 4-bromophenol and bromide transformation after exposure of supports
to 4-BP (0.1 mM) and UVB irradiation. .........................................................................61
Figure 4-6. Typical HPLC chromatogram of the products obtained ..............................62
Figure 4-7. (a) Tglass after NaOH treatment (scale 50 m) (b) Tglass after HCl treatment
(scale 50 m)...................................................................................................................64
Figure 4-8. EDX of Tglass surface after acid treatment. ...................................................65
Page 7 of 163
Figure 5-1. ESEM topography of typical particles of HRP-PG (left column) and HRPTglass (right column) before (top row) and after (bottom row) the polymerization process
with 4-BP, scale is 100 m for all images. .....................................................................68
Figure 5-2. The EDX spectra of HRP-Tglass surface (A). prior to the reaction, (B) after 4BP polymerization...........................................................................................................69
Figure 5-3. The EDX spectra of HRP-PG surface (A) prior to the reaction, (B) after 4BP polymerization...........................................................................................................70
Figure 5-4. ESEM imaging and EDX mapping of HRP-Tglass after the polymerization
process (a) scanned image, (b) titanium, (c) silicon, (d) bromine, (e) carbon, (f) oxygen.
All the elements are shown as bright spots, scale is 20 m for all images. ....................71
Figure 5-5. The effect of pH on the transformation and degree of adsorption of products.
HRP-Tglass treated 4-BP (0.1 mM) and H2O2 (0.1 mM) in a stirred batch reactor for 24 h.
.........................................................................................................................................72
Figure 5-6. The effect of temperature on the transformation and degree of adsorption.
HRP-Tglass treated 4-BP (0.1 mM) and H2O2 (0.1 mM) in a stirred batch reactor for 24 h.
.........................................................................................................................................73
Figure 5-7. Adsorption of end-products in an HRP-Tglass batch reactor .........................74
Figure 5-8 Linear regression of Langmuir equation for the adsorption of the reaction
products to the HRP-Tglass particles in a batch reactor....................................................75
Figure 5-9. Influence of UVB irradiation dose on the formation of H2O2 in the presence
of HRP-Tglass . .................................................................................................................76
Figure 5-10. Spectra after reaction with 20 mg L-1 pyrogallol (top to bottom); a) 20 mg
L-1 pyrogallol, b) UVB radiation for 30 min without HRP-Tglass, c) UVB radiation for 30
min with HRP- Tglass, d) UVB radiation for 30 min with Tglass, e) soluble HRP after
overnight reaction with 0.2 mM H2O2.....77
Figure 5-11. Absorbance spectra for products formed by HRP-Tglass after UVB
irradiation.. ......................................................................................................................78
Figure 5-12. Absorbance spectra after UVB irradiation of HRP-Tglass in the presence of
100 mg L-1 pyrogallol for various times. ........................................................................79
Page 8 of 163
Figure 5-13. Absorbance spectra after UVB irradiation of free HRP (40 g) with 100
mg L-1 pyrogallol for various times. ...............................................................................79
Figure 5-14. Influence of radiation dose on the conversion of 4-BP with HRP-Tglass ()
transformed 4-BP without HRP-Tglass (control), () transformed 4-BP, () remaining
immobilized enzyme activity (pyrogallol assay). ...........................................................81
Figure 5-15. Increasing adsorption of the polymerized products on the HRP-PG
particles in a plug flow reactor as increasing volumes of 4-BP (0.1 mM) and H2O2 (0.1
mM) are fed at 2 mL min-1 to the column (the HRP-Tglass column gave a similar
pattern). ...........................................................................................................................82
Figure 5-16. TOC removal (as adsorbed carbon) by HRP-PG and HRP-Tglass columns
from feed consisting of equimolar concentration of 4-BP (17.3 mg L-1) and H2O2 (3.4
mg L-1) at a flow rate of 2 mL min-1. ..............................................................................83
Figure 5-17. Concentration of bromide in effluent from HRP-PG and HRP-Tglass
columns resulting from feed consisting of equimolar concentration of 4-BP (17.3 mg L1
) and H2O2 (3.4 mg L-1) at a flow rate of 2 mL min-1. ...................................................83
Figure 5-18. Ratio of bromide/4-BP (calculated in mmoles) adsorbed to columns of

HRP-PG and HRP-Tglass from feed consisting of 4-BP (17.3 mg L-1) and H2O2 (3.4 mg
L-1) at a flow rate of 2 mL min-1. ....................................................................................84
Figure 5-19. Possible 4-bromophenol radicals formed (redrawn and amended according
to Dec and Bollag (1994) ................................................................................................85
Figure 5-20. ESI mass spectrum obtained for the soluble fraction after oxidation of 4BP with HRP-Tglass in a column reactor..........................................................................86
Figure 5-21. ESI mass spectra of the adsorbed fraction after oxidation of 4-BP with
HRP-PG and HRP-Tglass
in a column reactor (acetonitrile was used for solvent
extraction). ......................................................................................................................88
Figure 5-22. Gel permeation chromatograms for the products desorbed from the HRPPG and HRP-Tglass columns with TCB............................................................................89
Figure 5-23. HRP- Tglass particles coated with polymerized products prior to UVB
radiation (A) and after 96 hours UVB radiation (B). The 4-BP concentrations ranged
from 0.1 mM to 10 mM and are placed in increasing order from right to left................90
Page 9 of 163
Figure 5-24. HRP-PG particles coated with polymerized products prior to UVB
radiation (A) and after 96 hours UVB radiation (B). The 4-BP concentrations ranged
from 0.1 mM to 10 mM and are placed in increasing order from right to left................91
Figure 5-25. Influence of organic loading on the conversion of 4-BP with HRP-Tglass
() adsorbed carbon on the supports (mg) () residual activity of the particles after
initial reaction, () activity of the particles after 96 hours of UVB radiation. ...............92
Figure 5-26. Influence of organic loading on the conversion of 4-BP with HRP-PG ()
adsorbed carbon on the supports (mg) () residual activity of the particles after initial
reaction, () activity of the particles after 96 hours of UVB radiation...........................92
Figure 6-1. Carbon removal for HRP-PG for flow rates of 1.75 mL min-1 using a range
of 4-BP concentrations (2.1-161 mgC L-1) and H2O2 at 1:1 molar ratio........................96
Figure 6-2. Carbon accumulation for HRP-PG for the flow rates of 1.75 8 mL min-1
using a range of 4-BP concentration (2.9-161 mgC L-1) and H2O2 at 1:1 molar ratio..97
Figure 6-3. Carbon removal rate for HRP-PG for flow rates 1.75-8 mL min-1 using 4-BP
in a concentration range of 2.1-161 mgC L-1 and H2O2 at 1:1 molar ratio. ....................99
Figure 6-4. Correlation between flow rate and K, obtained from the slope of reaction
rate with initial substrate concentration). ......................................................................100
Figure 6-5. Degree of adsorption, maximum adsorption, measured adsorption rate and
calculated adsorption rate for HRP-PG at flow rates of 0.25 - 7.5 mL min-1, 4-BP and
H2O2 concentration 0.1 mM (7.2 mgC L-1, 3.4 mg L-1; respectively)...........................101
Figure 6-7. Carbon accumulation for HRP-Tglass for the flow rates of 1.75 8 mL min-1
using a range of 4-BP concentration (2.9-161 mgC L-1) and H2O2 in 1:1 molar ratio.
.......................................................................................................................................102
Figure 6-8. Carbon removal rate for HRP-Tglass for flow rates of 1.75-8 mL min-1 using
4-BP concentration ranged 2.1-161 mgC L-1 and H2O2 in 1:1 molar ratio. ...................104
Figure 6-9. Reciprocal plots of adsorption rate vs substrate for HRP-Tglass for a range of
flow rates (A: 1.75 mL min-1; B: 3 mL min-1; C: 6 mL min-1; D: 8 mL min-1;) ...........104
and Ks with flow rate................................................105
Figure 6-10. Relationship of app
max
Page 10 of 163
Figure 6-11. Comparison of experimental and calculated effluent TOC levels for flow
rate 4 mL min-1 and feed concentration 4-BP over the range 7.6-72 mgC L-1. ............106
Figure 6-12. Degree of adsorption, maximum adsorption, measured adsorption rate and
calculated adsorption rate for HRP-Tglass at flow rates of 0.25 - 7.5 mL min-1, 4-BP and
H2O2 concentration 0.1 mM (7.2 mgC L-1, 3.4 mg L-1, respectively)...........................107
Figure 6-13. Relationship between total carbon adsorption and feed volume. The feed
consisted of 4-BP (7.2 mgC L-1) and H2O2 (3.4 mg L-1) at a range of flow rates (1-8 mL
min-1).............................................................................................................................109
Figure 7-1. Influence of UV wavelength for a range of flow rates of MilliQ water (a)
UVA; (b) UVB. () hydrogen peroxide concentration exiting empty column (sampling
point A) () hydrogen peroxide concentration exiting column packed with HRP-Tglass
(sampling point B). .......................................................................................................114
Figure 7-2. Remaining TOC levels at sampling point B for 100 mL fractions of the
effluent after HRP-Tglass treatment of 0.1 mM 4-BP under UVA irradiation (A) and
under UVB irradiation (B). ...........................................................................................115
Figure 7-3. Bromide formed at sampling point B for 100 mL fractions of the effluent
after HRP-Tglass treatment of 0.1 mM 4-BP under UVA irradiation (A) and under UVB
irradiation (B)................................................................................................................116
Figure 7-4. Remaining 4-BP levels at sampling point B for 100 mL fractions of the
effluent after HRP-Tglass treatment of 0.1 mM 4-BP under UVA irradiation (A) and
under UVB irradiation (B). ...........................................................................................116
Figure 7-5. Molar ratio of bromide to 4-BP transformed for substrate flow rates of 0.5-9
mL min-1........................................................................................................................117
Figure 7-6. ESI-MS spectrum of the effluent of HRP-Tglass reactor after 2 hr of UVB
radiation on 0.5 mM 4-BP at a flow rate of 0.5 mL min-1. ..........................................118
Figure 7-7. Transformation of 0.1 mM 4-BP in a single column (containing 2.5 g HRPTglass) compared with two identical columns in parallel at the flow rate of 1.25 mL min1
. ....................................................................................................................................119
Figure 8-1. Extent of transformation of PCP and PBP (10 and 8 mg L-1, respectively) by
HRP-Tglass in a packed bed system at flow rates of 0.5 and 1.25 mL min-1..................122
Page 11 of 163
Figure 8-2. Extent of debromination of PCP and PBP mixture (10 and 8 mg L-1,
respectively), by HRP-Tglass in a packed bed system at flow rate of 0.5 and 1.25 mL min1
. ....................................................................................................................................124
Figure 8-3. Extent of dechlorination of PCP and PBP mixture (10 and 8 mg L-1,
respectively), by HRP-Tglass in a packed bed system at flow rate of 0.5 and 1.25 mL min1
. ....................................................................................................................................124
Figure 8-4. Average anions/moles of halogenated phenols released from PCP and PBP
mixture (10 and 8 mg L-1, respectively) by HRP-Tglass in a packed bed system at flow
rate of 0.5 and 1.25 mL min-1......................................................................................125
Figure 8-5. ESI mass spectrum obtained from PBP and PCP (10 mg L-1 and 8 mg L-1,
respectively) spiked reservoir water after treatment. ....................................................126
Figure 8-6. Results for Microtox tests performed on reservoir water spiked with PCP
and PBP (10 mg L-1 and 8 mg L-1, respectively) before and after treatment.. .............127
Figure 8-7. Results for Microtox tests performed on reservoir water spiked with PCP
and PBP (0.89 and 0.67 mg L-1, respectively) before and after treatment. ...................128
Figure A-1. An example of volume distribution according to the pore size that are
located on the surface of any support............................................................................152
Figure A-2 Example of data collected by the BET surface area analyzer according to
their pore size and surface area of any support. ............................................................153
Figure B-1. Typical HPLC chromatogram of a 0.1 mM standard solution of 4bromophenol on a C18 reverse phase column with monitoring at 280 nm....................154
Figure B-2. Calibration curve of 4-bromophenol on a C18 reverse phase column detected
at 280 nm.......................................................................................................................155
Figure C-1. Typical HPLC chromatogram of a 10 mg L-1 standard solution of
pentachlorophenol and pentabromophenol (retention time 11.4 and 12.3 min
respectively) on a C18 reverse phase column with monitoring at 240 nm. ...................156
Figure C-2. Calibration curve of PCP and PBP on a C18 reverse phase column detected
at 240 nm.......................................................................................................................157
Figure D-1. Calibration curve of bromide on an anion exchange column....................158
Page 12 of 163
Figure D-2. Calibration curve of chloride on an anion exchange column. ...................159

Figure E-1 Different Hydra responses to toxicity in a TES buffer and CaCl2. A: Normal
Hydra; B: Hydra with clubbed tentacles (first signal of toxicity, sub-lethal endpoint
used); C: Hydra in tulip stage (the lethal endpoint used)..............................................160
Figure F-1. Control test of the adsorption capacity of the enzyme active support in the
absence of H2O2. 4-BP concentration of 7 PPM of carbon was fed at a flow rate of 1 mL
min-1 at ambient condition.161
Page 13 of 163
List of Tables
Table 2-1 Toxicity data for bromophenol compounds....................................................24
Table 2-2 Toxicity data of chlorophenol compounds. ....................................................25
Table 2-3 Physical properties of rutile and anatase. ......................................................34
Table 4-1 The characteristics of the crystal properties of the TiO2 in Tglass. ..................56
Table 4-2 Surface characteristics of Tglass and PG and HRP activity after immobilization
.........................................................................................................................................64
Table 5-1 Proposed coupling reactions between free radicals generated during the
catalytic oxidation of 4-bromophenol. ...........................................................................85
Table 6-1 Apparent kinetic parameters for 4-BP removal in terms of TOC by HRP-Tglass
in plug flow mode. ........................................................................................................106
Table 7-1 Advantages and disadvantages of the reactor systems used in this project. .120
Table 8-1 Microtox sub-lethal (EC50) and lethal (LC50) endpoints for the tested
samples (95% confidence interval)a ............................................................................130
Table 8-2 Hydra lethal (LC50) and sub-lethal (EC50) endpoints for the tested samples
(95% confidence interval)a..........................................................................................130
Page 14 of 163
Summary
Halogenated phenolic compounds, especially polyhalogenated congeners, are highly
toxic pollutants generated by a variety of industries such as the manufacture of the
brominated flame-retardants used in the production of computers and consumer
electronics. A modern approach to deal with the halogenated phenol wastes is by the
use of enzymes. The most commonly applied enzymatic treatment for deactivation and
detoxification of halogenated phenols involves peroxidases, in particular horseradish
peroxidase (HRP).
In the presence of hydrogen peroxide, HRP can oxidize halogenated phenolic
compounds to phenoxyl radicals which then interact to create less soluble hydrophobic
polymeric products and cause partial dehalogenation. Treatment results in the
polymerization and sedimentation of the organic compounds, which may result in
reduction of toxicity of the solution and production of biodegradable products.
The overall objective of this research was to develop an improved reliable continuous
method of removing toxic phenolic substances from wastewater based on enzymatic
polymerization and dehalogenation through the use of immobilized HRP.
Normally there are two limiting factors for the peroxidase reaction: firstly, at high H2O2
concentrations, the peroxidase is eventually oxidised to a species with limited catalytic
activity. Secondly, in immobilized peroxidase systems, the oxidised hydrophobic
polymeric products are adsorbed to the immobilized enzyme due to hydrophobic
interactions, resulting in loss of enzyme activity.
In this study, a novel glass (Tglass) containing titanium dioxide (TiO2) was developed to
act as a support and also to utilize the TiO2 to minimize the two drawbacks of
peroxidase described above. TiO2, a known photocatalyst, under UV radiation oxidizes
organic material. The research hypothesis is that UV irradiation of HRP-Tglass in an
aqueous system will assist the HRP polymerization process by oxidizing the polymeric
products that are adsorbed and cover the enzyme, thus preventing deactivation of the
HRP. In addition, the TiO2 has a hydrophilic surface that can repel hydrophobic
oligomers, preventing the oligomers from being adsorbed to the enzyme and
subsequently leading its deactivation.
The Tglass surface was characterized using energy dispersive X-ray spectroscopy (EDXXray mapping) which showed a mosaic pattern of silica and titanium patches in a size
Page 15 of 163
range of 20 to 40 m2. The Tglass had the same properties of photo-oxidation as any
silica support coated with TiO2, which includes drawbacks such as a short life cycle and
the need to be regenerated after every use. It was shown that while the immobilized
HRP was spread evenly over the whole surface of the Tglass, its activity was detected
only on the SiO2 surface (to give an overall activity of 0.5 U g-1). The maximum surface
area that was achieved and used was 40 m2 g-1.
The kinetics of oxidation of a model substrate 4-bromophenol (4-BP) by HRP
immobilized onto Tglass (HRP-Tglass) was determined in a continuous flow regime. The
set-up for the kinetic experiment consisted of an immobilized enzyme-packed column
(plug flow reactor) which was fed with 4-BP and H2O2. The plug flow reactor
performance was studied under various operating conditions, i.e., substrate
concentration and flow rate; adsorption capacity of the column for a range of flow rates
was also studied.
The initial reaction mechanism seems to follow the Monod model; however, the
adsorption of the polymerized products overpowered that mechanism and eventually
reduced enzyme activity to zero. An inverse correlation between the flow rate and the
adsorption capacity was observed. Under optimal conditions, 80% carbon (as TOC)
removal of the initial substrate by adsorption was achieved.
The degree of polymerization of 4-BP in the plug flow reactor was ascertained from
analysis of the effluent and adsorbed material. The adsorbed product was extracted by
solvents (acetonitrile and 1,2,4-trichlorobenzene) and was analysed using mass
spectrometry and size exclusion chromatography; a range of oligomers (from dimers to
tetramers) was detected. The soluble carbon eluting from the reactor contained
unreacted substrate, 4-bromocatechol and bromide (35% of the theoretically available
bromine). After UVB irradiation of fully covered HRP-Tglass gave a recovery of 20% of
initial capacity of HRP-Tglass.
When the plug flow reactor was fed water under UVB radiation, low concentrations of
H2O2 (up to 1 mM) were generated. This was a direct consequence of UV irradiation of
the TiO2 in the glass matrix. The in situ generation of H2O2 was shown to be sufficient
to locally activate the HRP.
The plug flow reactor under UVB radiation was able to treat the 4-BP without adding
H2O2. It demonstrated a high 4-BP transformation capacity (75%) with a residence time
of 70 min. In contrast to the experiments in which H2O2 was added to the feed, the
Page 16 of 163
adsorption of the UVB reactor product was low, while the degree of dehalogenation
obtained was high (up to 95% of the initial available bromine was removed as bromide).
Analysis of the effluent using mass spectroscopy detected non-halogenated oligomers
(from benzo dimers up to benzo pentamers). The activity of the bound enzyme was
retained without loss for 16 hours at which time the experiment was terminated.
Pentachlorophenol (PCP) and/or pentabromophenol (PBP) spiked into a sample of
drinking water (8 and 10 mg L-1, respectively) was treated by the HRP-Tglass packed bed
reactor under UVB radiation. The treatment gave 98% transformation for both species
and up to 4 of the 5 available halogens (bromide and chloride) were removed.
Toxicity tests were performed on a solution feed containing PCP and/or PBP in MilliQ
water (1 mg L-1) before and after treatment. The toxicity (as measured by Microtox) of
the treated effluent showed that after treatment the lethal toxicity (LD50) was removed
while the effect toxicity (EC50) has reduced. Toxicity tests using Hydra hexactinella
showed a slight decrease in toxicity, however the dehalogenated PCP and PBP mixture
was shown to have a greater decrease in toxicity.
Dehalogenation plays an important rule in a reduction in toxicity; it was found that for a
partial transformation (up to 70% in both species) of the PBP and PCP mixture (0.67
and 0.89 mg L-1) in reservoir water, an increase in toxicity occurs.
The results of the present work clearly show the potential for continuous removal of
soluble phenolic pollutants from water and wastewater streams by immobilized
horseradish peroxidase using the novel Tglass developed in this study.
Page 17 of 163
List of Publications
Meizler, A., Roddick, F. A. & Porter, N. (2008) Horseradish peroxidase immobilized on
a novel support for the treatment of phenolic wastewater. Proceedings of IWA-LET
2008: 5th IWA Leading-Edge Technology Conference; Zurich (Switzerland), June
2008.
Meizler, A., Roddick, F. & Porter, N. (2008) A comparative study of enhanced photooxidation by adsorption of polymeric phenolic groups. Enviro08: Australias
Environmental & Sustainability Conference and Exhibition; Melbourne (Australia),
poster PWA39, May 2008, Peer reviewed.
Meizler, A., Roddick, F. A. & Porter, N. (2009) Continuous enzymatic polymerization
of 4-bromophenol initiated by UV irradiation. Proceedings of Reuse09: 7th IWA World
Congress on Water Reclamation and Reuse; Brisbane (Australia), September 2009 Peerreviewed.
Page 18 of 163
Nomenclature
Symbol
Description
Unit
Immobilized HRP activity
U g-1
a, b
Equation constants
BP0
Initial concentration of 4-BP
mM
Cads
Total adsorbed amount in terms of carbon
mg
Hcolumn
Empty column volume
mL
Substrate first order constant
mgC L-1
Kads
Adsorption constant
mg
Ks
Substrate utilization constant
mgC L-1
msb
Dry weight of immobilized HRP
Mass of material accumulated on the mg

particles
app
q max
Apparent maximum adsorbed capacity
mg
Flow rate
mL min-1
S0
Initial TOC concentration of the solution
mg L-1
Se
Effluent TOC concentration of the bulk mgC L-1

fluid
Enzyme activity unit
mg (pyrogallol) s-1
U g-1
Immobilized enzyme activity unit
mg (pyrogallol) s-1 g-1 (support)
Volume of the effluent
ads
Adsorption constant
app
max
Apparent maximum reaction rate
mgC L-1 min-1
app
rate
Apparent adsorption rate
mgC L-1 min-1
Hydraulic retention time
min
Page 19 of 163
Abbreviations
Symbol
Description
AOPs
Advanced oxidation processes
BFR
Brominated flame retardants
EC50
Half maximal effective concentration
EDX
Energy-dispersive X-ray
ESI
Electrospray ionization
ESEM
Environmental scanning electron microscopy
FTIR
Fourier transformation infra-red
GPC
Gel permeation chromatography
HPLC
High performance liquid chromatography
HRP
Horseradish peroxidase
LD50
Median lethal dose
Tglass
Novel support synthesized in this project
OSHA
Occupational Safety and Health Administration
PBR
Packed bed reactor
PG
Porous glass
TEF
Toxic equivalency factor
TOC
Total organic carbon
UV
Ultraviolet
US-EPA
United States Environmental Protection Agency
VOC
Volatile organic compound
XRD
X-ray diffraction
XPS
X-ray photoemission spectroscopy
Page 20 of 163
Chapter 1. Introduction
Halogenated phenolic compounds, especially the polyhalogenated congeners, are highly
toxic pollutants which originate from a variety of industries such as coal processing,
production of flame retardants, wood preservation, resins and plastics manufacture,
leather manufacturing, metal casting, and pulp and paper production (Dyer and
Mignone, 1983). These halogenated phenols have been found to accumulate in biota. In
the US, polychlorinated dibenzo-p-dioxins (PCDD) were detected in corn leaves
(Wagrowski and Hites, 1998). In Antarctica, 2,4,6-tribromophenol and other
brominated phenols were found in sponges (Vetter and Janussen, 2005). Traces of
pentabromophenol (870 g kg-1 wet weight) were found in sewage sludge in Germany
and Spain (Kuch et al., 2005). This shows that highly substituted halogenated phenols
have a long life span in the environment and are clearly absorbed by living cells. The
US-EPA (2004) reports that in the USA, up until 1993 at least 110 tons of
pentachlorophenol was released to the environment, and furthermore, that it gradually
entered the water supply. The US-EPA has set the Maximum Contaminant Level at 1
ppb. When a water source reaches this level, or is at 0.04 ppb for more than 3 months a
year, the public must be notified and the water source must be treated using active
carbon charcoal.
Lately, there is increasing interest in a specific group of halogenated phenols, the
brominated flame retardants. These compounds are primarily derived from electronic
and computer waste (E-waste), as more than 50 million tons per year of E-waste are
discarded to landfills around the world (Schwarzer et al., 2005). To emphasise the
problem with brominated flame retardants (BFR), an Oslo report (2007) stated that in
2001 worldwide market demand was 196,000 tons of BFR chemicals.
The authors of the Oslo report stated that they reported the data as production of BFR
rather than measure it in the environment due to the hydrophobic nature of BFRs. Most
compounds that contain a high number of halogens tend to be adsorbed and not
disperse, which leads to low levels of detection in aqueous media. Furthermore, a
complete and comprehensive inventory of information was not possible since in several
countries:
the necessary information is not routinely compiled by the relevant authorities.
legal restrictions (for example, business confidentiality rules) prevent authorities

from making data obtained from industry available for external use.
Page 21 of 163
The major groups emitted from E-waste are polychlorinated dibenzo-p-dioxins (PCDD)
and polychlorinated dibenzo-furans (PCDF); these compounds can lead to additional
major health risks as they are mutagenic and nephrotoxic (Dybinget al. 1980). With
daily exposure to the sun and microorganisms they can be converted to more toxic and
soluble products (Vollmuth et al., 1994), which can easily find their way into the water
supply or bioaccumulate. If their use cannot be eliminated, treating them before they are
discarded is the best way of preventing these phenolic compounds reaching the
environment and migrating or breaking down to form even more toxic by-products. The
current treatment recommended by the US-EPA is the use of activated carbon
adsorption (EPA 2004), This treatment varies for different compounds under different
conditions and as it changes the pollutant phase but not its nature, it is a non-destructive
method as the saturated activated carbon needs to be disposed of or regenerated.
A modern approach to deal with halogenated phenol wastes is by the use of enzymes.
Enzymes have advantages when compared to other conventional treatments such as
Fenton precipitation and microbial degradation. They can be used in low quantities with
minimal additives, they have short reaction times and they can work immediately
compared with treatment using microbes. The most commonly applied enzymatic
treatment for deactivation and detoxification of halogenated phenols involves
peroxidases, in particular horseradish peroxidase (HRP).
Due to the fact that enzymes are soluble, immobilization of the enzyme on a solid
support provides reusability; however, when a peroxidase is immobilized the products
formed tend to be adsorbed to the support surface and cover the active sites, thereby
deactivating them.
In this study, a novel glass containing titanium dioxide (TiO2) was developed both to act
as a support for HRP and to utilize the photocatalytic and superhydrophilic properties of
the TiO2. These properties become evident when TiO2 is exposed to UV radiation. The
enzyme-mediated part of the process is to target and treat halogenated phenols at low
concentration. The photo-mediated part assists the enzyme by providing it with H2O2
and preventing adsorption of the hydrophobic polymerized products that causes the
HRP deactivation.
These properties would potentially benefit the peroxidase by (1) providing hydrogen
peroxide which is required for activation of the enzyme and, (2) protecting against
deactivation of the enzyme by adsorption of the hydrophobic polymerized products.
Page 22 of 163
The synergy of the HRP and Tglass could be used to pre-treat industrial waste containing
halogenated phenols by converting them to non-toxic compounds that can be further
treated by microorganisms.
This thesis describes the use of HRP immobilized on this new support to treat a model
compound (4-bromophenol, 4-BP) and its application to the more highly halogenated
phenols such as pentabromophenol (PBP) and pentachlorophenol (PCP).
The major steps in this thesis were:
1) Investigation of the surface properties of the titanium dioxide containing support
(Tglass), including investigation of the Tglass as a photocatalyst and its capacity for
immobilizing HRP on its surface.
2) Comparison of treatment by HRP-Tglass with HRP immobilized on porous glass.
HRP-Tglass activation and activity regeneration was tested using UV irradiation.
3) A study of the HRP-Tglass and HRP-PG adsorption kinetics induced by the substrate
polymerization in the presence of H2O2, this included determination of the adsorption
capacity.
4) Finally, the HRP-Tglass reaction kinetics under UV irradiation were investigated in a
packed bed reactor, followed by PBP and PCP treatment and determination of the
toxicity of the effluent.
Page 23 of 163
Chapter 2. Literature review

In this chapter, a review of the halogenated phenols and their influence on the
environment, i.e. their toxicity, will be discussed. The proposed treatment of the
halogenated phenols is by using horseradish peroxidase immobilized on a support
incorporating TiO2. The biocatalyst will be reviewed in terms of advantages and
disadvantages, while the photocatalytic and hydrophilic properties of TiO2 will be
discussed. This thesis aims to use the TiO2 to assist the biocatalyst.
2.1
HALOGENATED PHENOLS
2.1.1 Toxicity of halogenated phenols

In this section the questions of the extent of toxicity and the contributing factors to that
toxicity will be discussed.
Table 2-1 and Table 2-2 show toxicity data for a range of simple halogenated phenolic
compounds. Generally, the greater the number of halogens bonded to a phenol, the
greater its toxicity (Ayude et al., 2009). As these compounds are highly hydrophobic
and tend to have low solubility in water, their toxicity values or ranking are not
consistent for the different toxicity tests. In addition to the toxicity values or rankings,
the effects of the compounds on a living organism are also important. For example, 4bromophenol (4-BP) and 2-bromophenol (2-BP) have close LD50 values, but, 2-BP is
nephrotoxic, while 4-BP is not (Howe et al., 2005). 4-BP is used in various industries
as a both pharmaceutical (Collette, 2009) and flame retardant. It contains a single
bromide and has a relatively moderate toxicity which makes it ideal as a working
substrate.
Table 2-1 Toxicity data for bromophenol compounds obtained from World Health Organization (Howe
et al., 2005)
4-BP
2-BP
2,4,6-TBP3
PBP4
1
LD50 (mg kg-1)

(rats, oral)
523 (mouse)
652
1486 to 5000
250300
EC50 (mg L-1)

(microalgae)
N.A.2
110
0.4 to 1.6
N.A. 2
PNEC1 (g L-1)
6
2
2
0.1
(PNEC) predicted no effect concentrations, 2( N.A.) not available, 3(2,4,6-TBP) 2,4,6-tribromophenol,

(PBP) pentabromophenol.
EC50 (mg L-1)

(daphnids)
0.9 to 6
0.9 to 6
0.3 to 5.5
N.A. 2
Page 24 of 163
Table 2-2 Toxicity data of chlorophenol compounds obtained from Laine et al. (1997)
2-CP
4-CP
2,4,6-TCP
PCP
3
LD50 (rats, mg EC50 (mg L-1)

kg-1) oral (MSDS (daphnids 48 h)
2009)
(Shim et al.,
2009)
670
82
670
42
820
21
200
N.A.
(2,4,6-TCP) 2,4,6-trichlorophenol, 4(PCP) pentachlorophenol.
EC50 values (mg L-1)

(luminescent Vibrio
bacteria)
(Zona et al., 1999)
21
1.2
5.2
0.2
The toxicity of the simple brominated or chlorinated phenols is high, but not as high as
their oxidized form (Vollmuth et al., 1994; Laine et al., 1997). When two halogenated
phenols are oxidized by solar photolysis or biodegradation they combine to form a
double aromatic ring compound. Since the number of halogens within the compound is
higher, so is its potential toxicity. Laine et al. showed that eight halogens on the two
phenyl rings results in the highest toxicity.
The most investigated products of PCP are the polychlorodibenzo-p-dioxins (PCDDs)
and polychlorodibenzofurans (PCDFs) which all are toxic while some are mutagenic or
teratogenic. In 1998, an international expert group convened by the World Health
Organization (WHO) revisited the existing allocated toxicity levels in terms of toxic
equivalency factors (`TEF) (Van den Berg et al., 1998). This new scheme expresses the
severity of these halogenated phenyl by-products (Fiedler et al., 1990; Vollmuth et al.,
1994). In this system, the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is set
at 1 and the toxicity of other PCDDs and PCDFs are expressed as a fraction of the
TCDD toxicity. This showed that an increase of a single chlorine on a compound raises
the toxicity level (TEF value) by 10 times (WHO, 2000).
The brominated phenols did not get the same attention as their chlorinated analogues.
The data available in the literature mainly classifies the brominated phenols in the
brominated flame retardant (BFR) group (Law et al., 2008). Some researchers consider
that PBP degradation is similar to PCP degradation (Mikesell and Boyd, 1986).
However, when a BFR is used to suppress fires, it can be oxidized to polybrominated
dibenzo-p-dioxins (PBDD) and polybrominated dibenzo-p-furans (PBDF), both are
similar in structure and regarded as highly toxic. According to Birnbaum et al. (2003)
the PBDD and PBDF tend to be more sensitive to UV degradation and the bromine can
be easily detached. In contrast to their chlorinated counterparts, these compounds
appear to be more biologically resistant. For example, Birnbaum (1985) demonstrated
that the half life in rats was twice as long as for the chlorinated analogues.
Page 25 of 163
In addition to their high toxicity levels, these compounds are hydrophobic and tend to
be adsorbed in the environment (Birnbaum 1985; Birnbaum et al., 2003). As was briefly
discussed in chapter 1, brominated phenols have been detected in sewage sludge. As can
be seen from reviewing the toxicity of these compounds, it is imperative that the wastes
containing these halogenated phenols be treated before reaching the environment,
otherwise they will be adsorbed to sediments and become a long term problem. Not
only are these compounds a problem if left untreated, they may become oxidized and so
increase in toxicity (Laine et al., 1997), causing more harm to the environment.
It is desirable that phenolic wastes are treated without the addition of chemicals and
with minimal energy consumption. Consequently, a biologically based treatment
process was chosen for this investigation.
2.1.2 The application of enzymes to treatment of wastewater containing halogenated
phenols
Conventional treatments for phenolic compounds include ozonation (Booth and Lester,
1995), Fenton precipitation (Bigda, 1996; Kavitha and Palanivelu, 2004) and adsorption
processes (Lin and Wang, 2002). These processes remove different compounds to a
greater or lesser extent. The main disadvantages of adsorption are that different
compounds require different conditions for different adsorbents such as different
activated carbons, and, while activated carbon does remove the compounds from the
water, it retains the compounds. This means that after saturation of the carbon it must
either be regenerated or disposed of to a secure landfill.
While ozonation destroys compounds, a toxicity and biodegradability study carried out
by Adams et al. (1997) showed that ozonation of halogenated phenols had different
effects dependent on the nature of the substituents. Ozonation of chlorophenols alone
and nitrophenols alone caused significant enhancement of their biodegradability. On the
other hand, ozonation of aminophenols alone caused a significant decrease in their
biodegradability. A second major disadvantage is that due to its instability, ozone must
be generated at the site of use and the equipment and operating costs can be quite high
(Wilkes University, 2010). A further disadvantage is that ozone is highly corrosive,
especially to steel and iron, and can even oxidize neoprene (Wilkes University, 2010).
Utilizing enzymes has many advantages over conventional microbially-mediated
treatments (Aitken et al., 1994; Dosoretz and Ward, 2004; Reihmann and Ritter, 2006
and Johannes et al., 2006):
Page 26 of 163
Treatment is applicable to a wide variety of compounds, whether they are toxic or

not.
Physical properties, (pH, temperature and salinity) of the waste stream have less
effect on the enzyme performance.
They have the ability to treat low and high contaminant concentrations with minimal
inhibition.
Sudden variations in concentrations of feed have only a small effect on the enzymes.
They have high conversion rates and short retention times (seconds to minutes)
compared to biological processes (hours, days).
There is no lag phase or need for biomass acclimation for enzymes.
The disadvantages of enzymatic processes are (Johannes et al., 2006; Reihmann and
Ritter, 2006):
Some enzymes are expensive.
Compared with using microorganisms which continue to multiply, the enzyme needs
to be replenished.
Enzymes often have a narrow range of substrates which they can transform.
Some enzymes require an activator such as H2O2 to be active.
2.2
PEROXIDASES
The most common enzymes used in the treatment of phenols are peroxidases.
Peroxidases (E.C. 1.11.1.7) are found in plants (horseradish, soybean), microorganisms
(white rot fungi) and animals (e.g. lactoperoxidase in milk). They can achieve a variety
of tasks such as participating in lignin breakdown (Call and Mucke, 1997) or effecting
the plant pigmentation process, where the peroxidase is responsible for the degradation
of the anthocyanin group which forms pigments ranging from pink and red to blue
(Kader et al., 2002).
In general, peroxidases are characterized as two superfamilies on the basis of similarity
of reaction mechanism sequences: mammalian peroxidases and plant peroxidases
(Welinder, 1992). The plant peroxidases can be further classified (Dosoretz and Ward,
2004):
Page 27 of 163
(i) Intracellular peroxidase (class I) which acts as an antioxidant to prevent damage to

mitochondria caused by H2O2 (Mliki and Zimmermann, 1992).
(ii) Peroxidase secreted by fungi (class II) which participate in the degradation of lignin
in wood (Tien and Kirk, 1983).
(iii) Peroxidase secreted by plants (class III) which allows the plant to adapt to seasonal
change and prevent stress to plant physiology and biosynthesis (Dunford, 1991).
2.2.1 Horseradish peroxidase
Horseradish peroxidase (HRP) is a glycoprotein enzyme isolated from the roots of the
horseradish plant. HRP is comprised of several distinct peroxidase isoenzymes with a
molecular weight of approximately 40 kDa (Veitch and Smith, 2001). The isoenzymes
have many functions in plant physiology such as crosslinking of cell wall polymers,
lignification and resistance to intracellular infection (Veitch, 2004).
HRP has received particular attention as a possible method to treat industrial wastewater
containing phenolic compounds (Klibanov et al., 1980; Nakamoto and Machida, 1992;
Nicell et al., 1992; Nicell et al., 1993; Tatsumi et al., 1996; Tong et al., 1998;
Bayramoglu and Arica 2008). The major advantages of HRP over other peroxidases are:
1. It has a high redox potential.
2. When the peroxidase is exposed to a high dose of H2O2, the peroxidase becomes a
form which has limited activity. For example, 0.4 mM H2O2 will deactivate HRP with
activity of 0.4 units per gram of PG (Meizler 2005). However, unlike other peroxidases
HRP can return from that state to an active form (Dunford, 1991).
3. HRP is available at a lower cost than other enzymes.
4. HRP can treat a wide range of phenol concentration from 5 to 200 mg L-1 (Vasileva
et al., 2009) and even as low as 1 g L-1 (Levy, 2003)
Klibanov et al. were the first to propose the treatment of wastewater with HRP to
remove over 30 different phenols and aromatic amines via precipitation, and recorded
removal efficiencies for some pollutants exceeding 99% (Klibanov and Morris, 1981).
Page 28 of 163
2.2.2 Catalytic cycle of peroxidases

The general process is that the peroxidase utilizes hydrogen peroxide to convert a
suitable substrate (phenolic derivative compound in most cases) into a free radical
(Figure 2-1). The reaction begins when the enzyme (Porphyrin-Fe3+) reacts with H2O2
to yield a high valence oxo-iron and porphyrin cationic radical (Porphyrin+-Fe4+=O)
compound I, releasing water from the reaction. The active site donates a positive charge
to the phenolic substrate; this charge assists in formation of a radical cation and the loss
of one electron from the oxidized enzyme, creating compound II (Porphyrin-Fe4+=O).
The reaction finishes as compound II transfers the last electron to a second substrate
returning the enzyme to its native state (Dunford, 1991; Veitch and Smith, 2001).
The radicals then undergo coupling to produce dimers (Klibanov et al., 1983; Aitken,
1993; Dec and Bollag, 1994; Duran and Esposito, 2000). Successive oxidation,
coupling, and a transfer reaction eventually results in the formation of a polymer. The
generation of radicals in this cycle is enzyme-dependent. However, radical-radical
coupling and transfer are controlled exclusively by phenoxyl radicals and solvent
chemistry (Ward et al., 2001).
Native enzyme
Compound II
Compound I
Figure 2-1. Catalytic cycle of a heme peroxidase. AH2 (any phenolic derivative compound) denotes a
reducing substrate. Source: http://metallo.scripps.edu/PROMISE/PEROXIDASES.html#reaction.
Page 29 of 163
2.2.3 Enzyme immobilization

The drawbacks often encountered in reactions catalyzed by soluble enzymes are:
The enzymes are not readily reusable and a supply of fresh enzyme is usually
required for continuous treatment (Levy et al., 2003).
Many enzymes are not sufficiently stable under most of the operational conditions
typical for wastewater treatment. This may result in a possible loss of catalytic
activity due to auto-oxidation and/or denaturation by the aqueous medium, the
solutes or mechanical shear forces (Hermanson et al., 1992).
As a rule of thumb, enzymes have a low productivity yield compared to whole
microorganisms whenever they are applied to industrial scale processes with high
concentrations of substrate(s) and product(s). With microbial treatment, the
organisms multiply and so maintain the biocatalytic process (Johannes et. al. 2005).
It seems that enzymes with selective mechanisms are better for treating low
concentration pollutants (practically, those that get little attention with conventional
treatments) than treating high concentrations that can be treated in other ways (Levy et
al., 2003).
Peroxidases may lose activity through two process-related mechanisms. The first is loss
of activity due to adsorption of oligomeric reaction products on their active sites. The
second relates to heme peroxidases which, although they are activated by H2O2, are
deactivated in the presence of excess H2O2 (Hiner et al., 1995; Van de Velde et al.,
2001). Certain additives, such as polyethylene glycol, can suppress the hydrophobic
interactions of the products with the enzyme, while others such as gelatin protect the
enzyme against peroxide deactivation (Wu et al., 1997; Buchanan and Nicell, 1998).
Enzyme immobilization was investigated as another strategy to increase enzyme
stability and to enable a continuous conversion process (Ganjidoust et al., 1996;
Tatsumi et al., 1996; Duran and Esposito, 2000).
Immobilization of enzymes, in particular HRP, includes a range processes such as:
attachment of the enzyme to a porous glass through covalent binding (Pappas et al.,
2002) or adsorption (Kondo et al., 1993) or chemical linkage between the enzyme
molecules to form insoluble macrostructures known as cross-linking (Levy et al., 2003).
Alternatively, the biocatalyst may be confined to a restricted area which it cannot leave
and where it remains catalytically active, eg., entrapment in a porous gel (Smith et al.,
Page 30 of 163
2002) or retained within a membrane-restricted compartment (Vasileva et al., 2009). As

a consequence, homogeneous catalysis using a native enzyme becomes heterogeneous
catalysis when immobilized biocatalysts are employed.
Depending on the immobilization technique, the properties of the enzyme such as
stability, selectivity, affinity, pH and temperature stability, may be significantly altered sometimes improved, sometimes diminished. Predictions of the effects of the
immobilization are very difficult to make (Hermanson et al., 1992; Johannes et. al.,
2005).
2.2.4 Covalent binding of enzymes
Covalent binding of an enzyme to a support leads to the formation of stable linkages,
thus reducing leakage of the enzyme from the reaction system compared with other
immobilization techniques, such as adsorption or cross linkage (Doretti et al., 1999).
Also, it is very important to allow easy access for the substrates and products to come
and go. Covalent binding (also adsorption) places the enzyme on the surface exposed to
the solution, compared to other immobilization techniques which limit substrate access
to the enzyme, such as sol-gel encapsulation or membrane entrapment.
However, a major disadvantage of this method is that the enzyme is subjected to the
rather reactive chemicals and the harsh environment often employed in the binding
process (Vishwamath et al., 1997; Johannes et. al., 2005), conditions which lead to a
loss of activity due to conformational changes or chemical denaturation of the enzyme
(Penosil et al., 2002). Another important drawback of the covalent binding of enzymes
onto solid carriers is that in many cases a specific technique needs to be developed.
Covalent binding can use a ligand to achieve a higher degree of immobilization. The
ligand anchors the enzyme to the support and also allows some flexibility (some
enzymes need to change their tertiary structure to commence a reaction) to the enzyme
(which depends on the ligands length) (Hermanson et al., 1992). The most commonly
used support is porous glass (rigid, durable and inert to physical changes) and the ligand
that is used for this support is an organic/inorganic molecule that consists of an amino
group at one end and silane at the other. It is possible to use the amino group to interact
directly with the enzyme using periodate (Pappas et al., 2002), or altering the amino end
to a carboxylic group using glutaraldehyde (Salinas et al., 2005) to contact with amino
groups on the enzyme surface.
Page 31 of 163
The functional groups of enzymes which are commonly involved in covalent binding
are nucleophilic: mainly N-terminal, carboxy-, sulfhydryl- and phenolic functions
(Hermanson et al., 1992; Penosil et al., 2002).
The ligand that was used in this thesis for immobilization of peroxidase on a glass
support was periodate. Periodate has several advantages:
(1) It is specific for glycosylated proteins such as HRP since binding occurs through the
sugar groups of the enzyme which are far away from the catalytic site. This can
allow immobilization of even crude (not pure) enzyme (Dosoretz and Ward 2004).
(2) The degree of immobilization of peroxidase does not depend on the freshness of
periodate, unlike the alternative ligand glutaraldehyde which forms impurities on
storage above 0C which interfere, as reported by Gilett and Gull (1972).
2.2.5 Disadvantages of immobilized peroxidase
Peroxidases are sensitive to the ratio of hydrogen peroxide (H2O2) to substrate. In the
absence of suitable reducing substrate or, at high H2O2 concentrations, the peroxidase is
oxidized to a species with limited catalytic activity (Dosoretz and Ward, 2004).
However when the peroxidase/substrate ratio is lower than the optimal ratio, only partial
transformation occurs (Nicell and Wright, 1997).
The oxidized hydrophobic polymeric products can be a problem as they are adsorbed to
the reactor bed after transformation, probably due to hydrophobic interactions, resulting
in enzyme deactivation and consequently a decrease in the activity of the enzyme
(Klibanov et al., 1980; Dordick et al., 1987; Nakamoto and Machida, 1992; Levy et al.,
2003). To overcome, or at least reduce this adsorption deactivation effect, additives or
solvents have been added to the reactor feed (Nakamoto and Machida, 1992). Although
its mode of action is not fully understood, polyethylene glycol (PEG) is thought to
protect the enzyme against deactivation by the product during reaction either by
diminishing the hydrophobic interaction of the polymerized products or by protecting
the enzyme active site from free radical oxidation. Nicell and coworkers claim that
PEG is better than the other additives as excess PEG has no negative effect on the
reaction and it has higher solubility in water than other additives (Nicell et al., 1995;
Buchanan and Nicell, 1998).
A different approach to overcoming the adsorption of the hydrophobic material is by
applying solvents. The role of solvents is to dissolve the product polymers so they can
Page 32 of 163
be removed from the reactor bed (Akkara et al., 1991). Although both methods affect
the rate of substrate transformation, they do not prevent the eventual formation of a
coating on the immobilized enzyme.
2.3
TITANIUM DIOXIDE
2.3.1 Physical properties of titanium dioxide

Photocatalysis using TiO2 may assist in dealing with the polymeric product adsorbed on
the support. TiO2 is used in industry not only as a support for sensor devices and as a
pigment in paints, but also as an adsorbent of high surface area and semiconductor with
high catalytic activity.
For a long time, the photoactivity of TiO2 has been considered a problem that needs to
be controlled. This photoactivity decomposes any organic materials that come in contact
with it in the presence of light. On the other hand, the photocatalytic properties of TiO2
play a positive and significant role in applications ranging from solar energy conversion
(Grtzel, 2001) through to self-cleaning (Paz et al., 1995) and anti-fogging functions
(Rong et al., 1997).
O
O
O
O
O
Figure 2-2. Crystal structures of titanium dioxide formation as (a) rutile (b) anatase (redrawn from Clark
(1968)).
TiO2 can exist as three different phases: anatase, rutile and brookite. Brookite, which
has an orthorhombic crystal structure, is usually found in minerals and has no practical
importance due to its low stability (Clark, 1968). Anatase and rutile can be formed as
oxidized products from titanium metalloids. Their structures are shown in Figure 2-2
and their physical properties are shown in Table 2-3. Anatase is thermodynamically
stable up to 800C where it is transformed irreversibly into the more thermodynamically
stable rutile (Clark, 1968). Both anatase and rutile are easier to produce than brookite
(Clark, 1968).
Page 33 of 163
Table 2-3 Physical properties of rutile and anatase (Clark, 1968)
Rutile
Anatase
Density (g cm-3)
4.3
3.8
Melting point (C)
1858
Changes to rutile at high

temperature
Dielectric constant
av = 110 -117
= 48 (powder)
Energy gap (ev)
3.0
3.2
2.3.2 Photo-induced hydrophilicity

Titanium dioxide has two industrial uses: photocatalytic activity which leads to the
degradation of organic compounds, and "super hydrophilicity" which involves higher
affinity to hydrophilic compounds (Rong et al., 1997). Even though these two
phenomena are intrinsically different, they are both effected by UV irradiation of the
TiO2 surface.
When a TiO2-coated support is irradiated by UV light, the surface tension of the water
droplet in contact with the surface decreases, which results in a decrease in the contact
angle between the liquid and the surface. A schematic illustration of the increased
hydrophilicity or photo-induced wetting for the water droplet on the support is shown in
Figure 2-3. According to Rong et al. (1997) the change in surface polarization is the
cause for the change in surface charge, and consequently is responsible for the change
in surface properties. This characteristic has various practical applications such as
repelling hydrophobic pollutants such as oil smudges or inducing an anti-fogging effect
on the TiO2-coated glass.
Page 34 of 163
UV
Dark
Figure 2-3. Photo-induced super hydrophilicity (redrawn from Rong et al. (1997)).
The mechanism of super hydrophilicity is still controversial to some extent. The most
accepted mechanism divides the process into three main stages (Rong et al., 1997) as
illustrated in Figure 2-4.
Oxygen Vacancies
H2O
OH-, H+
Ti
Ti
Dark
Ti
Ti
Ti
Dark
UV
UV
Ti
Ti
Ti
Ti
Ti
Ti
Ti
Figure 2-4. The mechanism of photo-induced super hydrophilicity (redrawn from Carp et al. (2004)).
In the first stage, a pair of electron-holes is produced by irradiation with supra-band gap
photons. The electrons reduce Ti4+ cations to the Ti3+ state, and the pair is oxidized to
form oxygen vacancies in the TiO2 crystal (Paz et al., 1995).
Water molecules can then fill these oxygen vacancies, providing OH extensions instead
of the oxygen, which make the surface more hydrophilic when a surface covered with
hydrophobic molecules is UV irradiated. The chemisorbed water layer appears again
and is able to wash away the pollutants.
TiO2 alone cannot remain super hydrophilic for a long period without UV irradiation.
However, TiO2 blended with SiO2 as the binder can maintain a low contact angle in the
dark for up to several weeks (Takata et al., 2003). This effect results from the great
ability of SiO2 to hold water molecules on its surface, and this stabilizes the overlying
water film.
Page 35 of 163
2.3.3 The photocatalytic mechanism

Photocatalytic semiconductors have been evaluated for air and water remediation
following incineration, air stripping and granular activated carbon adsorption (Williams,
1999; Li et al., 2004). The photocatalytic approach may be considered to come under
the umbrella of a number of new technologies known as advanced oxidation processes
(AOPs). These technologies rely mostly on the generation of reactive free radicals (e.g.,
OH) that are subsequently used to degrade organic pollutants or microorganisms. The
AOPs include (Oppenlnder, 2003; Gogate and Pandit, 2004; Parsons and Williams,
2004):
1. Homogeneous photolysis: employs direct UV photolysis of solutions containing
oxidants such as H2O2, ozone or combinations of both of these.
2. Radiolysis: utilizes high energy radiation (e.g., -rays) to produce reactive free
radicals during the irradiation of a target solution.
3. Indirect electrolysis: radicals such as OH are generated at a dark electrode/solution
interface containing additives such as H2O2.
4. Heterogeneous photolysis: involves the use of reusable semiconductor suspensions
that harness the light. The presence of water and oxygen molecules is required to
generate oxidizing species on the semi conductor surface.
Photocatalytic oxidation of low carbon content water and air with UV-irradiated
titanium dioxide has been considered promising. The main advantages of this process
are (Fujishima et al., 1997; Chen and Ray, 1999; Grtzel, 2001; Carp et al., 2004):
1. Chemical activation is provided by UVA (380 nm) irradiation, allowing significant
reactions to occur with minimal energy input.
2. There is potential for complete mineralization of a wide variety of contaminants.
3. The catalyst activity is insensitive to the presence of halogenated organics.
4. O2 and H2O are the primary triggers required for photooxidation and hence no
expensive chemicals are required.
Figure 2-5 shows the general scheme of the semiconductor reaction. On irradiation, UV
photons increase the semiconductor energy level forming supra-band gap photons and
electron hole (e-/h+) pairs. The electron hole pairs migrate to the surface of the
Page 36 of 163
photocatalyst where the holes may be trapped by H2O or OH- that are adsorbed to the
surface (Linsebigler et al., 1995). The H2O or OH- are transformed into peroxide
radicals which are responsible for oxidation of organic compound.
hEbg
A-ads
1
h
Ebg
VB
Aads
Recombination
CB
3
e-
2
Dads
h+
4
D+ads
Figure 2-5. Diagram of photoconductivity model (redrawn from (Fujishima and Honda, 1972; Grtzel,
2001)). (D) electron donor (i.e. organic compound), (A) electron acceptor (i.e. water or O2), (CB)
conduction band, (VB) valence band, (Ebg) energy level.
The diagram represents the conduction and valence bands (CB and VB, respectively) in
a nanoparticle of TiO2 and the energy levels of a trap state, and an electron scavenging
state, Aads and Dads on the surface of the nanoparticle. The arrows represent the different
possible electron transitions: (1) photogeneration (2) band-to-band recombination (3)
electron trapping (4) hole trapping (Eppler et al., 2002).
Since oxygen is the primary electron acceptor, H2O2 may be formed by reduction of two
oxygen electrons. This contributes to the degradation of the targeted pollutant by acting
as direct electron acceptor or as a direct source of hydroxyl radicals. H2O2 could
theoretically be formed via two different pathways in an aerated aqueous solution as
follows:
Page 37 of 163
O2 + 2e- + 2H+ H2O2
Equation 2-1
2H2O + 2h+ H2O2 + 2H+
Equation 2-2
The hydrogen peroxide production during the oxidation of low molecular weight
compounds has been shown to have pronounced Langmuir-Hinshelwood dependence of
the O2 partial pressure (Hoffman et al., 1994). This dependency has shown that the
adsorption of the electron donor (O2) was the limiting factor of the reaction, which
suggests that an increase in the adsorption of the O2 will increase the reaction rate.
Mao et al. (1991) observed that hydroxyl radicals are the principal reactive oxidants in
photoactivated TiO2. They also found that hydroxyl radicals or trapped holes are
directly available at the surface. These hydroxyl radicals are hydrogen peroxide
precursors, and their production can be limited by additives or competitive electron
donors as reported by Carraway et al. (1994). Grabner et al. (1991) demonstrated the
formation of Cl2- (which originated from the HCl introduced into the solution as HCl to
adjust the pH) contributed to the reduction of the hydroxyl radicals formed and thus the
formation of hydrogen peroxide.
2.3.4 Mineralization or formation of by-products
An ideal oxidation process would convert halogenated organic contaminants into the
complete oxidation products of carbon dioxide, water and inorganic acids. In the case of
chlorinated organics, complete mineralization would result in the additional
production of HCl.
In general, the issue of intermediates and of stable by-products has not been explored in
great detail. For all non-halogenated organic compounds, with the exception of
alcohols, complete oxidation has readily been observed (Zou et al., 2006). In most
published studies, carbon dioxide and water were not specifically tracked, but rather the
absence of gas chromatographic peaks upon analysis of the reactor effluents suggested
the absence of carbon-containing partial oxidation products (Ollis, 1985; Grabner et al.,
1991; Ollis et al., 1993). Kanki et al. used LC-MS to investigate the photo degradation
of phenol and bisphenol A in water, and they assumed the formation of CO2 and water
by the reduction in carbon levels measured by TOC. An exception was the work of
Suzuki et al. (1993) who used GC-MS to show that the carbon dioxide produced was
equal to the expected stoichiometric value for complete oxidation over a wide variety of
conditions.
Page 38 of 163
Nimlos et al. (1993) employed gas phase infrared spectroscopy and direct sampling
mass spectroscopy to detect gas-phase intermediates in the photocatalytic oxidation of
chlorinated VOCs and found trichloroethylene, dichloroacetyl and chloride. The
chlorine atom balance failed to close, and on this basis it was concluded that molecular
chlorine was formed. The observed products were typical for homogeneous photolysis
of trichloroethylene.
2.3.5 Oxidation of halogenated phenols by means of TiO2 photooxidation.
Photocatalysis using UV radiation and TiO2 has been used to deal with halogenated
phenol wastes. The most used system consists of direct UV radiation of a solution
containing a TiO2 slurry. Phenol and its derivatives can be oxidized by TiO2 in a
suspension to generate catechol and hydroquinone (Chen and Ray, 1999; Sobczynski et
al., 2004) which may eventually be completely mineralized, as shown in Figure 2-6.
Figure 2-6. Schematic representation of breakdown mechanism of halogenated phenol under UV radiation
in presence of TiO2 (Sobczynski et al., 2004).
Treatment of phenol and its halogenated derivatives using a UV/TiO2 system has
appeared in many guises. The treatments utilize a similar reaction mechanism
(LangmuirHinshelwood process, i.e., adsorption/desorption), with similar degradation
intermediates, i.e., catechol and hydroquinone. The initial stage in the reaction is
dehalogenation and then breakdown of the compounds (Ollis et al., 1991; Fujishima et
al., 1997; Chen and Ray, 1999).
A TiO2 slurry has the ability to provide a high photooxidation capability as it has a high
surface area. However, the higher the concentration of the slurry the lower light
penetration of the sample. The smaller the particle size the higher the surface area which
Page 39 of 163
causes higher catalytic ability, however, reusability is low due to small particle size.
Using any immobilization technique for the TiO2 greatly reduces its efficiency. For
example, complete degradation of 16 mg of 2-chlorophenol in less then 3.5 h was
achieved using 2 g L-1 TiO2 slurry (Ku et. al., 1996). In comparison, 4.41 mg L-1 of 4chlorophenol were transformed after 300 h when TiO2 pellets immobilized on a rotating
disk were used (Dionysiou et. al., 2000).
2.3.6 Catalyst lifetime
Under certain conditions (deprived of oxygen, high pH or increasing turbidity), steady
rates of photocatalytic oxidation are not obtained since the catalyst tends to be
deactivated over time (Peral and Ollis, 1997). Furthermore, the presence of water is
required to maintain photocatalytic oxidation activity (Vorontsov et al., 1999).
An inverse correlation between organic feed concentration and the onset time for
deactivation has been observed at higher concentrations of organics leading to a shorter
catalyst lifetime (Dibble and Raupp, 1992). Later, Raupps group (1997) tested a benchscale packed bed tubular photo-reactor on air contaminated with 5 ppm of organic
compounds for up to two weeks with no detectable deactivation. At 50 ppm, the onset
of deactivation occurred within 48 hours, and at 500 ppm, deactivation was observed
after only 6 hours of operation.
It is possible to regenerate the photocatalytic properties of TiO2 by calcining in a
furnace at 450C (Paz et. al., 2002). However, that method causes a concern as TiO2
photooxidation is normally regarded as an environmentally friendly method with a low
energy consumption; using a furnace for regeneration will tend to cancel that benefit.
2.4
RESEARCH AIMS
2.4.1 Overall aim

The overall aim of this project is to develop a reliable method of removing toxic
phenolic substances from wastewater (industrial effluent or E-waste leachate) by
enzymatic polymerization and dehalogenation.
The use of peroxidase to treat halogenated phenols in wastewater is considered by many
(Klibanov et al., 1980; Dordick et al., 1987; Nakamoto and Machida, 1992; Buchanan
and Nicell, 1998; Levy et al., 2003) to be the most appropriate course of action.
However, as discussed in section 2.2.5, immobilized horseradish peroxidase (and
Page 40 of 163
peroxidases in general) tends to be deactivated by the adsorption of its products. This

research investigates the use of the TiO2 as a super-hydrophilic and photo-oxidative
support to the HRP polymerization process. Under UV irradiation, the TiO2 can either
repel the hydrophobic products formed by the HRP and or degrade them and so obtain
hydrophilic compounds that may be also less toxic in the wastewater. The aim is that
the product is either harmless or can be dealt with minimal energy cost.
In Figure 2-7 the proposed reaction is shown. There are two possible benefits to come
from the combination of the oxidative reactions of the two catalysts. These are:
1. Controlled polymerization by means of hydrophilic repulsion. HRP polymerizes the
substrate without controlling the length, and the longer it gets the more hydrophobic
it becomes. However, when the surface is simultaneously irradiated by UV, the TiO2
increases in hydrophilicity, and so then can repel the more hydrophobic products.
This means prevention of formation of higher mass oligomers.
2. Controlled polymerization by photooxidation from TiO2 photocatalysis. When
irradiated by UV, TiO2 photooxidation can break down the products adsorbed to the
enzyme. This may result in halogen detachment and possible product oxidation,
which may further transform the product to be more hydrophilic and reduce its level
of toxicity.
Hydrophobic materials are generally considered hazardous. Although an increase in
hydrophobicity may cause a decrease in toxicity due to a reduction in their availability
to the environment, they are not easily utilized by microorganisms thus causing a long
term problem (Tabak and Govind, 1997; Semple et al., 2003). It is one of the objectives
of this research to transform the halogenated phenols to soluble products that have
lower toxicity which can then be treated by microorganisms.
Page 41 of 163
hv
H2O2
Phenolic
compound
dimer
HRP
H2O2
H2O2
trimer
HRP
NH
H2O2
tetramer
Products either repelled and

leave or stay to be
photooxidized
HRP
NH
NH
SiO2
TiO2
(hydrophilic)
Figure 2-7. Schematic representation of the reaction sequence for HRP polymerization and TiO2 support
approach.
2.4.2 Problems anticipated with the new system

HRP cannot be directly immobilized on the TiO2 surface since non-specific oxidation
by the TiO2 can affect any organic material, including enzymes (Scaiano et al., 2002).
Scaiano and Hancock-Chen (2000) showed that the activity of HRP decreased in the
presence of TiO2 and after a while became completely inactive. To counteract this
problem, in the present study a novel glass was synthesized, made of TiO2 incorporated
within fused silica. This support was developed to have properties to protect the HRP
from UV illumination and photocatalytic properties of the TiO2.
Several other issues regarding the new system need to be considered for such a concept
to succeed:
In addition to the possibility of deactivation of the enzyme, the new support may
prevent substrate access to the enzyme.
The affinity of product for the enzyme must not be too strong, otherwise the target
molecule will be too strongly adsorbed to the enzyme and will not diffuse towards
the TiO2 interface. However, there is a possibility of remote oxidation. Tatsuma et al.
(1999; 2001) reported the remote bleaching of methylene blue by UV-irradiated
titania in the gas phase. They came to the conclusion that the species oxidizing the
organic substrates in a moist environment was most likely OH. The rate of
degradation for remote oxidation is slow and complete decomposition can take
around 100 hours; however, significant changes can happen during the first 10 hours.
Page 42 of 163
Immobilization of the HRP may alter the balance between adsorption and surface
diffusion, altering the selectivity factor (Hermanson et al., 1992), meaning the
enzyme may be less efficient dealing with low concentrations. This research is based
on halogenated phenol transformation using HRP immobilized on porous glass
supports which is considered inert. The new support containing TiO2 can influence
the diffusion of the substrate to its surface.
2.5
THESIS OUTLINE
The aim of this work is to utilize HRP and TiO2 in a novel glass matrix to treat water
containing halogenated phenolic compounds by either removing or transforming the
toxic compounds to a biodegradable compound, subsequently reducing the overall
toxicity. The rest of the thesis is as follows:
Chapter 3 describes the materials and methods.
Chapter 4 describes the novel support (Tglass) in terms of physical properties and
compares its photocatalytic properties with range of TiO2-coated supports.
Chapter 5 provides an examination of the HRP immobilized on the Tglass support
(HRP-Tglass) and analysis of its behaviour under UV radiation, while comparing it to
free HRP and HRP immobilized on porous glass.
Chapter 6 details the kinetics of HRP immobilized on Tglass in a plug flow reactor (in
the absence of UV radiation) for the reaction of 4-BP and H2O2 (1:1 molar ratio) at
various flow rates and concentrations using HRP-Tglass or HRP-PG particles, and the
development of kinetic models for HRP on both supports.
In Chapter 7, HRP-Tglass was used to treat 4-BP at various flow rates in a continuous
flow reactor under UV irradiation.
Chapter 8 describes the treatment of PBP and PCP solutions (separately and as a
mixture) by UVB/HRP-Tglass and the determination of the change in toxicity by the
Microtox test and exposure to Hydra.
In Chapter 9 the overall conclusions are summarized.
Chapter 10 provides recommendations for future work.
Page 43 of 163
Chapter 3. Materials and methods

3.1
CHEMICALS
All chemicals used were of reagent grade or higher. 4-Bromophenol (4-BP), H2O2 (50%
v/v solution), acetylacetone, Triton X-100 and 1,2,3-trihydroxybenzene (pyrogallol)
were purchased from AJAX chemicals (Australia). Analytical grade dibasic and
monobasic potassium phosphate, sodium phosphate, boric acid, silicon dioxide, sodium
carbonate, aluminium oxide, titanium dioxide (synthesis grade, 99%) and trifluoroacetic
were purchased from Scharlau (Spain). Acetonitrile and methanol were purchased from
Honeywell (Australia). Horseradish peroxidase, 3-aminopropyltriethoxysilane (3APTES), pentabromophenol and pentachlorophenol were purchased from Sigma
(Australia).
3.2
SYNTHESIS OF SUPPORTS
3.2.1 Synthesis of porous glass (PG)

The following solids were ground together with a mortar and pestle and 7.5 g was
placed in a ceramic crucible: SiO2 55.2%, H3BO3 37.1% and Na2CO3 7.7% (w/w basis).
The crucible was heated at 1000C in a furnace (Barnstead/Thermolyne, 30400 model)
for 1 hour and then cooled rapidly by plunging into water. The glass so formed was
ground and a fraction of approximately 125-150 m diameter was collected by sieving.
The glass particles produced had a surface area of 25 m2 g-1 and average pore size of
62 .
3.2.2 Synthesis of Tglass
The following solids were ground together with a mortar and pestle and 7.5 g was
placed in a ceramic crucible: TiO2 50%, SiO2 35%, H3BO3 10%, Na2CO3 2.5% and
Al2O3 2.5% (w/w basis). The crucible was heated at a rate of 50C per minute to
1000C and held at this temperature for 2 hours and than rapidly cooled by plunging
into cold water and the resultant glass ground. The glass so formed was ground and the
125-150 m diameter fraction collected by sieving. The Tglass particles below that size
tended to break down to unusable powder.
The glass particles produced had a surface area of 9.4 m2 g-1 and average pore size of 70
.
Page 44 of 163
3.2.3 Synthesis of casamic

The casamic preparation method was adapted from Jones and Pascoe (2002). Regular
flat soda-lime glass was ground to powder (< 150 m). A mixture of 100 g of glass
powder, 10 g Ca(OH)2 and 10 mL MilliQ water was ground using a mortar and pestle.
An aliquot (40 g) was placed in a square die chamber (70.5 mm x 70.5 mm) and pressed
uniaxially at 500 kN for 2 min. The resultant plates were heated in an autoclave at
120C for 4 hours. The ceramic was equilibrated with the atmosphere overnight and
then ground (500-250 m).
3.2.4 Determination of surface properties of supports
The Brunauer-Emmett-Teller (BET) surface area and average pore size of the supports
were measured using an ASAP 2000 Gemini instrument (Micromeritics) at 77 K with
nitrogen as adsorbate. The data were processed by ASAP 2010 software version 5.02
(Micromeritics) (additional information is shown in Appendix A).
3.2.5 TiO2 coating procedure
This method was adapted from Paz et al. (1995). Titanium dioxide coating was prepared
by dipping a carrier, e.g., microscope slide, in a paste obtained by mixing TiO2 (45 g),
bi-distilled water (180 mL), acetylacetone (6 mL) and Triton X-100 (4 mL) and
subsequent calcination at 450 C for 30 min. Slow heating and cooling was important to
prevent cracking of the film.
3.2.6 Preparation of supports prior to enzyme immobilization
The PG and Tglass supports (50 g) were etched with 1 M NaOH for 1 hour under
sonication (Unisonics, Australia) at 50 kHz, 35.5 W to increase the pore size. After
washing thoroughly with water, the treated glass was dried at 110C. Silanization of the
supports was performed according to the method of Olsson and Ogren (1983). Acidwashed (boiling 5% nitric acid, 1 h) glass preparations were treated with 10% 3aminopropyltriethoxysilane at pH 3.45 (3 h, 75C).
The preparations were dried
overnight at 95C and stored dry for further use.

3.2.7 Technique of enzyme immobilization
This method was adapted from Pappas et al. (2002). HRP type I with RZ (A409/A280) of
1.0 and activity of 78 U mg-1 was purchased from Sigma (Australia). HRP (10 units)
was dissolved in 20 mL 0.1 M phosphate buffer (pH 7.0) and mixed with 5 mL of a
Page 45 of 163
fresh solution of 0.1 M sodium periodate. The mixture was stirred for 30 min at room
temperature. The silanized supports were suspended in 25 mL 0.2 M sodium borate
buffer (pH 8.6). Then, the two solutions were mixed and the reaction allowed to
proceed overnight at 4C. The supports were rinsed thoroughly with 0.1 M phosphate
buffer (pH 7.0) and stored in the same buffer at 4C until use.
3.3
ANALYTICAL METHODS
3.3.1 X-ray spectroscopy techniques

3.3.1.1 X-ray Diffraction (XRD)
The Tglass was ground to a fine powder (30 m) and analysed by X-ray diffraction
(Bruker AXS D8 advance) at 40 kV and 35 mA.
3.3.1.2 X-ray Photoemission Spectroscopy (XPS)
The XPS measurement was carried out with a Vg MICROLAB 310F spectrometer
using X-ray radiation (1486.5 eV). The emitted photoelectrons were detected using a
hemispherical analyser at a pass energy of 20 eV.
3.3.1.3 Environmental Scanning Electron Microscopy (ESEM)
Periodically, the immobilized HRP preparations were examined with a FEI Quanta 200
electron microscope equipped with energy-dispersive X-ray spectroscopy (EDAX Si(Li)
X-ray detector and Gatan Alto Cryo stage). ESEM is conducted under low vacuum and
so allows examination of wet specimens.
3.3.2 Characterization of products by mass spectrometry
The products of reaction were characterized by electrospray ionization (ESI) using a
Quattro Ultima Micromass LC/MS. The analysis was conducted using a feed rate of 10
L min-1, with settings for negative ions at a capillary energy of 2.5 kV and cone energy
of 80 V in MS scan mode. The source temperature was 120C and desolvation
temperature was 150C. Nitrogen was used as the desolvation gas at 550 L hr-1, and
Micromass MassLynx 3.5 software was used for data analysis.
3.3.3 Determination of carbon concentration in solution
The total organic carbon (TOC) was analyzed using a Sievers model 820 (Colorado,
USA) TOC analyzer. The TOC concentration was obtained by deduction of the
Page 46 of 163
inorganic carbon from the total carbon. The total carbon was obtained using 15%
ammonium persulfate (oxidizer) at a flow rate of 1 L min-1 and the inorganic carbon
using 6 M phosphoric acid solution (acid) at a flow rate of 0.75 L min-1. Limit of
detection of the carbon signal was determined to be 0.1 gC L-1 .
3.3.4 HPLC reverse phase chromatography
The extent of oxidation was determined by analysing the remaining substrate
concentration using Gilson instrumentation consisting of a Model GX-281 solventdelivery system with detection using a Model 156 UV-vis detector at 240 nm
(pentabromophenol and pentachlorophenol) and 280 nm (4-bromophenol). A
Lichrospher 100 RP-18 column (250 x 5 mm i.d., 5 m; Merck, Germany) was
employed. Analysis was performed using a gradient system, which increased the
relative amounts of methanol and acetonitrile present in aqueous 1 mM trifluoroacetic
acid (TFA) as described in sections 3.3.4.1 and 3.3.4.2.
3.3.4.1 4-Bromophenol analysis
The mobile phase consisted of component A (70% v/v distilled water, 15% v/v
methanol, 15% v/v acetonitrile in 1 mM TFA) and component B (20% v/v distilled
water, 40% v/v methanol, 40% v/v acetonitrile in 1 mM TFA) in the following program:
initially 100% A; linear gradient over 5 min to 100% B; held isocratically at 100% B for
5 min; linear gradient to 100 % A over 3 min; held isocratically at 100% A for 4 min.
The flow rate was maintained at 1 mL min-1 at ambient temperature (a typical
chromatogram and calibration curve are shown in Appendix B). Limit of detection of
the 4-BP signal area was determind as 0.01 M based on the signal:noise ratio
(approxamitly 3 to 1 respectively).
3.3.4.2 Pentabromophenol and pentachlorophenol analysis
The mobile phase for the pentahalogenated phenols consisted of component A (30% v/v
distilled water, 35% v/v methanol, 35% v/v acetonitrile in 1 mM TFA) and component
B (10% v/v distilled water, 45% v/v methanol, 45% v/v acetonitrile in 1 mM TFA) in
the following program: initially 100% A; linear gradient over 10 min to 100% B; held
isocratically at 100% B for 5 min; linear gradient to 100 % A over 6 min; held
isocratically at 100% A for 8 min. The flow rate was maintained at 1.5 mL min-1 at
ambient temperature (a typical chromatogram and calibration curve are shown in
Appendix C). Limit of detection of the PCP and PBP signal areas was determind as 1
M based on the signal:noise ratio (approxamitly 3 to 1 respectively).
Page 47 of 163
3.3.5 Characterization of products by gel permeation chromatography (GPC)

GPC analysis was performed at 140C using a Waters Alliance GPCV2000
chromatograph equipped with differential refractive index and viscometer detector.
1,2,4-Trichlorobenzene (TCB) was used as solvent at a flow rate of 1.00 mL min-1, the
system was calibrated with polystyrene sulfonate standards (925 185400 Da). The
chromatograms were processed using Millennium software.
3.3.6 Free and immobilized enzyme assay
The activity of soluble HRP was measured according to Sigma protocol SPPYO01 by
spectrophotometrically
monitoring
the
oxidation
of
1,2,3-trihydroxybenzene
(pyrogallol) at 420 nm. Pyrogallol is a colour assay substrate that produces long
coloured polymers which are easily monitored by absorbance at 420 nm.
For the immobilized HRP preparations, reaction mixtures (5 mL) consisted of 0.53 %
(w/v) pyrogallol, 0.027 % H2O2 in 14 mM sodium phosphate buffer pH 6.0 mixed with
10 mg immobilized HRP. After 1 minute (enough to produce a change in colour and
prevent the adsorption reaction overcoming the enzyme reaction) 3 mL of particle-free
solution was removed, the concentration of purpurogallin formed measured, and the
HRP activity calculated from:
A[U g -1 ] =
A 420s /60 sec - A 420B / 60 sec

12 m sb [g]
5[mL]
Equation 3-1
where: A 420S and A 420 B is the absorbance at 420 nm of sample and blank, respectively;
12 is the extinction coefficient (in a 1 cm cell) of purpurogallin at 420 nm, 5 mL is the
reaction mixture volume; msb is the dry weight of immobilized HRP.
3.3.7 Determination of anion concentration in solution
The extent of dehalogenation was determined by measuring anion concentration using
an ion conductivity chromatograph (ICC) (Dionex, USA). The anion exchange column
(MetroSEP anion dual column 15 cm; 3 mm i.d., 10 m) was run isocratically using 2.4
mM sodium bicarbonate, 2.5 mM sodium carbonate and 2% (v/v) acetone as eluent at
1.0 mL min-1 and 1430 psi (calibration curves are shown in Appendix D). Limit of
detection of the Cl- and the Br- signal areas was determind as 0.01 M and 0.05 M,
respectivly, based on the signal:noise ratio (approxamitly 3 to 1 respectively).
Page 48 of 163
3.3.8 Toxicity tests

The standard Microtox tests for the initial and treated solutions of PBP, PCP and 4-BP
were carried out by Geotechnical Pty Ltd and the Ecotoxicology Laboratory at RMIT
University. In the Microtox test, a bioluminescent bacterium (Vibrio fischeri) is used.
The luminescence emitted by the bacteria can be inhibited in the presence of toxic
compounds. The reduction in luminescence is regarded as a degree of toxicity.
Hydra toxicity tests for the initial and treated solutions of PBP and PCP were carried out
by the Ecotoxicology Laboratory at RMIT University. Hydra hexactinella is a
freshwater microinvertebrate with ubiquitous occurrence in freshwater environments.
The Hydra endpoint response to toxic solution is shown in Appendix E.
3.3.9 Analysis of H2O2
The concentration of the H2O2 was measured spectrophotometrically at 240 nm using an
extinction coefficient of 39.4 M-1 cm. The limit of detection (LOD - signal:noise ratio
was around 3:1) for H2O2 in MilliQ water was 0.001 mM while the instrument error was
1 M.
3.4
REACTOR SYSTEMS
The performance of the immobilized HRP in various reactors was evaluated in terms of
total organic carbon reduction and catalytic rate, as a function of the volumetric loading
rate and flow rate. Calculations were made according to the following equations:
S 0 -S e
100
S0
(S S e ) Q S 0 - S e
mg
app
)= 0
=
rate (
L min
H column
H
(min) = column
Q
Adsorbed carbon =
Equation 3-2
Equation 3-3
Equation 3-4
Where
Adsorbed carbon: degree of carbon reduction (%);
H column : empty reactor volume (mL);
: hydraulic retention time (min);
Q: feed flow rate (mL min-1);
S0, Se: TOC levels of initial and exit feed (mgC L-1), respectively.
Page 49 of 163
3.4.1 Continuously stirred batch reactor

The following batch experiments used a batch system which consisted of a glass beaker
with a stirrer. When UV radiation was used, the system was set up in a closed tank with
UV lamps mounted horizontally inside the lid, 30 cm above the surface of the samples.
The tank was cooled to 20C. The intensity of UV irradiation was measured at the
surface of the sample with a dosimeter (IL1400A, InternationalLight, Singapore).
3.4.1.1 Comparison of photocatalytic capacity of Tglass and a range of TiO2coated supports
Tglass (1 g) and a range of supports coated with TiO2 were placed in beakers (600 mL)
containing 50 mL 0.1 mM 4-BP and were irradiated with 2 UVB lamps (TL 20W/12,
320 nm) at an intensity of 1.4 mW cm-2 for 30 minutes. The supernatant was analysed
for TOC, 4-BP and pH. Before each run, the supports were activated in a furnace at
450C for 30 minutes.
3.4.1.2 Determination of pH and temperature range for HRP-Tglass
The activity of the HRP immobilized on Tglass particles was tested over pH range 2-13.
The reaction solution consisted of 0.1 mM 4-BP and 0.1 mM H2O2, and 1 M HCl and
1 M NaOH were used for pH adjustment. The reaction was at room temperature (22C)
and allowed to proceed for 24 h, after which TOC and 4-BP concentrations were
determined.
The activity of HRP immobilized on Tglass particles was tested over the temperature
range of 10-90C. The reaction solution consisted of 0.1 mM 4-BP and 0.1 mM H2O2 at
pH 7. The reactions proceeded for 24 h, after which TOC and 4-BP concentration were
measured.
3.4.1.3 Total adsorption capacity measurements
To determine the amount of 4-BP required to fully cover and deactivate the HRP-Tglass
by polymerization, 0.15 g HRP-Tglass was placed in a beaker and used to treat 250 mL
solution at a range of concentrations of 4-BP and H2O2 (0.04-0.22 mM, 1:1 molar ratio).
The system was run for 16 hours and the solution was monitored for TOC and 4-BP.
Page 50 of 163
3.4.1.4 Evaluation of UVB and UVA irradiation for H2O2 formation
The HRP-Tglass was suspended in 100 mL of MilliQ water in the batch reactor, agitated
and irradiated with UVB (460 W cm-2). Samples were taken for analysis at 0, 15, 45,
60 min. The concentration of the H2O2 was measured as in section 3.3.9.
3.4.1.5 Evaluation of UVB irradiation on product formation using pyrogallol
with free and immobilized HRP
Pyrogallol solutions (50 mL at concentration of 1-75 mg L-1) in 14 mM sodium

phosphate buffer pH 6.0 were irradiated with UVB (TL 20W/12, 320 nm) at a
received intensity of 460 W cm-2 (see 3.4.1.1).
HRP-Tglass was suspended in 100 mL of 0.1 mM 4-BP in the batch reactor, agitated and
irradiated with UVB (460 W cm-2) for different durations (5-960 mins). The solution at
the end of each run was analysed for 4-BP concentration and the activity of the
immobilized enzyme was then determined as described in section 3.3.6.
3.4.2 Plug flow reactor system
The plug flow system consisted of a 0.2 mL column (internal diameter 2.46 mm x
length 42.1 mm) filled with 0.15 g of particles of HRP immobilized on a support (PG or
Tglass). The column received a constant upflow feed of 4-BP mixed with H2O2 supplied
from a room temperature reservoir using a peristaltic pump (Gilson Miniplus 3) and the
effluent was passed to a sample collector (Gilson).
3.4.2.1 Evaluation of UVB irradiation for removal of adsorbed polymers
UVB irradiation was investigated for the removal of adsorbed oligomers from the
immobilized enzyme after treatment of 4-BP. Firstly, the immobilized preparations
(0.15 g) were loaded with polymer by placing them in a column (0.2 mL) and feeding
with 4-BP plus H2O2 at the same concentration over a range of 0.1-10 mM. The reaction
was allowed to proceed for 16 h after which the total organic carbon (TOC)
concentration of the effluent and the activity of the immobilized enzyme preparation
were determined. The immobilized preparations were then washed thoroughly to
remove non-adsorbed material and suspended in MilliQ water (25 mL). They were
subjected to UVB irradiation (TL 20W/12, 320 nm) at a received intensity of 460 W
cm-2 (see 3.4.1.1) for up to 96 hours. The activity of the immobilized enzyme was then
determined as described in section 3.3.6.
Page 51 of 163
3.4.2.2 Determination of influence of concentration on the system kinetics
Feed (100 mL) containing different concentrations of 4-BP (2-160 mgC L-1, 0.02-2.2
mM) with H2O2 at a molar ratio of 1:1 was run through 0.2 mL columns containing
either HRP-Tglass or HRP-PG at a range of flow rates between 1.75 and 8 mL min-1. The
effluent was collected in 20 mL fractions and the concentrations of TOC and bromide
were measured, providing 5 fractions for each combination of flow rate and
concentration.
3.4.2.3 Determination of influence of flow rate on the system kinetics
Feed (100 mL) containing 0.1 mM H2O2 and 0.1 mM 4-BP was run through a column
(0.2 mL) containing HRP-Tglass or HRP-PG at a range of flow rates between 0.25 and
7.5 mL min-1. The effluent was collected in 20 mL fractions and the concentrations of
TOC and bromide were measured, providing 5 fractions for each flow rate.
3.4.3 Packed bed under constant irradiation
Under constant UV irradiation feed was passed through an empty column (25 mL) and
then through a 10 mL column filled with HRP-Tglass particles (2.5 g). Equilibration of
the system with the feed was carried out with 2 system volumes. Analysis of the
effluent at sampling points A and B (Figure 3-1) was carried out.
HRP-Tglass column
UV Chamber
Peristaltic
pump
Empty column
Feed
reservoir
Sample
collection
B
Sample
collection
A
Figure 3-1. Schematic representation of the HRP-Tglass packed bed under UV radiation. The system
consisted of a 25 mL empty glass column followed by a 10 mL column filled with HRP-Tglass.
3.4.3.1 H2O2 production
To investigate the effect of UVA and UVB on the performance of the reactor with
regard to H2O2 production, a column (10 mL) was filled with 2.5 g of HRP-Tglass and
Page 52 of 163
run under UVA (intensity of 570 W cm-1), peak wavelength 365 nm, NEC) or UVB
(intensity of 1.4 mW cm-2, peak wavelength 320 nm, Sankyo Denki) irradiation at a
range of flow rates over 0.5-7.5 mL min-1. The concentration of the H2O2 exiting the
packed column was measured as in section 3.3.9.
3.4.3.2 4-BP transformation
The packed bed system as described in 3.4.3 (2.5 g HRP-Tglass) was fed with 500 mL of
4-BP (0.1 mM, 17.3 mg L-1 in MilliQ water) at flow rates of 0.5 - 9 mL min-1 in upflow
mode. The effluent (100 mL fractions) was analysed for 4-BP, TOC and bromide.
3.4.3.3 PCP and PBP mixture transformation experiments
The packed bed system as described in 3.4.3 (2.5 g HRP-Tglass) was fed with a mixture
of pentabromophenol and pentachlorophenol (8 and 10 mg L-1 respectively) in reservoir
water from Forest Reservoir (Victoria, Australia) at flow rates 0.5 and 1.25 mL min-1 in
upflow mode under UVB irradiation (1.4 mW cm-2). The effluent was measured for
halogenated phenol and bromide concentration, molecular mass of product, and toxicity
(MicrotoxTM) of the initial and treated water.
The experiment was repeated using the same conditions for concentrations of PBP and
PCP of 0.67 and 0.89 mg/L, respectively. The effluent was measured for toxicity.
3.4.3.4 PCP and PBP(separated and as a mixture) toxicity experiments
The packed bed system as described in 3.4.3 (2.5 g HRP-Tglass) was fed with
pentabromophenol and/or pentachlorophenol (1 mg L-1) in MilliQ water at a flow rate of
0.5 mL min-1 in upflow mode under UVB irradiation (1.4 mW cm-2). The effluent was
measured for concentration of halogenated phenol and toxicity (Microtox and Hydra)
of the initial and treated water.
Page 53 of 163
Chapter 4. A comparative study of the action of TiO2 in a

number of glass supports
4.1
INTRODUCTION
Among the many supports that are available for immobilization of enzymes, porous
glass containing boron has been favoured due to its tolerance to heat and pH, ease of
manipulation, its high surface area available for the designated enzyme and above all,
its lack of reactivity to the substrate and reaction products (Yazawa et al., 1999).
However, for HRP, it has not been very successful as the polymerized products are
adsorbed on the enzyme support, covering the enzyme as well as the glass. For this
project, glass containing TiO2, named Tglass, was developed to deal with the adsorption
problem. In section 2.3.3, the photooxidation capabilities of TiO2 were mentioned.
Before the HRP is immobilized on this support, it is imperative to determine the
photocatalytic properties of the Tglass.
A number of glass supports (porous glass, casamic and a microscope slide) were coated
with TiO2 and compared with the Tglass using UV irradiation to initiate
photodegradation. The system was investigated using 4-bromophenol (4-BP) as a model
substrate in a batch reactor. This treatment resulted in the formation of catechol and
hydroquinone (Figure 4-1), which are known to be readily treated by conventional
wastewater treatment methods such as anaerobic biodegradation (Latkar et al., 2003).
However, in addition to the biodegradable compounds this treatment also produced 2BP which has similar LD50 to the 4-BP and is nephrotoxic (see section 2.1.1).
Figure 4-1. The photooxidation products of 4-BP.
Further investigation of the newly synthesized Tglass was then conducted. EDX mapping
showed that the surface comprised patches containing silicon and titanium. Those
Page 54 of 163
patches were able to be modified using acid and base which influenced the level of
immobilized HRP activity.
4.2
4.2.1 Characteristics of the Tglass support

As discussed in section 2.4.1 Tglass was synthesized to assist the HRP with the treatment
of phenolic compounds. It was expected that the TiO2 incorporated in the glass would
photooxidize the adsorbed products caused by the HRP polymerization freeing the
enzyme to continue to be active. Therefore, the logical first step was to ensure that it
could be used for photocatalysis.
The steps that were taken to measure the photocatalyic capability of Tglass were:
1. determination of the anatase/rutile ratio.
2. investigation of the surface of the support to determine the amount of exposed TiO2.
3. determination of the photooxidative activity of TiO2 on the test supports.
4.2.1.1 The anatase/rutile ratio
As discussed in section 2.3.1, there are 2 major crystal components of TiO2: rutile and
anatase. Anatase has a higher band gap (3.26 eV) than rutile (3.05 eV) (Diebold, 2003)
and so a higher content of anatase makes the TiO2 a better photocatalyst (Linsebigler et
al., 1995). Today, the best known TiO2 photocatalyst is Degussa P25 which consists of
approximately 80% anatase and 20% rutile (Carp et al., 2004). This ratio makes it a very
good photocatalyst; nevertheless even with a low anatase content TiO2 can exhibit
photooxidation capability.
By employing X-ray diffraction (XRD) on the Tglass (see section 3.3.1.1), the crystal
coordinates and space groups (Table 4-1) were found to be primarily of rutile and
anatase structure (structure determination was achieved using the Bruker AXS D8
advance database). The distribution of these groups is shown in Figure 4-2. Using
these data, the ratio of the anatase to rutile was found to be 3:7. This proportion of
anatase was higher then expected, as at temperatures greater than 800C anatase is
converted to rutile (Clark, 1968). Toma et al. (2004) found that after 24 h at 700C
Degussa P25 TiO2 powder was completely converted to rutile. However, when the TiO2
Page 55 of 163
was doped with more than 20% SiO2, the conversion was inhibited; Kim et al. (2006)
claimed this was because the SiO2 is embedded in the TiO2 grains.
Table 4-1 The characteristics of the crystal properties of the TiO2 in Tglass.
Rutile
Anatase
Crystal structure
Tetragonal
Tetragonal
Unit cell a ()
4.6
3.8
Unit cell b ()
4.6
3.8
Unit cell c ()
2.9
9.5
Angle cell (alpha - beta 90
90
gamma)
Space group
Primitive-P42/mnm (136)-2
Body-centered-I41/amd
(141)-4
Figure 4-2. XRD spectrum of the Tglass particles (anatase rutile).

Page 56 of 163
This low ratio of anatase to rutile suggests that the photocatalytic capacity of the Tglass
may be limited; however, this does not imply that it will be inactive. TiO2 preparations
with nearly 100% rutile content have been reported to have photooxidative activity
(Watson et al., 2003).
4.2.1.2 The analysis of the topography of Tglass
The surface properties of TiO2 are also very important determinants of its photocatalyic
capacity: the higher the surface area, the higher the number of available active sites.
However, as the surface requires direct interaction with a light source, any deformation
in the surface, which although it increases the number of sites, can also shadow the
active sites. ESEM (see section 3.3.1.3) showed that the surface of the Tglass has two
major region types: smooth and rough (Figure 4-3 (A, B)). Comparison of Figure 4-3
(A) and (B) suggests that the rough area is comprised of TiO2. EDX mapping with XRD
confirmed that the rough region is made of Ti (Figure 4-3 (C)) and the smooth region of
Si (Figure 4-3 (D)).
Page 57 of 163
30 m
Figure 4-3. ESEM image of Tglass (A) and PG (B); EDX mapping based on ESEM image of Tglass (A),
clearly shows Ti (C) and Si (D). The bright areas show the location of the element (scale 30 m for all 4
images).
The mosaic pattern is caused by the synthesis process of the Tglass. The TiO2 melts at
1825C according to Sigma MSDS (2009) while the glass components were liquefied at
< 1000C, so the TiO2 particles become embedded in a glass matrix. The result of this
embedding is a reduction in the available sites that can be irradiated.
X-ray photoemission spectroscopy (XPS) (see section 3.3.1.2) of the Tglass surface (area
of 1 mm x 5 mm examined) showed that it was composed mostly of Ti, Si, Na and
oxygen (X-ray penetration length was 0.5 nm) (Figure 4-4). Traces of carbon and boron
were also observed, however, due to the signal to noise ratio their contribution to
overall element content was minimal. Based on these data and the knowledge that the
TiO2 did not melt within the glass component, the Ti/Si ratio was calculated by the XPS
software (Thermo Avantage V4.38) to be 0.13. This means that only 13% of the Tglass
surface is made of TiO2 and can be used as a photocatalyst. The proportion of anatase on
the surface can be calculated as 4% (the percentage of anatase times the TiO2
percentage on the surface), which is very low. The anatase/rutile distribution ratio was
Page 58 of 163
calculated as an average for the whole particle. It may also be that the anatase on the
surface was more readily converted to rutile due to direct exposure to the furnace heat,
meaning it may be possible that the proportion of anatase on the surface is even lower.
700000
O
Counts / s
525000
OKLL
Na
350000
NaKLL
Si
175000
B
Artifact
signal
Ti
C
0
0
350
700
1050
Binding energy (eV)
1400
Figure 4-4. XPS analysis of Tglass surface which clearly shows the presence of Ti, Si, Na and O (X-ray
penetration was 0.5 nm). NaKLL and OKLL are the element transitions obtained from their initial energy.
4.2.1.3 Determination of the photooxidative activity of TiO2 associated with

various supports
The photocatalytic efficiency of the Tglass and the original TiO2 was compared. As the
original TiO2 was in powder form (0.5-2 m) and so hard to recover, it was used as a
coating on various support structures (commercial glass and custom-made glasses). A
commercial microscope slide was also tested as a control support, with the knowledge
that it may be the least efficient support as it had the lowest surface area (0.002 m2 g-1).
The surface areas of the custom-made glasses were determined to give an indication of
the amount of TiO2 which could be held on the support (the greater the area, the greater
the amount of TiO2). The PG had a surface area of 25 m2 g-1, and the Tglass had a surface
area of 9.4 m2 g-1. The casamic (section 3.2.3), a newly developed material, holds great
interest as a possible support. Its synthesis from commercially available glass fragments
bound together at low temperature (120C) is advantageous in terms of cost. The use of
recycled broken window glass with minimal energy seems to sit well with the spirit of
this research. The casamic had half the surface area (12 m2 g-1) of the PG, which makes
it a reasonable alternative for the PG in terms of cost of production and energy use.
Page 59 of 163
Tglass (1 g) and the TiO2 coated supports (holding approximately 0.5 g of TiO2,
according to section 3.2.5) were investigated for their photocatalytic efficiency
according to the method described in section 3.4.1.1 using UVB irradiation at an
intensity of 1.4 mW cm-2 for 30 minutes. The solution was maintained as a thin layer (1
mm deep) over the support in each beaker, hence each would experience almost similar
intensity. The irradiation control comprised a solution without support.
The extent of reaction of the photooxidation of 4-BP was measured as the concentration
of the remaining 4-BP and bromide ions using HPLC and ICC, respectively (no 4-BP
adsorption to the support was detected). Under UVB irradiation of the solution only
there was sufficient energy to transform up to 40% of the 4-BP and release up to 30% of
the bromine (Figure 4-5). The results show that casamic, the Tglass and porous glass
were the best supports for the transformation of 4-BP. The casamic support was
problematic as the concentration of bromide released to solution was higher than the
amount potentially available from the 4-BP, which suggests that some contamination
came from the support. That can be explained by the weakening of the ceramic
structure, resulting in crumbling, loss of the TiO2 coating, and the release of ions from
the casamic which interfered with the analysis of bromide release from reaction.
Page 60 of 163
100
Debromination
4-BP transformed
pH levels
80
8
6
4
pH
60
40
2
20
0
0
A
blank
Figure 4-5. pH, 4-bromophenol and bromide transformation after exposure of supports to 4-BP (0.1 mM)
and UVB irradiation (average results, n=3) A: Tglass, B: casamic, C: porous glass, D: low surface area
glass particles (ground microscope slide), E: microscope slide, blank: no support (just UVB irradiation of
the solution).
Further analysis of the results suggests that bromine detachment is the main process in
the reduction of 4-BP concentration. The pH results showed that, as expected, the pH
decreased from its original level (5.77). However the pH is related to the bromide
concentration, the lower the bromide level, the lower the pH. This may be attributed to
formation of products that raise the pH, such as 2-bromophenol (2-BP) which was
detected by HPLC after UVB radiation of 4-BP using PG and Tglass (Figure 4-6). The
importance of this is that after the bromide was released, it reacted with the
dehalogenated substrate and formed the 2-BP. Although the 2-BP has a similar toxicity
as 4-BP as shown in section 2.1.1, it has much more devastating effects as 2-BP is
nephrotoxic. This may result in increasing, instead of reducing, toxicity.
Of all the supports, the Tglass produced the higher concentrations of 2-BP and catechol.
This shows that the Tglass, although a better photocatalyst than the other two, cannot be
used alone for detoxification of 4-BP.
Analysis using ESI-MS and HPLC confirmed that the products of the UV-mediated
reaction were hydroquinone and catechol. Similar results were observed by Sivalingam
et al. (2004) after irradiation of chlorophenols in the presence of Degussa P-25 TiO2
under similar conditions.
Page 61 of 163
Detector Response Units
36000
Hydroquinone
casamic
PG
4-BP
27000
Catechol
18000
Tglass
2-Bromophenol
4-Bromophenol
9000
0
0
10
Time (min)
15
20
Figure 4-6. Typical HPLC chromatogram of the products obtained according to the method described in
section 3.3.3.1 and Appendix B. These 4 traces are for treated and untreated 4-BP using TiO2 coated PG
and casamic after UVB irradiation for an hour.
Although the Tglass had a low proportion of anatase and a low surface area, it
demonstrated a greater capacity for 4-BP transformation (95%) than any of the other
supports coated with TiO2. The TiO2-coated supports retained their coating integrity
whereas the casamic tended to crumble in solution. Consequently, casamic and
microscope slide (poor results) were not used further.
Many researchers have investigated TiO2 supported on glass in various reactors for the
treatment of phenols and halogenated phenols. For example, Matthews and McEvoy
(1992), Trillas et al. (1996) and Yawalkar et al. (2001) investigated phenol; Theurich et
al. (1996) and Pelizzetti et al. (1990) investigated 2-chlorophenol, 4-chlorophenol, 2,4dichlorophenol. The photocatalytic activity of these systems was determined by
transformation of these compounds.
Overall, the Tglass possesses photocatalytic capability, however that ability is low
compared with other researchers results, such as those of Sun et al. (2003). They treated
phenol (0.1 g in 800 mL distilled water) in a fluidized bed reactor, achieving full
mineralization in 250 minutes using only 0.2 g of Degussa P25 TiO2 spiked with
platinum (0.1% Pt).
Page 62 of 163
4.2.2 Tglass as a support for enzyme immobilization

Whenever a support is designed for enzyme immobilization, there are several
requirements (Penosil et al., 2002). Mainly it must have a high surface area, and be
sufficiently robust to withstand changes in the environment such as flow and
temperature, as well as resist structural degradation. Furthermore, if a ligand is to be
used to link the enzyme to the support, the support must have minimal interaction with
the enzyme (Hermanson et al., 1992),
Tglass was designed to satisfy these requirements. The surface area results (measured
according to section 3.2.4 and Appendix A) for the Tglass particles were significantly
lower than for the PG (Table 4-2). Consequently the activity of immobilized HRP was
also low: HRP-Tglass activity was 0.01 U g-1 compared with 0.095 U g-1 for HRP-PG.
In addition to the surface area, the pore size also contributes to the amount of enzyme
which can be immobilized on the support. The pore size determines the access of the
enzyme, and the diffusion of the substrate, into the support structure and so affects the
activity of the immobilized enzyme. There is a narrow pore size range over which
optimal activity can be obtained (Ralph, 1974; Stratilov et al., 1989): a small pore size
will prevent the access of the enzyme, while a larger pore size will reduce the area
available for enzyme. The molecular weight of HRP is 44,000 Da (Veitch, 2004) and
this corresponds to a protein molecule of approximately 53 diameter (University of
Bremen, 2009). However, both Ralph and Stratilov found that the pore size for
maximum activity is approximately 2-3 times the size of the enzyme, hence the pore
size for maximum HRP activity should be 120-180 . This is close to what Thibault et
al. (1981) found, they reported that the pore diameter was between 15-25 nm for the
highest loading yield of HRP on the PG. As the average pore sizes for the supports
immediately after synthesis were too low for maximum activity (Table 4-2), they were
treated to see if the size of the pores could be increased.
To increase the pore size, the supports were subjected to alkaline etching (section 3.2.6)
which led to smoothing of the SiO2 and TiO2 surfaces. Acid treatment (similar to
method in section 3.2.6, 1 M HCl was used instead of 1M NaOH) led to increasing
roughness and the formation of crevices in the SiO2 patches which may increase the
number of binding sites, but reduced the PG particle size from an average of 125 m to
approximately 90 m. The surface characteristics of the supports and the HRP activity
(section 3.3.6) after immobilization are shown in Table 4-2.
Page 63 of 163
Table 4-2 Surface characteristics of Tglass and PG and HRP activity after immobilization
Original state
Surface
area
(m2 g-1)
Tglass 9.4
25
PG
Alkaline etched
Acid etched
Average
pore
size ()
Enzyme
activity
(U g-1)
Surface
area
(m2 g-1)
Average
pore
size ()
Enzyme
activity
(U g-1)
Surface
area
(m2 g-1)
Average
pore
size ()
Enzyme
activity
(U g-1)
70
0.01
28
170
0.6
42
100
0.3
62
0.095
40
121
0.13
N.T.
N.T.
N.T
N.T. Not tested as acid treatment of PG resulted in particle breakdown.
Alkaline etching of the Tglass led to an increase in both surface area and pore size, and
thus greater increase in the activity of the immobilized HRP compared with acid
etching. The higher activity of HRP immobilized on PG compared with Tglass after
alkaline etching was attributed to the higher surface area of the PG.
50 m
50 m
a
Figure 4-7. (a) Tglass after NaOH treatment (scale 50 m) (b) Tglass after HCl treatment (scale 50 m)
Sodium hydroxide smooths the entire surface as it etches the surface of both the TiO2
and the SiO2 patches (Figure 4-7(a)), while the HCl attacks the SiO2 leaving large
crevices and tunnels up to 2 m wide Figure 4-7(b).
A drawback was detected for the HCl treatment. Using EDX (Figure 4-8) on the surface
of acid-modified Tglass, the presence of high levels of chlorides was detected. The
problem is that chloride can become an electron donor like O2 and water, forming
chloride radicals instead of hydroxyl radicals (Grabner et al., 1991) and which can then
form chlorinated products that may have higher toxicity (section 2.1.1). Tatsuma et al.
(2001) elaborated on this idea further by theorizing that chloride competes with O2 and
water for a spot on the surface of the photocatalyst, thus reducing the number of
hydroxyl radicals which can either continue to react with water to form H2O2 or migrate
to the surface of the support and provide remote oxidation of target pollutants ().
Page 64 of 163
Detector response %
100%
Chlorine peaks
Binding energy (eV)

Figure 4-8. EDX of Tglass surface after acid treatment.
4.3
In this chapter, the photocatalytic ability of Tglass was examined and its suitability for the
immobilization of active HRP was determined.
ESEM imaging showed that the Tglass consists of TiO2 patches surrounded by glass.
Although the content of TiO2 on a weight basis was 50%, XPS measurement showed
that TiO2 comprised only 13% of the surface area. The TiO2 itself consists of 70% rutile
and 30% anatase, this in combination with the low area of the TiO2 on the surface
suggested that the Tglass would have low photocatalytic properties. However using 4bromophenol as a substrate for photooxidation, the Tglass behaved better than the other
supports surface-coated with TiO2. The reaction products were catechol, hydroquinone,
2-bromophenol and bromide, as expected. Overall, the Tglass has photocatalytic
properties but is less effective than the best current available photocatalysts (i.e.,
Degussa P25 or Pt-TiO2).
It was shown that the Tglass can act as a support for HRP. The surface area of Tglass was
increased using acid or base treatment. Although the alkali-treated Tglass had a lower
Page 65 of 163
surface area (28 m2 g-1), it had a larger pore size (170 ) which gave higher HRP
activity after immobilization (0.6 U g-1) than the acid treated Tglass (0.3 U g-1).
In conclusion, the Tglass can act as a support for the HRP and can also act as a
photocatalyst. This combination was further studied in the next chapter in terms of
HRP-Tglass characteristics and behaviour with and without UVB irradiation.
Page 66 of 163
Chapter 5. Determination of the role of HRP immobilized

on Tglass with and without UV radiation
5.1
INTRODUCTION
In chapter 4, Tglass was described with regards to its photocatalytic properties and its role
as a support for HRP. It was demonstrated that it can carry the enzyme and allow the
enzyme to be active in the same manner as a regular PG support. In section 2.2.5 it was
explained how the immobilization of peroxidase leads to its deactivation as the
polymerized products are adsorbed and cover the active sites.
This chapter details investigation of the activity of horseradish peroxidase immobilized
on Tglass (HRP-Tglass) under UV irradiation. HRP-Tglass was used to treat halogenated
phenols with and without UV irradiation, and the ability to protect the enzyme against
deactivation was tested. The UV irradiation assists by hydrophilic repulsion to remove
the hydrophobic polymeric products formed by the enzymatic polymerization by
increasing the hydrophilicity of the surface of the titanium dioxide in close proximity to
the enzyme, and thus assists in restoration of the enzyme activity.
In this chapter two types of supports: PG and Tglass, were compared. The HRP
immobilized on these supports was used to treat pyrogallol and 4-bromophenol (4-BP)
which were used as model substrates. The reaction involved the conversion of the
model substrates in either the presence of H2O2 or UVB radiation to polymeric form,
this reaction is accompanied by dehalogenation of the 4-BP.
Two types of reactors were used, a plug flow reactor (3.4.2) and a stirred batch reactor
(3.4.1). The plug flow reactor was used to measure the transformation of the substrate
with time, and the batch reactor was used to study the reaction in a steady state
condition.
5.2
5.2.1 Analysis of support after enzymatic reaction

Two HRP supports were used to treat 4-BP (0.1 mM) plus H2O2 (0.1 mM) in a beaker
for two hours.
The support characteristics before and after the polymerization were investigated by
ESEM (Figure 5-1) and XRD (Figures 5-2 and 5-3) (section 3.3.1.3 and 3.3.1.1,
Page 67 of 163
respectively). After polymerization the smooth surface of the HRP-PG had a layer that
contained fractures (Figure 5-1 c), this was not so for HRP-Tglass for which the surface
was too rough to distinguish any differences.
100 m
Figure 5-1. ESEM topography of typical particles of HRP-PG (left column) and HRP-Tglass (right column)
before (top row) and after (bottom row) the polymerization process with 4-BP, scale is 100 m for all
images.
By using EDX imaging of the HRP-Tglass (Figure 5-2) and HRP-PG (Figure 5-3) clear
visualization of the elements that were present on the surface (within a depth of 1 m)
after polymerization was possible. These results were obtained for many particles and
so reflect an average for the sample. These results were obtained for many particles and
so reflect an average for the sample. As the enzyme polymerizes the 4-BP, the product
increases in bromine content. It was stated in the literature review that the polymeric
product tends to be adsorbed and cover the enzyme active sites, thus where there is an
increase in organic mass that is where the enzyme is most active. EDX is not very
sensitive for measuring carbon, however bromine can represent the area where the
enzyme is most active.
Page 68 of 163
Figure 5-2. The EDX spectra of HRP-Tglass surface (A) prior to the reaction, (B) after 4-BP
polymerization.
Page 69 of 163
Figure 5-3. The EDX spectra of HRP-PG surface (A) prior to the reaction, (B) after 4-BP polymerization.
Using EDX analysis (an ESEM option) on the HRP-Tglass after polymerization,
distribution of the elements on the surface was obtained (Figure 5-4). Mapping for
Page 70 of 163
carbon showed that it was evenly distributed across the HRP-Tglass, as expected due to
the presence of the immobilized enzyme. Bromine was found concentrated on the SiO2
regions, indicating the presence of adsorbed polymer and thus the location of active
enzyme. The limited size of the SiO2 regions, combined with the hydrophilicity of the
TiO2 regions which should repel the nascent hydrophobic polymer, means that the size
of the polymer would be somewhat limited. Silicon, oxygen, carbon and bromine were
uniformly distributed across the HRP-PG samples, consistent with uniform distribution
of the active enzyme.
20 m
Figure 5-4. ESEM imaging and EDX mapping of HRP-Tglass after the polymerization process (a) scanned
image, (b) titanium, (c) silicon, (d) bromine, (e) carbon, (f) oxygen. All the elements are shown as bright
spots, scale is 20 m for all images.
5.2.2 Determination of pH and temperature ranges of HRP immobilized on Tglass

In a stirred batch reactor HRP-Tglass particles were used to treat 0.1 mM 4-BP plus 0.1
mM H2O2 for 24 h over a range of pH and temperature conditions (Figure 5-5 and
Figure 5-6, respectively), the solutions were analysed for TOC and 4-BP concentration
(section 3.4.1.2). The enzyme worked best in the pH range 3-11 for 4-BP transformation
and degree of polymeric product adsorption (measured as the loss in carbon content,
section 5.2.3). Temperature had only a slight effect on the enzyme performance up to
60C after which the activity declined. Previous research (Meizler, 2005) showed that
HRP immobilized on porous glass demonstrated high activity in transforming 4-BP at
the pH and temperature range of 7-9 and 10-60C, respectively. The HRP on Tglass has a
wider pH range, as discussed below.
Examination of the literature showed that greatest activity of free HRP is at pH 8-9 (Lai
and Lin, 2005) and temperature 25-30C (Vasileva et. al., 2009). Thus immobilization
Page 71 of 163
of the HRP on Tglass increased the range of pH and temperature over which the enzyme
is active. This would be advantageous in industrial applications.
HRP immobilized on Tglass has activity over a wider pH range than obtained by other
researchers. Lai and Lin (2005) used HRP immobilized on PG and obtained a pH range
of 7-9 for pyrogallol conversion, Cheng et al., (2006) used HRP immobilized on clay
and obtained a pH range 5-9 for converting phenol, and Bayramoglu and Arica (2008)
used HRP immobilized on magnetic beads and obtained a pH range of 5-8 for treating
4-chlorophenol. As discussed by Penosil et al. (2005) the pH normally alters the free
enzyme oxidation number and its structural configuration. However when the enzyme is
covalently bound to a rigid surface, the change is dependent on the strength of the bond
and the diffusion layer as the mass transfer through a liquid diffusion film from the bulk
liquid to the surface of the biocatalyst has a significant effect on biocatalyst activity.
With regards to temperature, all the previously mentioned researchers showed that the
activity of their immobilized HRP was highest over a temperature range of 10-60C,
which is similar to the results shown here. As free enzymes are proteins they are
typically denatured at temperatures higher then 40C (Johannes et al., 2006). The
immobilization increased that limit to 60C as the enzymes are able to
retain their
native geometry and microenvironment, and are protected against changes in the bulk
Degree of transformation (%)
system (temperature, pH, ionic strength, mechanical stress) (Penosil et al 2002).
100
75
50
25
Transformed 4-BP
Removed TOC
0
0
10
15
pH
Figure 5-5. The effect of pH on the transformation and degree of adsorption of products. HRP-Tglass
treated 4-BP (0.1 mM) and H2O2 (0.1 mM) in a stirred batch reactor for 24 h.
Page 72 of 163
Degree of transformation (%)
90
80
70
60
50
40
30
20
10
0
Transformed 4-BP
Removed TOC
20
40
60
Temperature (C)
80
100
Figure 5-6. The effect of temperature on the transformation and degree of adsorption. HRP-Tglass treated
4-BP (0.1 mM) and H2O2 (0.1 mM) in a stirred batch reactor for 24 h.
There was approximately 10% difference between the transformation of 4-BP and the
degree of adsorption. That difference represents the soluble portion of transformed 4BP, i.e., the portion which is not adsorbed to the HRP-Tglass, which will be investigated
in the following sections.
5.2.3 Determination of adsorption capacity of immobilized HRP at equilibrium
To determine the HRP-Tglass adsorption capacity (section 3.4.1.3), HRP-Tglass particles
(0.15 g) were used to treat a range of 4-BP concentrations (0.04-0.22 mM) with H2O2 in
a 1:1 molar ratio in a stirred batch reactor (50 mL) until no further activity was detected
(i.e., they were fully covered by adsorbed product).
Figure 5-7 shows the correlation between the adsorbed mass at equilibrium (no further
activity detected) and the initial concentration of 4-BP. The difference between the mass
of carbon adsorbed and the mass of carbon as transformed 4-BP is relatively constant
and must equate to the soluble fraction of carbon (4-BP transformed but not adsorbed).
In a normal immobilized enzyme reaction the products are quickly released to the
aqueous medium, allowing new substrate compounds to enter the enzyme active site,
thus providing a constant reaction. However, the peroxidase reaction produces higher
mass polymers that are adsorbed to the matrix (as explained in section 2.2.5). Due to the
constant difference between the masses of the transformed to the adsorbed 4-BP, it may
be concluded that following the formation of soluble product, a local saturation of those
Page 73 of 163
products must occur prior to their adsorption. The total 4-BP mass was calculated by
multiplying the initial concentration and the feed volume.
Adsorbed 4-BP
Transformed 4-BP
Theoretical 4-BP mass
-1
Mass on particles (mg g )
140
105
70
35
0
0
0.05
0.1
0.15
0.2
Initial 4-bromophenol concentration (mM)
0.25
Figure 5-7. Adsorption of end-products in an HRP-Tglass batch reactor
The maximum adsorption and transformation was measured to be 36 and 42 mg of

substrate per g HRP-Tglass, respectively.
The equilibrium adsorption of 4-BP and transformation exhibited an isotherm pattern
which was described by Giles and co-workers (1960) as a unimolecular adsorption
which normally applies to microporous adsorbents. The model that can express this
pattern was developed by Langmuir (1916), the Langmuir equation is expressed below
(Equation 5-1):
q=
app
BP0 ads q max
1 + ads BP0
Where:
q: mass of material accumulated on the particles (mg);
app
q max
: apparent maximum adsorbed capacity (mg);
BP0: initial concentration of 4-BP (mM);

ads: adsorption constant.
Page 74 of 163
Equation 5-1
Although there are factors which indicate that this model is not appropriate, the
Langmuir isotherm describes the adsorption and desorption of a substrate in
equilibrium. Here the products are adsorbed to the support but they are not desorbed,
and the reaction depends on the enzyme activity (producing the adsorbed products).
Despite this, the data fitted the model. Linear regression of the Langmuir isotherm i.e.,
BP0 BP0
1
= app +
app
q
q max ads q max
Equation 5-2
provided values for the maximum adsorption capacity of q app

max = 34 mg and ads = 89 for
app
the constant (Figure 5-8). The calculated q app
max (34 mg) and the measured q max (36 mg)
were found to be relatively similar.
4-BP/total mass on particles

(mM mg-1 g)
0.008
0.007
0.006
y = 0.02927x + 0.00033
R2 = 0.9916
0.005
0.004
0.003
0.002
0.001
0
0
0.05
0.1
0.15
0.2
Initial 4-BP concentration (mM)
0.25
Figure 5-8 Linear regression of Langmuir equation for the adsorption of the reaction products to the HRPTglass particles in a batch reactor.
5.2.4 Generation of hydrogen peroxide by HRP-Tglass under UVB irradiation

UV irradiation of TiO2 surfaces in the presence of water produces hydroxyl radicals that
can produce H2O2 (as described in section 2.3.3). HRP in the presence of H2O2 can
transform halogenated phenols (section 2.2.2). Hence the next step was to see if under
UV irradiation H2O2 can be produced in sufficient quantities to activate the HRP.
To determine how much H2O2 was generated at the surface of the TiO2 particles
incorporated within the Tglass under UVB and UVA irradiation, HRP-Tglass was
suspended in MilliQ water in a batch reactor without agitation and irradiated for varying
periods (see section 3.4.1.4). H2O2 production was low for UVB and none occurred for
Page 75 of 163
UVA (Figure 5-9) due to low transmission of the light through the glass reactor walls
(measured intensity for UVB was 290 W cm-2). However, the measured concentration
of H2O2 reflects a bulk value for the solution. Due to the close proximity of the TiO2 to
the enzyme on the Tglass, it is hypothesized that the peroxide concentration in the
immediate vicinity of the enzyme would have been markedly higher, possibly sufficient
to activate the enzyme for successful transformation and thus treatment of the phenolic
pollutant. Not only would this reduce the amount of H2O2 required to be added to the
system, but the formation of H2O2 in situ would have the advantage of reducing HRP
deactivation due to the presence of excess H2O2 (Hiner et al., 1995) as may occur when
it added as a reactant.
0.025
H2O2 (mM)
0.02
0.015
0.01
0.005
0
0
20
40
60
80
Radiation dose (min)

Figure 5-9. Influence of UVB irradiation dose on the formation of H2O2 in the presence of HRP-Tglass .
5.2.5 The effect of UVB/HRP oxidation on pyrogallol polymerization with and

without Tglass
UV irradiation affects not only the production of H2O2, but can photooxidize organic
material (substrate and product alike). The absorbance spectrum of a 50 mL solution of
pyrogallol (20 mg L-1) after contact with HRP-Tglass (0.1 g, 0.25 U g-1) with 30 min
UVB irradiation (320 nm at intensity of 460 W cm-2) was compared with that for
reaction with the free enzyme (0.25 U) with H2O2 in the dark for 5 min (see 3.4.1.5).
When treated by HRP in the presence of H2O2 pyrogallol gives the oxidized product
purpurogallin. The major difference between the two spectra was a shift in the peak at
Page 76 of 163
300 nm to 320 nm for HRP-Tglass (Figure 5-10). This may be related to the formation of
a different product from purpurogallin by the UVB irradiated HRP-Tglass.
300 nm
320 nm
0.25
Absorbance units
1.2
Pyrogallol
0.2
Irradiated pyrogallol
Tglass with UV irradiation

HRP-Tglass
0.8
Tglass with UV irradiation

Tglass
Free THRP
glass
0.15
0.6
420 nm
0.1
0.4
0.05
Absorbance units
0.3
0.2
0
200
300
400
500
Wavelength (nm)
600
Figure 5-10. Spectra after reaction with 20 mg L-1 pyrogallol (top to bottom); a) 20 mg L-1 pyrogallol, b)
UVB radiation for 30 min without HRP-Tglass, c) UVB radiation for 30 min with HRP- Tglass, d) UVB
radiation for 30 min with Tglass, e) soluble HRP after overnight reaction with 0.2 mM H2O2, all reactions
performed in 10 mM phosphate buffer pH 6.0.
The addition of Tglass to the 20 mg L-1 pyrogallol solution compared to without the Tglass
slightly reduced the spectrum intensity at 300 nm, This provides clear evidence that
HRP-Tglass leads to greater substrate transformation than UVB/Tglass alone, and a
different reaction product.
To investigate the effect of substrate concentration on the polymerization process, HRPTglass (0.1 g, 0.25 U g-1) was exposed to various pyrogallol concentrations under UVB
radiation for 30 min. The absorbance spectra of the photooxidized pyrogallol solutions
(1-100 mg L-1) after subtracting the spectra for irradiated pyrogallol solutions without
HRP-Tglass are shown in Figure 5-10.
Page 77 of 163
pyrogallol
(mg L-1)
0.4
1
5
20
50
75
0.3
0.2
Product absorbance at
320nm
Absorbance units
0.5
0.5
0.4
0.3
0.2
0.1
0
y = 0.0058x + 0.013
R2 = 0.9987
20
40
60
80
-1
Initial pyrogallol concentration (mg L )
0.1
0
200
300
400
Wavelength (nm)
500
600
Figure 5-10. Absorbance spectra for products formed by HRP-Tglass after UVB irradiation.(The spectra
were obtained by subtraction of the spectra for irradiated pyrogallol solutions of the same concentration in
the absence of HRP-Tglass). Pyrogallol (0-75 mg L-1), phosphate buffer pH 6.0.
From Figure 5-10 there appears to be a direct correlation between the initial
concentration and pyrogallol transformation (R2 = 0.9987); however, adsorption of
polymerized products to the Tglass was observed at the end of the run as the particles
changed colour.
5.2.6 Influence of UVB dose on free and immobilized HRP in transforming pyrogallol
The influence of irradiation time on the activity of free HRP and HRP-Tglass was
investigated. HRP-Tglass particles (0.1 g, 0.25 U g-1) and 40 g soluble HRP (3.2 U)
were placed in separate beakers with 100 mg L-1 pyrogallol and irradiated for specified
periods (5-60 min) (section 3.4.1.5). Those two quantities were used as they had a
similar change in absorbance of purpurogallin at 420 nm when H2O2 was added. The
absorbance spectra of the supernatants were obtained and normalized against an
irradiated reference solution containing the same solutions without the HRP-Tglass or the
free enzyme. Figure 5-11 and Figure 5-12 show that the absorbance of the products
increases with UVB radiation dose. The spectra for HRP-Tglass had higher intensity than
for the free HRP system. This can be related to the increased production of H2O2 due to
the presence of TiO2 and the protected environment for the immobilized enzyme.
Page 78 of 163
Radiation Dose
(min)
1.6
5
10
1.2
20
0.8
30
60
0.4
320nm
Absorbance units
2
1.5
1
0.5
0
0
20
40
60
80
0
200
300
400
Wavelength (nm)
500
600
Figure 5-11. Absorbance spectra after UVB irradiation of HRP-Tglass in the presence of 100 mg L-1
pyrogallol for various times. (The spectra were obtained by subtracting the spectra for pyrogallol in the
absence of HRP-Tglass irradiated for the same time).
0.7
Absorbance units
0.6
0.5
10
0.4
20
0.3
30
60
0.2
320nm
Radiation Dose
(min)
0.5
0.4
0.3
0.2
0.1
0
0
20
40
60
80
0.1
0
200
300
400
Wavelength (nm)
500
600
Figure 5-12. Absorbance spectra after UVB irradiation of free HRP (40 g) with 100 mg L-1 pyrogallol
for various times. (The spectra were obtained by subtracting the spectra for pyrogallol in the absence of
enzyme irradiated for the same time).
The degree of pyrogallol transformation increased with increased radiation dose for
both the immobilized and free enzyme systems. However, the extent of reaction tended
Page 79 of 163
to increase proportionally with time for the immobilized enzyme, whereas it tended to
decrease with time for the free enzyme. Hence, extended exposure to UVB resulted in
the reduction of free enzyme activity while the Tglass support prevented the reduction.
Thus it indicates that the surface tunnels and crevices in the Tglass (shown in section
4.2.2, Figure 4-7) offer protection of the enzyme from deactivation by the UVB
radiation. In addition to enzyme protection, the production of H2O2 in situ due to the
presence of the TiO2 in the Tglass was observed to be sufficient to activate the enzyme.
5.2.7 Influence of UV dose on the activity of HRP-Tglass in transforming 4-BP
Evaluation of the behaviour of HRP-Tglass under UVB irradiation was carried out in
section 5.2.6 where pyrogallol was used as a model substrate and product formation was
monitored. However, pyrogallol is not a brominated phenol, so 4-BP was used as a
model substrate in all subsequent experiments.
The HRP-Tglass was suspended in a solution of 4-BP in the batch reactor, agitated and
irradiated with UVB (460 W cm-2). Although transformation was low over the first 30
mins, after 120 mins 90% of the 4-BP was transformed (Figure 5-13). Enzymatic
activity decreased over the first 120 mins, but then remained at approximately 75% of
the initial value. Thus the system of HRP-Tglass activated by hydrogen peroxide
produced by UVB irradiation alone was shown to both withstand UVB irradiation and
to have increased enzyme stability compared to a reaction where H2O2 was added
(section 5.2.3, and will further be shown in chapter 6). A control experiment conducted
in the absence of HRP-Tglass was run in parallel and gave low removal (15%) of 4-BP.
Page 80 of 163
100
80
80
Transformed 4-BP
60
60
Transformed 4-BP (control)
40
40
Remaining activity
20
20
0
0
200
400
600
800
Time (min)
1000
Remaining activity (%)
4-BP transformed (%)
100
0
1200
Figure 5-13. Influence of radiation dose on the conversion of 4-BP with HRP-Tglass () transformed 4-BP
without HRP-Tglass (control), () transformed 4-BP, () remaining immobilized enzyme activity
(pyrogallol assay).
The transformation of 0.09 mmole L-1 of 4-BP (90% transformed) in two hours
compares favourably with the results obtained by Chen and Ray (1999) who used a
TiO2 slurry under UVA irradiation for the treatment of 4-chlorophenol and obtained a
transformation of 0.16 mmole L-1 in 2 hours. They used a high quality photocatalyst
powder (Degussa P25) at 2 g L-1; as this photocatalyst powder is expensive, it is
unlikely that it would be used commercially.
The HRP-Tglass system showed resilience to UVB radiation and worked continuously
over 16 h with only 18% activity loss and was able to transform 98% of 4-BP.
5.2.8 Plug flow reactor studies
The concept that the HRP-Tglass can work in the presence of UVB irradiation has been
proved with the batch system. The HRP-Tglass remained active and produced a sufficient
concentration of H2O2 in situ to activate the enzyme. However, it is preferable to use
this new concept in a continuous regime, i.e., a plug flow reactor.
The immobilized HRP preparations were placed in columns (0.2 mL, 0.15 g) and their
efficacy evaluated (in terms of TOC reduction, concentration of bromide in the effluent,
and rate of reaction) as functions of volumetric loading rate and flow rate (section
Page 81 of 163
3.4.2). The adsorption pattern of the products was monitored visually and a plug flow
pattern for both the HRP-PG and the HRP-Tglass was obtained (Figure 5-14).
0 mL
40 mL
80 mL
160 mL
280 mL
480 mL
Figure 5-14. Increasing adsorption of the polymerized products on the HRP-PG particles in a plug flow
reactor as increasing volumes of 4-BP (0.1 mM) and H2O2 (0.1 mM) are fed at 2 mL min-1 to the column
(the HRP-Tglass column gave a similar pattern).
When the feed flow rate was 2 mL min-1, removals of 2.04 mg C for the HRP-Tglass and
2.31 mg C for the HRP-PG were obtained from the 7.0 mg C applied, representing
removals of 29% and 33% from 480 mL feed, respectively, as seen in Figure 5-15.
Figure 5-15 shows that as the feed volume increases there is a reduction in adsorption,
this contributes to reduction in activity. Similar behaviour was detected by most
researchers as they looked at the reduction in substrate transformation (Lai and Lin,
2005; Cheng et al., 2006; Bayramoglu and Arica, 2008).
Calculations were made according to Equation 3-2 in section 3.3.
Adsorbed carbon =
S o -S e
100
So
Where:
Adsorbed carbon: degree of carbon reduction (%);
S0: initial concentration of carbon (mgC L-1);
Se: effluent concentration of carbon (mgC L-1).
Page 82 of 163
Equation 3-2
Figure 5-15. TOC removal (as adsorbed carbon) by HRP-PG and HRP-Tglass columns from feed
consisting of equimolar concentration of 4-BP (17.3 mg L-1) and H2O2 (3.4 mg L-1) at a flow rate of 2 mL
min-1.
Br- concentration (mM)
0.030
HRP-PG
0.025
HRP-Tglass
T glass
0.020
0.015
0.010
0.005
0.000
0
125
250
375
500
Feed volume (mL)

Figure 5-16. Concentration of bromide in effluent from HRP-PG and HRP-Tglass columns resulting from
feed consisting of equimolar concentration of 4-BP (17.3 mg L-1) and H2O2 (3.4 mg L-1) at a flow rate of
2 mL min-1.
Page 83 of 163
Br-/ adsorbed 4-BP (mole %)
60
HRP-PG
HRP-Tglass
T glass
45
30
15
0
0
125
250
Feed volume (mL)
375
500
Figure 5-17. Ratio of bromide/4-BP (calculated in mmoles) adsorbed to columns of HRP-PG and HRPTglass from feed consisting of 4-BP (17.3 mg L-1) and H2O2 (3.4 mg L-1) at a flow rate of 2 mL min-1.
The effluent concentration of bromide decreased (Figure 5-16), and the TOC adsorbed
also decreased (Figure 5-15), with increasing feed volume. This decrease in both
dehalogenation and adsorption indicates that the enzyme activity can be correlated to
adsorption rather than 4-BP transformation (Figure 5-17), the latter being usual in the
literature. The net result of dehalogenation is reflected in the ratio of the concentration
of bromide ion to the concentration of the adsorbed carbon. The ratio is higher for HRPPG than HRP-Tglass for a feed volume up to 250 mL after which it becomes similar. This
suggests that the difference in the properties of the two supports is diminished as they
become covered by the adsorbed products
Since dehalogenation occurs along with the polymerization process, it may also occur
for the low mass product oligomers (dimers and trimers). These molecules are soluble
and can serve as electron acceptors and so be processed by the enzyme in a similar
fashion to the substrate.
This secondary dehalogenation appears at low ratios of
4-BP:H2O2 and leads to the release of additional bromide (Ward et al., 2002). Thus
there may be a correlation between the size of the oligomers produced and the amount
of bromide released. With HRP-Tglass the ratio of bromide in the effluent to the
equivalent amount of 4-BP adsorbed to the column averaged 35%, whilst for the HRPPG it averaged 40%.
Table 5-1 shows the range of dimer derivatives that could possibly be formed upon
oxidation of 4-BP, and the coupling reactions leading to their formation based on the
Page 84 of 163
possible radicals that can be formed following formation of a phenoxyl radical by

peroxidase (Figure 5-18).
Figure 5-18. Possible 4-bromophenol radicals formed (redrawn and amended according to Dec and
Bollag (1994)
Table 5-1 Proposed coupling reactions between free radicals generated during the catalytic oxidation of 4bromophenol (redrawn and amended according to Dec and Bollag (1994)
Free radicals
O*
Result
Loss of
M.W.
Br
(Dalton)
--
342
Br
265
--
344
2Br
186
Br
265
--
344
O*
Br
Br
Br
O*
Br
Br
HO
C*
Br
Br
O*
Br
C*
+
Br
Br
HO
C*
Br
OH
HO
C*
Br
Br
O
C*
Br
HO
C*
Br
Br
O
C*
Br
Page 85 of 163
HO
Br
Br
C*
+
Br
OH
OH
If it is assumed that all these free radicals can participate in oxidative coupling
reactions, there are 6 possible combinations (the two ortho positions are identical, Table
5-1). Three of these combinations involve free radicals with a bromine atom attached to
the aromatic carbon carrying the unpaired electron. The coupling of these radicals
should result in the release of bromide ion. Overall, the hypothetical coupling of 4-BP
results in the release of 5 bromide ions from 12 molecules engaged in dimerization,
which means that 41% debromination is possible.
This dimerization model, however, seems insufficient because further stages of
polymerization are not considered. Furthermore, it is cannot be assumed that all rates of
coupling are equal and that only the monomer radicals lose bromide atoms when
coupling with the oligomer radicals (as Ward et al., (2002) pointed out).
5.2.9 Analysis of products from enzymatic polymerization
5.2.9.1 Analysis of soluble products

The soluble products in the column effluent were collected and subjected to ESI mass
spectrometry (section 3.3.2). The spectrum (Figure 5-19) showed two main peaks with
molecular weights corresponding to that of 4-bromophenol (171 Da) and 4bromocatechol (187.1 Da). Although bromide was detected, no debrominated oligomers
were found, probably due to their low concentration.
OH
Br
OH
OH
Br
Figure 5-19. ESI mass spectrum obtained for the soluble fraction after oxidation of 4-BP with HRP-Tglass
in a column reactor.
Page 86 of 163
5.2.9.2 Analysis of adsorbed products

Examination of an acetonitrile extract of the polymers from the spent column packings
by ESI mass spectrometry (section 3.3.2) showed that the polymers on the HRP-Tglass
were of smaller mass than those on the HRP-PG (Figure 5-21). For HRP-Tglass, dimers
and 4-bromocatechol were the major compounds detected, while for HRP-PG, dimers,
trimers and tetramers were detected. This is consistent with the data for debromination
suggesting that a different range of products was formed with the HRP-Tglass system.
Using the less polar solvent ethyl acetate, Levy et al. (2003) obtained similar end
products of dimers and trimers from the polymerization of 4-BP using HRP
immobilized on cellulose.
Page 87 of 163
HRP-Tglass
m/z
HRP-PG
Figure 5-20. ESI mass spectra of the adsorbed fraction after oxidation of 4-BP with HRP-PG and HRPTglass in a column reactor (acetonitrile was used for solvent extraction).
The masses of the adsorption products extracted from the HRP-Tglass and HRP-PG
columns were allocated to structures.
Page 88 of 163
The structures were: 4-BP (MW 173.2), 4-bromocatechol (MW 186.9) dimer of 4-BP
(MW 343.1), trimer of 4-BP (MW 513.2) and tetramers of 4-BP (MW 685.2). Although
bromide was detected in the effluent, the debrominated oligomers were hard to detect
due to their complex structure, but the unidentified masses (E) can be attributed to the
debrominated compounds.
Detector Response
0.06
HRP-PG
0.05
HRP-Tglass
0.04
0.03
0.02
0.01
0
100
1000
Molecular Weight (Dalton)
10000
Figure 5-21. Gel permeation chromatograms for the products desorbed from the HRP-PG and HRP-Tglass
columns with TCB.
To further investigate the characteristics of the polymers, two column reactors

containing HRP-PG or HRP-Tglass were run until loaded with 3.6 mg C, and the column
contents were extracted with trichlorobenzene (TCB). The extracts were analysed by gel
permeation chromatography (see 3.3.5) which showed the presence of higher
concentrations of the higher oligomers for the HRP-PG compared with the HRP-Tglass
column (Figure 5-21), consistent with the results for the acetonitrile extraction and ESI
mass spectrometry.
The polymers desorbed from the HRP-PG column had a higher content of tetramers
(686 Da) and higher molecular mass products than did those from the HRP-Tglass reactor
which had mainly trimers (515 Da). In section 5.2.8 it was concluded that bromide
release can occur during the formation of higher mass oligomers, resulting in an overall
lower mass, however previous measurement showed only a small difference in bromine
removal between HRP-Tglass and HRP-PG (35% and 40%, respectively). Impurities and
monomers would appear in the peak at <200 Da, and mask any smaller mass peaks
Page 89 of 163
which may have contained the bulk of the HRP-Tglass adsorbed products such as 4bromocatechol and fragments of catechol polymers due to the TCB extraction (Ward et
al., 2004).
5.2.10 Removal of adsorbed oligomers
The polymerization reaction was run in 0.2 mL columns filled with catalyst (0.15 g)
overnight with a range of concentrations of 4-BP and H2O2 (1:1 molar ratio) (section
3.4.2.1). Both the HRP-Tglass and the HRP-PG particles lost their activity and the
adsorbed mass loaded on the support was measured as the reduction in the effluent
concentration of the feed concentration multiplied by the volume treated. After this time
the particles had changed in colour from beige to dark red for HRP-PG and to grey for
HRP-Tglass. The particles were washed thoroughly to remove non-adsorbed material,
covered with 25 mL MilliQ water, and then exposed to UVB radiation (460 W cm-2)
until they returned to their original colour. This took 12 hours for HRP-Tglass, whereas
the HRP-PG was not fully decolourised, even after 96 hours (Figure 5-22 and Figure
5-23).
Figure 5-22. HRP- Tglass particles coated with polymerized products prior to UVB radiation (A) and after
96 hours UVB radiation (B). The 4-BP concentrations ranged from 0.1 mM to 10 mM and are placed in
increasing order from right to left.
Page 90 of 163
Figure 5-23. HRP-PG particles coated with polymerized products prior to UVB radiation (A) and after 96
hours UVB radiation (B). The 4-BP concentrations ranged from 0.1 mM to 10 mM and are placed in
increasing order from right to left.
Determination of the enzyme activity (pyrogallol assay) of the UV-treated immobilized

HRP preparations showed that the HRP-Tglass (Figure 5-24) preparation recovered up to
56% of its activity, whilst the HRP-PG (Figure 5-25) preparation remained inactive.
This was attributed to the formation of hydroxyl radicals on the TiO2 surfaces of the
Tglass resulting in the oxidation and removal of the oligomers from the covered active
sites (see section 2.4.2 regarding remote oxidation). The remaining sites may still have
been covered by adsorbed oligomer or have been destroyed by the radiation. This raised
the possibility of using the continuous production of hydroxyl radicals from TiO2 which
would obviate the need for solvents such as those used by Dordick (1987) and Ward
(2004) to release the polymeric products from the immobilized enzyme.
Page 91 of 163
60
50
40
30
20
10
Activity Units remaining

(%)
Adsorbed carbon (mg)
0
0
2.5
5
7.5
4-bromophenol concentration (mM)
10
Figure 5-24. Influence of organic loading on the conversion of 4-BP with HRP-Tglass () adsorbed carbon
on the supports (mg) () residual activity of the particles after initial reaction, () activity of the particles
10
0
0
2.5
5
7.5
4-bromophenol concentration (mM)
Activity Units remaining

(%)
Adsorbed carbon (mg)
after 96 hours of UVB radiation.
10
Figure 5-25. Influence of organic loading on the conversion of 4-BP with HRP-PG () adsorbed carbon
on the supports (mg) () residual activity of the particles after initial reaction, () activity of the particles
after 96 hours of UVB radiation.
Page 92 of 163
5.3
In this chapter HRP-Tglass was characterized and investigated under different conditions,
and the results were compared to HRP-PG.
The surfaces of HRP-PG and HRP-Tglass were initially characterized using ESEM and
EDX. EDX mapping of HRP-Tglass after a polymerization reaction showed that although
the enzyme was immobilized over the whole surface of the Tglass particle, it was only
active on the silica section.
Batch experiments showed that the HRP-Tglass has a wider range of pH stability (pH 311) than any examples of immobilized HRP found in the literature, although the
temperature stability range (10C - 60C) was found to be the same.
The adsorption by HRP-Tglass of the reaction products of 4-BP followed the Langmuir
model. The calculated maximum adsorption capacity for these products was 34 and 36
mg per g support, respectively.
HRP-Tglass particles in water were irradiated with UVB, resulting in the formation of
H2O2. When pyrogallol was transformed in the UVB irradiated HRP-Tglass system a new
product was detected. The absorption peak shifted from 300 nm (for normal
polymerization) to 320 nm (for transformation induced by UVB). The HRP-Tglass
transformed the pyrogallol at a higher rate than either the free enzyme or the Tglass
support alone. It also appeared to provid protection for the enzyme against the UVB
irradiation, unlike the free enzyme which lost its activity during its exposure to UVB
irradiation.
When treating 4-BP, the HRP-Tglass system showed resilience to UVB radiation and
worked continuously over 16 h with only 18% activity loss, and was able to transform
98% of 4-BP (0.1 mM).
A plug flow reactor was filled with HRP-PG or HRP-Tglass and treated with an
equimolar (0.1 mM) solution of 4-BP with H2O2. At a flow rate of 2 mL min-1 HRPTglass and HRP-PG adsorbed 29% and 33% TOC and removed 35% and 40% of bromide
from the adsorbed products, respectively. Analysis of the adsorbed products from HRPPG and HRP-Tglass treatment of 4-BP showed that the polymers were smaller and less
debrominated for HRP-Tglass, indicating a difference in the reaction mechanisms.
Page 93 of 163
It was demonstrated that UVB irradiation can be used to regenerate fully loaded HRPTglass to 56% of activity, whilst the HRP-PG become inactive.
Thus the Tglass can also provide a means of partial restoration of enzyme activity
through photooxidative breakdown (and thus removal) of the organic material blocking
the active sites of the immobilized enzyme.
The kinetics of HRP-PG and HRP-Tglass in a plug flow reactor are described in the next
chapter. The kinetics were investigated as the adsorption of the formed products rather
than the substrate transformation.
Page 94 of 163
Chapter 6. Kinetics of HRP immobilized on PG and Tglass

6.1
INTRODUCTION
The kinetics of HRP immobilized on various supports (i.e., clay, cellulose and porous
glass PG) have been described by equations such as the Michaelis-Menten equation,
and subsequent Lineweaver-Burk plots of the data have been used to provide the model
coefficient values. In using the Michaelis-Menten equation these researchers (Nicell et
al., 1993; Tatsumi et al., 1996; Caza et al., 1999; Ward et al., 2002) have assumed that
in the initial stages of the reaction no denaturation, inhibition or adsorption occurs. This
assumption arose from the way they monitored the system, i.e., they measured the
substrate transformation rather than the adsorption of product which plays a significant
role in reducing the enzyme activity.
In the present study, the focus and the novelty was to determine the adsorption kinetics
of the polymerized products produced by the enzymatic reaction at the initial and final
stages. The system described in this chapter treated 4-bromophenol (as model substrate)
with HRP-PG or HRP-Tglass in a plug flow reactor. The adsorbed polymer concentration
was determined by measuring the change in total organic carbon in the treated stream.
The efficiency of the removal of 4-BP was evaluated as a function of substrate loading
rate under constant flow, and as a function of flow rate at a constant H2O2:substrate
ratio. Measurements to determine the maximum adsorption capacity using a range of
flow rates were also conducted.
6.2
6.2.1 The influence of substrate concentration and flow rate on HRP-PG

As described in section 2.2.5, the HRP-PG reaction mechanism has two components,
polymerization followed by adsorption. Solutions containing a range of 4-BP
concentrations were fed to a column containing 0.2 mL HRP-PG at a constant flow rate,
and the first 100 mL of effluent for each run was collected in 20 mL aliquots. Initially,
the kinetics were investigated in terms of adsorbed carbon (section 3.4.2.2).
Calculations were made according to Equation 3-2 in section 3.4.
Page 95 of 163
Adsorbed carbon =
S o -S e
100
So
Equation 3-2
Where:
Adsorbed carbon: degree of carbon reduction;
S0: initial concentration of carbon (TOC);
Se: effluent concentration of carbon (TOC).
Figure 6-1 demonstrates clearly how increase in substrate concentration led to decreased
enzyme activity. At substrate concentrations up to 8.8 mgC L-1 the enzyme produced a
limited amount of polymerized products that were adsorbed without reducing its
activity, thus the active sites were still free to operate. There is a problem with the HRP
(both immobilized and free) polymerization reaction when dealing with low
concentrations of phenols as noted by Maloney et al., (1986) and Tong et al., (1997).
These researchers concluded that as the level of substrate decreases, H2O2 tends to react
with the enzyme and reduce its performance (section 2.2.3). Thus this would be the
cause of the partial adsorption seen in Figure 6-1 for 2.86 mgC L-1.
At 4-BP concentration greater than 22.6 mgC L-1 deactivation of the enzyme occurred
due to adsorption of polymerized products to active sites.
Figure 6-1. Carbon removal for HRP-PG for a range of flow rates (A: 1.75 mL min-1; B: 3 mL min-1; C: 6
mL min-1; D: 8 mL min-1;) using a range of 4-BP concentrations (2.1-161 mgC L-1) and H2O2 at 1:1
molar ratio.
Page 96 of 163
Calculation of the mass of product adsorbed to the HRP-PG particles showed that the
mass adsorbed was higher at lower flow rates than at higher flow rates (Figure 6-1).
This indicates that as the flow rate increases, the enzyme becomes deactivated more
quickly and accumulates less product. It also shows that as the initial substrate
concentration (i.e., mass of potential product) increases the accumulation of mass is not
linear.
This suggests that the adsorption of the products may be dependent on the formation of
different length oligomers, resulting in a different degree of hydrophobicity and
consequently different affinity for adsorption to the support.
Accumulated mass (mgC)
0.2
0.16
Flow rate
(mL min-1)
0.12
1.75
0.08
3
6
0.04
0
0
10
20
30
-1
S0 (mgC L )
40
50
Figure 6-1. Carbon accumulation for HRP-PG for the flow rates of 1.75 8 mL min-1 using a range of 4BP concentration (2.9-50 mgC L-1) and H2O2 at 1:1 molar ratio.
Figure 6-1 shows the reduction of carbon after the HRP-PG reaction with a range of
substrate concentrations at different flow rates: the faster the flow rate, the greater the
reduction in reaction velocity. Figure 6-2 emphasises that reduced adsorption capability
as there is an increasing difference in the carbon accumulation for each flow rate.
6.2.2 Development of a model for HRP-PG
To predict the loss of enzyme activity due to product adsorption, a kinetic model needs
to be established. As the system was monitored for substrate removal and not for
Page 97 of 163
substrate transformation, the Michaelis-Menten model cannot be used to describe the

system behaviour. As can be seen in Figure 6-2, it is apparent that correlation between
2
the app
rate and the initial carbon concentration (i.e., applied mass) is almost linear (R
values of 0.989-0.998) and appears to follow a first order reaction:

app
rate = K S0
Equation 6-1
Where:
-1
-1
app
rate : specific rate of product adsorption on the HRP-PG particles (mgC L min );
S0: concentration of substrate (mgC L-1);

K: substrate first order constant (mgC L-1).
The app
max was obtained according to Equation 3-3 and 3-4 in section 3.4
(S S e ) Q S 0 - S e
mg
)= 0
=
L min
H column
H
(min) = column
Q
app
rate (
Equation 3-3
Equation 3-4
Where
H column : empty reactor volume;
: hydraulic retention time;

Q: feed flow rate;
S0, Se: TOC levels of initial and exit feed (mg L-1), respectively.
Page 98 of 163
app `
rate (mgC L-1 min-1)
1200
Flow Rate
(mL min-1)
800
400
y = 5.740x + 25.511
R2 = 0.989
1.75
y = 8.821x + 42.272
R2 = 0.993
y = 23.056x - 68.723
R2 = 0.998
y = 21.545x + 5.935
2
R = 0.997
0
0
50
100
150
S0 (mgC L-1)
200
250
Figure 6-2. Carbon removal rate for HRP-PG for flow rates 1.75-8 mL min-1 using 4-BP in a
concentration range of 2.1-161 mgC L-1 and H2O2 at 1:1 molar ratio.
Based on that correlation, the slope (K) was obtained for each flow rate and compared
(Figure 6-3), resulting in a correlation (R2 = 0.991) of:
K= 3.01 Q + 0.79
Equation 6-2
This result describes the enzyme product accumulation behaviour, whereby the HRP
reaction controls the overall kinetics, but only for the initial phase (in which the
adsorption has not yet deactivated the enzyme). After the initial phase the adsorption
reaction is more dependent on the initial concentration of the substrate.
Page 99 of 163
K (min-1)
30
y = 3.011x + 0.792
R2 = 0.991
20
10
0
0
10
Flow rate (mL min-1)

Figure 6-3. Correlation between flow rate and K, obtained from the slope of reaction rate with initial
substrate concentration).
6.2.3 Validation of the HRP-PG model

The effect of a change in flow rate with a constant feed on the kinetics was investigated.
The HRP-PG column, pre-saturated with 4-BP solution for column equilibration, was
fed a solution containing 7.2 mgC L-1 4-BP (17.3 mg L-1) and 3.4 mg L-1 H2O2
(corresponding to a 1:1 molar ratio) (section 3.4.2.3). Column efficiency and the point
at which maximum transformation occurred are presented as a function of the loading
rate in Figure 6-4. The curve for maximum adsorption was obtained by multiplying the
adsorbed carbon (%) and the specific adsorption rate. This shows the conditions for
maximum carbon removal occur for 4-BP at 7.2 mgC L-1 at a flow rate of 2 mL min-1
for HRP-PG.
Figure 6-4 shows the combined effect of flow rate and concentration on the maximum
reaction rate for HRP-PG. The first order model fits the adsorption rate results up 2 mL
min-1 above which there is no change in the adsorption capacity; this may be due to the
rapid saturation of the HRP-PG particles and their deactivation.
Page 100 of 163
70
-1
-1
rate (mgC L min )
100
Adsorption rate measured

Maximum adsorption
60
Adsorption rate calculated
50
Degree of adsorption
80
60
40
40
30
20
20
10
0
Adsorbed carbon (%)
80
0
0
4
6
-1
Flow Rate (mL min )
Figure 6-4. Degree of adsorption, maximum adsorption, measured
8
adsorption rate and calculated
-1
adsorption rate for HRP-PG at flow rates of 0.25 - 7.5 mL min , 4-BP and H2O2 concentration 0.1 mM
(7.2 mgC L-1, 3.4 mg L-1; respectively).
Overall, the maximum apparent rate obtained was 45 mgC L-1 min-1, further comparison
of the performance of HRP-PG will be discussed in section 6.2.6.2.
6.2.4 The influence of substrate concentration and flow rate on HRP-Tglass
There are three main mechanisms that occur for the HRP-Tglass reaction when treating 4BP in the presence of H2O2. The first is the enzyme conversion reaction in which HRP
polymerizes the 4-BP. This is followed by adsorption of the product via hydrophobic
binding to the support and finally, due to the proximity to the TiO2, hydrophilic
repulsion of highly hydrophobic products which is expected to cause the product to be
repelled from the adsorption sites into the nearby medium.
The column system was fed with range of carbon concentrations of 4-BP (1-164 mgC L-1)
at different flow rates (1.75, 3, 6 and 8 mL min-1). The first 100 mL of effluent for each
run was collected in 20 mL aliquots (section 3.4.2.2). Figure 6-5 shows the results for
different 4-BP concentrations (i.e., masses) at the same flow rate.
It is evident that the behaviour of the HRP-Tglass is similar to the HRP-PG in that
increasing substrate concentration leads to reduced enzyme activity. For 4-BP
concentrations up to 8.8 mgC L-1 the enzyme adsorbs a relatively constant amount of
Page 101 of 163
product without reducing its activity ( Figure 6-5). For concentrations of 22.6 mgC L-1
and higher, deactivation of the enzyme commences as the enzyme is smothered with
polymerized products.
Figure 6-5. Carbon removal for HRP-Tglass for a range of flow rates (A: 1.75 mL min-1; B: 3 mL min-1; C:
6 mL min-1; D: 8 mL min-1) using a range of 4-BP concentrations (2.1-161 mgC L-1) and H2O2 at 1:1
molar ratio.
As before, there is a slope indicating enzyme deactivation for each concentration and
flow rate above which adsorption overpowers the enzyme kinetics. In terms of the
accumulated mass on the particles, there is little difference between the HRP-PG
(Figure 6-1) and the HRP-Tglass (Figure 6-5). In both cases it was apparent that the
adsorbed mass accumulated on the particles is higher at lower flow rates than at higher
flow rates, there being lower transformation of low substrate concentrations at high flow
rates.
Page 102 of 163
Accumulated mass (mgC)
0.2
0.15
Flow rate
(mL min-1)
0.1
1.75
3
6
8
0.05
0
0
10
20
30
-1
S0 (mgC L )
40
50
Figure 6-6. Carbon accumulation for HRP-Tglass for the flow rates of 1.75 8 mL min-1 using a range of
4-BP concentration (2.9-40 mgC L-1) and H2O2 in 1:1 molar ratio.
6.2.5 Development of a model for HRP-Tglass

For the HRP-Tglass system the enzyme reaction is followed by two other processes and
so the Michaelis-Menten model cannot describe this system. A more appropriate model,
which describes the growth and death of microorganisms, was derived by Monod
(1949). In this case, it can describe the growth of the polymeric mass on the particles
until cessation or the death of the enzyme polymerization reaction. Equation 6-3
displays the Monod equation:
app
rate
app
max
S0
K S + S0
Equation 6-3
Where
app
rate
: specific growth rate of the polymer on the particles (mgC L-1 min-1);
app
max
: apparent maximum specific growth rate (mgC L-1 min-1);
S0: concentration of growth limiting substrate (mgC L-1);

Ks: substrate utilization constant (mgC L-1).
Page 103 of 163
app
Using the data obtained from 6.2.4, the rate
was obtained according to Equations 3-3
and 3-4 in section 3.4 and plotted against the initial substrate (Figure 6-8). As can be
seen in Figure 6-8, it is apparent that the flow rate has an effect on the adsorption rate.
Flow rate
-1
(mL min )
240
1.75
-1
-1
rate (mgC L min )
320
160
app
80
0
0
50
100
150
200
-1
S0 (mgC L )
Figure 6-7. Carbon removal rate for HRP-Tglass for flow rates of 1.75-8 mL min-1 using 4-BP concentration
ranged 2.1-161 mgC L-1 and H2O2 in 1:1 molar ratio.
Plotting
1
S
gives Figure 6-8, which is a line with a slope K s and intercept of
B
-1
0.12
y = 0.128x + 0.003
R2 = 0.998
0.08
0.04
0
0
0.3
0.6
1/S0 (L mg-1)
0.9
-1
y = 0.051x + 0.003
R2 = 0.979
0.01
0.005
0
-0.1
1 .
app
max
0.1
0.2
-1
1/S0 (L mg )
0.3
0.4
y = 0.052x + 0.006
R2 = 0.929
0.008
0.004
0.02
0.02
0.015
0.012
0
-0.025
1.2
1/rate (min L mgC )
-1
1/rate (min L mgC )
0.025
app
max
0.016
A
1/ rate (min L mgC )
-1
1/rate (min L mgC )
0.16
vs.
0.025
0.075
1/S0 (L mg-1)
0.125
0.175
0.016
0.012
y = 0.037x + 0.002
2
R = 0.986
0.008
0.004
0
-0.025
0.075
0.175
0.275
-1
1/S0 (L mg )
0.375
Figure 6-8. Reciprocal plots of adsorption rate vs substrate for HRP-Tglass for a range of flow rates (A:
1.75 mL min-1; B: 3 mL min-1; C: 6 mL min-1; D: 8 mL min-1;)
Page 104 of 163
Plotting the Ks values and app

max against flow rate shows that the Ks remained constant
while the app
max increased with the flow rate (Figure 6-9). This relationship can be
explained if the products formed are more readily washed away due to the increasing
flow rate. This, in turn, makes more sites available for further reactions. The increased
flow rate also reduces the retention time for reaction which may reduce the production
of larger polymers. The equation in Table 6-1 expresses the dependency of app
max on the
flow rate.
500
300
9
200
Ks
4.5
app
max
100
-1
-1
400
13.5
(mgC min L )
-1
Ks (mgC L )
18
0
1
-1
Q (mL min )
Figure 6-9. Relationship of app
and Ks with flow rate.
max
While the increasing app

max value with the flow rate suggests a greater capacity for
substrate removal at higher flow rates, the system will be limited by increasing
backpressure. The catalytic performance of the HRP-Tglass plug flow reactors, as
calculated according to Monod kinetics, is presented in Table 6-1.
Page 105 of 163
Table 6-1 Apparent kinetic parameters for 4-BP removal in terms of TOC by HRP-Tglass in plug flow
mode.
Kinetic Parameter
Unit
Value
app
max {1.75<Q<8}
mgC L-1 min-1
app
max (Q) = 62Q + 56
KS
mgC L-1
15 1
6.2.6 Validation of HRP-Tglass model
6.2.6.1 The effect of concentration

The model was tested by running the system at a flow rate of 4 mL min-1 and comparing
the results with predictions made using the Monod model (Figure 6-10). 4-BP
concentrations were in the range of 7.6 to 72 mgC L-1. The adsorption rate was
measured and correlated well with the results for the Monod model, particularly for S0
less than 36 mgC L-1.
240
app
-1
-1
rate (mgC L min )
320
160
Measured
80
Calculated
0
0
20
40
60
80
S0 (mgC L-1)
Figure 6-10. Comparison of experimental and calculated effluent TOC levels for flow rate 4 mL min-1 and
feed concentration 4-BP over the range 7.6-72 mgC L-1.
Page 106 of 163
6.2.6.2 The effect of flow rate

In this validation experiment, the effect of flow rate with a constant feed concentration
was investigated. The reactor, pre-saturated with 4-BP solution, was fed a solution
containing 7.2 mgC L-1 4-BP and 3.4 mg L-1 H2O2 (corresponding to a 1:1 molar ratio)
(see section 3.4.2.3). Column efficiency and the point at which maximum
transformation occurred are presented as a function of the loading rate in Figure 6-11
(HRP-Tglass). The maximum adsorption was obtained by multiplying the degree of
adsorption (T(%)) and the adsorption rate.
80
135
60
90
40
Adsorption rate measured
Maximum adsorption
Adsorption rate calculated
Degree of adsorption
45
0
0
2
4
6
-1
Flow Rate (mL min )
Figure 6-11. Degree of adsorption, maximum adsorption, measured
20
Adsorbed carbon (%)
100
-1
-1
rate (mgC L min )
180
0
8
adsorption rate and calculated
-1
adsorption rate for HRP-Tglass at flow rates of 0.25 - 7.5 mL min , 4-BP and H2O2 concentration 0.1 mM
(7.2 mgC L-1, 3.4 mg L-1, respectively).
The predicted adsorption values (adsorption rate calculated) from the Monod equation
obtained previously closely corresponded to the measured adsorption rate in the range 2
- 6 mL min-1 (Figure 6-11). Increasing the flow rate resulted in lower adsorption of the
polymeric products while at 7.5 mL min-1 there was an increase in column backpressure
due to the presence of adsorbed polymer, which caused leaks to occur in the system.
Increasing the loading rate in this way resulted in an increased rate of transformation,
with a corresponding decrease in degree of transformation. The maximum point of the
maximum adsorption curve is shown to be at the flow rate of 6 mL min-1, which means
Page 107 of 163
at that flow rate in a 0.2 mL column the system can adsorb up to 40 g of substrate in a
minute.
Overall HRP-Tglass performs better than the HRP-PG system. At the flow rate of 2 mL
min-1 the HRP-Tglass had a higher apparent maximum rate (120 mgC L-1 min-1)
compared with HRP-PG (45 mgC L-1 min-1). As in this work the kinetics of the HRP are
based on carbon adsorption it is not possible to correlate it with kinetics that are based
on substrate transformation. However, it can be speculated how well the HRP-Tglass
system works compared with other immobilized HRP systems. For example, results
obtained by Levy et al. (2003) showed that the transformation of 4-BP at a flow rate of
1.5 mL min-1 by HRP immobilized on cellulose (0.1 g) was 378 mgC L-1 min-1.
6.2.7 Product adsorption capacity of HRP-Tglass
The model described in the preceding section represents the behaviour of the HRP-Tglass
at the flow rate at the point when maximum conversion (degree of adsorption) occurs.
However, this does not predict the end point for HRP-Tglass adsorption. Furthermore,
this mechanism cannot be described by either the Langmuir or Freundlich isotherm,
since the system is not in equilibrium. The Monod equation is valid for the effect of
concentration on the initial rate of growth of the polymeric products, but does not
indicate the point of deactivation of the enzyme when all active sites become covered.
The production of the adsorbate is dependent only upon the enzyme reaction since no
substrate is adsorbed without enzyme activation by H2O2 (Appendix E). The adsorption
capacity of the particles is limited and the adsorption process will gradually
predominate over the enzyme reaction as the polymeric products occupy the enzyme
sites.
This experiment is similar to the previous experiment (effect of the flow rate on the
HRP-Tglass performance), but here the effect of a longer exposure of substrate feed on
the performance of the HRP-Tglass particles and uptake capacity was measured. The
polymeric product adsorption was calculated by subtracting the concentration of TOC in
the effluent from the concentration of TOC in the influent and multiplying by the
volume added, so the units were measured in mass of TOC (mgC). The total mass of
available 4-BP was calculated as the added loading volume (V= 0.48 L) multiplied by
the initial substrate concentration (S0 = 7.2 mgC L-1) to give 3.45 mgC. After 500 mL
had been passed through the system, the mass of carbon being adsorbed was below
detection for each flow rate used. Figure 6-12 shows that the lower the flow rate, the
greater the total mass of carbon adsorbed.
Page 108 of 163
2.5
Flow rate
(mL min-1)
Cads(mgC)
1
3
3.5
4
6
8
1.5
1
0.5
0
0
100
200
300
400
Feed volume (mL)
500
600
Figure 6-12. Relationship between total carbon adsorption and feed volume. The feed consisted of 4-BP
(7.2 mgC L-1) and H2O2 (3.4 mg L-1) at a range of flow rates (1-8 mL min-1)
Since very little detectable adsorption occurred after passing 500 mL of 7.2 mgC L-1 of
4-BP through the system, the active sites must have become inactive, probably through
being covered by the polymeric products formed. The difference in amount adsorbed
suggests that the size of the polymeric products formed at the lower flow rates was
greater than at higher flow rates, leading to the adsorption of a greater mass.
The performance of the HRP-Tglass and HRP on magnetite (0.1 U per g support) as
reported by Kenji et. al. (1996) were compared. HRP on magnetite was able to adsorb
1.68 mgC per g support of 4-chlorophenol. The HRP-Tglass (0.6 U per g support) was
able to achieve a removal of 13.3 mgC per g support of 4-BP in a plug flow system and
36 mgC per g support in a batch reactor.
6.3
The Michaelis-Menten model, and variations of this model, for the transformation of a
substrate by HRP (including any loss for adsorption) have been reported in the
literature. It was shown in section 5.2.8 that the polymerization and adsorption rate of
the products for HRP-PG and HRP-Tglass can be conveyed also as the deactivation rate,
as the enzyme is progressively loses its activity through the adsorption of polymeric
products, and adsorption gradually decreases as the solution is fed to the reactor.
Page 109 of 163
This work is the first to report on carbon loss after polymerization with immobilized
HRP in the presence of H2O2 in a plug flow system. Flow rate and 4-BP concentration
governs the adsorption kinetics, for both HRP-PG and HRP-Tglass there is a point at
which adsorption overpowers the enzymatic reaction.
The maximum concentration of 4-BP that the HRP-Tglass and HRP-PG were able to treat
without any reduction in transformation was 8.8 mgC L-1. Higher concentrations fed to
the system showed a decline in the enzyme activity as shown by the decline in
adsorption percentage.
The kinetic model that fitted the HRP-PG system was first order, however it was only
valid for flow rates below 2 mL min-1.
The Monod model describes the behaviour of the HRP-Tglass system for the initial stages
of 4-BP removal. The enzyme is activated by the presence of substrate and peroxide and
the higher the flow rate, the higher the rate of adsorption. The apparent optimal flow
rate for maximum adsorption was shown to be 6 mL min-1 (for 4-BP concentration of
7.2 mgC L-1 or 17.3 mg L-1). It also appears that the mass of the polymeric products
differs depending upon the flow rate, with lower flow rates enabling time for larger
mass polymers to form and be adsorbed.
The kinetic parameters for adsorption for HRP-PG and HRP-Tglass were different. The
maximum apparent rate of carbon adsorption at a flow rate of 2 mL min-1 was measured
for HRP-PG as 45 mgC L-1 min-1, which was lower than for HRP-Tglass (120 mgC L-1
min-1). In addition, the adsorption constant for the reaction of HRP-Tglass (15 mgC L-1) was
lower than for HRP-PG, which theoretically allows the enzyme immobilized on Tglass to
treat lower concentrations.
Compared with other immobilized HRP systems the HRP-Tglass appeared to have
relatively similar efficiency. However, it showed a higher capacity to adsorb the
polymerized products than the HRP-PG.
In conclusion, the use of a plug flow system to treat 4-BP with added H2O2 showed that
HRP-Tglass is a better option than HRP-PG. Under the conditions used, adsorption and
consequential deactivation of the enzyme cannot be overcome. The plug flow system
led to faster adsorption than the batch system described in section 5.4.3, but the amount
of adsorbed material was lower (3.2 mgC batch, 2.4 mgC plug flow).
Page 110 of 163
The kinetics and the limitations of 4-BP treatment using HRP-Tglass and H2O2 have been
determined in this chapter. Investigation of a plug flow system with UV irradiation to
produce H2O2 in situ instead of adding it to the feed will be discussed in the next
chapter.
Page 111 of 163
Chapter 7. Kinetics of HRP immobilized on Tglass with

UV irradiation
7.1
INTRODUCTION
In previous chapters two situations involving HRP-Tglass that showed promise were
discussed: 1) UVB regeneration after the HRP-Tglass particles were fully covered
(section 5.2.10) and 2) plug flow reactor where the optimal conditions allow faster
transformation with lower adsorbed mass on the HRP-Tglass particles before complete
deactivation (chapter 6).
However, the objective of this thesis is to develop a stable system in which the phenolic
compounds are transformed in a continuous regime. The overall objective of this
chapter is to study 4-BP transformation using UV irradiated HRP-Tglass in a continuous
regime (i.e., plug flow) at a range of flow rates.
The HRP-Tglass particles were packed inside a narrow diameter column and were
subjected to either UVA or UVB irradiation. The use of UVA irradiation of TiO2 for
photooxidation purposes has been reported consistently in the literature, some examples
being Carraway et al., (1994); Linsebigler et al., (1995); Chen and Ray, (1999) and
Zeng et al., (2003). UVA is preferable to UVB in terms of cost of the UV lamps and in
terms of health and safety (although UVA wavelengths at 315-340 nm are considered
possibly skin cancer-causing (Yang et al., 2003)), and they have the lowest energy
consumption. However, the wavelength of UVA irradiation (365 nm) is longer than for
UVB (320 nm), resulting in a lower energy level and thus capacity to initiate a narrower
range of reactions.
The preliminary steps taken to determine the system efficiency in terms of substrate
transformation with UVA and UVB comparison were to:
1) Pass MilliQ water through the HRP-Tglass column at a range of flow rates and
determine the production of H2O2.
2) Supply 4-bromophenol (4-BP) in the feed and monitor the effluent for 4-BP
transformation, TOC reduction, bromide formation and toxicity.
Page 112 of 163
7.2
RESULTS
7.2.1 H2O2 production in the packed bed reactor

To assess the amount of H2O2 produced by the HRP-Tglass particles in the presence of
UVA and UVB radiation, MilliQ water was passed through the system depicted in
section 3.4.3. The H2O2 concentration of samples collected after passing through the
empty column (sample point A) and the column packed with the HRP-Tglass particles
(sample point B) was determined (section 3.4.3.1). Figure 7-1a shows that UVA
irradiation of the HRP-Tglass promoted little H2O2 production. An inverse correlation
between the flow rate and H2O2 production was shown, indicating that retention time is
an important factor in the activation of TiO2 in the Tglass.
Rao et al. (1980) reported higher levels of H2O2 (0.4 mM) when using UVA (60 minutes)
on a TiO2 slurry (30 g P25 Degussa, 700 mL of deionised water) in a batch system.
Calculation based on molar ratio showed that this was 9 times higher than the
concentration obtained here for UVB at a flow rate of 0.5 mL min-1; this is
understandable as they used a high concentration of TiO2.
Higher H2O2 production was obtained from the HRP-Tglass under UVB radiation (Figure
7-1b). There was markedly higher production of H2O2 at low flow rates than at higher
flow rates for UVB compared with UVA. This result was expected as mentioned before
that the UVB radiation has higher energy, which allows the TiO2 in the Tglass to promote
more electrons to the conduction band (section 2.3.3) which provides electron to
catalyse the water to form hydroxyl radicals which can then form H2O2. It is also noted
that there are similar H2O2 concentrations at sampling point A in both the UVA and
UVB systems.
Page 113 of 163
Sampling point A
0.8
Sampling point B
0.6
H2O2 production (mM)
H2O2 production (mM)
1.2
0.4
0.2
0
0
5
-1
Flow Rate (mL min )
10
1.2
1
Sampling point A
0.8
Sampling point B
0.6
0.4
0.2
0
0
5
Flow Rate (mL min-1)
10
Figure 7-1. Influence of UV wavelength for a range of flow rates of MilliQ water (a) UVA; (b) UVB. ()
hydrogen peroxide concentration exiting empty column (sampling point A) () hydrogen peroxide
concentration exiting column packed with HRP-Tglass (sampling point B).
7.2.2 4-BP transformation in a continuous regime initiated by UV irradiation

The ability of HRP-Tglass to transform 4-BP under UVB irradiation was discussed in
section 5.2.7. The effect of flow rate, ie., the retention time in the column, on the 4-BP
transformation and TOC reduction under UVA and UVB irradiation is shown in Figure
7-2 (effluent was collected from sample point B). The reaction was allowed to proceed
for 16.7 hours and thus 500 mL of feed was treated. The best results were obtained at
the flow rate of 0.5 mL min-1 under UVB irradiation for which there was 74%
transformation of the 4-BP, the release of 70 M bromide and 21% reduction in TOC;
under UVA irradiation there was 32% 4-BP transformation, 25 M bromide released
and 13% reduction in TOC. No H2O2 was added to the feed and activity loss of less than
1% during the reaction was detected for both UVA and UVB systems. This result for
the UVB irradiated system compares well with that of Bayramoglu and Arica (2008)
who treated 4-chlorophenol using HRP immobilized on beads of ferric-poly glycidyl
methacrylate linked with methylmethacrylate and measured the operational stability in a
fluidized bed reactor. They added H2O2 to the feed and obtained 67% degradation of
their initial 4-chlorophenol concentration (1 mM) at a flow rate of 1 mL min-1. Their
system lost 12.5% of its activity after 16.7 hours, whereas the HRP-Tglass system
retained its activity over the same period.
From Figure 7-2 the reduction in carbon was greater under UVB irradiation than under
UVA irradiation at low flow rate (0.5 mL min-1), while at higher flow rate the carbon
reduction appears similar. Taking into consideration that at low flow rate the production
of H2O2 under UVB irradiation is greater than under UVA, and the main reaction
Page 114 of 163
products of the HRP tend to be adsorbed, this confirms that the enzymatic reaction has
greater activity under UVB. Although these results provide no information about any
photocatalytic effect of TiO2 on the organic material (substrate or products), it may
contribute to the observed reduction of carbon seen in Figure 7-2.
100
Flow rate
(mL min )
9
6
0.5
90
85
80
75
Flow rate
95
-1
TOC (%)
TOC (%)
95
100
-1
(mL min )
85
80
75
70
9
6
2.5
1.25
0.5
90
70
150 300 450 600 750

Effluent Volume (mL)
150 300 450 600 750

Figure 7-2. Remaining TOC levels at sampling point B for 100 mL fractions of the effluent after HRPTglass treatment of 0.1 mM 4-BP under UVA irradiation (A) and under UVB irradiation (B).
It is essential to point out that in comparison with section 6.2.7, the degree of adsorption
detected here is lower than for the reaction initiated by added H2O2. This corresponds
with the one of the objectives of this work which is prevention of the adsorption of the
oligomeric products to the particles.
Debromination at low flow rates was greater under UVB irradiation than under UVA
irradiation (Figure 7-3). Furthermore, it was also greater than for HRP-Tglass with H2O2
added to the feed. The HRP-Tglass UVB system produced 50% of the available bromide
whereas the HRP-Tglass H2O2 system gave 35% using the same conditions of flow rate (1
mL min-1) and 4-BP concentration (0.1 mM). This is the first indication that removal of
the halogen group can exceed the theoretical value of 41% debromination discussed in
section 5.2.8. Hence the UVB/HRP-Tglass should be more effective in the treatment of
highly halogenated compounds than the HRP mediated system alone.
Page 115 of 163
Flow rate
-1
(mL min )
60
Bromide formed (M)
Bromide formed (M)
80
9
6
0.5
40
20
0
0
80
60
Flow rate
-1
(mL min )
9
6
2.5
1.25
0.5
40
20
0
0
150 300 450 600 750

150 300 450 600 750

Figure 7-3. Bromide formed at sampling point B for 100 mL fractions of the effluent after HRP-Tglass
treatment of 0.1 mM 4-BP under UVA irradiation (A) and under UVB irradiation (B).
The reductions in 4-BP shown in Figure 7-4, and the results for bromide and the carbon
content, support the claim that this system provides continuous polymerization with less
adsorption. In section 5.2.3, when H2O2 was added to the reaction in a batch system, the
carbon mass in the effluent was around 10% of the total transformed 4-BP, here it was
20% for UVA irradiation and 50% for UVB irradiation at the flow rate of 0.5 mL min-1.
This supports the hypothesis that the UV radiation causes the TiO2 to repel the
hydrophobic compounds and so prevents them from blocking the active sites of the
immobilized enzyme.
100
Flow rate
100
Flow rate
-1
(mL min )
75
9
6
0.5
50
25
A
0
0
150 300 450 600 750

4-BP (%)
4-BP (%)
-1
(mL min )
9
6
2.5
1.25
0.5
75
50
25
B
0
0
150 300 450 600 750

Figure 7-4. Remaining 4-BP levels at sampling point B for 100 mL fractions of the effluent after HRPTglass treatment of 0.1 mM 4-BP under UVA irradiation (A) and under UVB irradiation (B).
These results for TOC reduction, 4-BP transformation and bromide formation
demonstrate that the HRP-Tglass catalyst functions well in the UVB environment. Under
UVA irradiation, the molar ratio of bromide formed to TOC transformed was calculated
for each of the flow rates. In the UVA system as well the UVB system the lowest flow
rate had the highest ratio, and as the flow rate increased the ratio decreased. The ratio
calculated for the UVA system decreased from 0.9 to 0.75, whereas for the UVB
Page 116 of 163
irradiated system it was 0.95 at a low flow rate and decreased to 0.37 (Figure 7-5).
Overall, because a high percentage of dehalogenation occurs at the lowest H2O2
concentration produced (section 7.2.1, Figure 7-1), it can be concluded that the
dehalogenation occurs prior to the enzymatic reaction, i.e., UV photooxidation plays an
important role.
This means that the role of halogen in the system is complex. On one hand, the release
of the halogen allows the enzyme to treat more highly halogenated compounds and so
potentially reduces toxicity as discussed previously in this section and in section 5.2.8.
On the other hand, the presence of halogen can interfere with the production of
hydroxyl radicals and form a less reactive radical, as discussed in section 2.3.3. This
means that increasing concentration of anion in the system will lead to less in situ
formation of hydrogen peroxide that is available to the enzyme.
Molar ratio bromide released

/4-BP transformed
1
0.75
0.5
0.25
UVA
UVB
0
0
4
6 -1
Flow Rate (mL min )
10
Figure 7-5. Molar ratio of bromide to 4-BP transformed for substrate flow rates of 0.5-9 mL min-1.
7.2.3 Analysis of products from HRP-Tglass system activated by UVB

The ratio between the bromide and 4-BP transformation under UVB irradiation is low
at high flow rate (short retention time) (Figure 7-5) meaning greater dehalogenation
occurs at high retention times due to photooxidation. Thus at high flow rates, the main
contributor to the production of bromide is the HRP polymerization process, while at
low flow rates photooxidation becomes a greater contributor. The concentration of the
polymeric products from the transformation of 4-BP was too low to measure, however
Page 117 of 163
the bromide formation and the TOC reduction are two good indicators of the efficiency
of the system.
The soluble products eluting from the column were collected and subjected to ESI mass
spectrometry. The spectrum (Figure 7-6) showed untreated 4-BP and a range of
dehalogenated oligomers corresponding to dimers up to pentamers (there are two dimers
as one of them also lost a hydroxy group, 232.9 Da).
Figure 7-6. ESI-MS spectrum of the effluent of HRP-Tglass reactor after 2 hr of UVB radiation on 0.5 mM
4-BP at a flow rate of 0.5 mL min-1.
7.2.4 The effect of amount of HRP-Tglass on the system performance.

In Figure 7-7 the results are shown for the system in which two columns, each packed
with 2.5 g HRP-Tglass, were run singly and also arranged in parallel thus effectively
increasing the volume but maintaining the same UV exposure of the packed column.
The system was run under UVB irradiation with a feed flow rate of 1.25 mL min-1 and
the results compared with those in section 7.2.3 for the single column. As expected the
dual column reactor performed better than the single column, giving an increase of 20%
in 4-BP transformation. The 4-BP transformation increase was not proportional to the
increase in amount of HRP-Tglass particles. Thus with appropriate design, the system can
be scaled up.
Page 118 of 163
100
1.25 mL min (single column)
4-BP (%)
80
1.25 mL min (dual column)
60
40
20
0
0
100
200
300
400
Feed volume (mL)
500
600
Figure 7-7. Transformation of 0.1 mM 4-BP in a single column (containing 2.5 g HRP-Tglass) compared
with two identical columns in parallel at the flow rate of 1.25 mL min-1.
7.2.5 Comparison of the HRP-Tglass systems performance

Three main types of reactors have been investigated:
1) batch reactor in which the HRP-Tglass polymerized the 4-BP became deactivated and
then UVB was used to regenerate it (section 5.2.10);
2) plug flow system in which the HRP-Tglass packing was fed with 4-BP and H2O2
(chapter 6) and
3) UVB irradiated system in which the plug flow system packed with HRP-Tglass
particles was fed with only 4-BP (chapter 7).
For each reactor there are advantages and disadvantages as seen in Table 7-1.
Page 119 of 163
Table 7-1 Advantages and disadvantages of the reactor systems for HRP-Tglass used in this project.
Reactor type
Advantages
Batch reactor with UV Greater adsorption
regeneration
capacity, easy to use. Can
reteive a substantial amount
of initial activity (20-56%).
Plug flow system
Can be used for low

concentrations. Can be
used at a high flow rate.
Better performance than
HRP-PG.
UVB/HRP-Tglass in a plug Does not require addition

flow system
of H2O2 or enzyme
protection against
denaturation due to
adsorbed products or
excess H2O2.
7.3
Disadvantages
Deactivates quickly, the
regeneration is partial.
Agitation can cause particle
breakage. At least 12 h of
UVB irradiation are needed
to remove the adsorbed
polymers.
Deactivates gradually with
increasing volume of feed.
Adsorption rate has an
inverse correlation with the
flow rate. Lower adsorption
than the batch reactor.
High retention time and
large amount of HRP-Tglass
are required for
transformation.
A reactor system was designed to enable the treatment of halogenated phenolic

compounds by HRP immobilized on Tglass under UV irradiation without the need for the
addition of H2O2. UVB irradiation was shown to be more effective then UVA due, at
least in part, to the generation of higher concentrations of H2O2 produced on the surface
of TiO2 and so close to the enzyme immobilized on the Tglass. The prototype reactor
system gave 74% transformation of 4-BP (initial concentration 0.1 mM) with the release
of 70 M bromide and 21% reduction in TOC. The system operated for 17 hours with
no apparent decline in activity. Greater debromination was measured for the UVB/HRPTglass system than in the absence of UVB irradiation, thus indicating potential for greater
detoxification of halogenated phenols. This was attributed to the combination of photomediated and enzyme-mediated dehalogenation.
The UVB/HRP-Tglass system has shown promise in transforming the 4-BP, however it is
important to determine the reduction of the effluent toxicity to confirm if the treatment
is really effective. By treating more highly halogenated phenolic compounds, the system
can be put to the test as described in chapter 8.
Page 120 of 163
Chapter 8. Detoxification of highly halogenated phenolic

compounds by HRP immobilized on Tglass
8.1
INTRODUCTION
As discussed in section 2.1.1, increasing halogen content leads to increasing toxicity of

phenolic compounds. Furthermore, it has been observed that the presence of both
chlorine and bromine in phenolic compounds results in higher toxicity (Laine et al.,
1997). Such compounds are a direct result of the oxidation of man-made chemicals
(Vetter and Janussen, 2005).
Although both PCP and PBP are highly halogenated, they are not generally considered a
highly toxic threat to biota as they are rapidly adsorbed onto sediments. It is when they
start to degrade and become oxidized (even when adsorbed) that they become a serious
problem, which is why adsorption e.g., onto activated carbon, without further treatment
does not solve the problem.
The overall objective of this chapter is to reduce the toxicity of some example
polyhalogenated phenols: pentachlorophenol (PCP) and pentabromophenol (PBP).
Separately they are toxic, but if together in the environment they can oxidize and form a
highly toxic chloro/bromo phenolic compound (Laine et al., 1997). In this chapter the
aim is to determine the reduction of the toxicity of 4-BP, PCP, PBP, and a mixture of
PCP and PBP using the UVB/HRP-Tglass system.
Water obtained from a reservoir was spiked with PCP and/or PBP and treated with the
UVB/HRP-Tglass system. The effluent was analysed for the transformation of PBP and
PCP, bromide and chloride formation as appropriate. The mass spectra of the effluent
products and toxicity were also monitored.
The toxicity was determined by Geotechnical Services Pty Ltd and the Ecotoxicology
Laboratory at RMIT University using Microtox and Hydra tests (see section 3.3.7 and
Appendix E).
8.2
RESULTS
8.2.1 Treatment of high concentration of PCP and PBP

The packed bed system (2.5 g HRP-Tglass) was fed with PBP and PCP (8 and 10 mg L-1,
equivalent to 16 M and 38 M, respectively) in a natural water sample (Forest
Page 121 of 163
Reservoir, Victoria) at flow rates of 0.5 and 1.25 mL min-1 in upflow mode under UVB
irradiation (1.4 mW cm-2) (section 3.4.3.3). The reason for this high concentration was
due to the limits of detection for concentrations of halogenated phenol, bromide and
molecular mass of the product in the treated water.
By using the PBP and PCP together the system can be truly tested, as the UV irradiation
can lead to bromine and chlorine on the same compound which can significantly
increase the toxicity (US-EPA, 2009). If soluble oligomers with both halogens were
formed, it was expected that the toxicity would increase after exposure to UVB
irradiation.
It was expected that the PBP and PCP would be adsorbed to the HRP-Tglass particles
(25% - 35%), however when the transformation reaction reached a plateau at 0.5 mL
min-1 the particles became slightly coloured (brownish), this colour normally occurs
with polymerization of phenols as observed in section 5.2.10 and by Chance and
Maehly (1955). The results in Figure 8-1 show the transformation of PBP and PCP
using HRP-Tglass in a UVB environment. At a low flow rate (0.5 mL min-1) the
transformation of both species was almost 98%, while at a higher flow rate (1.25 mL
min-1) the system was able to treat 60% of the PBP and PCP.
PCP, PBP (transformed %)
100
75
Flow rate
(mL min-1)
PCP at
0.5
PBP at
0.5
PCP at
1.25
PBP at
1.25
50
25
0
0
50
100
150
200
250
300
350
Figure 8-1. Extent of transformation of PCP and PBP (10 and 8 mg L-1, respectively) by HRP-Tglass in a
packed bed system at flow rates of 0.5 and 1.25 mL min-1. A volume of 300 mL was treated under UVB
radiation.
Page 122 of 163
It was shown in section 7.2.2 that there was a correlation between dehalogenation and
retention time: the longer the pollutant stays in the system, the more halogens are
released. This is very important, as the remaining halogen content determines the
potential toxicity level of the treated stream.
The concentration of the released bromide and chloride were fairly constant throughout
the run (Figure 8-2). At a low flow rate (0.5 mL min-1) the bromide concentration in the
effluent was an average of 70% and the chloride was 65% of their available
corresponding halogen, while at the higher flow rate (1.25 mL min-1) the bromide was
almost 22% (Figure 8-2) and the chloride was 37% of the available halogen content
(Figure 8-3). However, from comparison of the molar ratio of halogen released to the
theoretically available halogens in the transformed PBP and PCP, it appears that the
chlorine is more easily removed, as an average of 3 of the available 5 chlorines occurs
in the effluent for both the flow rates. The bromine is harder to remove and the HRPTglass requires longer exposure to UVB to remove 3 of the available 5 bromines from the
PBP (Figure 8-4). This may be why brominated phenols have been reported in sludge
and polluted river basins (NICNAS, 2001; Kuch et al., 2005) while chlorinated phenols
appear to be degraded by soil bacteria (Apajalahti and Salkinoja-Salonen, 1987).
The UVB/HRP-Tglass was compared against a control using only UVB/Tglass to evaluate
the contribution of photo-mediated dehalogenation. The control was run at a flow rate of
0.5 mL min-1 for 2 h. There was a reduction in the concentration of PCP (69%) and PBP
(51%); however, bromide and chloride were 8% and 9%, respectively. The ESI detected
the
original
compounds
alongside
tetrabromophenol,
tetrabromocatechol,
tetrachlorophenol and tetrachlorocatechol. While it is clear that UVB/Tglass can treat

halogenated phenols, it has been shown repeatedly in this work that the combination of
photo-mediated and enzyme-mediated reaction greatly increases dehalogenation.
Page 123 of 163
Bromide formed (M)
80
60
Flow rate
(mL min-1)
40
0.5
1.25
20
0
0
50
100
150
200
250
Effluent volume (mL)
300
350
Figure 8-2. Extent of debromination of PCP and PBP mixture (10 and 8 mg L-1, respectively), by HRPTglass in a packed bed system at flow rate of 0.5 and 1.25 mL min-1. A volume of 300 mL was treated
under UVB radiation.
Chloride formed (M)
160
Flow rate
(mL min-1)
120
0.5
1.25
80
40
0
0
100
200
300
400
Figure 8-3. Extent of dechlorination of PCP and PBP mixture (10 and 8 mg L-1, respectively), by HRPTglass in a packed bed system at flow rate of 0.5 and 1.25 mL min-1. A volume of 300 mL was treated
under UVB radiation.
Page 124 of 163
anions released/mole of
halogenated phenols
Flow rate
(mL min-1)
4
3
2
Cl- at
Cl- at
0.5
Br- at
Br- at
0.5
1.25
1.25
1
0
0
100
200
300
400
500
Figure 8-4. Average anions/moles of halogenated phenols released from PCP and PBP mixture (10 and 8
mg L-1, respectively) by HRP-T glass in a packed bed system at flow rate of 0.5 and 1.25 mL min-1. A
volume of 300 mL was treated under UVB radiation.
8.2.1.1 Analysis of products from the treatment of PCP and PBP

PCP and PBP are hydrophobic and so tend to adsorb on surfaces or sediments in nature
(Laine et al., 1997). Unlike the transformation of 4-BP and the resultant adsorption of
products (when H2O2 was added in section 5.2.3), PCP and PBP do not need to be
transformed to be able to adsorb on the immobilized enzyme particles. The HRP-Tglass
system was able to break down the adsorbed substrate to produce soluble products. The
ESI mass spectrum of the polymerized and dehalogenated products of PBP and PCP in
the effluent is presented in Figure 8-5 and the compounds were identified.
Page 125 of 163
Cl
A-2Cl
Cl
Cl
Cl
Cl
OH
A-Br
Br
A-3Cl
Br
Br
Br
Br
OH
A-3Br
B-Cl,Br
B-Cl,2Br
B-6Cl
C-Cl,2Br
C-4Br
Figure 8-5. ESI mass spectrum obtained from PBP and PCP (10 mg L-1 and 8 mg L-1, respectively)
spiked reservoir water after treatment. A volume of 300 mL was treated at a flow rate of 0.5 mL min-1
under UVB radiation. A- monomers; B- dimers; C- trimers.
The effluents for PCP and PBP were analysed for their relative peak intensity in
isotopic clusters of ions and their fragmentation patterns. These data and the knowledge
of the initial substrate mass and form allowed identification of dehalogenated
derivatives from the oxidation of the parent PCP (MW 265.0) and PBP (MW 486.5) as:
A- Monomers: dichlorophenol (MW 163.0), bromophenol (173.0), trichlorophenol
(MW 195.0), tribromophenol (MW 330.3).
B- Dimers: dichloro bromo phenolic dimer (MW 337.1), chloro dibromo phenolic
dimer (MW 379.1), hexachloro phenolic dimer (MW 393.2).
C- Trimers: chloro dibromo phenolic trimer (MW 508.7), tetrabromo phenolic trimer
(MW 593.2).
This mass and ion analysis confirmed the previous statement in section 7.2.3 that the
polymerization initiated by UVB radiation produces a greater variety of soluble
dehalogenated oligomers than the reaction of the free enzyme (Kazunga et al., 1999). It
was concluded that near complete dehalogenation is essential and must come prior to
the polymerization.
Page 126 of 163
8.2.1.2 Toxicity of treated solutions according to Microtox test

The degree of treatment required to reduce the toxicity is not only dependent on the
amount that has to be converted, but also on the toxicity of the products relative to the
initial compounds. As a wide range of polymerized dehalogenated phenols appear in the
product stream (section 8.2.2) they may be more toxic than the original solution.
Consequently, the toxicity of the treated halogenated phenols was determined. The
results for the MicrotoxTM toxicity test for PBP and PCP pre- and post-treatment are
shown in Figure 8-6, along with those for the unspiked water samples treated by the
UVB irradiated HRP-Tglass system as a control.
100
Level of toxicity
80
water sample
60
before treatment
40
after treatment
20
0
0
25
50
75
Concentration (%) of effluent
100
Figure 8-6. Results for Microtox tests performed on reservoir water spiked with PCP and PBP (10 mg L1
and 8 mg L-1, respectively) before and after treatment, MilliQ water was used for dilution. The reaction
was carried out in a UVB irradiated HRP-Tglass system at a flow rate of 0.5 mL min-1, unspiked water
sample was used as a control.
8.2.2 Toxicity of treated PCP and PBP solutions in low concentrations according to
Microtox and Hydra tests
The toxicity of the original mixture of PBP and PCP (8 and 10 mg L-1) was measured as
100%. The toxicity of the diluted solution gave an exponential reduction which shows
that the toxin levels of this concentration overwhelm the test bacteria. The same pattern
occurred when the mixture was treated by the HRP-Tglass system at a flow rate of 0.5
mL min-1 which also gave 100% toxicity. Clearly the concentrations of PCP and PBP in
Page 127 of 163
the effluent were too high, and so lower concentration of PBP and PCP (0.67 and 0.89
mgL-1, respectively) were treated and the toxicity tested (Figure 8-7).
Level of toxicity
100
water sample
80
-1
1.25 mL min
0.5 mL min-1
60
before treatment
40
20
0
0
25
50
75
Concentration (%) of effluent
100
Figure 8-7. Results for Microtox tests performed on reservoir water spiked with PCP and PBP (0.89 and
0.67 mg L-1, respectively) before and after treatment. MilliQ water was used for dilution. The reaction
was carried out in a UVB irradiated HRP-Tglass system at flow rates of 0.5 and 1.25 mL min-1, unspiked
water sample was used as a control.
The untreated PBP and PCP mixture gave a toxicity of 54%. At the flow rate of 1.25
mL min-1 the toxicity level increased to 73% while at the flow rate of 0.5 mL min-1 the
toxicity was reduced to 35%. Previously it was demonstrated (Figure 8-2; Figure 8-3)
that higher levels of halogen were released at 0.5 mL min-1 compared with 1.25 mL
min-1. It was also demonstrated (Figure 8-5) that the products contain both bromine and
chlorine, which are known for their toxicity (Birnbaum et al., 2003). All this suggests
that if the HRP-Tglass UVB system only partially dehalogenates the halogenated phenols
(especially when there is a mixture of different halogens), the toxicity will increase.
However, the system was shown to be more effective for dehalogenation at lower flow
rates resulting in reduced toxicity. This result compares well with that of Kazunga et al.
(1999) for PCP oxidation with free HRP in the presence of H2O2. They showed that
there is only one primary product (2,3,4,5,6-pentachloro-4-pentachlorophenoxy-2,5cyclohexadienone) that precipitates and can only be extracted using strong solvents such
as ethyl acetate. That compound, according to Van den Berg et al. (1998), is extremely
toxic (more than 10,000 times that of its parent compound).
Page 128 of 163
In addition to the transformed products, the system produces H2O2 and anions, both of
which can contribute to toxicity. The concentration of H2O2 is low and consumed
immediately in the presence of a reductive substrate (e.g., phenolic compound). The Brand Cl- concentrations are low and add to the salt pool; both chloride and bromide (with
sodium) have an oral rat LD50 of more then 3 g kg-1 (MSDS 2010); however bromide is
considered to be a mutagen, and long term use of bromide-based sedatives can result in
bromism (a psychiatric disorder due to impaired neuronal transmission)(Van Leeuwen
and Sangster 1987).
The UVB/HRP-Tglass system was used to treated 1 mg L-1 of PCP, PBP, PCP with PBP
and 4-BP at 17.3 mg L-1 in MilliQ water (section 3.4.3.4). HPLC measurements of the
after solutions treatment showed less than 5% of the substrate remained in any of the
solutions, and due to the detection limit, the analysis by ICC detected only traces of
bromide and chloride in PCP and PBP solution (90% of removed bromine was detected
for the 4-BP). The Microtox EC50 values for before and after treatment were measured
and are shown in Table 8-1. According to Coleman and Qureshi (1985) the ratings of
toxicity for assessing the toxicity of environmental samples using Microtox are: EC50
(%) =
<25 = highly toxic;
2550 = moderately toxic;
5175 = toxic;
>75 = slightly toxic;
>100 = Non-toxic.
Overall, the toxicity of all the solutions was nullified after treatment. The only
exception was the mixture of PBP and PCP, which after 24 h exposure to the
bioluminescent bacterium had a slight reduction in toxicity. This is expected due to
incomplete dehalogenation, the more halogen that remains on the compounds the more
toxic it can be (see section 2.1.1).
Page 129 of 163
Table 8-1 Microtox sub-lethal (EC50) and lethal (LC50) endpoints for the tested samples (95%
confidence interval)a
PBP before treatment

PBP after treatment
PCP before treatment
PCP after treatment
PCP with PBP before
treatment
PCP with PBP after
treatment
Microtox
EC50 15 min (CI b)
EC50 30 min (CI b)
LC50 24 h (CI b)
34.1
(30.9-36.7)
not toxic
35.4
(30.9-38.3)
-
56.12
(45.14-69.78)
-
51.2
(50.8-52.8)
not toxic
51.9
(50.9-52.9)
-
80.6
(79.4-83.8)
80.7
(79.8-84.0)
>100
2.18
(1.99-2.23 )
4BP after treatment
not toxic
a
All data expressed as %, v/v.
b
CI-confidence interval at 95%.
4BP before treatment
2.20
(2.00-2.33)
-
not toxic
not toxic
4.42
(0-0)
32.73
(28.31-37.85)
-
The LC50 and EC50 values for the Hydra test results of the UVB/HRP-Tglass treatment of
1 mg L-1 of PCP, PBP, and PCP with PBP (before and after treatment) were measured
and are shown in Table 8-2. The toxicity of the effluent was reduced for all the
solutions, however not to the extent indicated in the Microtox tests. According to Lusia
et al. (2000) the Daphnia and Hydra are the most sensitive tests when measuring
aquatic life mortality. The results are very satisfactory for the mixture of PBP and PCP
as the toxicity after treatment is greatly reduced.
Table 8-2 Hydra lethal (LC50) and sub-lethal (EC50) endpoints for the tested samples (95% confidence
interval)a
Hydra hexactinella
LC50 48 h (CI b) LC50 72 h (CI b)
LC50 96 h (CI b)
EC50 96 h (CI b)
PBP before
treatment
PBP after
treatment
16.37
(14.15-18.93
20.23
(14.29-28.62)
16.4
(14.15-18.93)
20.18
(14.59-22.49)
15.15
(12.51-18.36)
20.10
(16.76-22.08)
11.14
(8.96-13.85)
12.02
(9.08-14.03)
PCP before
treatment
PCP after
treatment
9.55
(7.14-9.55)
12.99
(10.33-16.34)
9.70
(7.87-11.98)
11.76
(10.73-12.77)
2.58
(2.13 -3.12)
9.55
(8.56-11.04)
PCP with PBP

2.86
before treatment (2.12-3.87)
PCP with PBP
24.86
after treatment
(23.97-25.07)
a
All data expressed as %, v/v.
b
CI-confidence interval at 95%.
2.21
(0-0)
19.09
(16.51-22.08)
1.90
(0-0)
17.68
(0-0)
Page 130 of 163
cannot be calc.
cannot be calc.
1.48
(0-0)
14.03
(11.28-17.45)
Note: EC50 values from Microtox tests were calculated using the Microsoft Excel
workspace EC50 developed by M.J. McCormick (RMIT). All calculated values for the
Hydra were obtained using the Trimmed Spearman-Karber method (US EPA, 2006).
The reduction in PCP toxicity after using the UVB/HRP-Tglass system compares well
with the results obtained by Aitken et al. (1994). They reported a large drop in toxicity
during the treatment of 0.05 mM (13.25 mg L-1) PCP by HRP (with 0.05 mM H2O2)
only at pH 4, at higher pH the toxicity remained at the same levels. They hypothesized
that this toxicity reduction was caused by PCP adsorption onto reaction products at this
low pH. Their results were contradicted by Zhang and Nicell (2000) who reported that
while adsorption can occur, the remaining solution toxicity remained high.
Wagner and Nicell (2002) found that the HRP treatment of separate phenolic solutions
(phenol, 2-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol and 2-methylphenol)
doubled in toxicity. However, they reported that they did achieve detoxification by
using UV irradiation for 5 mins regardless of whether H2O2 was present or not.
This thesis has shown that the halogen attached to the compound is the cause for the
increase in toxicity. It may be that the other researchers have decreased the amount of
their pollutant by adsorption or precipitation, however the remaining soluble products
contain a high number of halogens which makes them toxic. The UVB/HRP-Tglass
system targets the halogen, as seen in this chapter, and so reduces the toxicity.
8.3
The system using HRP-Tglass under UVB radiation was able to transform 98% of PBP
and PCP (initial concentration of 8 and 10 mgL-1, respectively) and to release 70% of
the constituent bromine and 65% of chlorine, respectively. It was also found that the
chloride is more readily released during the reaction than the bromide, which explains
the appearance of more brominated intermediates in the environment. In addition, it was
found that although the system was able to treat high concentrations of PBP and PCP (8
and 10 mg L-1, respectively) the toxicity (as measured by the Microtox test) was not
greatly reduced. However when the system treated a lower concentration of PBP and
PCP (0.67 and 0.89 mgL-1, respectively) the toxicity could be markedly reduced, but
this depended on the flow rate. At a flow rate of 1.25 mL min-1 the treated effluent had a
higher toxicity level than the initial mixture. This was attributed to the low
dehalogenation effect, and the formation of mixed brominated and chlorinated
oligomers. However, at the flow rate of 0.5 mL min-1 the toxicity of the treated effluent
Page 131 of 163
was lower than the mixture, this result and the result obtained regarding the
dehalogenation values confirms the formation of less toxic products with lower halogen
content.
Toxicity tests were carried out on 4-BP, PBP, PCP and a mixture of PBP and PCP
solutions in MilliQ water (1 mgL-1) before and after treatment with the UVB/HRP-Tglass
system. When the UVB/HRP-Tglass system was run at 0.5 mL min-1 (as it was shown
previously that high halogenation can occur at that flow rate) the sub-lethal toxicity
(Microtox) was completely removed, whereas the lethal toxicity (Hydra) was changed
from highly toxic to moderately toxic. Thus the UVB/HRP-Tglass system successfully
reduces the toxicity of halogenated phenols.
Overall the results showed that the UVB irradiated HRP-Tglass system can produce
soluble treated products that have lower toxicity, in contrast to treatment using with
enzyme (free and immobilized) and H2O2 which can produce insoluble, potentially
highly toxic compounds.
Page 132 of 163
Chapter 9. CONCLUSIONS
The overall aim of this thesis was to develop a method of removing toxic halogenated
phenolic substances from an aqueous solution by enzymatic polymerization and
dehalogenation in a continuous system.
The initial focus of investigation was the glass support which incorporated TiO2 (Tglass).
The Tglass was characterized in terms of its photocatalytic properties, followed by an
investigation of its use as an adequate support for HRP.
ESEM imaging showed that the Tglass consists of TiO2 patches surrounded by glass.
XPS measurement showed that TiO2 comprises 13% of the surface area and consists of
70% rutile and 30% anatase. The Tglass had greater capacity for photooxidizing 4bromophenol than any of the supports surface-coated with TiO2 tested. The reaction
products were catechol, hydroquinone, 2-bromophenol and bromide.
As a support for HRP the Tglass gave an immobilized HRP preparation with an activity
0.6 U g-1. The HRP-Tglass showed a wider range of pH stability (3-11) than reported in
the literature, although the temperature stability range (10 - 60C) was found to be the
same.
HRP interaction with the Tglass support was studied under various conditions. The results
were compared to HRP immobilized on PG. EDX measurements of HRP-Tglass after
polymerization showed that although the enzyme was immobilized over the entire
surface of Tglass particle, it was only active on the silica section.
During UVB irradiation of the HRP-Tglass particles low concentrations of H2O2 were
formed and were sufficient to activate the enzyme locally. When treating 4-BP, the
HRP-Tglass had high resilience toward the UVB radiation and worked continuously over
16 h with no more than 1% activity loss and was able to transform 98% of 4-BP (0.1
mM). This system operated better than 4-BP transformation with added H2O2 with
either HRP-PG or HRP-Tglass. Both the latter systems showed similarity in activity loss
(after 8 h) and a total of 28.5% of carbon removal (as TOC) over the duration of the
reaction. Bromide was detected leaving the H2O2-spiked system and it was noted that
debromination decreased in parallel with decreased adsorption, demonstrating that
dehalogenation occurred with the polymerization process.
Page 133 of 163
Analysis of the adsorbed products after 4-BP treatment by the HRP-PG and HRP-Tglass
preparations showed that the HRP-Tglass products differed, with the polymers being
smaller and less debrominated than for the HRP-PG, indicating a difference in the
reaction mechanisms.
To evaluate the kinetics of carbon loss after polymerization in the presence of H2O2, the
HRP-PG and HRP-Tglass preparations were used in a plug flow reactor and the effluents
were monitored for carbon removal. Since flow rate and substrate concentration govern
the adsorption kinetics, in both plug flow systems there was a point at which the
adsorption overpowered the enzymatic reaction. The HRP-Tglass and the HRP-PG were
able to treat concentrations as high as 8.8 mgC L-1 before a reduction in transformation
was noticed. At higher than 22 mgC L-1, the adsorption of the polymerized products
started to deactivate the enzyme.
In the plug flow reactor with HRP-Tglass the higher the flow rate, the higher the rate of
adsorption. However, as the enzyme is progressively inactivated through the adsorption
of polymeric products, adsorption gradually decreases. It also appears that the mass of
the polymeric products differs depending upon the flow rate, with lower flow rates
enabling time for larger mass polymers to form and be adsorbed.
Kinetic models were devised for the HRP-PG and the HRP-Tglass. A first order model
fitted the reaction mechanism of HRP-PG for flow rates of less than 2 mL min-1, while
the Monod model described the behaviour of the HRP-Tglass system for the initial stages
of 4-BP removal.
A system was designed to enable the treatment of halogenated phenolic compounds in a
continuous regime. The HRP immobilized on Tglass was packed in a column and was
subjected to UV irradiation without the addition of H2O2. UVB irradiation was shown to
be more effective than UVA for producing H2O2 and transforming 4-BP. The prototype
reactor system with UVB gave 74% transformation of 4-BP (initial concentration 0.1
mM) with the release of 70% bromide and only a 21% reduction in TOC indicating that
less adsorption causes less deactivation of the enzyme. The system showed stability for
16 h, treating 4-BP with no apparent decline in activity. This compares well against 4BP dehalogenation using the HRP-mediated reaction (up to 41%).
The system using HRP-Tglass particles under UVB radiation was able to treat a mixture
of PBP and PCP (initial concentration of 8 and 10 mgL-1, respectively). At a flow rate
of 1.25 mL min-1 60% of initial concentration was transformed, 35% of chlorine and
Page 134 of 163
22% of the bromine was released. At a flow rate of 0.5 mL min-1 98% of the initial
concentration was transformed releasing 70% of bromine and 65% of chlorine. When
the system treated a lower concentration of PBP and PCP (0.67 and 0.89 mgL-1,
respectively) the toxicity was reduced but this depended on the flow rate. At a flow rate
of 1.25 mL min-1 the treated effluent had higher toxicity levels than the initial mixture.
This was attributed to the low degree of dehalogenation, and the formation of more
toxic mixed brominated and chlorinated oligomers; direct measurements were
unsuccessful as the halide concentration was too low to be detected. However, at the
flow rate of 0.5 mL min-1 the toxicity of the treated effluent was lower than for the
mixture, confirming the formation of less toxic products with lower halogen content.
The UVB/HRP-Tglass system was used to treat 1 mg L-1 of PCP, PBP, PCP plus PBP,
and 4-BP at 17.3 mg L-1 in MilliQ water at a flow rate of 0.5 mL min-1. The sub lethal
Microtox toxicity showed total detoxification, while the lethal toxicity for both
Microtox and Hydra toxicity tests showed a moderate reduction in toxicity. These
results are better than any other reports of HRP-treated phenolic compounds.
In terms of scientific contribution, in this work HRP was immobilized on a new support
(to become HRP-Tglass) and was used to treat halogenated phenols. The biocatalyst was
packed in a plug flow reactor and the continuous flow system operated under UVB
radiation.
This work is the first to report the operation of immobilized HRP under UVB radiation
whilst retaining enzyme activity. The UVB radiation caused the TiO2 part of the support
to become more hydrophilic and prevent the precipitation of the polymerized products
on to the support, and so prevented the enzyme from being covered and deactivated.
The HRP was active on the silica part of the support and transformed a range of
halogenated phenol concentrations (as low as ppt levels and as high as ppm levels), and
reduced the toxicity of the water sample.
In terms of industrial contribution, this biocatalyst system could be used as an
alternative to adsorption by activated carbon which is used to remove halogenated
phenols from water (US-EPA, 2004). The conversion of the halogenated phenols to
soluble polymeric products with low toxicity would allow the effluent to be treated by
microbial means.
In conclusion, the results of the present work clearly show the potential of continuous
removal of soluble phenolic pollutants from water and wastewater streams by
Page 135 of 163
peroxidase immobilized in close proximity to TiO2 under UV irradiation. Although

further work is required for optimization of the system, the findings presented highlight
the potential of such a system for a detoxification.
Page 136 of 163
Chapter 10. RECOMMENDATIONS FOR FURTHER

WORK
It is imperative that halogenated phenolic waste be treated before it reaches the
environment, otherwise it can be a long term problem. The potential of the UVB
irradiated HRP-Tglass system for the treatment of water containing phenolic compounds
is shown to be high and further research into constructing a reactor with a better design
is recommended.
The future challenges for optimization of this type of system are determination of the
right type of reactor, the UVB intensity required, and determination of whether further
treatment is necessary.
Another approach is the use of the UVB/HRP-Tglass system as a pre-treatment to
biological treatment. As shown in this study, the toxicity level of pentabromophenol and
pentachlorophenol was reduced and their products are soluble. Dehalogenation of the
phenols allows certain enzymes to use them as substrate, as previously electrophilic
attack was prevented by the halogen group.
While proof of concept of the UVB/HRP-Tglass system was demonstrated, improvement
can be achieved by: 1) replacing the reactor glass walls with quartz wall to increase
penetration of UV radiation, 2) designing the system so it may use other radiation
sources such as solar radiation.
It is desirable to treat phenolic wastes without additives and with minimal energy
consumption. The system may prove to be an attractive alternative to adsorption or
reverse osmosis with advantages such as the use of renewable energy, reduction in
carbon footprint and reduction in costs.
This study demonstrates that the UVB/HRP-Tglass system greatly influenced the reaction
of HRP, and was able to increase halogen removal along with the reaction. Knowing
that, it may be beneficial to explore the immobilization of other enzymes that can
benefit from high halogen removal or need H2O2 on Tglass.
Page 137 of 163
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Appendix A. Surface area and pore size measurement for supports.

Surface area and pore size measurement using BET analysis were described in section
3.2.4. Figure A-1 shows a typical pore size distribution according to pore volume
obtained by nitrogen gas desorption. Figure A-2 shows a data page from a typical
surface analysis of a support. The BJH desorption average pore size and BET surface
area were used (shown as highlighted results in Figure A-2)
Figure A- 1 An example of volume distribution according to the pore size on the surface of a support.
Page 153 of 163
Figure A- 2 An example of data collected by the ASAP 2000 Gemini instrument according to their pore
size and surface area of a support.
Page 154 of 163
Appendix B. Determination of 4-bromophenol using HPLC-UV

The procedure for the determination of 4-BP concentration using HPLC-UV was
described in section 3.3.4.1. Figure B-1 shows a typical HPLC chromatogram of 4-BP
with detection at 280 nm, the 4-BP elutes at a retention time of 9.2 minutes. Figure B-2
shows the standard calibration curve, showing the linear relationship between the area
of 4-BP peak and molar concentration of 4-BP. The trend line was obtained from three
experimental data sets.
Detector response
25
20
15
10
5
0
0
6
9
Retention time (min)
12
15
Figure B-1 Typical HPLC chromatogram of a 0.1 mM standard solution of 4-bromophenol on a C18
reverse phase column with monitoring at 280 nm.
Page 155 of 163
7000000
y = 6209034.29x + 24625.39
R2 = 1.00
detector response units
6000000
5000000
4000000
3000000
2000000
1000000
0
0
0.2
0.4
0.6
0.8
1.2
4-BP concentration (mM)
Figure B-2 Calibration curve of 4-bromophenol for a C18 reverse phase column detected at 280 nm.
Page 156 of 163
Appendix C. Determination of PBP and PCP using HPLC-UV

The procedure for the determination of PBP and PCP concentration using HPLC-UV
was described in section 3.3.4.2. Figure C-1 shows a typical HPLC chromatogram of
PBP and PCP detected at 240 nm, the PBP and PCP elute at retention times of 11.4 and
12.3 min, respectively. Figure C-2 shows the standard calibration curve, for which there
was a linear relationship between the areas of the PBP and PCP peaks and their molar
concentration. The trend line was obtained from three experimental data sets.
Detector response
140
PBP
105
PCP
70
35
0
0
10
15
20
25
Retention time (min)

Figure C-1 Typical HPLC chromatogram of a 10 mg L-1 standard solution of pentachlorophenol and
pentabromophenol (retention time 11.4 and 12.3 min respectively) on a C18 reverse phase column with
detection at 240 nm.
Page 157 of 163
2500000
y = 20026x + 2335.5
R2 = 0.9998
detector responce area
2000000
1500000
PCP
PBP
1000000
y = 18929x - 1344.2
R2 = 0.9996
500000
0
0
20
40
60
80
100
120
Substrate concentration (mM)
Figure C-2 Calibration curves of PCP and PBP for a C18 reverse phase column detected at 240 nm.
Page 158 of 163
Appendix D. Ion conductivity chromatography and standard calibration curves

Calibration curves for the determination of chloride and bromide by ion
chromatography were prepared as described in section 3.3.7. The curves are shown in
Figures D-1 and D-2 with their corresponding gradients and R2 value.
There was a linear response for anion concentrations in the range 0.01-1 mM (Figure D1, Figure D-2)
Detector response units
300000
250000
y = 2,837,919x
2
R = 0.9996
200000
150000
100000
50000
0
0
0.02
0.04
0.06
0.08
Bromide concentration (mM)

Figure D-1 Calibration curve for bromide.
Page 159 of 163
0.1
0.12
Detector response units
800000
700000
y = 6,822,143x
600000
R = 0.9997
500000
400000
300000
200000
100000
0
0
0.02
0.04
0.06
0.08
Chloride concentration (mM)

Figure D- 2 Calibration curve for chloride.
Page 160 of 163
0.1
0.12
Appendix E. Determination of toxicity using Hydra hexactinella.

The procedures for the determination of toxicity using Hydra were obtained from the
Ecotoxicology Laboratory at RMIT University:
Hydra hexactinella (Southern Biological Pty. Ltd.) was cultured in freshly prepared
Hydra medium in glass bowls. They were fed daily on <24 h old brine shrimp Naulplii
sp. The Hydra were held unfed for 24 h prior to testing as well as during test exposures.
Seven test concentrations from the samples plus a control solution composed of Hydra
medium were prepared. The serial dilution series 100, 50, 25, 12.5, 6.25, 3.125 and
1.156% v/v were prepared from the test samples. All the dilutions were performed in
Hydra medium using the method of Trottier et al. (1996) with the exception that no
EDTA was added, supplying the 100% sample with TES buffer and CaCl2.
All Hydra tests were conducted in 12 well microplates at 20 1.0C in a 16 hour light/8
hour dark diurnal regime. The organisms were initially observed at 0 h after
introduction into the wells of the microplate. Changes in Hydra morphology were
observed at 6-10 X magnification using an Olympus dissecting microscope after 24, 48,
72 and 96 h of exposure to test solutions.
Adult Hydra (Figure E-1 A) undergo drastic changes in morphology as toxicity
intensifies. When exposed to toxic substances, Hydra show signs of slight intoxication
(clubbed tentacles) (Figure E-1 B). The next stage, the tulip phase, will lead in most
cases to the death of the organisms (Figure E-1 C). The clubbed tentacle stage was
selected as the sub lethal endpoint, while the tulip phase was selected as lethal endpoint.
Figure E-1 Different Hydra responses to toxicity in a TES buffer and CaCl2. A: Normal Hydra; B: Hydra
with clubbed tentacles (first signal of toxicity, sub-lethal endpoint used); C: Hydra in tulip stage (the
lethal endpoint used).
Page 161 of 163
Appendix F. Determination of 4-BP adsorption on supports in the absence of

peroxidase reaction using TOC.
To determine the removal of 4-BP on the column packings in the absence of enzyme
activity (i.e., addition of H2O2) glass columns were packed with 0.20 mL (0.15 g) of
either HRP-Tglass or HRP-PG and operated in single pass mode. The feed solution
containing 4-BP in MilliQ water (0.1 mM) was maintained at ambient temperature (22
1C) and fed at a constant flow rate (1 mL min-1) by a peristaltic pump (Gilson
Minipuls 3). The product was collected in 20 mL fractions and analysed for bromide
and TOC.
The TOC results are depicted in Figure F-1. No bromide was detected.
TOC (mgC L-1)
4-BP
HRP-PG
HRP- Tglass
7.5
7
6.5
6
20
40
60
80
100
120
Volume (mL)
Figure F-1 Control test of the adsorption capacity of the enzyme active support in the absence of H2O2. 4BP concentration of 7 ppm of carbon was fed at a flow rate of 1 mL min-1 at ambient conditions.
Page 162 of 163

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Treatment of wastewater containing toxic

halogenated phenolic compounds by immobilized

A thesis submitted in fulfilment of the requirements for

School of Civil, Environmental and Chemical Engineering

MATERIALS AND METHODS

CHAPTER 4. A COMPARATIVE STUDY OF THE ACTION OF TIO2 IN A

CHAPTER 5. DETERMINATION OF THE ROLE OF HRP IMMOBILIZED ON

KINETICS OF HRP IMMOBILIZED ON PG AND TGLASS

CHAPTER 7. KINETICS OF HRP IMMOBILIZED ON TGLASS WITH UV

SUMMARY FOR CHAPTER 7

CHAPTER 8. DETOXIFICATION OF HIGHLY HALOGENATED PHENOLIC

SUMMARY FOR CHAPTER 8

RECOMMENDATIONS FOR FURTHER WORK

APPENDIX A. SURFACE AREA AND PORE SIZE MEASUREMENT FOR

APPENDIX D. ION CONDUCTIVITY CHROMATOGRAPHY AND STANDARD

APPENDIX F. DETERMINATION OF 4-BP ADSORPTION ON SUPPORTS IN

) and H2O2 (3.4 mg L-1) at a flow rate of 2 mL min-1. ...................................................83

Figure 5-18. Ratio of bromide/4-BP (calculated in mmoles) adsorbed to columns of

in a column reactor (acetonitrile was used for solvent

Figure D-2. Calibration curve of chloride on an anion exchange column. ...................159

Immobilized HRP activity

Initial concentration of 4-BP

Total adsorbed amount in terms of carbon

Empty column volume

Substrate first order constant

Substrate utilization constant

Dry weight of immobilized HRP

Mass of material accumulated on the mg

Apparent maximum adsorbed capacity

Initial TOC concentration of the solution

Effluent TOC concentration of the bulk mgC L-1

Enzyme activity unit

Immobilized enzyme activity unit

mg (pyrogallol) s-1 g-1 (support)

Volume of the effluent

Apparent maximum reaction rate

mgC L-1 min-1

Apparent adsorption rate

mgC L-1 min-1

Hydraulic retention time

Advanced oxidation processes

Brominated flame retardants

Half maximal effective concentration

Environmental scanning electron microscopy

Fourier transformation infra-red

Gel permeation chromatography

High performance liquid chromatography

Median lethal dose

Novel support synthesized in this project

Occupational Safety and Health Administration

Packed bed reactor

Toxic equivalency factor

Total organic carbon

United States Environmental Protection Agency

Volatile organic compound

X-ray photoemission spectroscopy

the necessary information is not routinely compiled by the relevant authorities.

legal restrictions (for example, business confidentiality rules) prevent authorities

Chapter 2. Literature review

2.1.1 Toxicity of halogenated phenols

LD50 (mg kg-1)

EC50 (mg L-1)