Impact of Biological Nutrient Removal Process Operating and Design Conditions On The Removal of Micropollutants From Wastewater

Impact of Biological Nutrient Removal Process
Operating and Design Conditions on the Removal
of Micropollutants from Wastewater
by
Olumuyiwa Omotola Ogunlaja
A thesis
presented to the University of Waterloo
in fulfillment of the
thesis requirement for the degree of
Doctor of Philosophy
in
Civil Engineering
Waterloo, Ontario, Canada, 2015
©Olumuyiwa Omotola Ogunlaja 2015

AUTHOR’S DECLARATION
I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis,
including any required final revisions, as accepted by my examiners.
I understand that my thesis may be made electronically available to the public.
ii
Abstract
The efficacy of three different wastewater treatment configurations, conventional
activated sludge (CAS), nitrifying activated sludge (NAS) and biological nutrient
removal (BNR) for removal of selected micropollutants from authentic wastewater was
investigated. The processes were also characterized based on their proficiency to reduce
the estrogenic activity of the influent wastewater using the in-vitro recombinant yeast
assay. The selected micropollutants (MPs) covered a broad spectrum of therapeutic
classes, i.e non-prescription analgesic (Ibuprofen (IBU)), anti-convulsant/epileptic
(meprobamate (MEP) and carbamazepine (CBZ), lipid lowering drug (Gemfibrozil
(GEM)), antibiotic (trimethoprim (TMP) and sulfamethoxazole (SMX)), steroid hormone
(androstenedione (ADR)), estrogen (estrone (E1)) and estrogenic compounds (nonyl
phenol (NP) and bisphenol A (BPA)). The removal efficiency of TMP improved with the
complexity of the three treatment process configurations. IBU, ADR, SMX, NP, E1 and
BPA had moderate to high removals (> 65%) while CBZ and MEP remained recalcitrant
in the three treatment process configurations. The removal of GEM was better in the
NAS than in BNR and CAS treatment configurations. The YES assay analyses showed
an improvement in estrogenicity removal in the BNR and NAS treatment
configurations as compared to the CAS treatment configuration. Comparing the
estrogenic responses from the three treatment configurations, the removal efficiencies
followed the order of BNR = NAS > CAS and all were greater than 81%.
iii
The removal of estrogenicity in a University of Cape Town- biological nutrient
removal (UCT-BNR) wastewater treatment processes was investigated using pilot and
bench scale systems, batch experiments and mathematical modeling. In the pilot BNR
process, 96 ± 5% of the estrogenicity exerted by the EDCs in the wastewater was
removed by the treatment process. The degradation efficiencies in the anaerobic, anoxic
and aerobic zones of the pilot BNR bioreactor were 11± 9%, 18 ± 2% and 93 ± 10%
respectively. In order to further understand the performance of the BNR process in the
removal of EDCs from wastewater, a bench scale BNR process was operated with
synthetic wastewater dosed with E1 and E2. The removal of estrogenicity in the bench
scale system (95 ± 5%) was comparable to the pilot BNR process and the degradation
efficiencies were estimated to be 8± 0.8%, 38 ± 4% and 85 ± 22% in the anaerobic, anoxic
and aerobic zones. A biotransformation model developed to predict the fate of E1 and
E2 in batch tests using the sludge from the BNR process was calibrated using the data
from the experiments. The biotransformation rate constants for the transformation of E2
to E1 were estimated as 71 ± 1.5, 31 ± 3.3 and 1 ± 0.9 L.gCOD-1d-1 for the aerobic, anoxic
and anaerobic batch tests respectively while the corresponding biotransformation rate
constants for the transformation of E1 were estimated to be 7.3 ± 1.0, 3 ± 2.0, and 0.85 ±
0.6 L.gCOD-1d-1. A steady state mass balance model formulated to describe the
interactions between E2 and E1 in BNR activated sludge reasonably described the fate
of E1 and E2 in the BNR process.
iv
A combination of pilot scale biological nutrient removal (BNR) process, batch
experiments and modeling exercises were employed to investigate the removal and
biotransformation of trimethoprim (TMP) in a BNR activated sludge. The
concentrations of the active microbial groups- ammonia oxidizing bacteria (AOB),
ordinary heterotrophic organism (OHO) and polyphosphate accumulating organism
(PAO) in the BNR bioreactor were estimated to be 40, 780 and 2710 g COD/m3
respectively. TMP was biotransformed in all the redox zones of the BNR bioreactor. The
TMP biotransformation efficiencies in the anaerobic, anoxic and aerobic sections were
13 ± 12%, 17 ± 10% and 24 ± 4% respectively. Batch tests with and without nitrification
inhibition showed that AOB played a role in the biotransformation of TMP in BNR
activated sludge. A pseudo first order model that incorporated the contributions of
PAO, OHO and AOB to the overall biodegradation of TMP was found to describe the
biodegradation of TMP in batch tests with and without nitrification inhibition. The
estimated biotransformation rate constant with respect to PAO, OHO and AOB were
0.32 ± 0.06, 0.58 ± 0.06 and 13.7 ± 0.06 L/gCOD/d respectively. This model showed that
PAOs, OHOs and AOBs contributed towards the biotransformation of TMP in BNR
activated sludge with the trend AOBs = PAOs > OHOs.
v
Acknowledgements
There is an African proverb that says “A great thinker will always appreciate the
importance of thanksgiving”. First and foremost, the completion of this research work,
especially this thesis, will not be possible without the excellent tutelage and financial
assistance of my supervisor, Prof. Wayne J. Parker. I count it as a rare privilege to have
him as a supervisor. I am very grateful for all he has invested in me and I hope that I
have made a contribution to his research group.
I would like to appreciate the support of my advisory committee members, Profs.
Mark Servos, Peter Huck and Sheree Pagsuyoin for reading my thesis and for their
suggestions and constructive feedbacks. I would like to specially thank Prof. Mark
Servos for his advice on analysis of estrogens in wastewater and granting me
unhindered access to his laboratory. I am also grateful to my external examiner, Prof.
Ramesh Goel, of the University of Utah, for reading my thesis and for his invaluable
feedback.
I would like to appreciate the financial support of Canadian Center for Inland
Waters – Wastewater Technology Center (WTC). The facility contributed the pilot
plants, bench scale equipment, chemical and material supplies and maintenance repairs
for the pilot plants. I especially would like to thank Dr. Peter Seto, Scott Dunlop and
Sam Dith for their extensive invaluable support during the first three years of my study.
vi
I also would like to appreciate the friendship and support of Dr. Chen Xia, who
introduced me to the world of wastewater treatment processes when I joined WTC.
I would like to thank Prof. Chris Metcalfe of Trent University and his staff for
providing the protocol used for the solid phase extraction and YES assay method. I
sincerely appreciate the assistance of Brenda Mcllelan and Dr. Ehsanul Hoque of Trent
University for training me on solid phase extraction method and for analyzing the
wastewater samples collected during this study respectively.
I would like to appreciate the friendship and support of some of my colleagues
working in Prof. Mark Servos’ Lab, Leslie Bragg, Maricor Arlos and Patricia Marjan for
providing assistance and advice on YES assay procedure. I appreciate the assistance of
the lab technicians of the department of Civil and Environmental Engineering,
University of Waterloo, Mark Sobon and Mark Merlau, who went extra mile in making
sure that I had all I needed to run smooth experiments. I am also grateful for the
assistance of Ryan Lacharity of the City of Waterloo for granting me access to Elmira
wastewater treatment plant.
I would like to thank my group members who made this journey interesting and
exciting, Dr. Martha Dagnew, Vince Pileggi, Qirong Dong, Daniella Conidi, Kyle
Murray and Gillian Burger. I am grateful for the friendship and administrative
assistance of the project manager of the Center for Control of Emerging Contaminants-
University of Waterloo, Thomas Sullivan.
vii
I am very grateful for the financial support provided during my study by
Canadian Water Network (CWN), EnviroSim, Civil and Environmental Engineering
Department-University of Waterloo, Center for Control of Emerging Contaminant
(CCEC)-University of Waterloo and Graduate Student Office (GSO) - University of
Waterloo.
This thesis will not be complete without acknowledging the steadfast love and
support of my wife, Sileola Ogunlaja, who stood by me through thick and thin. She
gave me the timely and necessary dose of pep talks when I lose focus of the big picture
and encouraged me to keep my hopes high no matter the obstacle. The role she played
in the completion of this project seemingly latent, made me get to this point of writing
the acknowledgement for my thesis, a landmark that I am excited about. Every man
goes through ups and downs, but the difference between those that succeeds and those
that fails lies within the voice of the people around them. She has always been the voice
of reason and encouragement that feeds my impetus to succeed. Words cannot express
how grateful I am but I hope that I would be able to show her in everything I do for the
rest of our lives.
Finally, there is an African proverb that says “a river that forgets his source will
eventually dry”. Hence, I give all glory and honor to the God of all ages, the source of
all wisdom, knowledge and understanding. He who gives wisdom to the wise and
knowledge to the discerning. The revealer of deep and hidden things and the knower of
viii
all things that lies in the darkness. I thank Him and praise Him for giving me life and
wisdom to achieve this feat. To Him alone I give all the glory.
ix
Dedication
To my wife Sileola Ogunlaja (Abebi) and my children, Daniel Ireoluwa Ogunlaja
(Sunshine) and Victoria Aanuoluwa Ogunlaja (Angel) who endured the hard times,
supported and encouraged me throughout my program.

Table of Contents
AUTHOR’S DECLARATION ...................................................................................... ii
Abstract ..................................................................................................................... iii
Acknowledgements ....................................................................................................vi
Dedication ................................................................................................................... x
Table of Contents ........................................................................................................xi
List of Figures ...........................................................................................................xvi
List of Tables.............................................................................................................. xx
Nomenclature ......................................................................................................... xxii
Chapter 1 Introduction ................................................................................................ 1
1.1 BACKGROUND ...................................................................................................................1
1.2 SCOPE AND OBJECTIVES ....................................................................................................8
1.3 THESIS OUTLINE................................................................................................................9
Chapter 2 Literature Review...................................................................................... 10
2.1 MICROPOLLUTANTS ........................................................................................................10
2.2 BIOLOGICAL NUTRIENT REMOVAL PROCESSES IN WASTEWATER TREATMENT ...........11
2.2.1 Summary of BNR Processes in Wastewater Treatment ......................................19
2.3 BNR PROCESS CONFIGURATIONS ..................................................................................19
2.3.1 Summary on BNR Process Configurations ...........................................................27
2.4 BIOLOGICAL PHOSPHORUS REMOVAL IN BNR PROCESS ..............................................28
2.4.1 VFA Potential of the Influent Stream.....................................................................30
2.4.2 SRT/HRT ....................................................................................................................32
2.4.3 Temperature ..............................................................................................................34

xi
2.4.4 Effect of pH ................................................................................................................35
2.4.5 Dissolved Oxygen (DO) ...........................................................................................36
2.4.6 Internal Mixed Liquor Recycle Flows (IMLR) ......................................................37
2.4.7 Summary of Biological Phosphorus Removal in BNR Process ..........................40
2.5 MICROPOLLUTANT REMOVAL MECHANISMS IN ACTIVATED SLUDGE SYSTEMS ..........41
2.5.1 Sorption ......................................................................................................................42
2.5.2 Biodegradation of Micropollutants ........................................................................45
2.5.3 Micropollutant Biodegradation Models ................................................................51
2.5.4 Summary of Micropollutant Removal Mechanisms in Activated Sludge
System .....................................................................................................................................53
2.6 REVIEW OF MICROORGANISMS INVOLVED IN MICROPOLLUTANT DEGRADATION ......53
2.6.1 Summary of Microorganisms involved in Micropollutant Degradation .........59
2.7 INFLUENCE OF REDOX CONDITIONS ON MICROPOLLUTANT REMOVAL .......................60
2.7.1 Summary of Influence of Redox Condition on Micropollutant Removal ........69
2.8 ENDOCRINE DISRUPTING COMPOUNDS IN WASTEWATER TREATMENT PLANT
EFFLUENTS .................................................................................................................................70
2.8.1 Bioassays for Detection of EDCs ............................................................................72
2.9 SUMMARY OF LITERATURE REVIEW ...............................................................................75
Chapter 3 Impact of Activated Sludge Process Configuration on Removal of
Micropollutants and Estrogenicity ............................................................................ 78
3.1 INTRODUCTION ...............................................................................................................80
3.2 METHODOLOGY ..............................................................................................................82
3.2.1 Process Description ..................................................................................................82
3.2.2 Sample Collection and Extraction ..........................................................................86
3.2.3 Solid Phase Extraction ..............................................................................................87
3.2.4 Biological Testing ......................................................................................................89

xii
3.2.5 Target MPs .................................................................................................................91
3.2.6 Statistical Analysis ....................................................................................................92
3.3 RESULTS AND DISCUSSIONS ............................................................................................94
3.3.1 Conventional Parameters ........................................................................................94
3.3.2 Micropollutant Removal during Treatment .........................................................96
3.3.3 Micropollutant Removal in BNR Treatment Configuration ............................105
3.3.4 Biological Activity ..................................................................................................110
CONCLUSIONS ..........................................................................................................................112
Chapter 4 Assessment of the Removal of Estrogenicity in Biological Nutrient
Removal Wastewater Treatment Processes .............................................................. 114
4.1 INTRODUCTION .............................................................................................................114
4.2 APPROACH ....................................................................................................................118
4.2.1 Pilot Scale BNR Wastewater Treatment Process ................................................119
4.2.2 Bench Scale BNR Wastewater Treatment Process .............................................121
4.2.3 Monitoring and Sampling–Pilot and Bench Scale BNR System ......................124
4.2.4 Batch Experiments ..................................................................................................125
4.2.5 Batch Experiment Monitoring and Sampling .....................................................127
4.2.6 Sample Extraction ...................................................................................................127
4.2.6.1 Solid Phase Extraction ....................................................................................128
4.2.6.2 Extraction Recovery ........................................................................................128
4.2.7 Chemical Analysis ..................................................................................................129
4.2.8 YES Assay ................................................................................................................129
4.2.8.1 Data Processing ...............................................................................................131
4.2.8.2 Determination of Relative Potency between E1 and E2-Eq (y) .................132
4.2.9 Statistical Analysis ..................................................................................................133
4.3 RESULTS AND DISCUSSIONS ..........................................................................................133

xiii
4.3.1 Performance of Pilot and Bench Scale BNR System – Conventional
Pollutants..............................................................................................................................134
4.3.2 Evaluation of Extraction Recovery .......................................................................135
4.3.3 Estrogenicity Removal in Pilot and Bench Scale BNR ......................................136
4.3.4 Interstage Comparison of Estrogenicity Removal in Pilot and Bench Scale
BNR ...................................................................................................................................139
4.3.5 Biotransformation of E2 and E1 in Batch Tests ..................................................143
4.3.6 Modeling of Biotransformation of E1 and E2 in Batch Tests ............................148
4.3.7 Relative Potency between E1 and E2-Eq (𝐲) .......................................................151
4.3.8 Modeling Results ....................................................................................................152
4.3.9 Biotransformation Dynamics between E2 and E1 With Respect to E2-Eq .....155
4.3.10 Model Verification ..................................................................................................158
CONCLUSIONS ..........................................................................................................................163
Chapter 5 Biotransformation of Trimethoprim in Biological Nutrient Removal
Treatment System – The Role of Active Microbial Groups ..................................... 166
5.1 INTRODUCTION .............................................................................................................166
5.2 APPROACH ....................................................................................................................170
5.2.1 BNR Pilot Plant Description ..................................................................................171
5.2.2 Pilot Monitoring and Sampling ............................................................................174
5.2.3 Estimation of Active Biomass Fractions ..............................................................174
5.2.4 Batch Experiments ..................................................................................................176
5.2.5 Batch Experiment Monitoring and Sampling .....................................................178
5.2.6 Biotransformation Model ......................................................................................179
5.2.7 Chemical Analysis ..................................................................................................181
5.2.8 Statistical Analysis ..................................................................................................182
5.3 RESULTS AND DISCUSSIONS ..........................................................................................183

xiv
5.3.1 Conventional Contaminants Removal ................................................................184
5.3.2 Pilot Plant Calibration and Active Biomass Estimation ....................................185
5.3.3 TMP Removal in BNR Pilot Plant ........................................................................186
5.3.4 Performance of Batch Reactors with respect to Conventional Pollutants ......192
5.3.5 Performance of Batch Reactors with respect to Trimethoprim Removal .......195
5.3.6 Estimation of Biotransformation Rate Constants ...............................................197
5.3.7 Contribution of Active Biomass Groups towards Trimethoprim
Biotransformation ...............................................................................................................201
CONCLUSION ...........................................................................................................................203
Chapter 6 Conclusions and Recommendations for Future Research ........................ 206
6.1 CONCLUSIONS ...............................................................................................................206
6.2 RECOMMENDATIONS FOR FUTURE RESEARCH .............................................................209
Bibliography ........................................................................................................... 212
Appendix A............................................................................................................. 237
Appendix B ............................................................................................................. 238
Appendix C ............................................................................................................. 248
Appendix D ............................................................................................................ 250
Appendix E ............................................................................................................. 259
Appendix F ............................................................................................................. 263
Appendix G ............................................................................................................ 266
xv
List of Figures
Figure 2-1. Events in a typical BNR process bioreactor................................................18
Figure 2-2. Flow schematic of MLE process. ..................................................................21
Figure 2-3. Flow schematic of A2O process. ..................................................................21
Figure 2-4. Flow schematic of Bardenpho process ........................................................22
Figure 2-5. Flow schematic of modified Bardenpho process. ......................................23
Figure 2-6. Flow schematic of UCT-BNR process. ........................................................24
Figure 2-7. Flow schematic of modified UCT process. .................................................25
Figure 2-8. Schematic of Step Feed process. ...................................................................25
Figure 2-9. Schematic of Sequencing Batch Reactor......................................................26
Figure 2-10. Schematic of Oxidation Ditch process.......................................................27
Figure 3-1. Flow diagram of the pilot-scale BNR treatment process. .........................84
Figure 3-2. Flow diagram of the pilot-scale CAS and NAS treatment process. 1.
Primary clarifier; 2. Aerobic bioreactor; 3. Final clarifier. ............................................85
Figure 3-3. Summary of multi-residue extraction procedure. .....................................88
Figure 3-4. Box plot and average of influent and effluents concentrations from CAS,
NAS and BNR treatment A- Ibuprofen, B- Gemfibrozil, C- Trimethoprim, D-
Carbamazepine .................................................................................................................102
xvi
Figure 3-5. Box plot and average of influent and effluents concentrations from CAS,
NAS and BNR treatment. E- Meprobomate, F- Sulfamethoxazole, G- Androstedione,
H-Estrone. .........................................................................................................................103
Figure 3-6. Box plot of influent and effluents concentrations from CAS, NAS and BNR
treatment. I- Nonyl-phenol, J- Bisphenol-A. ................................................................104
Figure 3-7. Micropollutant removal efficiencies in pilots. .........................................105
Figure 3-8. Ibuprofen concentrations in the influent and stages of BNR process ..108
Figure 3-9. Micropollutant concentrations in the influent and stages of BNR process
...........................................................................................................................................109
Figure 3-10. Mass balances of Ibuprofen in BNR pilot plant. (mass flow rate in µg/d).
...........................................................................................................................................109
Figure 3-11. Box plot comparing the E2-Eq responses of influent and effluents from
CAS, NAS and BNR (n = 4-5). ........................................................................................112
Figure 4-1. Flow schematic of pilot BNR activated sludge treatment system. .......121
Figure 4-2. Flow schematic of the bench scale BNR system. .....................................123
Figure 4-3. E2-Equivalent profiles along pilot and bench scale BNR bioreactors. AN-
Anaerobic, AX-Anoxic, AO-Aerobic. ............................................................................142
Figure 4-4. Mass balances (µg/d) of estrogenicity around pilot BNR bioreactor. ..142
Figure 4-5. Mass balances (µg/d) of estrogenicity around bench scale BNR bioreactor.
...........................................................................................................................................143
xvii
Figure 4-6. E2 equivalent profiles for batch tests dosed with E1 or E2. O2-aerobic, AX-
anoxic, AN-anaerobic. .....................................................................................................147
Figure 4-7. Correlation between E1 concentrations and E2 Equivalents as measured by
YES assay. ..........................................................................................................................152
Figure 4-8. Measured and predicted E2-Eq in E1 and E2 dosed batch tests (aerobic
(AO), anoxic (AX) and anaerobic (AN)). ......................................................................155
Figure 4-9. Simulated and observed estrogen concentration vs time in E2 dosed
aerobic batch tests. ...........................................................................................................157
Figure 4-10. Simulated and measured estrogen concentrations vs time curve in E2
dosed anoxic batch tests. .................................................................................................157
Figure 4-11. Simulated and measured estrogen concentrations vs time in E2 dosed
anaerobic batch tests. .......................................................................................................158
Figure 4-12. Flow and mass balance schematic for prediction of E2-Eq in BNR
bioreactor...........................................................................................................................159
Figure 4-13. Measured E2-Eq and predicted E2, yE1 and E2 + yE1 in the influent and
along the bioreactor in bench scale BNR process. .......................................................163
Figure 5-1. Overview of study approach. .....................................................................171
Figure 5-2. Flow schematic of pilot BNR activated sludge treatment system. .......173
Figure 5-3. Schematic diagram of batch test. ...............................................................178
xviii
Figure 5-4. Trimethoprim concentrations along BNR pilot plant. (deviation bar
represents standard deviation of measurements (n=8)). ............................................191
Figure 5-5. Mass balances of Trimethoprim in BNR pilot plant (TMP mass flow rate in
µg/d). ..................................................................................................................................191
Figure 5-6. Profile of conventional pollutant concentrations in batch experiment
without AOB inhibition (Aerobic-1). ............................................................................194
Figure 5-7. Profile of conventional pollutant concentrations in batch experiment with
AOB inhibition (Aerobic-2).............................................................................................195
Figure 5-8. TMP fraction remaining in the water phase in the batch reactors: Aerobic
1- without AOB inhibition, Aerobic 2- with AOB inhibition. ....................................197
Figure 5-9. Simulated and measured fractions of Trimethoprim in aerobic batch
reactors without AOB inhibition. ..................................................................................200
Figure 5-10. Simulated and measured fractions of Trimethoprim in aerobic batch
reactors with AOB inhibition. ........................................................................................200
Figure 5-11. Contribution of PAO, OHO, and AOB to the overall TMP removal rate.
...........................................................................................................................................203
xix
List of Tables
Table 2-1. BNR process reactions and mediating organisms.......................................18
Table 2-2. Typical design parameters for commonly used BNR processes ...............40
Table 2-3. Kd of selected Micropollutants in contact with secondary sludge ............45
Table 2-4. Micropollutant removal attributed to biodegradation ...............................49
Table 2-5. Biodegradation models for micropollutants removal ................................52
Table 2-6. Summary of reports on role of microorganisms in MP removal from
wastewater ..........................................................................................................................56
Table 2-7. Summary of influence of redox condition on Micropollutant removal ...63
Table 2-8. Pseudo first order biodegradation rate coefficient (kd) (Lgss-1d-1) of selected
micropollutants during biological wastewater treatement ........................................68
Table 3-1. BNR, NAS and CAS operating and design information ............................85
Table 3-2. Physicochemical properties of selected micropollutants ...........................93
Table 3-3. Influent and effluent concentrations of conventional responses (mg/L)
(Mean (S.D)) ........................................................................................................................96
Table 3-4. Statistical significant testing (p ≤ 0.05) results .............................................98
Table 3-5. Biotransformation efficiency of MPs in BNR bioreactor ..........................109
Table 4-1. Pilot BNR operating and design conditions...............................................120
Table 4-2. Composition of influent synthetic wastewater ..........................................123
Table 4-3. Feed composition for batch tests .................................................................126

xx
Table 4-4. Conventional pollutants in BNR influent and effluent (Mean ± SD.) ....135
Table 4-5. Solid phase extraction recovery of E2 .........................................................136
Table 4-6. Model Performance and biotransformation rate constants with 95%
confidence intervals .........................................................................................................154
Table 5-1. Pilot BNR operating and design conditions...............................................173
Table 5-2. Average influent parameter values for BNR pilot plant simulation ......176
Table 5-3. Initial concentrations of chemicals in batch reactors ................................178
Table 5-4. Measured and predicted effluent concentrations (g/m3) from the pilot BNR
process ...............................................................................................................................186
Table 5-5. Estimated biotransformation rate constant for TMP with respect to PAO,
OHO and AOB .................................................................................................................201
xxi
Nomenclature
A2O Anaerobic/Anoxic/Aerobic
AOB Ammonia Oxidizing Bacteria
AR Anoxic Recycle
AN Anaerobic
AX Anoxic
AO Aerobic
BOD Biochemical Oxygen Demand
BNR Biological Nutrient Removal
CAS Conventional Activated Sludge
Ca Calcium
CSTR Continuously Stirred Tank Reactor
CFSTR Continuous Flow Stirred Tank Reactor
COD Chemical oxygen demand
DO Dissolved Oxygen
DPAO Denitrifying Phosphorus Accumulating Organism
EBPR Enhanced Biological Phosphorus Removal
EDC Endocrine Disrupting Compounds
HRT Hydraulic Residence Time
IMLR Internal Mixed Liquor Recycle

xxii
MBR Membrane Biological Reactor
MLSS Mixed Liquor Suspended Solids
MLVSS Mixed Liquor Volatile Suspended Solids
MP Micropollutant
N Nitrogen
NAS Nitrifying Activated Sludge
NH3 Ammonia
NO3 Nitrate
NO2 Nitrite
NR Nitrified Recycle
O2 Oxygen
OHO Ordinary Heterotrophic Organism
P Phosphorus
PhAc Pharmaceutically Active compounds
PAO Phosphorous Accumulating Organism
PHA poly-β-hydroxyalkanoates
PHB Poly-β-hydroxybutyrate
PolyP Polyphosphate
PO4-P Orthophosphate
RAS Return Activated Sludge
xxiii
rbCOD Readily Biodegradable COD
SCFA Short Chain Fatty Acid
SRT Solids Residence Time
TAN Total Ammonia Nitrogen
TKN Total Kjeldahl Nitrogen
TN Total Nitrogen
TP Total Phosphorous
TrOC Trace Organic Compounds
TSS Total Suspended Solids
UCT University of Cape Town
VFA Volatile Fatty Acid
VIP Virginia Initiative Process
VSS Volatile Suspended Solids
GC Gas Chromatography
LC-MS Liquid Chromatography Mass Spectrometer
WAS Waste Activated Sludge
WWTP Wastewater Treatment Plant
xxiv
List of Chemical Compounds
IBU Ibuprofen
MEP Meprobamate
CBZ Carbamazepine
GEM Gemfibrozil
TMP Trimethoprim
SMX Sulfamethoxazole
ADR Androstenedione
E1 Estrone
E2 17-β-Estradiol
NP Nonyl-phenol
BPA Bisphenol-A
EE2 Ethinylestradiol
xxv
Chapter 1 Introduction
1.1 Background
Micropollutants (MPs) in wastewater have been reported as early as the
1950’s (Stumm-Zollinger and Fair, 1965). However, recent interest of the presence
of these compounds in the aquatic environment (Heberer, 2002; Kolpin et al.,
2002; Joss et al., 2005) was borne out of the advancement in analytical
technologies available to detect trace levels of these compounds in different
environmental matrices and the potential ecotoxicological effects of these
compounds on aquatic organisms. This has led to the detection of MPs in
wastewater treatment plants (WWTPs) effluents, surface waters, ground water,
sediments and soil in Europe (Heberer, 2002; Carballa et al., 2004) North America
(Kolpin et al., 2002) and Asia (Nakada et al., 2006). These prior studies have
recommended further investigation into the occurrence, fate, effect and
attenuation of MPs in the environment.
Micropollutants such as human and veterinary pharmaceuticals and their
metabolites, natural and synthetic endocrine disrupting compounds are usually
excreted via urine and feces or inappropriately disposed in the septic system and
can subsequently enter into the aquatic environment through municipal
wastewater treatment plant effluents (Daughton and Ternes, 1999). Appendix A
1
presents a summary of the sources and distribution of pharmaceuticals in the
environment.
Wastewater treatment plant effluents have been identified as an important
point of discharge for MPs into the aquatic environment (Koplin et al., 2002; Joss
et al., 2004; Clara et al., 2005a) because WWTPs are not typically designed to
remove MPs. The limited removals of MPs that have been reported from
wastewater treatment processes are considered to be due to cometabolism
because the concentrations at which the MPs exist in the WWTP influent are too
low to sustain microbial growth or metabolic activities like conventional soluble
organic substrates. The complex chemical structure of some MPs also enhances
their persistence and recalcitrance during wastewater treatment.
The concern about MPs has been accentuated by reports of gonad and
reproductive abnormalities in various trophic levels of aquatic organisms at very
low concentrations (Purdom et al., 1994; Gagne and Blaise, 1998; Fent et al., 2006).
Thereafter, several questions have been raised concerning the chemical
persistence, microbial resistance and synergistic effects of the cocktail of MPs
present in the effluents of WWTP.
Tertiary wastewater treatment technologies such as advanced oxidation
process (using UV, hydrogen peroxide or ozone) and membrane technologies
(using MBR, nano-filtration and reverse osmosis) have been reported to deliver
2
higher removal of MPs from wastewater (Ternes et al., 2003) as compared to
conventional treatment methods. However, there are concerns of producing
more toxic oxidation by-products or metabolites during the use of advanced
oxidation process to treat wastewater (Magdeburg et al., 2012; Maletz et al.,
2013). In addition, these tertiary treatment processes are energy intensive,
resulting in increased operational cost to the plant owners. Therefore, the
optimization of the operation of existing biological wastewater treatment
processes to remove MPs is desirable.
It is hypothesized that advanced biological wastewater treatment process
such as BNR process could be optimized to deliver higher removal efficiencies of
MPs as compared to conventional activated sludge treatment processes. The
BNR processes develops a complex microbial consortia in unique reactor
configurations to remove COD, nitrogen and phosphorus from wastewater. The
important fundamental metabolic processes that occur in a typical BNR process
include; nitrification, denitrification and biological phosphorus removal. These
processes are mediated by diverse microorganisms that exists under different
redox conditions and these organisms may cometabolically degrade MPs along
with the utilization of macropollutants as growth substrates. Hence, the BNR
process has the potential to degrade MPs in wastewater.
3
The microbial populations that exist in a BNR process vary, but based on
the metabolic processes mediated by these organisms, three major groups can be
identified, namely ammonia oxidizing bacteria (AOBs), ordinary heterotrophic
organisms (OHOs) and phosphorus accumulating organism (PAOs). The AOBs
mediates nitrification reaction while utilizing ammonia for growth and cellular
maintenance, the OHOs mediate a wide range of processes including COD
removal, hydrolysis, ammonification (conversion of organic nitrogen to
ammonia), fermentation in anaerobic zones, and so on. The PAOs are responsible
for the biological phosphorus removal from the process. Hence, the PAOs, OHOs
and AOBs collectively co-exist in BNR activated sludge but function differently
depending on the prevailing redox condition (Tchobanoglous et al., 2003).
The role of the microbial communities in the biotransformation of MPs in
activated sludge systems has been previously treated as a “black box”, where all
the microbial communities are lumped together in one single term as mixed
liquor suspended solid (MLSS) or mixed liquor volatile suspended solid
(MLVSS) (Cowan et al., 1993-WWTreat; Monteith et al., 1995-ToxchemTM; Plotz et
al., 2010-WEST®). This approach has been employed because of the difficulty in
determining the active fractions of the various biomass groups, thus it is usually
practiced in the modeling of the biotransformation of MPs in activated sludge
system. The disadvantage of using MLSS or MLVSS for predicting the
4
biotransformation kinetics of MPs is that MLSS or MLVSS contains both active
and inactive fractions of the biomass. While the inactive fraction does not
contribute to the biotransformation of MPs, the active biomass is dependent on
the process operating and design conditions (Layton et al., 2000). The dynamics
of the microorganisms present in an activated sludge varies depending on the
operating condition and configuration of the bioreactor and the ability of an
activated sludge to degrade MPs strongly depends on the presence of
appropriate microbial population (Tchobanoglous et al., 2003). Therefore, the use
of the active biomass concentration rather than MLSS concentration should give
a better description of the biotransformation kinetics of MPs in activated sludge
systems.
There are very few reports on the contribution of heterotrophic organisms
to the biotransformation of MPs in activated sludge. Previous studies have
identified some of the microorganisms responsible for MPs’ biotransformation in
activated sludge systems (Shi et al., 2004; Gaulke et al., 2008; Khunjar et al., 2011),
although this issue still remains debatable. These studies suggest that
heterotrophic organisms co-contribute or predominantly contribute to the
biotransformation of some MPs in activated sludge systems (Gaulke et al., 2008;
Khunjar et al., 2011; Majewsky et al., 2011). However, no study has specifically
5
investigated the role of PAOs, OHOs and AOBs in the biotransformation of MPs
in BNR activated sludge systems.
The presence of different redox conditions is important to the operation of
a BNR process because the alternating redox zones proliferate PAOs for
biological phosphorus removal. Previous studies have shown that combined
anaerobic/anoxic/aerobic processes may create favorable conditions for the
removal of MPs in WWTPs (Li et al., 2010; Joss et al., 2004; Dytzak et al., 2008).
Biodegradation of MPs like E1, E2 and EE2 has been found to depend on redox
conditions, for example, a configuration of anaerobic/aerobic/anoxic yielded 18%
overall removal of E2 while a sequence of anoxic/anoxic/aerobic and
anaerobic/anoxic/aerobic yielded overall removal of 80% and 97.6% of E2
respectively. These results suggest the possibility of a relationship between the
redox zones in a BNR process and the removal of MPs as well as the importance
of the zone sequencing in a BNR system because of the variability in the removal
efficiencies in the different redox zones and with different redox zone
arrangements in a multi-redox zone system. However, the data in literature on
which this premise was based is limited. Therefore, further study is required to
elucidate the contribution of redox conditions to the removal of MPs in BNR
wastewater treatment processes.
6
Assessing the biological effects of MPs on the flora and fauna of the
aquatic ecosystem is an indispensable tool for conducting detailed and
appropriate risk assessment of MPs in the environment. Unlike chemical
analyses that can provide a quantitative measure of the compounds present in a
sample, biological analyses such as in vitro bioassays can provide a qualitative
and quantitative measure of the estrogenic potential or estrogenicity of all the
endocrine disrupting compounds (EDCs) that are present in an effluent (Leusch
et al., 2010). Previous studies have investigated the removal of MPs in
wastewater using different wastewater treatment technologies, but it has not
been conclusively established whether an improved removal of MPs will
translate into a reduction in the biological effects or estrogenicity. Hence, the
investigation of the removal of MPs in a wastewater treatment process in relation
to the degree of the estrogenic response in the effluents will provide a holistic
approach in determining the performance of the treatment process.
A comprehensive review conducted by Pomies et al (2013) on modeling of
MPs in biological wastewater treatment recommended the need for further
research on the effects of oxidation-reduction conditions on biodegradation of
MPs, the need for authors to clearly specify the operating conditions that guides
the domain of validity of their models and the estimation of the active biomass
fraction that mediates biodegradation or biotransformation of MPs. Therefore,
7
there is the need to elucidate the role of PAO, OHO and AOB in the removal of
MPs from wastewater, investigate the effects of redox conditions on the
biotransformation of MPs in activated sludge systems and ultimately assess the
treated effluent quality with respect to the removal of estrogenicity.
1.2 Scope and Objectives
The overall scope of this study was to assess the impact of BNR process
design and operating conditions on the removal of micropollutants from
wastewater. The specific objectives of the research conducted over the course of
this study were to:
 Compare the performance of different activated sludge process
configurations in terms of the removal of MPs and estrogenicity.
 Assess the removal of endocrine disrupting compounds in BNR wastewater
treatment process.
 Elucidate the effects of redox conditions on the removal of MPs in a BNR
process.
 Evaluate the role of the active microbial groups in a BNR process
bioreactor in the biodegradation of micropollutants.
A portion of this research work was carried out at the Wastewater
Technology Center (Science and Technology branch of Environment Canada), in
8
Burlington, Ontario. Existing pilot scale activated sludge treatment processes
were employed in this research work while bench-scale BNR and batch tests
were designed and operated in the University of Waterloo laboratory. The
Wastewater Technology Center was suitable for this research study because the
pilot plants had access to authentic wastewater from the Burlington Skyway
WWTP and the center provided the logistics and technical support for the
operation of the pilot plants.
1.3 Thesis Outline
This thesis is divided into six chapters, references and seven appendices.
Chapter one presents the introduction to the problem under investigation, the
scope and objectives of the research work. Chapter two contains the literature
review on micropollutants, BNR process, BNR process configurations, operating
conditions necessary for biological phosphorus removal in BNR process,
activated sludge microorganisms involved in MPs degradation, MPs removal
mechanisms, MPs removal from wastewater and endocrine disrupting
compounds in sewage treatment plant. Chapters three, four and five are
presented in paper format investigating the specified objectives. Chapter six
contains the conclusions of the research, recommendations and suggestions for
future research.
9
Chapter 2 Literature Review
This chapter reviews the important processes and the mediating
organisms in a typical BNR system. The background information required to
understand the operation of a typical BNR process was reviewed in this section.
MP removal mechanisms and the role of the bacterial community in the removal
of MPs were reviewed. Endocrine disrupting compounds in wastewater and the
available biological analytical techniques were reviewed to determine the
appropriate bioassay for qualitative and quantitative analysis.
2.1 Micropollutants
Micropollutants (MPs) can be broadly defined as any synthetic or
naturally occurring chemical that is not commonly monitored in the environment
but has the potential to enter the environment and cause known or suspected
adverse ecological and or human health effects (USEPA, 2013) . These pollutants
are usually organic compounds that persist in the environment because they do
not biodegrade. The term MPs is believed to envelope a wide array of
compounds which includes but not limited to human and veterinary MPs and
their metabolites, personal care products - detergent, cosmetics, fragrance,
10
natural and synthetic hormones, fire retardants, surfactants, plasticizers, anti-
microbial compounds, endocrine disrupting compounds, ultraviolet absorbing
compounds, domestic products and recently nanotechnology residuals. These
compounds are typically detected at low concentrations, usually in the range of
nanogram per liter or microgram per liter, hence the name “Micropollutants”.
Other terms that are commonly used to refer to these compounds are “xenobiotic
compounds” because most of these compounds are of anthropogenic origin;
“emerging contaminants” because of the recent technological advancement in
analytical methods that are sufficiently sensitive to detect the MPs at
environmentally relevant trace concentrations ;“Trace organic compounds”
because of their detectable low concentrations in different environmental
matrices; “Persistent Organic Compounds” because of their non-biodegradable
nature in the environment.
2.2 Biological Nutrient Removal Processes in Wastewater
Treatment
Biological nutrient removal (BNR) processes are advanced biological
wastewater treatment processes for the removal of COD, nitrogen and
phosphorus from wastewater. Despite the complexity of their design and
operation, they have favor among researchers and operators as an economic and
11
effective method of treating municipal wastewater. Some of the benefits of BNR
systems (Randall et al., 1992) are:
 Reduction or elimination of chemical addition for phosphorus removal
 Decrease in oxygen requirements as compared to nitrifying activated
sludge
 Decrease in sludge production as compared to conventional activated
sludge
 Recovery of alkalinity by the denitrification process (denitrification
produces alkalinity)
 Reduction in filamentous growth and improvement in sludge settleability.
A typical BNR system is an adaptation of a basic activated sludge system
that involves: 1) a slurry of microorganisms, 2) suspended solid recycle, 3)
quiescent sedimentation and 4) solid retention time (SRT) control. In addition,
the bioreactor of a BNR is divided into different redox zones; anaerobic (AN),
anoxic (AX) and aerobic (AO), and typically includes mixed liquor recirculation.
These zones are defined in terms of the electron acceptor that is utilized. Oxygen
is the electron acceptor in aerobic zone, nitrate-N is the electron acceptor in
anoxic zone and neither oxygen nor nitrate-N is present in the anaerobic zone.
The uniqueness of the BNR system is the presence of the alternating redox
condition that enhances the cultivation of different microbial species. The
12
anaerobic zone is essential for phosphorus removal, the anoxic zone is necessary
for nitrogen removal and the aerobic zone is a pivotal component of all BNR
systems (Grady et al., 1999).
The biological nutrient removal (BNR) process involves the utilization of
three different biochemical processes for the removal of nutrients (N, P) in a
wastewater treatment facility. These three key processes are nitrification,
denitrification (both processes are termed biological nitrogen removal) and
biological phosphorus removal.
Nitrification is a two-step process that involves two obligate aerobic
bacteria consortia namely, Ammonia Oxidizing Bacteria (AOB) and Nitrite
Oxidizing Bacteria (NOB). Ammonia is initially oxidized to hydroxylamine, a
reaction that is catalyzed by ammonia monooxidase (AMO). Hydroxylamine is
then oxidized to nitrite, catalyzed by hydroxylamine oxidoreductase (HAO).
This process is summarized in Equation 2-1.
(2-1)
13
Nitrification converts nitrogen from a reduced form (ammonia) to an
oxidized form (nitrate). It is not in itself a nitrogen removal mechanism,
therefore; denitrification is employed after nitrification to achieve complete total
nitrogen removal.
Denitrification involves the utilization of readily biodegradable organic
matter (rbOM) by specific heterotrophic bacteria under anoxic conditions to
reduce nitrate to nitrogen gas as shown in equation 2-3;
𝑁𝑂2− + 𝑟𝐵𝑂𝑀 → 𝑁2 (𝑔) + 𝐶𝑂2 + 𝐻2 𝑂 + 𝑂𝐻 − + 𝑁𝑒𝑤 𝑐𝑒𝑙𝑙𝑠 (2-3)
Biological phosphorus removal is achieved by cycling the mixed liquor
between anaerobic and aerobic conditions to cultivate phosphorus accumulating
organisms (PAOs) e.g Accumulibacter phosphatis (Gu et al., 2008), which are then
removed through excess sludge removal. Figure 2-1 simplifies the events that
occur in a typical BNR bioreactor with respect to poly-p organism (PAOs) and
other non-poly-p accumulating organisms; ordinary heterotrophic organism
(OHO) and ammonia oxidizing bacteria (AOB).
14
 In the anaerobic stage, PAOs do not grow because they cannot utilize
or degrade the fermentative compounds in an anaerobic condition, but
they convert short chain fatty acids (SCFAs) to intracellular energy rich
carbon polymers, poly-β-hydroxybutyrate (PHB). The fatty acids
(particularly acetate and propionate) are produced through the
anaerobic activity of fermentative bacteria (anaerobic OHOs) or
supplied externally by chemical addition. The polymerization of fatty
acids requires an expenditure of cellular energy by the PAO. This
energy is obtained from the breakdown of intracellular poly-
phosphates to release orthophosphates from the PAO into the bulk
solution of the anaerobic tank. As a result of the release of
orthophosphate from the poly-P bacteria, the anaerobic tank contains
two pools of phosphorus; the phosphorus in the influent wastewater
(feed phosphorus) and the released phosphorus by the poly-P bacteria.
Magnesium and potassium ions are concurrently released to the
anaerobic tank along with phosphate (WEF, 2005). Previous studies
have shown that anaerobic phosphorus release is a precursor to
effective aerobic phosphorus uptake by the PAOs (Barker and Dold,
1996).
15
 Under anoxic condition, where nitrate (NO3) become available, it is
believed that OHOs continues to mediate hydrolysis of particulate
organic substrates while a fraction of the PAOs that is capable of
utilizing nitrate as an electron acceptor (denitrification) oxidizes
previously stored PHBs to obtain energy for growth and maintenance
requirements (Barker and Dold 1996). However, the anoxic
phosphorus uptake per intracellular PHB oxidized is less efficient
when compared to aerobic phosphorus uptake and phosphorous
uptake per unit PHB oxidized seems to occur simultaneously with
phosphorus release per PHB storage when SCFAs are available under
anoxic condition.
 In the aerobic zone, where sufficient external electron acceptors (NO3
and O2) become available, PAOs oxidize the previously stored PHBs to
obtain energy for growth and maintenance requirements. PHB in
PAOs serves two important functions. First, it helps the bacteria to
grow and rebuild polyphosphates by taking up soluble phosphate.
Second, PHB along with polyphosphates help aerobic poly-P bacteria
to survive in an anaerobic condition. Concurrently, the poly-P bacteria
restore intracellular energy reserves through absorption of
16
orthophosphate to form intracellular polyphosphates granules or
volutins.
The energy obtained from the degraded PHB is high enough for the PAOs
to absorb not only the released phosphorus but also a significant quantity of feed
phosphorus. Phosphorus removal is achieved when the bacteria (sludge) are
wasted from the secondary clarifier. Thus a low effluent phosphorus
concentration is achieved from the BNR reactor (WEF, 2005). Sludge that is not
wasted is returned to the anaerobic tank where the BNR process is repeated. By
exposing the poly-P bacteria to alternating anaerobic and aerobic conditions, the
poly-P bacteria are stressed and take up phosphorus in excess of normal cellular
requirements.
Table 2-1 summarizes the processes occurring and the mediating
organisms in the different zones of a typical BNR system. From Table 2-1, it is
quite clear that there are different processes that proceed in the different zones
and the organisms responsible for these processes could either be heterotrophic
or autotrophic. These organisms are present in all of the zones due to the internal
recycles but growth and metabolic activity is however dependent on the redox
conditions of the zones.
17
Figure 2-1. Events in a typical BNR process bioreactor (adapted from
EnviroSim 2008).
Table 2-1. BNR process reactions and mediating organisms

Zone Process Mediating Organism
Anaerobic Phosphorus release and PHB storage Heterotrophs (PAOs)
Fermentation: Complex organics converted to Heterotrophs (non-
VFAs. PAOs)
Pre-anoxic Denitrification: Nitrate to nitrogen gas via the: Heterotrophs (non-
-Use of influent substrate-BOD removal, and PAOs)
-Use of stored substrate (PHB) – phosphorus Heterotrophs (DPAOs)
uptake
Post Anoxic Denitrification: Nitrate to nitrogen gas via the:
(if -Use of cellular substrate (endogenous Heterotrophs (non-
provided) reactions), or PAOs)
-Use of methanol
Aerobic BOD removal Heterotrophs (non-
PAOs)
Ammonification: Organic nitrogen to Heterotrophs (non-
ammonia nitrogen PAOs)
Nitrification: Ammonia nitrogen to nitrate Autotrophs (AOB &
nitrogen NOB)
PHB degradation and excess phosphorus Heterotrophs (PAOs)
uptake
Source: Jeyanayagam (2005).
18
2.2.1 Summary of BNR Processes in Wastewater Treatment
Important fundamental processes that occur in a typical BNR system
include 1) Nitrification, 2) denitrification and 3) biological phosphorus removal.
Even though other processes like hydrolysis, fermentation and ammonification
also occur in a typical BNR process, the former three directly relate to the
existence of the different redox zones present in a BNR process. The
microorganisms that mediate these processes are ammonia oxidizing organisms
(Nitrosomonas and Nitrobacter), denitrifying bacteria and phosphorus
accumulating organism all existing in the activated sludge but functioning
differently depending upon the redox zones (anaerobic/anoxic/aerobic),
assuming all other operating conditions are appropriate
BNR systems can be configured in various designs with differing zone
sequence. The type of configuration employed may have an effect on the
degradation or removal of micropollutants in wastewater treatment. The next
section examines the various BNR basin configurations that have been explored
for the treatment of wastewater.
2.3 BNR Process Configurations
Various combinations and modifications of the BNR system have been
developed to meet economic and regulatory demands that have been placed
upon wastewater treatment. BNR process configuration can be designed to

19
achieve either total nitrogen (TN) removal or both TN and total phosphorus (TP)
based on the incorporation of either two of the three redox conditions
(anoxic/aerobic) or a combination of the three (anaerobic/anoxic/aerobic)
(Jeyanayagam, 2005). There are a variety of configurations of BNR systems and
the selection of a configuration depends on the influent characteristics, effluent
limits and desired operating conditions (WEF, 2005). Although this list is not
exhaustive, it presents common BNR system configurations, their process
diagrams and brief descriptions. Detailed descriptions of the BNR process
configurations have been reported by WEF (2005).
1) Modified Ludzack-Ettinger (MLE) Process – continuous-flow suspended-
growth process with an initial anoxic stage followed by an aerobic stage, to
enable denitrifying bacteria first access to the influent substrate. An
increase in internal mixed liquor recycle (IMLR) rate increases the rate of
denitrification, but a rate beyond 400% of influent, provides no added
benefits (WEF, 2005). The absence of an anaerobic zone prevents MLE from
being suitable phosphorus removal. Figure 2-2 presents the flow schematic
of the MLE process.
20
Figure 2-2. Flow schematic of MLE process.
2) Phoredox (A2O) Process – MLE process preceded by an initial anaerobic
stage; used to remove both TN and TP. Here the introduction of the
anaerobic zone before the anoxic and aerobic zone enables the cyclic pattern
required for proliferation of PAOs (phosphorus removal) and the IMLR from
aerobic to anoxic zone enables nitrogen removal, although the system is
sensitive to nitrate and dissolved oxygen recycle to the anaerobic zone. Figure
2-3 presents the flow schematic of the A2O process.
Internal mixed liquor recycle
Influent Anaerobic Anoxic Aerobic Effluent
Return Activated Sludge

WAS
Figure 2-3. Flow schematic of A2O process.
21
3) Bardenpho Process (Four-Stage) – continuous-flow suspended-growth
process with alternating anoxic/aerobic/anoxic/aerobic stages. Excellent for
total nitrogen removal but no capability for phosphorus removal due to the
absence of an anaerobic zone, a selector for PAOs. Figure 2-4 presents the
flow schematic of Bardenpho process.
Influent Prim Anoxic Aerobic Sec Anoxic Re-aer Effluent
WAS
Figure 2-4. Flow schematic of Bardenpho process.
4) Modified Bardenpho Process – Bardenpho process with addition of an
initial anaerobic zone, a selector for PAOs enrichment in the sludge. It
provides excellent nitrogen removal and good phosphorus removal. Figure
2-5 shows the flow schematic of modified Bardenpho process.
22
Influent Anaerobic Prim Anoxic Aerobic Sec Anoxic Re-aer Effluent
WAS
Figure 2-5. Flow schematic of modified Bardenpho process.
5) Virginia Initiative Process (UCT-BNR) – A variation of the A2O
configuration with the addition of an internal mixed liquor recycle from the
anoxic zone to the anaerobic zone and the RAS returned to the anoxic zone
rather than the anaerobic zone. This configuration minimizes the adverse
effects of the nitrate return to the anaerobic zone. Here PAOs are given the
selective advantage of full access to all available readily biodegradable
organic matter or acetate. It is good for both total nitrogen and total
phosphorus removal. Figure 2-6 shows the flow schematic of a UCT-BNR
process.
23
WAS
Figure 2-6. Flow schematic of UCT-BNR process.
6) Modified University of Cape Town (UCT) Process – To further minimize
the effects of the nitrate recycle to the anaerobic zone from the anoxic zone,
a second anoxic stage is introduced, where the internal nitrate recycle is
returned. The first anoxic zone provides the mixed liquor recycle to the
anaerobic zone, and under appropriate design and operating conditions, no
nitrates are returned to the anaerobic zone. It provides good nitrogen
removal and excellent phosphorus removal. Figure 2-7 shows the flow
schematic of a modified UCT process.
24
Influent Anaerobic Anoxic 1 Anoxic 2 Aerobic Effluent
WAS
Figure 2-7. Flow schematic of modified UCT process.
7) Step Feed Process – alternating anoxic and aerobic stages, influent flow is
split to several feed locations and the recycle sludge stream is sent to the
beginning of the process; used to remove TN. Figure 2-8 shows the flow
schematic diagram of step feed process.
Figure 2-8. Schematic of Step Feed process.
8) Sequencing Batch Reactor (SBR) Process – SBRs are fill‐and‐draw reactors
that operate sequentially through the different redox conditions by
adjusting the mixing and aeration. The anaerobic/anoxic/aerobic
25
progression is necessary for removal of phosphorus and total nitrogen.
Because of the fill‐and‐draw nature of SBRs, it is necessary to remove the
nitrates remaining from the previous aerobic cycle before anaerobic
conditions can be established, thus the typical treatment progression
becomes anoxic/anaerobic/aerobic as depicted in the Figure 2-9.
Figure 2-9. Schematic of Sequencing Batch Reactor.
9) Oxidation Ditch – continuous-flow process using looped channels to
create time sequenced anoxic, aerobic, and anaerobic zones, cycling
pattern essential for the proliferation of PAOs in the activated sludge. It
provides both total nitrogen and total phosphorus removal. Figure 2-10
shows flow schematic of the oxidation ditch process.
26
Figure 2-10. Schematic of Oxidation Ditch process.
2.3.1 Summary on BNR Process Configurations
Various configurations or basin arrangements of the BNR system have
been developed to achieve efficient nutrient removal. The use of three zones,
anaerobic/anoxic/aerobic is common in the configurations with exceptions that
employ only two of the three. Two major features differentiate one configuration
from the other; 1) zone sequencing and 2) recycle stream locations. The
proliferation and enrichment of PAOs in a BNR bioreactor requires the selection
of an appropriate BNR configuration. This is essential so as to ensure a stable
reactor performance in terms of meeting the effluent quality while
simultaneously enriching the population of PAOs in the bioreactor. The UCT-
BNR process was selected for the research study because the process is amenable
to changes in operating conditions and the configuration of the reactor supports
proliferation of PAOs while maintaining good effluent quality.
27
It has been proposed that subdivision of the reactor volume into a cascade
of compartments, significantly improves the removal of biodegradable MPs
compared to a single mixed reactor (Joss et al., 2006), suggesting that, different
configurations of BNR systems may provide different MP removal. Therefore,
the estimation of the biotransformation constants of MPs under different redox
condition would provide a platform that could be extrapolated to other BNR
configurations. The next section examines the operating conditions for ensuring
efficient biological phosphorus removal in a typical BNR process.
2.4 Biological Phosphorus Removal in BNR Process
The previous section identified the importance of selecting a BNR
configuration that includes sequencing of anaerobic, anoxic and aerobic zones,
creating the appropriate conditions for the enrichment of PAOs. In addition to
having proper zone sequencing, appropriate design and optimum operating
conditions such as optimum recycle ratio, influent carbon to phosphorus ratio
and solids retention time must be employed so as to sustain the redox conditions
and ultimately establish a healthy biomass in the reactors (WEF, 2005). Therefore,
an understanding of the factors that affect the performance and stability of the
BNR process is needed to effectively design, maintain, control and optimize the
performance of the process. This section summarizes the design and operating
28
conditions required for operating a BNR system for enhanced biological
phosphorus removal.
PAOs are organisms capable of storing phosphate as intracellular
polyphosphate, leading to phosphorus (P) removal from the bulk liquid phase
via PAO cell removal in the waste activated sludge. Unlike most other
microorganisms, PAOs can take up carbon sources such as volatile fatty acids
(VFAs) under anaerobic conditions, and store them as intracellular carbon
polymers, called poly-β-hydroxyalkanoates (PHAs). The energy for this anabolic
build-up is generated by the cleavage of polyphosphate leading to the release of
phosphate from the cell. Reducing power is also required for PHA formation,
which is produced largely through the glycolysis of internally stored glycogen
(Mino et al., 1998). Aerobically, PAOs use their stored PHA as the energy source
for biomass growth, glycogen replenishment, P uptake and polyphosphate
storage. Net P removal from the wastewater is achieved through the removal of
waste activated sludge containing high polyphosphate content. While the
majority of P removal from the BNR process is often achieved through
anaerobic–aerobic cycling, anaerobic–anoxic operation also allows P removal to
occur, due to the ability of some PAOs (i.e. denitrifying PAOs or DPAOs) to use
nitrate or nitrite instead of oxygen as electron acceptors and, therefore, perform P
uptake and denitrification simultaneously. Maximizing the fraction of P removal
29
in the anoxic zone can reduce process operational costs, due to savings in
aeration as well as in the amount of carbon sources needed for denitrification.
The design of any BNR process configuration to optimize biological
phosphorus removal requires that the microbial reaction proceed in favor of the
PAO population (WEF, 2005). There are a number of operational conditions that
affect PAO development and growth in a BNR activated sludge. These
conditions include:
 VFA potential of the influent stream (Jonsson et al., 1996;
Mulkerrins et al., 2004)
 SRT/HRT (Shao et al., 1992; Smoulder et al., 1995)
 Temperature (Chang and Park, 2008; Whang and Park, 2001)
 pH (Filipe et al., 2001; Mulkerrins et al., 2004)
 Dissolved O2 (Jonsson et al., 1996)
 Internal Mixed Liquor Recycle Rate (Jonsson et al., 1996)
2.4.1 VFA Potential of the Influent Stream
The performance of biological phosphorus removal processes has been
reported to depend on the composition of the readily biodegradable organic
matter (acetates) in the influent (Comeau et al., 1986). These acids may be in the
feed, produced through the fermentation of municipal wastewater or can be
added as a commercial or waste product. As previously mentioned, PAOs

30
transforms short chain VFAs (acetate and propionic) into intracellular PHAs.
Therefore, a high concentration of VFAs in the influent stream will result in their
rapid phosphorus uptake by the PAOs (Copp, 1998). Jonsson et al. (1996)
indicated that 14 mg of VFA-potential is required to remove 1 mg of phosphorus.
VFA-potential was defined as the concentration of VFA in a fermented sample of
wastewater. Abu-Ghararah and Randall (1991) reported similar VFA-potential
values within the range of 10 to 20 mg VFA/Premoved.
Comeau et al. (1996) indicated that a critical factor essential for optimizing
phosphorus removal in a BNR system is the amount of VFA in the influent
stream. Barnard and Steichen (2006) reported that 7 – 9 mg of VFA was needed to
remove 1 mg of phosphorus, while Oldham et al. (1994) used VFAs to achieve
effluent phosphorus level as low as 0.2 – 0.3 mg/L. Typically, to achieve
phosphorus removal to an effluent concentration less than 1.0mg/L, the COD: TP
ratio should be 40 or more (U.S. EPA, 2009).
Another group of organisms, glycogen accumulating organisms (GAOs)
e.g Competibacter and Defluviicoccus, also have the ability to take up acetate in the
anaerobic zone, not by using energy in phosphate bonds but by using stored
glycogen as the energy source (Kong et al., 2006). The VFA are stored as a
complex carbohydrate containing polyhydroxyvalerate (PHV), instead of PHB
formed with poly‐phosphorus as the energy source. Under certain conditions,
31
such as high temperatures over 28oC, high SRT, low pH in the aerobic zone or
longer anaerobic HRT, GAOs may out‐compete PAOs for the VFAs, which
would result in less or no release of phosphorus in the anaerobic zone. This in
turn will result in fewer fractions of PAOs in the biomass and ultimately
deterioration of the BNR system (Filipe et al., 2001; Saunders et al., 2003).
Oehmen et al. (2005) indicated that PAO can have a competitive
advantage over GAO when the VFA consists of roughly equal proportions of
acetic and propionic acid as substrate. This is because PAOs are able to switch to
propionate much more quickly and effectively than GAOs. Therefore to
stimulate optimal growth of PAOs, the strategy is to feed the BNR system with
an equal amount of acetic acid and propionic acid (Oehmen et al., 2005; Bott et
al., 2007).
2.4.2 SRT/HRT
The SRT represents the average length of time a particulate constituent
stays in a bioreactor. HRT determines the contact time between the solution
phase that contains the substrate and nutrients and the biomass, which is made
up of the micro-organisms scavenging food materials for growth. Phosphorus
release and uptake rates in anaerobic and aerobic zones must be considered in
selecting the overall system and individual zone HRT values. The HRT value can
be controlled by appropriate flow adjustments at constant volume.

32
The effect of SRT on the BNR process has been widely investigated (Shao
et al., 1992; Smoulder et al., 1995). An increase in SRT causes an increase in the
concentration of ordinary heterotrophs that produce VFAs in the anaerobic zone
and therefore proliferation of PAOs and subsequent P-removal may be enhanced
in processes with low VFAs in the feed. However, the SRT increase could lead to
reduction in P-removal due to reduced rate of sludge wastage.
BNR systems can operate at SRT values greater than 3 days. However, at
SRT values greater than 4 days and at temperature greater than 15oC, nitrification
will become active and nitrates should be denitrified. As the SRT is increased to a
level where endogenous reactions become significant, secondary release of
phosphorus may lead to decreased performance at constant feed VFA and COD
values (WEF, 2005). Although the performance of other heterotrophic reactions
rely more on the system sludge age, in BNR, performance cannot be defined
solely based on the SRT and HRT. The feed COD: P ratio shapes the microbial
composition of the BNR sludge and the effluent levels that can be attained (WEF,
2005).
Previous studies have shown that the ratio of HRT in the anaerobic zone
to the HRT in the aerobic zone is important for the optimal operation of a BNR
system. Sufficient time should be allowed for the formation of VFAs and storage
of intracellular PHAs in the anaerobic zone. If the time is too short, phosphorus
33
uptake in the aerobic zone will be lower than achievable because insufficient
PHAs were stored in the anaerobic zone. Neethling et al. (2005) reported a ratio
of between 3 and 4 for aerobic HRT to anaerobic HRT led to optimal
performance of a BNR system.
Grady et al. (1999) suggested that at temperatures above 15oC, it may be
very difficult to operate at an aerobic SRT sufficiently high to allow PAOs to
grow without also populating nitrifying bacteria. Under such circumstances, the
UCT-BNR process has distinct advantages.
2.4.3 Temperature
High temperature can have an adverse effect on phosphorus removal.
Whang and Park (2006) and Lopez-Vazquez et al. (2007) reported that
temperatures lower than 20oC could favor PAO over GAO, resulting in a stable
BNR process, while the opposite could occur at temperature greater than 20oC.
Bott et al. (2007) reported predominance of GAOs in a BNR operated at
temperatures greater than 28oC.
Full and pilot scale studies have shown that BNR can be affected by low
temperature. PAOs outcompete GAOs at temperatures as low as 5oC, the GAOs
practically disappeared in the 5oC reactor (Erdal et al. (2002). However,
fermentation in the collection system will decrease with decrease in temperature,
34
thus leading to insufficient VFA in the feed, thereby interfering with overall
phosphorus removal.
2.4.4 Effect of pH
Many studies have shown that a higher ambient pH in enriched PAO
sludges has resulted in a higher anaerobic P release (Smolders et al., 1994; Liu et
al., 1996; Bond et al., 1999; Filipe et al., 2001). Smolders et al. (1994) found that the
ratio of anaerobic P release to acetate uptake varied linearly from 0.25 to 0.75 P-
mol/C-mol when pH rose from 5.5 to 8.5. The reason for this variation was
explained as follows: under the assumption that the internal pH of the cell is kept
constant, there is an increased pH gradient and a corresponding increase in
electrical potential difference across the cell membrane at a high ambient pH.
Therefore, more energy is needed for acetate transport through the membrane
when external pH is high. This increased energy is generated through an
increase in polyphosphate degradation.
However, the acetate uptake, glycogen degradation and PHA
accumulation rates of PAOs have been shown in batch tests to be independent of
pH over the range 6.5–8.0 (Filipe et al., 2001), indicating that the higher energy
requirements to take up acetate does not negatively affect their ability to
metabolize VFA. Aerobically, a series of batch tests has shown that P uptake,
PHA utilization and biomass growth were all inhibited by a low pH (6.5),
35
suggesting that a higher aerobic pH (7–7.5) would be more beneficial for PAOs
(Filipe et al., 2001).
It has been postulated that an anaerobic pH of 7.25 is a critical point,
whereby GAOs are able to anaerobically take up VFA faster than PAOs at pH
values below 7.25, and PAOs take up acetate faster above this pH value (Filipe et
al., 2001). However, Liu et al. (1996) indicated an optimum pH of 6.8 ± 0.7 for
anaerobic acetate metabolism (acetate uptake rate coupled with P-release rate).
An improved level of P removal was observed when the anaerobic pH set point
was increased from 6.8 to 7.25 (Filipe et al., 2001). Other studies have also shown
higher P removal when the anaerobic and/or aerobic pH level was increased
(from pH 7 to 7.5–8.5) (Bond et al., 1998; Serafim et al., 2002). The reason for the
improved performance was hypothesized to be a shift in the microbial
competition from GAOs to PAOs.
2.4.5 Dissolved Oxygen (DO)
A BNR process needs to satisfy different oxygen demands from the
different bacterial populations present in the system. BNR systems designed for
COD removal and nitrification typically require DO levels greater than 2 mg/L
(Louzeiro et al., 2002). In a BNR system, the anaerobic zone must be kept devoid
of oxygen (0 - 0.2 g/L O2) as the presence of oxidizing substances such as oxygen
and nitrate interfere with the efficiency of the BPR process.

36
The presence of low levels of oxygen or other oxidizing agents affects the
redox potential and thus negatively impacts on the rate of phosphate release.
(Shehab et al., 1996). Maintaining an oxygen concentration of between 3.0 and 4.0
mg/l in the aerobic zone has been recommended (Shehab et al., 1996). It has also
been reported that, for successful BNR, a DO concentration 3.0 - 4.0 mg/l is
essential. Brdjanovic et al. (1998) revealed that excess aeration can have a
negative impact on the BPR process as cessation of P-uptake occurs due to
depletion of poly-hydroxy-butyrate (PHB) in an over aerated process.
The presence of nitrate in the anaerobic zone has also been reported to
affect the BPR process (Shehab et al., 1996). Residual nitrate in the anaerobic
phase results in consumption of rbCOD by denitrifiers, thus decreasing the
availability of organic matter for PAOs.
2.4.6 Internal Mixed Liquor Recycle Flows (IMLR)
Typical internal recycle flows employed in BNR processes include:
 Recycle from aerobic zone to anoxic zone (nitrified mixed liquor
recirculation-NR)
 Recycle from anoxic zone to anaerobic zone (anoxic mixed liquor
recirculation-AR), and
A low NR flow limits the amount of DO and nitrate available for the
PAOs to uptake phosphorus and could lead to secondary release of phosphorus.

37
Increasing the NR recycle flow will reduce the retention time in the anoxic zone
and increase the amount of nitrates and dissolved oxygen in the anoxic zone.
This will stop secondary release of phosphorus thus reducing the mass of
released phosphorus that must be taken up in the aeration basin. One of the
major factors influencing the occurrence of DPAO and associated anoxic P
uptake appears to be the nitrate load into the anoxic reactor, i.e, the nitrate load
should be large enough or exceed the denitrification potential of ordinary
heterotrophic organisms (OHO), i.e non-PAOs in the anoxic reactor to stimulate
DPAO in the system (Hu et al., 2002). If the nitrate load into the anoxic reactor is
less than the denitrification potential of OHO, the OHO will outcompete PAO for
the use of the limited nitrate, while if the nitrate load exceeds the denitrification
potential of OHO, the PAO will utilize the “excess” nitrate and thus develop in
the system (Hu et al., 2002).
The AR rate should be established such that oxygen and nitrates are not
recycled to the anaerobic zone. The presence of oxygen and nitrate in the
anaerobic zone inhibits fermentation of the soluble organics to acetate thereby
starving the PAOs. Also, denitrifying bacteria and OHOs will compete with
PAOs for rbCOD/VFA substrate, reducing the selective advantage for the PAOs
in the zone. This inadvertently interferes with the efficiency of biological
phosphorus removal.
38
In the UCT process, an increase in the anoxic mixed liquor recycle (AR)
provides for increased organic utilization in the anaerobic stage. In this
configuration the AR contains soluble BOD but little or no nitrate, thereby
providing optimal conditions for fermentation and VFA uptake in the anaerobic
zone. Because the mixed liquor rather than the RAS is recycled to the anaerobic
zone, the MLSS concentration in the zone is lower than the MLSS concentration
in the remainder of the bioreactor. Therefore, a longer anaerobic zone HRT (1 to 2
hrs) is needed to achieve the desired SRTs. The anoxic recycle (AR) and the
nitrified mixed liquor recirculation rates (NR) are typically two times the process
influent flow rates (Grady et al., 1999).
Phosphorus may also be released when there is a low nitrate
concentration in the mixed liquor to the final clarifier. When a deep sludge
blanket develops in the final clarifier, nitrates will be denitrified, clarifier
becomes anaerobic and hence phosphorus can be released. This release may not
affect the effluent phosphorus but may return a large portion of the released
phosphorus back to the anoxic/aerobic zone, where there may not be enough
VFA for uptake of this additional released phosphorus (WEF, 2005).
Typical operating parameters used in the design of various BNR systems
are shown in Table 2-2. The operating parameters vary depending on the BNR
configuration. The BNR system selected for investigating MPs removal should be
39
able to accommodate the removal of conventional wastewater pollutants (COD,
N, P) as well as the removal of MPs. As previously stated, the UCT-BNR design
was selected for this study because it satisfies the requirements for the research
work.
Table 2-2. Typical design parameters for commonly used BNR processes
Process/ SRT, d MLSS, Anaerobi Anoxic Aerobic RAS, % IMLR, % of
Design mg/L c HRT, h HRT, h HRT, h of Influent
Parame Influent
ters
UCT 10-25 3000-4000 1-2 2-4 4-12 80-100 100-300
(aero)
200-
400(anoxic)
A2O 5-25 3000-4000 0.5-1.5 0.5-1 4-6 25-100 100-400
A/O 2-5 3000-4000 0.5-1.5 - 1-3 25-100
PhoStrip 5-20 1000-3000 8-12 4-10 50-100 10-20
SBR 20-40 3000-4000 1.5-3 1-3 2-4

UCT- 5-10 2000-4000 1-2 1-2 4-6 80-100 100-
BNR 200(anoxic)
100-
300(aero)
Modifie 10-20 3000-4000 0.5-1.51-3 4-12 50-100 200-400
d (1st (1stStage
Bardenp Stage) )
ho 2-4 0.5-1
(2 Stag (2ndStag
nd
e) e)
General Design Considerations: Aerobic zone DO > 2 mg/L, pH > 6.5
Adapted from Tchobanoglous et al., 2003.
2.4.7 Summary of Biological Phosphorus Removal in BNR Process
One of the objectives of this research work was to investigation the role of
PAOs in the removal of MPs in wastewater. PAO enrichment in a typical BNR
system requires careful selection and operation of the system to achieve desired
40
objectives, given the fact that BNR operation is more onerous to maintain than
conventional activated sludge system.
Factors that may affect the proliferation of PAOs in the anaerobic basin of
a BNR include temperature, SRT, and pH. The selected experimental SRT was
expected to favor PAOs over GAOs in the pilot plant because the operating SRT
(18-20 days) was beyond the range that could affect the competition of PAOs and
GAOs (3 to 5 days; Whang and Park, 2001). pH has been found to be one the key
factors that affect the metabolic rates and competition between PAOs and GAOs
(Filipe et al., 2001). Acetate uptake rate by both GAOs and PAOs has been shown
to increase with pH up to 6.5, but while it starts to decrease at pH 7.5 for GAOs,
it remains nearly constant from 7.0 to 8.5, and starts to decrease at pH 8.5 for
PAOs (Filipe et al., 2001; Schuler and Jenkins, 2002). Therefore, the operating
conditions of a BNR process should be carefully monitored and maintained in
order to proliferate PAOs for enhanced biological phosphorus removal.
2.5 Micropollutant Removal Mechanisms in Activated Sludge

Systems
Sorption to solids (dependent on solid–water distribution coefficient),
biodegradation (aerobic/anoxic/anaerobic), volatilization (influenced by Henry’s
Law constants) and chemical transformation (hydrolysis, acid base, photolysis,
and precipitation) are possible mechanisms of removal of MPs. However, the
41
former two are typically considered as the most significant for many non-volatile
MPs and personal care products (PPCP) (Ternes et al., 2004). In this section, the
fundamental mechanisms (sorption and biodegradation) that impact MPs in
activated sludge systems are reviewed.
2.5.1 Sorption
The process by which chemicals become associated with the solid phase is
generally referred to as sorption. The attachment of the molecule to a two-
dimensional surface is termed adsorption while molecular penetration into a
three dimensional matrix is called absorption (Schwarzenbach et al., 2003). In
wastewater treatment, sorption onto reactor biomass can be an important
removal mechanism depending on the propensity of MPs to partition to the
sludge. The sorption potential of a given compound is largely dependent on the
distribution coefficient, Kd. The distribution coefficient is the ratio between the
concentration of a compound in the sorbed phase and the liquid phase. Under
equilibrium conditions, the concentration sorbed onto sludge (X) is assumed to
be proportional to the concentration in solution (S) (Ternes et al., 2004) :
𝑋𝑝𝑎𝑟𝑡 𝑋
𝐾𝑑 = = (2-4)
𝑆 𝑋𝑠𝑠 .𝑆
Where,
42
X= concentration sorbed onto sludge, per unit reactor volume [µg.L-1]
Xpart= concentration sorbed onto sludge, per amount of sludge dry matter [µg.g SS-
1]
Kd= solid-water distribution coefficient [L.gSS-1]
Xss= suspended concentration in raw wastewater or production of suspended
solids in primary and/or secondary treatment per L of wastewater [g SS.L-1]
S= dissolved concentration [µg.L-1]
The total concentration (C) of a compound in a solution is given as C=X+S and
hence under equilibrium conditions,
C = S. (1+Xss.Kd) (2-5)
In a situation when Kd cannot be determined experimentally, Dobbs et al. (1989)
presented an expression to estimate Kd of a substance from its Kow. This
expression has been reported to be valid for determining the sorption of
compounds in wastewater.
Log Kd = 1.14 + 0.58 Log Kow (2-6)
43
The Kow (octanol-water partition coefficient) is defined as the ratio of the
concentration of a chemical in octanol to the concentration of the chemical in
water at equilibrium in a two phase water/octanol system at a specified
temperature.
Table 2-3 shows solid-liquid partitioning coefficient, Kd values reported by
different studies suggesting the existence of variability between secondary
sludge which ultimately affects sorption. However, removal by sorption in
municipal WWTP is typically considered to be negligible (<10%) for compounds
with log Kd values ≤ 2.5 L.kgSS-1 (Joss et al., 2006; Ternes et al., 2004). Most
hydrophilic MPs have log Kd values ≤ 2.5 L.kgSS-1. Therefore, removal efficiencies
observed for most hydrophilic MPs in WWTP are attributed to biodegradation
and not sorption (Ternes et al., 2004). Based on this premise, this study will be
conducted with an assumption that sorption does not contribute significantly to
the removal of the target MPs, since their log Kd values are ≤ 2.5 L.kgSS-1.
44
Table 2-3. Kd of selected Micropollutants in contact with secondary sludge
Compound log Kd pKa References
(Secondary sludge)
L.kgSS-1
Carbamazepine 1.4 Jones et al., 2002
0.09 <1,13.9 Ternes et al., 2004
1.8 Urase and Kikuta, 2005
Trimethoprim 2.3 Gobel et al., 2005
0.28 Batt et al., 2006
Estrone 3 Joss et al., 2004
2.9 Carballa et al., 2007
17-β-Estradiol 2.8 Clara et al., 2004
17-α-Ethinylestradiol 2.5 Ternes et al., 2004
Gemfibrozil 1.9 4.8 Urase and Kikuta, 2005
Ibuprofen 2.7 Jones et al., 2002
0.85 4.5-5.2 Ternes et al., 2004
1.9 Urase and Kikuta, 2005
Sulfamethoxazole 2.4 1.8/5.6 Gobel et al., 2005
2.5.2 Biodegradation of Micropollutants
Biodegradation is considered the most important elimination process for
MPs in activated sludge (Quintana et al. 2005). This process can occur through
direct metabolism or cometabolism. Direct metabolism involves microbial use of
MPs as primary or secondary carbon and energy sources while cometabolic
transformation involves microbial energy gain from non-limiting biodegradable
organic compounds (Volodymyr, 2011), while indirectly transforming MPs.
Another facet of cometabolism, called ‘‘commensalism,’’ involves sequential
transformation of a substance by a group of microbial species with some species
of the community specialized in early and other in later stages of
biotransformation (Volodymyr, 2011).
45
Various studies have examined MP removal by biodegradation in many
different systems, including WWTPs, membrane bioreactors (MBRs), sequencing
batch reactors (SBRs), and constructed wetlands. Some of these studies focus
solely on biodegradation as a removal process (Layton et al., 2000; Quintana et
al., 2005; Yu et al., 2006) , while others examined overall removal due to a
combination of different processes including biodegradation (Andersen et al.,
2003; Carballa et al., 2005; Gobel et al., 2007). These studies have expanded the
knowledge base regarding the fate of MPs in various treatment systems (Lab
scale, pilot scale and WWTP). However, there exists a wide variation in removal
efficiencies across therapeutic class, treatment processes, and even among
separate studies for the same individual compounds (Onesios et al., 2009) as
shown in Table 2-4. This variability prevents the generalization of compound
behavior in engineered or natural systems thus making the characterization of
the fate and risks associated with MPs in the environment challenging. However,
these studies are useful as they provide insight into the potential for
biodegradation or biotransformation of MPs in the environment. The results
shown in Table 2-4 provide an overview of the removal efficiency of different
processes where biodegradation or biotransformation has been assumed to be
the predominant removal mechanism.
46
The differences in removal of the compounds have been attributed to
various factors (Xue et al., 2010; Onesios et al., 2009; Joss et al., 2006) including;
the use of different definitions of removal by various studies investigating the
removal of MPs, the physical and chemical properties of the MPs, the degree of
biodegradation or biotransformation in different studies (operating condition of
the system used), type of biological treatment employed, redox conditions of the
reactors, initial MP concentrations, primary substrate concentrations, incubation
times for batch experiments, and source or type and concentration of microbial
inoculums. Therefore there is the need for more research to critically assess the
effects of these factors on the removal of MPs and to investigate the optimization
potential of these factors in improving the removal of MPs in treatment systems.
It is noted that the disappearance of the parent compound of MPs cannot
be solely considered synonymous with complete biodegradation. The loss of the
parent compound indicates biotransformation to an unknown degree, and not
necessarily mineralization. Without monitoring of metabolites or mineralization
end products, the extent or degree of biodegradation cannot be accurately
determined. In order to determine the actual biodegradability of MPs, detailed
biodegradation studies, such as mineralization experiments and biodegradation
pathway studies, are employed (Onesios et al., 2009). Therefore, it is
47
recommended that proper nomenclature should be encouraged among
researchers to avoid confusion or report variability.
48
Table 2-4. Micropollutant removal attributed to biodegradation/biotransformation
Removal System
Compound efficiency Studied Reference comments
Anticonvulsant
Carbamazepine Anaerobic Anaerobic digester: Pilot scale,
digester, pilot Carballa et al. mesophilic (M) and thermophilic (T)
0 (M), 0(T) scale (2006) conditions, 4–400 µg L-1 PPCP
Hormone
EE2 MBR: Lab scale, % mineralization

<1 MBR, lab scale Cirja et al. (2007) reported, 100 µg L-1 PPCP
85 ± 5
(M), Anaerobic Anaerobic digester: Pilot scale,
75 ± 15 digester, pilot Carballa et al. mesophilic (M) and thermophilic (T)
(T) scale (2006) conditions, 4–400 µg L-1 PPCP
Batch: Nitrifying activated sludge
inoculum, 6 d incubation, 50 µg L-1
a
Vader et al. (2000); PPCP; 72 µg L-1 PPCP, 24 h incubation,
a b
~ 100 , Layton et al. WWTP biosolids inoculum; %
20.2 ± 11b Batch (2000) mineralization presented
Lipid regulator
and statin
Gemfibrozil WWTP: 24 h composite influent and
>99 Batch Yu et al. (2006) effluent samples
Batch: Activated sludge inoculum, 50 d
incubation, 1, 10, and 50 µg L-1 PPCP
NSAID
Ibuprofen 40 ± Anaerobic Anaerobic digester: Pilot scale,
15(M), digester, pilot Carballa et al. mesophilic (M) and thermophilic (T)
73 ± 9(T) scale (2006) conditions, 4–400 µg L-1 PPCP
49
Table 2.4 continued. Micropollutant removal attributed to biodegradation/biotransformation
Removal System
Compound efficiency Studied Reference comments
WWTP: Pilot scale, activated sludge
Zwiener and inoculum, 10 µg L-1 PPCP + 30 mg L-1
64-701 Lab columns Frimmel (2003) acetone;
Lab column: Activated sludge
inoculum, 10 µg L-1 + 35 mg L-1
17-212 acetone, (1)oxic, (2)anoxic
WWTP: 24 h flow proportional
samples, influent to biological stage and
treated effluent sampled; Batch: WWTP
influent inoculum and activated sludge,
97- >99 Batch Buser et al.(1999) incubated for 8 h
WWTP: 24 h composite samples from
WWTP MBR, mean removals reported;
Batch: sludge inoculum, mean removals
by transformation reported, 28 d
incubation; (1)20 mg L-1, PPCP as sole
Quintana et al. carbon source, (2)5 mg L-1 PPCP and 50
ND1, 1002 (2005) mg L-1 milk
WWTP: 24 h composite influent and
effluent samples
Batch: Activated sludge inoculum, 50 d
>99 Yu et al. (2006) incubation, 1, 10, and 50 µg L-1 PPCP
Antibiotic
Sulfamethoxazole Anaerobic Anaerobic digester: Pilot scale,
99 ± 1(M), digester, pilot Carballa et al. mesophilic (M) and thermophilic (T)
99 ± 1(T) scale (2006) conditions, 4–400 µg L-1 PPCP
Adapted from Onesois et al. (2009)
50
2.5.3 Micropollutant Biodegradation Models
Several models have been postulated to fit the biodegradation data for
MPs in a biological reactor. Table 2-5 shows model equations that have been
reported for modeling biodegradation of MPs. The table shows the common use
of a pseudo-first order formulation which is dependent on the liquid phase
concentration of the MPs and the mixed liquor biomass concentration, “X”.
Different models have used different values for “X”. For example using the
mixed liquor suspended solids (MLSS) concentration as the value of X in the
model would lead to a linear increase in the removal rate from a system with a
low value of MLSS to a system with a high value of MLSS without considering
the sludge activity.
Some studies have used the volatile suspended solid (VSS) concentration
for the value of “X” in their attempt to account for the active biomass
concentration but there are concerns involved in the use of VSS as an estimate of
the active bacterial population because it does not differentiate between active
and inactive biomass. In fact, other protozoa and metazoan may contribute to the
volatile content of the solid. It is not unreasonable to assume that not all the
organic fraction of the biomass contributes to substrate degradation, only the
active portion of the organic fraction does. Therefore the use of viable biomass
concentration is more appropriate.

51
Furthermore, it is also reasonable to assume that there are different groups
of viable biomass that participate in the biodegradation of MPs in the activated
sludge. For example, some studies have suggested that nitrifying bacteria and
heterotrophic bacteria could be responsible for MP biotransformation or co-
metabolic transformation (Shi et al., 2004; Gaulke et al., 2008; Khunjar et al.,
2011). Therefore, the model developed for this study incorporates the
concentration of specific groups of heterotrophs; poly-phosphate accumulating
organisms (PAOs, XPAO) and ordinary heterotrophic organisms (OHOs, XOHO)
rather than the concentration of the total organic fraction of the biomass, Xvss.
Table 2-5. Biodegradation models for micropollutants removal

Process Model Biodegradation rate equation References
1a 1st order KmC Zhao et al., 2008
1b 1st and pseudo order KmC(1+KdXv) Gang et al., 2007
2 Two 1storder KmC + Km.Xv Cowan et al., 1993
3a Pseudo-1st order KmXvC Schwarzenbach et al., 2003
3b Pseudo-1st order KmXTC Joss et al., 2006
3c Pseudo-1st order KmXHC Jacobsen et al., 1996
3d Pseudo-1st order KmXcC Lee et al., 1998
4 Monod Model KmC/(C+Ks) Marfil-Vega et al., 2010
Source: adapted from Banihashemi and Drote (2011)
Km-biodegradation rate coefficient; C-concentration of dissolved Micropollutants; Xv-

volatile suspended solid concentration (mg/L); XT-total concentration of solids (mg/L);
XH-concentration of heterotrophy biomass (mg/L); Xc-concentration of specific
Micropollutants degrader (mg/L); Ks-half velocity constant for soluble degradable
substrate (ML-3) and K1-inhibition constant.
52
2.5.4 Summary of Micropollutant Removal Mechanisms in
Activated Sludge System
Biodegradation has been suggested as the most important removal
mechanism for MPs, especially for compounds with Log Kd ≤ 2.5 LkgSS-1.
Micropollutants degradation by biomass in the activated sludge process is
considered to be achieved through co-metabolic degradation due to the low
concentrations at which the MPs exist in wastewater. A pseudo first order kinetic
model is the most commonly used model to describe the degradation of MPs in
wastewater. A modified form of this model was used in this research.
2.6 Review of Microorganisms involved in Micropollutant

Degradation
The source and type of the microbiological populations is an important
factor in the removal of MPs, be it biotransformation, biodegradation or
mineralization. This was demonstrated by Layton et al. (2000) when they
reported that the removal efficiency of E2 in biosolids obtained from municipal
plants was 80% greater than removal efficiency found in biosolids obtained from
industrial plants.
Several previous studies investigating the removal mechanism of MPs in
wastewater treatment have attributed their degradation to the presence of
autotrophic ammonia oxidizing bacteria in nitrifying activated sludge (Clara et
al., 2005b). Although an isolated culture of heterotrophic bacteria has been
53
grown under a high concentration of estrogens as primary substrate for carbon
and energy (Shi et al., 2002; Haiyan et al., 2007), there has been very few reports
on the role of heterotrophs in the removal of MPs at the concentrations typical of
WWTPs.
A few recent studies (Table 2-6), have reported that heterotrophs play a
role in the overall degradation of MPs.
The important findings from these reports are;
 Both autotrophic and heterotrophic organisms contribute to the removal
of MPs from wastewater.
 The relationships between autotrophic organisms and heterotrophic
organisms in MP degradation in wastewater could lead to commensalism
(Khunjar et al., 2011). Commensalism refers to the sequential
transformation of a substance by a group of microbial species with some
species of the community specialized in early and other in later stages of
biotransformation. It is a relationship between biotic elements in which
only one element benefits. (Volodymyr, 2011).
 There are a variety of heterotrophic organisms that are involved in MP
degradation but no report has investigated the role of PAOs.
54
 Co-metabolic enzymes produced by heterotrophic bacteria seem to cover
broader range than ammonia oxidizing bacteria’s enzyme (Khunjar et al.,
2011).
 There is insufficient information in the literature to conclusively link
either autotrophic or heterotrophic organisms to MP degradation in
wastewater treatment.
55
Table 2-6. Summary of reports on role of microorganisms in MP removal from wastewater
Dominant Organisms/
Compound Degradation Environment Biomass involved in degradation Comments Reference
AOB biotransformed EE2 but
Ammonia Oxidizing Bacteria not TMP, AOB biotransformed High degree of transformation
(AOB) Culture EE2 (≤ 1mg/L) was observed for
better than heterotrophs EE2 and TMP in the presence of
Khunjar et al.,
EE2,TMP heterotrophic dioxygenase
Heterotrophs mineralized EE2, 2011
enzyme. AOB and heterotrophs
Heterotrophic culture devoid of biotransforms TMP and may cooperatively enhance the
nitrifiers mineralized EE2-derived efficiency of EE2 removal.
metabolites generated by AOBs
Strong relationship between

nitrification and co metabolic
Batch test with enriched Yi and Harper
EE2 AOB EE2 degradation. Suggested
Autotrophic Ammonia Oxidizer (2007)
heterotrophic organisms might
play a role but not investigated.
Results suggested EE2 removal
at concentrations found in
WWTP is not due to co
N. europaea & Nitrospira Gaulke et al.,
EE2 Batch test with AOB metabolic degradation by AOB
multiformis 2008
or abiotic nitration, but most
likely due to heterotrophic
bacteria.
Suggested Heterotrophs are
Gaulke et al.,
EE2 Batch Test at low and High SRT Autotrophs and Heterotrophs capable of PhAc degradation at
low and high SRT 2009
Suggested there are other

E1, E2, EE2 Batch Test with a culture of AOB N. europaea organisms involved in Estrogen Shi et al., 2004
degradation apart from AOB
56
Table 2-6 (continued). Summary of reports on role of microorganisms in MP removal from wastewater.
Dominant Organisms/
Compound Degradation Environment Biomass involved in degradation Comments Reference
Reported faster degradation of
CAF,CBZ,
PhAc in the presence of high Majewsky et al.,
DCF,SMX, Batch Test with heterotrophs Heterotrophs
concentration of heterotrophs 2011
PCT
and low SRT
High removal rate (>90% were

reported for the compounds at
4-NP, TCS, Batch experiment using sludge the 3 SRTs, suggesting Stasinakis et al.,
Autotrophic and Heterotrophic Org.
BPA from 3, 10 and 20 d SRT heterotrophs also play a 2010
significant role in
Micropollutants degradation
BNR achieved better removal of

EDC and PhA/cs than other
Various EDCs Pilot scale experiment with NAS, methods suggesting the Ogunlaja et al.,
Autotrophic and Heterotrophic Org.
and PhAcs CAS, BNR involvement of other (2013)
heterotrophs in the removal of
Micropollutants.
Rhodococcus erythropolis Reported rapid degradation of Yoshimoto et al.,
estrogen 2004
E1, E2, E3, Batch test with isolated The microorganism grew on
EE2 Heterotrophic bacteria EE2 (30mgL-1) as the sole
Haiyan et al.,
Sphingobacterium sp. carbon and energy source (87%
metabolized) within 10 days and 2007
30oC
CAF-caffeine, CBZ-carbamazepine, DCF-Diclofenac, SMX-Sulfamethoxazole, BPA-Bisphenol A, 4-NP-4 Nonyl phenol,TCS-Triclosan,
PhAc-pharmaceutically active compounds, TMP-Trimethroprim, E1- Estrone, E2- Estradiol, EE2- Ethinylestradiol, PCT- Paraceutamol.
57
Research into novel methods of isolating and culturing MP degrading
inocula found in WWTP and introducing them in bulk concentration into the
biological reactors of wastewater treatment plants could potentially reduce the
concentrations and estrogenic activities of MP released into the environment.
However, isolation of the microbial species in the activated sludge of a BNR
process is not a trivial task.
Currently, investigation of the microbial ecology in BNR systems has
relied on molecular based techniques such as fluorescence in situ hybridization
(FISH) or PCR-based methods (Seviour et al., 2003; Oehmen et al., 2010). BNR
performance, kinetics and metabolic characterization has typically been based on
measurements of the substrates and products using enrichment mixed cultures
due to the unsuccessful attempts to obtain any PAO isolates. As a result, BNR
population characterization has been limited to the species whose phylogenetic
information has been obtained.
However, it is recognized that the phylogenetic diversity of PAOs in full-
scale BNR systems are likely larger than those known (Gu et al., 2008; Seviour et
al., 2003). On the other hand, phylogenetic identification and quantification do
not necessarily reflect cell metabolic state and their activities and so, linking the
58
BNR performance activities with the relative population composition/structure
has been quite difficult.
Advanced techniques such as FISH combined with microautoradiography
are useful to identify and understand the in vivo physiology, diversity and
activity of functional microbial groups in biological processes (Nielsen et al.,
1999), but again, this method can reveal organic substrate assimilation and
phosphate uptake by only those bacteria that can be targeted by available probes
(Kong et al., 2004). Recently, flow cytometry was used as a novel fluorescent
staining technique that allows reliable quantification of PAOs and
polyphosphate accumulation dynamics revealing growth activities of not-yet
cultivable bacteria in BNR (Gunther et al., 2009). However, these methods are
very laborious, time consuming and require specialized technical knowhow.
Therefore, quantification of active biomass species would not be carried out
during this study but their abundance will be monitored through their metabolic
activities in the BNR process.
2.6.1 Summary of Microorganisms involved in Micropollutant
Degradation
Microorganisms that co metabolize or commensalize MP in wastewater
treatment plants are diverse but can be grouped as either autotrophic or

59
heterotrophic organisms. Previous attention has been given to autotrophic
organisms as the dominant contributors to MP degradation with little insight
into the likely contribution of heterotrophic organisms. Currently, there is no
consensus on the type of active organism responsible for the biotransformation
of MPs in activated sludge systems. Hence, further study is required to elucidate
the active microorganisms involved in the biotransformation of the MPs in
wastewater. It is believed that the current study is the first to investigate the role
of PAOs, OHOs, and AOB in the removal of MPs in a BNR activated sludge
treatment configuration.
2.7 Influence of Redox Conditions on Micropollutant Removal
The removal mechanisms for conventional contaminants under different
redox conditions are well understood and have been efficiently employed in
most full-scale WWTPs. The impact of redox conditions on the removal of MPs in
WWTPs is however still unclear.
The relatively few studies that have investigated the influence of redox
condition on MP removal from wastewater are summarized in Table 2-7. Many
of the studies were conducted using synthetic wastewater in batch experiments
and the sludge samples were either taken from an existing full scale wastewater
treatment plant or a lab scale continuous system. Typically the MPs were spiked
60
into the reactor and the degradation rate in each redox zone or the overall
removal from the system was monitored. Some of the findings from the literature
review summarized in Table 2-7 are itemized below;
 There has been more research on estrogen (E1, E2, and EE2) removal
under different redox condition than other MPs. Apart from estrogens,
few studies have been conducted to investigate the removal of MPs under
anaerobic conditions.
 Due to the scarce research data describing removal of MPs under different
redox condition, it is difficult to make broad comparisons or
generalizations.
 With the exception of a few studies, most reports that describe the effect of
different redox condition on MP removal, have reported only the overall
removal without considering the contribution of each zone.
 MP removal seems to be affected by the different redox conditions, some
positively, with better removal along the reactor like Ibuprofen,
Roxithromycin, Estrone, E1+E2, Estradiol (E2) and Ethinylestradiol (EE2),
some negatively, with worse removal along the reactor like
Carbamazepine, Citalopram, Triclosan and Sulfametazine and some in
between like Sulfamethoxazole and Sulfadiazine.
61
 Different redox conditions do not seem to have an effect on the adsorption
of MPs to activated sludge biomass (log Kd).
 Except for E1 and E2, there is no consistency in the reported values from
the various studies of the removal of compounds under different redox
conditions.
 Aerobic and anoxic zones seem to be the dominant zones that contribute
to the removal of MPs from wastewater. Except for SDZ, SMR and SMX,
the greatest removals of MPs were observed under aerobic conditions.
Since most of the literature reviewed investigated the removal of
estrogens under different redox conditions, listed below are some important
findings in relation to estrogen degradation;
 E1 could be degraded under all redox condition but at different rates.
 E2 could be degraded under all redox condition but at different rates.
 E1, E2, E3 and EE2 could be significantly degraded under anaerobic
digestion.
 Overall removal of E1 and E2 does not seem to depend on the redox zone
sequence but similar conclusions cannot be drawn for EE2.
62
Table 2-7. Summary of influence of redox condition on Micropollutant removal
Removal % due to Transformation
during batch experiment
Redox Overall
Compound Comb. Log Kd (L kgss-1) Ao Ax An removal Reference
(An/Ax/Ao)
4-NP An/Ax/Ao 4.6/4.9/5.1 75.6 Xue et al. (2010)
4-Op An/Ax/Ao 4.9/4.5/4.2 79.3 Xue et al. (2010)
AHTN Ao/Ax 99.9 82.9 Levine et al. (2006)
BPA An/Ax/Ao 4.6/5.1/5.4 >90 Xue et al. (2010)
Bromoform Ao/Ax 93 96 Levine et al. (2006)
Caffeine An/Ax/Ao 2.5/2.8/2.9 >90 Xue et al. (2010)

Caffeine Ao/Ax 90 -68.2 Levine et al. (2006)
CBZ Ao/Ax 68.6 21.4 Levine et al. (2006)
CBZ Ao/Ax 0.1 6±12 1±10 Suarez et al (2010)

CBZ An/Ax/Ao 3.6/3.8/3.0 <20 Xue et al. (2010)
CTL Ao/Ax 2 60±17 44±9 Suarez et al (2010)
63
Redox Overall
(An/Ax/Ao)
DZP Ao/Ax 1.3 17±11 16±17 Suarez et al (2010)
E1 An/Ax/Ao 3±2 96±1 Joss et al. (2004)

E1 An/Ao/Ax 99 0 70 99 Pholchan et al. (2008)
E1 An/Ao 90 75 100 Pholchan et al. (2008)
E1 An/Ax/Ao 4.3/4.5/4.7 >90 Xue et al. (2010)
E1 Ao/Ax 83.8 9.6 Levine et al. (2006)
E1 + E2 Ao/Ax 99±0 72±2 Suarez et al (2010)

E1 + E2 Ax/Ax/Ao 96.5 -96.6/85.4 97.8 Anderson et al.(2003)
E2 An/Ax/Ao 2.8±1.3/2.7±1.1 97±0.1 37±1 10±0.5 99.99± 0.0002 Li et al (2010)

E2 Ax/Ao 94 85 Dytzak et al (2008)
E2 An/Ax/Ao 2.4±2.2 >97 Joss et al. (2004)
E2 An/Ax/Ao 4.4/4.5/4.7 >90 Xue et al. (2010)
E2 An/Ao 100 70 100 Pholchan et al. (2008)
E2 An/Ao/Ax 99 0 90 100 Pholchan et al. (2008)
17α-E2 An/Ax/Ao 4.8/4.6/5.3 >90 Xue et al. (2010)
E3 An/Ax/Ao 2/1/1.5 >90 Xue et al. (2010)

64
Redox Overall
(An/Ax/Ao)
EE2 Ax/Ax/Ao 71.4 -14.3/-42.9 80 Andersen et al.(2003)
EE2 An/Ax/Ao 4.1/5.3/5.2 97.6 Xue et al. (2010)
EE2 Ao/Ax 2.5 87±11 20±10 Suarez et al (2010)
2.8±0.95/2.8±0.85
EE2 An/Ax/Ao /2.8±0.5 27±2 10±0.9 n.r 79.13±0.77 Li et al (2010)
EE2 Ax/Ao 22 5 Dytzak et al (2008)
EE2 An/Ax/Ao 2.5±1.6 94±2 Joss et al. (2004)
EE2 An/Ao -58 0 0 Pholchan et al. (2008)
EE2 An digester 89.6 41.9 Esperanze et al. (2006)
EE2 An/Ao/Ax 45 30 -98 18 Pholchan et al. (2008)
ERY Ao/Ax 2.2 89±2 20±10 Suarez et al (2010)
Fluoxetin Ao/Ax 0.7 92±3 88±15 Suarez et al (2010)
HHCB An/Ax/Ao 3.8/3.5/3.6 59 Xue et al. (2010)
Ibuprofen Ao/Ax 0.9 95±4 37±26 Suarez et al (2010)
METOP An/Ax/Ao 1.3/1/1 Xue et al. (2010)
65
Redox Overall
(An/Ax/Ao)
ROX Ao/Ax 2.2 91±0 15±17 Suarez et al (2010)
SDZ Ax/An/Ao 41 49 -27 Hong et al. (2008)
SMR Ax/An/Ao 0 73 47 Hong et al. (2008)
SMX Ao/Ax 2.4 22±5 n.a Suarez et al (2010)

SMX Ax/An/Ao 40 43 -89 Hong et al. (2008)
SPD Ax/An/Ao 33 24 -91 Hong et al. (2008)
TMP Ao/Ax 2.3 14±10 n.a Suarez et al (2010)
Triclosan Ao/Ax 11.8 -17.7 Levine et al. (2006)

SMX; Sulfamethoxazole, CBZ;Carbamazepine, TMP;Trimethroprim, DZP;Diazepam, NPX; Naproxen, TCS; Triclosan, ERY;Erythromycin,
CRL; Citalopram, ROX; Roxithromycin, 4-NP; 4Nonyl phenol, 4-OP; 4 Octyl phenol, BPA; Bisphenol A, E1; Estrone, E2; Estradiol, EE2;
Ethinylestradiol, DCF; Diclofenac, METP; Metoprolol, CAF; Caffeine, SDZ; Sulfadiazine, SPD; Sulfapyridine, SMR; Sulfamerazine,
IBU;Ibuprofen, BRMF;Bromoform,
n.d-non detect; n.a- not analysed; n.r- not recorded. An-Anaerobic; Ax-Anoxic; Ao- Aerobic.
66
 Oxidation of E2 to E1 was observed under aerobic and anoxic conditions
(Dytczak et al., 2008).
 Reduction of E1 to E2 could occur under anaerobic condition (Joss et al.,
2004).
 EE2 seems to be significantly degraded under aerobic conditions.
 EE2 is more resistant to degradation than E1 and E2.
 Slight EE2 degradation could occur under anoxic condition (Dytczak et al.,
2008; Li et al., 2011 and Suarez et al., 2010).
Based on these findings it is clear that there is need for more information on the
effects of redox conditions on the removal of MPs from wastewater.
For contaminant fate modeling, estimation of the values of biodegradation
rate constants of the MPs is essential in order to assess the kinetics of the MPs in
the environment. Suarez et al. (2010) reported faster degradation under aerobic
condition for over 90% of the investigated MPs compared to the anoxic
condition. Table 2-8 shows the reported biodegradation rate constants under
different redox conditions and treatment conditions for the target MPs. It is
obvious from Table 2-8 that the different redox zones contributed to the overall
degradation of the MPs with the aerobic zone contributing the largest
percentage. It also shows that few reports exist that present the biodegradation
rate coefficients of MPs with respect to varying redox condition. Therefore it is

67
recommended that a comprehensive data base be generated to present the
biodegradation rate coefficient of various MPs of environmental concern with
respect to varying redox conditions since these chemicals has the tendency of
existing under different redox conditions in the natural environment.
Table 2-8. Pseudo first order biodegradation rate coefficient (kb) (Lgss-1d-1) of
selected micropollutants during biological wastewater treatement
MP log Kow AO AX AN CAS MBR Reference
CBZ 2.45 <0.06 <0.03 Suarez et al., 2010
E1 3.13 162 ± 25 30 ± 10 10 ± 1 Joss et al., 2004

32/77 Lust and Stensel, 2011
32/66 Lust and Stensel, 2011
GEM 4.77 6.4-9.6 0.5-1.8 Joss et al., 2006

2.26 ± 1.35 Urase and Kikuta, 2005
IBU 3.97 20 1.5 Suarez et al., 2010

21-35 9.0-22 Joss et al., 2006
1.33 Abegglen et al., 2009
10.75 ± 13.08 Suarez et al., 2010
3.04 ± 1.59 Urase and Kikuta, 2005
15.38 ± 14.95 Clara et al., 2005a
SMX 0.89 0.3 n.a Suarez et al., 2010

5.9-7.6 3.2-5.0 Joss et al., 2006
0.2 ± 0.01 Abegglen et al., 2009
TMP 0.91 0.15 Suarez et al., 2010

0.22 Abegglen et al., 2009
0.56 ± 0.46 Xue et al., 2010
AO-Aerobic; AX-Anoxic, AN- Anaerobic
68
2.7.1 Summary of Influence of Redox Condition on Micropollutant
Removal
Previous studies have investigated the effects of redox condition on the
removal of MPs from wastewater. Most of these studies have focused on
estrogens, with very few investigations of other MPs. The degradation of E1, E2
and EE2 has been found to depend on redox condition but the overall removal of
E1 and E2, yielded consistent results (> 96%) regardless of the sequence of the
redox condition while EE2 removal seems to be affected by the redox zone
sequencing, for example, a configuration of anaerobic/aerobic/anoxic yielded
18% overall removal while a sequence of anoxic/anoxic/aerobic and
anaerobic/anoxic/aerobic yielded overall removal of 80% and 97.6% respectively.
Many researchers have also indicated that the aerobic zone contributed
the largest removal of the three redox zones investigated for MP removal.
Although the transformation of MPs has been reported to occur under anoxic
and anaerobic conditions, the studies are few and there is considerable
variability between studies. The proposed work will seek to address this
knowledge gap and hence one of the objectives of this study is to investigate the
effects of redox condition on the removal of MPs from wastewater. Investigating
the effects of redox conditions on the removal and biodegradation of MPs will
give vital information required to optimize the efficiency of the treatment
method employed.
69
2.8 Endocrine Disrupting Compounds in Wastewater Treatment
Plant Effluents
Endocrine disrupting compounds (EDCs) are defined by their ability to
mimic or interfere with the mechanisms that govern the biosynthesis, transport
or availability, and metabolism of hormones (Lister et al., 2001). The Canadian
Environmental Protection Act of 1999, article 3, subsection 43 defined “hormone
disrupting substance” also known as EDC as “a substance that has the ability to
disrupt the synthesis, secretion, transport, binding action or elimination of
hormones in an organism, or its progeny, that are responsible for the
maintenance of homeostasis, reproduction, development or behavior of an
organism”.
Steroid sex hormones and related synthetic compounds such as those
used in contraceptive pills, have been shown to be present in the aquatic
environment, mainly as a result of inefficient removal in sewage treatment
plants. The concentrations of these compounds, although very low, at ng/L, are
sufficient to induce estrogenic responses and alter the normal reproduction and
development of wildlife and human endocrine system.
A number of previous studies have investigated the concentrations of
EDCs in treated wastewater effluents (Ternes, 1998; Jones et al., 2002; Kolpin et
al., 2002) and some studies have investigated the ecotoxicological effects of EDCs
in treated wastewater (Cleuvers, 2003; Koh et al., 2009). In these studies, the
70
acute toxicity in the environment was found to be very rare except during spills.
However, there are concerns that chronic or synergistic effects may exist due to
their persistence in the environment (Fent et al., 2006).
Although various studies have been conducted to investigate the
ecotoxicological effects of wastewater effluents on the environment, it is still not
clear whether the cost that would be required to upgrade existing WWTPs to
accommodate EDCs removal along with conventional pollutants removal would
eventually reduce the intrinsic effects of the EDCs on the environment. The
investigation of the relationship between the removal of EDCs from wastewater
and the reduction of effects when discharged into the environment is challenging
because there are various mechanism by which these chemicals may exert effects
on organisms (e.g., interference with various receptors which affect enzymatic
processes and antagonistic effects of mixture of EDCs). In addition, it is difficult
to link EDC concentrations and biological effects in the environment because of
the weak correlation between intrinsic gene expression and physiological effects
on organisms after exposure. One of the objectives of this study is to compare
treated effluents that are generated under different treatment configurations with
respect to MPs and estrogenicity removal.
71
2.8.1 Bioassays for Detection of EDCs
One of the methods used to detect EDCs in the environment is the use of
biologically based assays (Chang et al., 2009). They usually provide either
qualitative or quantitative responses especially in the presence of multiple EDCs.
The use of mass-based analytical methods to quantify EDCs has provided
accurate and precise measurements. However, these methods are limited in their
ability to describe overall estrogenic effects, such as the synergistic or anti-
estrogenic influences in the presence of multiple EDCs (Liu et al., 2010).
Therefore, biological assays are useful methods in studying the overall effects of
EDCs as well as the identification of other xenobiotic compounds that exhibit
endocrine disrupting behaviors similar to EDCs.
Several mechanisms have been employed in biological assays that are
used to measure EDCs and these include; cell proliferation, luciferase induction,
ligand binding, vitellogenin induction, or antigen–antibody interactions.
 Cell proliferation utilizes the estimation of cell growth and
reproduction in different samples, while ligand binding quantifies
the number of specific estrogen binding sites (Soto et al., 2004;
Hamers et al., 2008).
 Luciferase induction measures the amount of luciferase induced
from estrogen receptors and response elements with luminescence
72
after cell lysing and the addition of luciferin (Legler et al., 2002;
Michallet-Ferrier et al., 2004).
 Vitellogenin induction quantifies the amount of vitellogenin in the
plasma of female fish liver after extraction, which is secreted as a
response to estrogens. In addition, the production of vitellogenin in
male fish can be seen as an indication of endocrine disruption
(Jimenez, 1997; Saaristo et al., 2009).
 Antigen–antibody interactions use the principle of immunoassays
based upon the non-covalent binding of antigen to antibodies
(Gascón et al., 1997; Alyea and Watson, 2009).
Biological assays could either be “in vivo” or “in vitro”. “In vivo” assays
utilize the endocrine disruption process in amphibians, fishes, birds, and insects
in order to monitor the EDCs in aquatic environments. The responses in the
organisms are determined by deformities, reproductive deficiencies, egg and
offspring development, and serum protein production, such as vitellogenin
(Chang et al., 2009). Many “In vivo” assays have been used to investigate the
effects of EDCs on fishes, some of the species used include rainbow trout
(Oncorhynchus mykiss), fathead minnow (Pimephales promelas), sheephead
minnow (Cyprinodon variegates), and zebrafish (Brachydanio rerio) to mention but a
few (Fenner-Crisp et al., 2000; Folmar et al., 2000; Legler et al., 2002; Micael et al.,
73
2007; Warner and Jenkins, 2007; Soares et al., 2008). In addition, the populations
of wild leopard frogs (Rana pipiens) have been known to be particularly sensitive
to the exposure of EDCs, based on a study of their gonadal abnormalities (Hayes
et al., 2002; Opitz et al., 2006).
An advantage of using “In vivo” assays in the detection of EDCs is the
ability to quantify the actual effects of EDCs on a target species as well as the
usage of the species as a representative biological indicator in their natural
habitats. In addition, the method may provide a cumulative estrogenic effect
caused by exposure to a mixture of EDCs in a given environment. The major
disadvantage of this method is associated with the deficiency of a specific
organism response to certain EDCs (Chang et al., 2009).
The “in vitro” assays can be categorized into three types (Kinnberg, 2003):
1) Estrogen receptor (ER) competitive ligand binding assays: measure the
binding affinity of a chemical to ERs
2) Cell proliferation assays: measure the increase in the number of target
cells during the exponential phase of proliferation (i.e. E-screen assay) and
3) Reporter gene assays: measure ER binding-dependent transcriptional and
translational activity (i.e. ER-CALUX assay, MVLN cell assay, Yeast
estrogen screen (YES) assay).
74
The aforementioned in-vitro assays use a protein expression system,
representing the estrogen response formed or stimulated by a dimer that is
produced from the binding between the estrogen and the estrogen receptors.
Luciferase, quantified with a luminometer after cell lysis and β-galactosidase,
measured with a spectrophotometer, using a back-calculation from the amount
of colored products after the enzyme-catalyzed reaction process has been
completed, are some examples of these types of response proteins (Chang et al.,
2009). Out of all the methods available for measuring the estrogenic activity of
water samples, the yeast assay has been reported to be fast, sensitive and reliable.
Therefore, the YES assay method was used to estimate the estrogenicity of the
pilot plant effluents in relation to the different operating conditions.
2.9 Summary of Literature Review
A detailed understanding of the active microbial groups involved in the
removal of MPs from BNR wastewater treatment process will enhance the ability
to design treatment methods which would lead to a reduced load and ultimately
reduced effects of MPs on the environment. Presently, there is no general
consensus among researchers on the role of various active microbial groups in
activated sludge with respect to the removal of MPs from wastewater. Some
studies have suggested that nitrifying organisms contributed significantly to
biodegradation while other studies suggested that heterotrophs played a

75
dominant role in the biotransformation of MPs in wastewater. Other studies
have suggested that nitrifying and heterotrophic organisms work together as co-
contributors to biotransform MPs. These inconsistencies impede the
development of a robust model for the removal of MPs in activated sludge
wastewater treatment processes. Hence, further study is required to elucidate the
role of active microbial groups in the biotransformation on MPs.
Previous studies have shown that high removals of some MPs can be
achieved through biological wastewater treatment processes operated under
different redox conditions. However, these studies are few and the variability
between studies prevents the generalization of conclusions. Therefore, the
investigation of the removal of MPs under different redox conditions will
contribute to the general data base of knowledge while presenting the
opportunity for optimization of existing biological wastewater treatment
processes.
The YES assay is a useful biological tool for detecting EDCs present in
different matrices in the environment. This method is appropriate for estimating
qualitative or quantitative estrogenic responses of environmental samples. It is
unique in its mode of operation because it quantifies the overall estrogenic effects
(synergistic or anti-estrogenic) induced by the WWTP effluents. However, the
76
YES assay has limitations in that it can produce false negative results due to the
presence of antagonistic compounds or toxicants in the extracted samples.
77
Chapter 3 Impact of Activated Sludge Process Configuration on
Removal of Micropollutants and Estrogenicity
A version of this chapter was published as the paper: Ogunlaja, O. O.; Parker, J. W.; Metcalfe, C.;
Seto, P. (2013) “Impact of activated sludge process configuration on removal of Micropollutants
and estrogenicity” Proceedings of the 86th Annual Water Environment Federation
Technical Exposition and Conference (CD-ROM), Illinois, Chicago, Oct. 5-9. Water
Environment Federation: Alexandria, Virginia.
Other collaborative publications involving the study in this chapter are;
1) Pileggi V., M. Ogunlaja, X. Chen, J. W. Parker, P. Yang, S. Kleywegt, N.
Feisthauer, S. Tabe, J. Schroeder, T. Fletcher and P. Seto (2013). Comparison
of effluent conventional and microcontaminant chemistry in three pilot
wastewater treatment processes during winter and summer simulated
conditions, Proceedings of the 86th Water Environment Federation Technical
Exposition and Conference, Chicago, IL.
2) Wojnarowicz, P., O. Ogunlaja, C. Xia, W. Parker, C. Helbing. (2013). Impact of
Wastewater Treatment Configuration and Seasonal Conditions on Thyroid
Hormone Disruption and Stress Effects in Rana catesebeiana Tailfin. Enviro. Sci.
Technol., 47(23), 13840-13847.
3) Parker W.J., V. Pileggi, P. Seto, X. Chen, M. Ogunlaja, G. Van Der Kraak, J.
Parrott. (2014). Impact of Activated Sludge Configuration and Operating
Conditions on In Vitro and In Vivo Responses and Trace Organic Compound
Removal. Sci. of Total Environ., 490,360-369.
Summary of contributions to aforementioned publications;
 Conducted daily checks and measurements to maintain the smooth
operations of the pilot plants.
78
 Collected and prepared samples before other chemical quantifications.
 Conducted YES assay analysis on collected samples.
 Prepared samples before sending to laboratories for chemical analysis
 Collated conventional pollutants and chemical analysis results for ease of
comparison between treatment trains.
 Participated in writing a portion of article #2.
79
3.1 Introduction
Wastewater treatment plant (WWTP) effluents have been identified as the
primary route of entry for micropollutants (MPs) into the aquatic environment
(Joss et al., 2004). The concern with respect to MPs in the environment results
from the potential deleterious effects of these chemicals on the aquatic
ecosystem. These chemicals have been shown to cause developmental and
reproductive abnormalities in various trophic levels of organisms when present
at very low concentrations (Purdom et al., 1994; Fent et al., 2006; Parrott and
Blunt, 2005, Parrott et al., 2009) and their occurrence has been measured in both
effluent and surface water in North America (Kolpin et al., 2002, Metcalfe et al.,
2003; Lishman et al., 2006).
Wastewater treatment plants are generally designed to remove
conventional pollutants such as nitrogen, phosphorus, BOD and TSS and the
limited removal of MPs from WWTPs is generally fortuitous. A multi-level
approach with layers of different removal mechanisms (sorption,
biodegradation, size exclusion, oxidation, etc.) has been suggested for
improvement in the removal of MPs from wastewater (Koh et al., 2009; Qian et
al., 2011). However, there is still uncertainty as to whether upgrading wastewater
treatment for enhanced conventional pollutant removal (i.e. nitrification,
80
denitrification, enhanced biological phosphorous removal) will yield improved
removal of MPs.
Assessing the biological effects of MPs on the flora and fauna of the
aquatic ecosystem is an indispensable tool required to conduct detailed and
appropriate risk assessment of MPs in the environment. Unlike chemical
analyses that can provide a quantitative measure of the compounds present in a
sample, biological analyses such as in vitro bioassays can provide a qualitative
and quantitative measure of the net endocrine-disrupting potential of all the
endocrine-active substances that are present in an effluent (Leusch et al., 2010).
Studies that have compared the removal of micropollutants and the potential for
endocrine disruption in the effluents between different wastewater treatment
technologies are few. Specifically, it has not been established whether improved
removal of MPs will translate into a reduction in the biological effects on an
aquatic ecosystem.
In this study, the removal of 10 MPs classified according to EU directive
93/67/EEC as either toxic (gemfibrozil, ibuprofen, trimethoprim), harmful
(carbamazepine), non-toxic (sulfamethoxazole, trimethoprim), estrogenic
(estrone, bisphenol-A and nonyl-phenol) or androgenic (androstendione) in three
different wastewater treatment technologies (conventional activated sludge
(CAS), nitrifying activated sludge (NAS) and biological nutrient removal (BNR))
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was investigated. The assessment was conducted at steady state in pilot scale
processes treating authentic municipal wastewater as an influent. In addition, the
potential biological effects of the effluents were assessed using a recombinant
yeast estrogen screen (YES) assay to quantify their net estrogenicity. The
reduction of estrogenic activity was estimated by calculating E2-Equivalent
concentration (E2-Eq) in the effluents of the treatment trains. It was hypothesized
that as the sophistication of the treatment methods increased from CAS to BNR,
there would be an improvement in MP removal and reduction in estrogenic
activity in the effluents.
3.2 Methodology
3.2.1 Process Description
Three pilot wastewater treatment plants located at the Environment
Canada Wastewater Technology Centre (WTC) in Burlington, Ontario were
employed for this study. The WTC receives raw municipal wastewater from
Burlington Skyway Wastewater treatment plant. Schematics of the pilot plants
are shown in Figures 3-1 and 3-2. The conventional activated sludge (CAS),
nitrifying activated sludge (NAS) and biological nutrient removal (BNR) pilots
were fed from a common primary clarifier that received the raw wastewater. The
activated sludge reactors were segmented into six cells (60 L each) to simulate
82
pseudo-plug flow. Coarse bubble aerators were used for aeration and mixing (in
a cyclic pattern; high air flow at 40 Lpm and low air flow at 10 Lpm) in the CAS,
NAS and the aerated zones of the BNR. The operating and design conditions are
presented in Table 3-1. An insulated water jacket with automatic temperature
controller was installed around the bioreactors to control the temperature of the
bioreactors. Di-potassium phosphate (K2HPO4) (11 g/L at the rate of 14.4 L/d) and
sodium bicarbonate (NaHCO3) (22 g/L at the rate of 14.4 L/d) were added to the
primary effluent before entering into the reactors. This was done to ensure the
system was not phosphorus limited and to provide a pH buffer for the system.
The CAS pilot was a single sludge aerobic system operated at sludge
retention time (SRT) of 3 days to facilitate BOD removal without nitrification.
The NAS was a single sludge aerobic system operated at SRT of 10 days to
facilitate BOD removal and nitrification. The longer SRT enabled the
proliferation of autotrophic nitrifying bacteria which mediates nitrification
reactions. The BNR process consisted of anaerobic, anoxic and aerobic zones and
was operated at a SRT of 20 days. This mode of operation was designed to
achieve nitrification, denitrification and enhanced biological phosphorous
removal. To achieve an appropriate range of COD/P ratio that would support a
healthy BNR activated sludge, the clarified influent wastewater stream to the
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BNR bioreactor was augmented with an exogenous source of readily
biodegradable organic substrate (150 mg/L of acetate).
The pilot plants were operated for over 365 days with monitoring for over
180 days to ascertain stable operation in terms of biomass and effluent
characteristics so as to enable a comparison among the three treatment trains.
Plant performance was ascertained by monitoring SRT, temperature, COD, NH3-
N, TP, soluble PO4-P, NO3-N, TSS, VSS and pH.
Figure 3-1. Flow diagram of the pilot-scale BNR treatment process.
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Figure 3-2. Flow diagram of the pilot-scale CAS and NAS treatment process. 1.
Primary clarifier; 2. Aerobic bioreactor; 3. Final clarifier.
Table 3-1. BNR, NAS and CAS operating and design information
Size/description Unit
Flow rate (Q) 1282 L/d
Primary settler Area = 0.46 m2
Depth = 1.56 m
Activated sludge Pass volume = 6 X 60 L
Depth = 1.28 m
Dissolved Oxygen CAS-DO = 2-2.5 mg/L
NAS-DO = 4-4.5
BNR
DO(aerobic) = 4-5
DO(anoxic) = 1-2.5
DO(anaerobic) = 0-0.2
Final settling tank Area = 0.204 m2
Depth = 1.4 m
Underflow = 962 L/d
Waste flow CAS = 107 L/d
NAS = 34
BNR = 18
CAS-BNR Aerobic = 2Q L/d
Internal recirc. split factor Anoxic = Q
SRT CAS = 3 days
NAS = 10
BNR = 20
HRT Average nominal = 7 h
Temperature 18 ± 2 oC
85
3.2.2 Sample Collection and Extraction
A total of 5 sampling campaigns were conducted over three months
during the steady state plant operation (July 2011 – Sept. 2011). The influent
samples were collected after it had passed through primary clarification while
the effluent from each train was collected at the outlet of the secondary clarifier.
Eight hour composite grab samples were also collected from the anaerobic,
anoxic and aerobic zones of the bioreactor during the same time as when the 24 h
composite samples were collected. To reduce the influence of diurnal changes, 24
h composite samples of the influent and effluent were collected in 10 L stainless
steel canisters using flow proportional (150 mL / 30mins) refrigerated auto
samplers (HACH Company, Loveland USA).
Immediately after sample collection, the samples were centrifuged at 4000
rpm for 5 minutes and the supernatant filtered through 1.5 µm glass microfiber
filters (Whatman, Toronto, Ontario, Canada). The filtered samples were split into
two sub-samples each with a volume of 120 mL. The sub-samples were acidified
to pH 3 and stored in amber glass bottles at 4oC until they were extracted and
analyzed. One set of sub-samples was extracted using the subsequently
described multi-residue solid phase extraction technique (SPE) before shipping
to Trent University for chemical quantification using LC/MS-MS. The second set
of samples was extracted using the same procedure as the first set but without
86
spiking with deuterated compounds and processed for the yeast estrogen screen
(YES) assay.
The samples were also analyzed for conventional wastewater parameters
(as per Standard Methods for the Examination of Water and Wastewater (Eaton,
2005)) including, chemical oxygen demand (COD), biochemical oxygen demand
(BOD), total Kjeldahl nitrogen (TKN), ammonia, nitrate, total suspended solids
(TSS), alkalinity and pH.
3.2.3 Solid Phase Extraction
A multi-residue extraction procedure was used for extraction of the
compounds from the water samples (Li et al., 2010) prior to the MP analysis and
the YES assays. The samples extracted for MP analysis were initially spiked with
surrogate standard solution (100 µL of 0.5 ppm) of the selected compounds
prepared in methanol before the extraction. The samples extracted for
characterization by the YES assay did not include the spiked standards. The SPE
process is summarized in Figure 3-3.
In the extractions, Visiprep solid-phase extraction (SPE) vacuum
manifolds coupled with Oasis® MAX SPE 6 mL (500 mg) SPE cartridges were
used to concentrate the filtered samples (sample pH was initially adjusted to 8
by adding 0.1M NaOH). The HLB tubes were conditioned with 6 mL of
methanol, 6 mL 0.1M NaOH in Milli-Q water, and 6 mL of Milli-Q water, taking

87
care not to let the cartridge go dry. The filtered wastewater sample was loaded
onto the cartridge at a flow rate of approximately 5 mL/min to ensure good
recovery and the sample bottles were rinsed with 10 mL of pH 8 distilled water.
After loading, the cartridges were rinsed with 2 mL of pH 8 water and dried
under vacuum for 10-20 minutes. The compounds were then eluted from the
cartridges with 2 mL of MeOH at a flow rate of about 1 mL/min and rinsed thrice
with 3 mL of 2 % formic acid in methanol at a flow rate of 1 mL/min, waiting six
minutes between each elution. After elution, the cartridge was aspirated to
dryness for about 10 min under vacuum. The eluted fractions were collected in
amber glass tubes and dried under a gentle stream of nitrogen gas (N2). The
dried residue was dissolved in 0.4 mL of methanol and stored at 4oC before using
it for YES assay or LC-MS-MS analysis.
Figure 3-3. Summary of multi-residue extraction procedure.
88
3.2.4 Biological Testing
The estrogenic activity of the extracted samples was evaluated using the
recombinant yeast screen for estrogenicity bioassay (YES) according to the
methods developed by Routledge and Sumpter (1996) with adjustments
described by Citulski and Farahbakhsh, (2010). A recombinant yeast strain
(Saccharomyces cerevisiase) containing the human estrogen receptor was used for
the assay. The strain contains an expression plasmid carrying the lac-Z reporter
gene. When the cells are incubated for 3 days at 32oC in the presence of
estrogenic compounds, the lac-Z product, β-galactosidase, is secreted into the
medium and causes the chromogenic substrate, chlorophenol red-β-D-
galactopyranoside (CPRG), to turn from yellow to red.
In the YES assay, the samples were initially evaporated to dryness under
N2, after which the evaporated residue was re-dissolved in 400 µL of ethanol.
The sample was then transferred to a vial which was stored at 4°C. A 100 µL
aliquot of each E2 sample was transferred to a 96 well micro titer plate (the
dilution plate) and a dilution series was produced (using 100 µL of absolute
ethanol). Standard curves for each test were prepared from positive-control
rows of 17-β-estradiol (E2) in ethanol, diluted from 10 nM to 5x10-3 nM nominal
concentration. The dilution factor in each titre plate was 2 times with ethanol
across the 12 wells to produce a final dilution of 1/2048 (i.e 211 times dilution).
89
A 10 µL aliquot of each dilution was transferred to a new micro titer plate
(the assay plate) in a laminar air flow bench. The assay plate contained one row
of standard solutions (17β-estradiol as positive control), one row of blank
samples (absolute ethanol as negative control) and three rows of serially diluted
environmental samples (80 µL of sample extract diluted with 80 µL of ethanol).
The assay plates were allowed to dry in the laminar air flow bench after which
200 µL of combined growth medium/CPRG/yeast mixture were added.
Subsequently, the assay plates were incubated at 32°C with the shaker tray set to
150 rpm for 72 hours to promote suspension of the yeast cells and constant
mixing with the assay medium.
The absorbance (AB) at 540 nm and 620 nm was determined using a micro
titer plate reader (Sunrise Basic TECAN). In order to adjust the absorbance of
chlorophenol red at 540 nm for the extent of yeast growth (turbidity), the
absorbance measurements for E2 standards and environmental samples were
transformed as per equation 3-1 (Fent et al., 2006):
Adj. Absorbance = AB540nm (sample) – [AB 620nm (sample) – AB 620nm (blank, average)] 3-1
The dose response curve for the standards and environmental samples
were calculated using GraphPad Prism 6 (v. 6.02) that employed a four-
90
parameter sigmoidal Hill equation to calculate the EC-50. To obtain the
estrogenicity values for the extracted sample, the logarithm of the extraction
dilution that yield a 50% response was computed from the fitted curve. The E2-
Eq was calculated as the ratio between the amount of E2 in the incubation well at
EC50 in the standard curve and the equivalent volume at EC50 as per equation 3-
2.
𝑛𝑔 𝐸2 𝐸𝐶50( 𝑛𝑔⁄𝐿)
E2 − 𝐸𝑞 ( )= ( ) 3-2
𝐿 𝐸𝐶50 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟∗0.4
The value 0.4 accounted for the air dried 80 µL sample extract applied to the titer
well and reconstituted with 200 µL of the growth medium containing yeast cells
(80/200 = 0.4). The method detection limit based on E2 standard was determined
as 1 ng/L.
3.2.5 Target MPs
The micropollutants investigated in this study are presented along with
their physicochemical characteristics in Table 3-2. The target compounds
included a broad range of substances including acidic, basic and neutral drugs
and estrogenic compounds and were selected on the basis of their detection
frequency in WWTP effluents (Metcalfe et al., 2003; Lishman et al., 2006) and the
91
ability to detect low concentrations (ng/L) using LC-MS/MS (Miao and Metcalfe,
2002)
3.2.6 Statistical Analysis
The conventional and chemical data were analyzed for outliers using the
Grubb’s test. The regressions used to construct the YES assay response curves for
the samples were compared to the response curves of estradiol (E2) standard
using the F-test. To check the yeast growth absorbance (AB620 nm) for the
presence of toxic effects resulting from the wastewater extracts, the yeast growth
was compared to the average turbidity (± 3 standard deviations) of 12 ethanol
only negative-control wells that were incubated in the same plate as the samples.
Samples that had yeast growth with turbidity values below the average minus 3
standard deviations of the negative control were removed from the dose-
response curve analysis. The final conditioned data were employed to generate
the dose-response curve for the estimation of the EC50. A paired-t-test was
employed to compare the MPs’ concentrations and the E2-Eq concentrations in
the influent and pilots’ effluent streams at the 95% confidence level using
Microsoft excel 2013.
92
Table 3-2. Physicochemical properties of selected micropollutants
Henry’s law
Molecular Water solubility @
constant @ (Log Kow)- (Kd)*
Micropollutants mass 25oC (mg/L) - Biological or Clinical effects
25oC(atm m3 /mol)- Bioconcentration L/KgSS
(g/mol) Dissolving
Evaporation
Ibuprofen 206.29 21 (moderate) 1.5E-07 (moderate) 3.97 (moderate) <30 Non-prescription analgesic
Meprobamate 218.25 4700 (high) 1.8E-10 (high) 0.7 (low) <30-190 Anti-convulsant
Nonyl phenol 220.35 7 (low) 3.4E-05 (low) 5.76 (high) 7k-13kc Estrogenic
Bisphenol A 228.29 120 (moderate) 1E-11 (high) 3.32 (moderate) 314-502 Estrogenic
Carbamazepine 236.27 17.7 (moderate) 1.08E-10 (high) 2.45 (low) 36-65 Anti-epileptic
Gemfibrozil 250.33 10.9 (moderate) 1.19E-08 (high) 4.77 (high) 30-45 Cholesterol lowering Drug
610 @ 37oC
Sulfamethoxazole 253.28 6.42E-13 (high) 0.89 (low) 200-400 Antibiotic
(moderate)
Estrone 270.37 30 (moderate) 3.8E-10 (high) 3.13 (moderate) 607-645 Estrogen
Androstenedione 286.41 57.8 (moderate) 3.68E-08 (high) 2.75 (moderate) 134-174 Steroid hormone
Trimethoprim 290.32 400 (moderate) 2.39E-14 (high) 0.91 (low) 119-251 Antibiotic
Physprop Database. http://www.syrres.com/esc/physdemo.htm *Stevens-Garmon et al., 2011; c Xue et al.,2010
93
3.3 Results and Discussions
3.3.1 Conventional Parameters
The pilot plants were monitored with respect to the removal of
conventional wastewater pollutants such as biochemical oxygen demand (BOD)
and nitrogen species. This data was employed to establish whether the treatment
plants were operating within normally established ranges and to provide insight
into the types of microbial metabolisms (i.e. aerobic heterotrophic growth, anoxic
heterotrophic growth, aerobic autotrophic growth) that were active in the
bioreactors. The measured responses are presented in Table 3-3.
Considering the inherent variability of processes treating raw municipal
wastewater, the effluents from the pilot plants as indicated in Table 3-3, were
relatively consistent with time. Carbonaceous BOD5 was consistently removed in
all pilot plants and most final concentrations were less than 10 mg/L. This was
considered indicative of good removal of biodegradable organic matter. The
BNR and NAS processes produced the lowest effluent BOD concentrations while
the effluent concentrations from the CAS process were slightly elevated (average
of 13.3 mg/L).
94
Based upon the operating conditions that were employed in this study it
was expected that the effluents from the CAS pilot would have higher
concentrations of TKN and TAN and low concentrations of NO3-N and NO2-N as
compared to NAS and BNR pilot effluents. From Table 3-3 it can be observed
that the CAS pilot effluent NO3-N concentrations were consistently low and the
effluent TKN and TAN concentrations remained elevated. The NAS pilot was
expected to have low concentrations of TKN and TAN and elevated
concentrations of NO3-N in the effluent. From Table 3-3 it can be seen that these
patterns were consistently observed. Hence, it was concluded that the CAS pilot
was not nitrifying while the NAS pilot was nitrifying effectively. The BNR pilot
was expected to have low effluent concentrations of PO4-P, TKN, TAN, NO3-N
and NO2-N. A review of Table 3-3 indicated that these patterns were consistently
observed. Hence, it was concluded that the BNR process was effectively
nitrifying, denitrifying and biologically removing phosphorus. Collectively, it
was concluded that the pilots were achieving levels of treatment that are typical
of their respective operations at technical scale (Tchobanoglous et al., 2003).
95
Table 3-3. Influent and effluent concentrations of conventional responses
(mg/L) (Mean (S.D))
TAN NO3-N NO2-N TKN cBOD5 PO4-P
Influent 20.9(6.6) 0.62(0.9) 0.16(0.23) 27.1(10.4) 79.8(56.4) 5.15(1.6)
CAS 21.1(6.4) 1.7(4) 0.4(0.6) 21.4(5.7) 13.3(10.1) 5.38(1.4)
NAS 0.076(0.039) 22(2.6) 0.013(0.018) 1.2(0.3) 3.5(1.7) 5.24(1.4)
BNR 1.3(2.1) 5.4(2.2) 0.3(0.4) 2.4(2.3) 6.4(6) 0.19(0.4)
3.3.2 Micropollutant Removal during Treatment
All 10 compounds were detected in the influent wastewater. The observed
average influent concentrations were generally lower than previously reported
studies (Lishman et al., 2006, Lajeunesse et al., 2012). The influent data was
conditioned by using Grubb’s test to determine any outliers which were
removed before conducting other statistical analysis. There were some overall
variability associated with the influent concentrations of the selected compounds
with a median relative standard deviation (RSD) of 25% across all influent
samples for the five sampling campaigns. The variability was primarily
attributed to temporal changes in the influent concentrations.
The effluents from the three pilots were compared to examine whether
any of the pilot out performed each other by examining the responses of 10
representative MPs. The effluent data was conditioned by using Grubb’s test to
96
determine any outliers which were removed before conducting other statistical
analysis. A paired-t-test was employed to compare the mean concentrations of
the MPs in the influent to the mean concentrations of the MPs in the effluent
streams from CAS, NAS and BNR treatment configurations. A paired-t-test was
also used to compare the mean concentrations of the MPs in the BNR to the mean
concentrations of the MPs in the effluent from NAS and CAS treatment
configuration. This analysis was conducted in order to determine the statistical
significance of the differences in the mean concentrations between the influent
and the effluent streams from the treatment configurations. Hence, Table 3-4
summarizes the results from the statistical analysis.
Table 3-4 shows the p-values of the paired t-tests that compared the mean
concentrations in the streams from the pilot plants. The results of the paired-t-
test showed a statistically significant difference between the mean concentrations
in the influent and CAS, NAS and BNR effluents for IBU, CBZ, GEM, ADR, E1,
NP and BPA. There was no statistically significant difference between the mean
concentrations in the influent and CAS, NAS and BNR for MEP. The result
showed that the mean concentrations in the influent and the NAS effluent for
GEM were statistically significant. The mean of the difference in the
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concentrations of TMP in the influent and in the BNR effluents were also
statistically significant. In addition, there were no statistical difference between
CAS and BNR effluents or NAS and BNR effluents at the 95% confidence level
for all the investigated compounds except for TMP. These results show that
majority of the responses of the MPs in the influent and the effluent from the
CAS, NAS and BNR treatment configurations were significant. Hence, the data
were employed for further technical comparisons among the three treatment
configurations.
Table 3-4. Statistical significant (p ≤ 0.05) testing results – P values

Comparison IBU MEP GEM CBZ TMP SMZ ADR E1 NP BPA
IN-CAS 0.002 0.252 0.425 0.025 0.219 0.020 0.006 0.038 0.042 0.004
IN-NAS 0.002 0.429 0.001 0.006 0.201 0.003 0.005 0.017 0.035 0.01
IN-BNR 0.002 0.402 0.091 0.043 0.008 0.067 0.005 0.018 0.036 0.004
BNR-CAS 0.07 0.64 0.23 0.62 0.03 0.62 0.13 0.06 0.24 0.08
BNR-NAS 0.09 0.99 0.08 0.41 0.03 0.15 0.35 0.72 0.19 0.19
IN-influent
Box plots and averages of the concentrations of the compounds in the
influent and effluents from CAS, NAS and BNR are presented in Figures 3-4, 3-5,
and 3-6. These plots show that 5 MPs (IBU, ADR, E1, NP and BPA) had
comparable effluent concentrations for CAS, NAS and BNR. Two MPs (CBZ,
MEP) had effluent concentrations equal or higher than the influent
concentrations for all the treatment processes. TMP, GEM and SMX effluent
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concentrations followed the trend of BNR < NAS < CAS, NAS < BNR < CAS and
NAS < CAS = BNR respectively.
The removal efficiencies of each compound across the treatment processes
were calculated as the percent difference in concentration from influent to
effluent. The term removal used in this study described the loss of the target
compound and did not necessarily imply mineralization. The removal of the
target compound could have proceeded via different mechanisms including
chemical and physical transformation, biodegradation or biotransformation and
sorption. The MPs removal efficiencies across the three treatment processes are
presented in Figure 3-7. It is apparent from Figure 3-7 that 6 MPs (IBU, ADR,
SMX, E1, NP and BPA) were consistently removed with removal efficiency
greater than 65% across the three treatment processes while no removal was
observed for 2 MPs (CBZ and MEP) regardless of the treatment process utilized.
The moderate to high removal efficiencies for the IBU, ADR, SMX, E1, NP
and BPA were consistent with the removal efficiencies previously reported in the
literature (USEPA 2010). Treatment processes with SRTs ranging from 2 – 68
days in a lab scale and 22- 82 days in WWTP pilot scale were reported to have
IBU removals ranging from 80% to 100% (Onesios et al., 2009). The average
99
reported removal efficiency for BPA in a variety of treatment processes and SRTs
was reported to be 83% (Melcer and Klecka, 2011). Clara et al. (2005) reported
BPA and IBU average removals of 82 to 98% at SRTs as low as 2 days in an
activated sludge system. Reported values for ADR, NP and E1 removal efficiency
in various aerobic activated sludge systems and SRTs have ranged from 98 to
100% with an average of 99%. These results suggest that the removals of IBU,
ADR, SMX, E1, NP and BPA in the CAS, NAS and BNR were independent of the
process configuration or system’s SRT.
The apparent negative removals for CBZ and MEP (Figure 3-7) reflected
the increased effluent concentrations of CBZ and MEP from the treatment
processes as compared to the influent concentrations (Figures 3-4D and 3-5E).
This suggests that CBZ and MEP were not removed by any of the treatment
processes. This may have resulted from de-conjugation of conjugated versions of
the parent compounds by the action of the microorganisms during the treatment
process (Lindqvist et al., 2005). Gobel et al. (2007) suggested that the increase in
effluent concentration of some MPs as compared to their influent concentration
could be due to the encasing of the influent MPs in fecal particles leading to an
apparent increase in concentration during treatment as the fecal particles are
100
degraded. Previous studies have shown that recalcitrant compounds like CBZ
rarely sorb or degrade in a variety of wastewater treatment processes (Ternes et
al. 2004; Clara et al. 2005a). The recalcitrant behavior of CBZ and MEP could be a
useful characteristic in applications where they might be employed as an
anthropogenic tracer.
Figure 3-7 shows an increase in the removal of TMP as the treatment
process complexity progressed from a simple non-nitrifying CAS to a complex
BNR configuration. Longer solids retention times (SRTs) accommodate slower
growing bacteria like nitrifiers and can also support the proliferation of a wider
range of bacteria species. The presence of a broad array of bacteria likely allows
for a wider range for biotransformation of TMP thereby leading to improved
removal at longer SRTs (CAS-3 days, NAS-10 days and BNR- 20 days).
Correlations between treatment efficiency and SRT have previously been
established (Clara et al., 2005a) and an SRT longer than 10 days has been
suggested as being more effective in removing micropollutants such as estrogens
(Carballa et al., 2007). Gobel et al., 2007 investigated the removal of MPs by
activated sludge and showed an increase in the removal of TMP from 50% to
90% when SRTs were increased from 16 ± 2 to 60 days. Therefore, the increased
101
removal efficiency of TMP in the BNR could be due to microbial diversity and
SRT of the process as compared to the CAS and NAS treatment configurations.
Figure 3-4. Box plot and average of influent and effluents concentrations from
CAS, NAS and BNR treatment A- Ibuprofen, B- Gemfibrozil, C-
Trimethoprim, D- Carbamazepine.
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CAS, NAS and BNR treatment. E- Meprobomate, F- Sulfamethoxazole, G-
Androstedione, H-Estrone.
103
CAS, NAS and BNR treatment. I- Nonyl-phenol, J- Bisphenol-A.
104
120
100
Removal Efficiency (%)
80
60
40
20
0
-20
-40
-60
-80
IBU MEP GEM CBZ TMP SMX ADR E1 NP BPA
CAS NAS BNR
Figure 3-7. Micropollutant removal efficiencies in pilots.
3.3.3 Micropollutant Removal in BNR Treatment Configuration
The bioreactors of the CAS and NAS pilots were operated as single
aerobic reactors but the BNR bioreactor was divided into three different zones
operated at different redox conditions. Therefore, it was expected that the
different zones would contribute differently to the biotransformation of the MPs
in the BNR treatment configuration. This expectation was based on the fact that
the amount of energy that is captured by the microorganisms in aerobic
conditions is usually higher than the energy captured in anoxic and anaerobic
conditions (Tchobanoglous et al., 2003). This bioenergy could be instrumental to
the biotransformation of the MPs in the BNR bioreactor. Hence, the contribution
105
of the redox conditions to the removal of the MPs that were removed in the BNR
treatment configuration was assessed.
The MPs concentrations in the influent and the intermediate stages of the
BNR bioreactor are presented in Figures 3-8 and 3-9. It is apparent from Figures
3-8 and 3-9 that the concentrations of the MPs decreased through each stage of
the bioreactor, which suggested that each of the zones contributed to the overall
MPs’ removal. However, during the operation of the pilot BNR process, mixed
liquors were recycled from the aerobic zone to the anoxic zone and from the
anoxic zone to the anaerobic zone. The returned activated sludge was also
recycled from the final clarifier to the anoxic zone of the bioreactor. These recycle
flows can have dilution effects on the pilot plant influent at different points of the
treatment system. Therefore, a set of mass balances were employed to
characterize the fate of the MPs that were removed in the pilot BNR treatment
configuration.
In the mass balances, the biotransformation efficiencies of the MPs in each
zone were calculated as the difference between the mass flow entering and
leaving the zone, divided by the mass flow entering the zone. The difference
between the mass inflow and outflow in the aqueous phase across each zone of
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the bioreactor was assumed to be due to microbial biotransformation within the
zone of the bioreactor. Figure 3-10 shows a representative schematic of the mass
flows employed for the MPs that were removed in the BNR treatment
configuration. The biotransformation efficiencies of the MPs in the anaerobic,
anoxic and aerobic sections of the BNR bioreactor are presented in Table 3-5.
Table 3-5 shows that the biotransformation efficiencies in the bioreactor of the
BNR process increased from the anaerobic to the anoxic and aerobic zone for IBU
and TMP while only the aerobic zone degraded SMX, ADR, E1 and GEM.
Hence, it was concluded that out of the 6 MPs that were removed in the BNR
treatment configuration, only IBU and TMP were biotransformed in all the redox
zones of the BNR bioreactor at different percentages while the other 4 MPs were
biotransformed only in the aerobic zone.
The energy that is captured by the microorganisms in aerobic conditions is
higher than the energy captured in anoxic and anaerobic conditions
(Tchobanoglous et al, 2003). This difference in energy could explain why the
aerobic zone had the highest and in some cases only biodegradation efficiency
among the three stages for the pilot BNR treatment configuration. The high
biotransformation efficiencies in the aerobic zones as compared to the anoxic and
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aerobic zones suggests the potential for significant removal of MPs in aerobic
environments. It was concluded that the aerobic environment is an important
condition for significant biotransformation and removal of MPs in wastewater
treatment. However, further study is recommended to investigate ways of
improving the removal of MPs in anoxic and anaerobic environments.
1600
1400
1200
1000
ng/L
800
600
400
200
0
Influent Anaerobic Anoxic Aerobic
Figure 3-8. Ibuprofen concentrations in the influent and stages of BNR process
(deviation bar represents standard deviation of measurements (n = 5)).
108
140
Gemfibrozil
120
Trimethoprim
100 Sulfamethoxazole
80 Androstendione
ng/L
Estrone
60
40
20
0
Figure 3-9. Micropollutant concentrations in the influent and stages of BNR
process (deviation bar represents standard deviation of measurements (n = 5)).
Figure 3-10. Mass balances of Ibuprofen in BNR pilot plant. (mass flow rate in
µg/d).
Table 3-5. Biotransformation efficiency of MPs in BNR bioreactor

Biodegradation efficiency (%)
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Micropollutant Anaerobic Anoxic Aerobic
Ibuprofen 15 ± 1 45 ± 1 83 ± 5
Trimethoprim 13 ± 12 17 ± 10 24 ± 4
Sulfamethoxazole n.d n.d 48 ± 11
Androstendione n.d n.d 100 ± 14
Estrone n.d n.d 95 ± 0.2
Gemfibrozil n.d n.d 67 ± 23
n.d – not degraded.
3.3.4 Biological Activity
Due to constraints on chemical analysis, it is unlikely that the suite of
chemicals selected for characterization will account for the entire range of
endocrine disrupting compounds (EDCs) present in the effluent. Hence, chemical
quantification may not present a holistic picture of the potential for endocrine-
disrupting activity of the effluents. In fact, differences in endocrine-active
potency amongst different chemical species, or possible antagonism or
synergism of chemical mixtures cannot be accounted for by chemical analysis.
Therefore, the application of in-vitro bioassays to determine the estrogenicity of
environmental samples (Leusch et al., 2006; Fernandez et al., 2009) has been
developed and were examined in this study.
The YES assay was employed to assess the performance of the treatment
processes in terms of estrogenicity removal. The YES assay E2-Eq values from the
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treatment processes are summarized in Figure 3-11. The influent estrogenic
equivalence (E2-Eq) ranged from 32 to 44 ng/L with an average concentration of
37.6 ± 5 ng/L and relative standard deviation (RSD) of 14% across all influent
samples. The effluent E2-Eq values ranged from 4.2 - 8.5 ng/L for the CAS, 0.6 -
8.6 ng/L for the NAS and 0.2 - 2.1 ng/L for the BNR. The average effluent
concentrations for the three processes were 6.3, 4.7 and 0.84 ng/L for the CAS,
NAS and BNR processes respectively.
A paired-t-test was used to compare the E2-Eq in influent to the E2-Eq in
the three effluent streams from CAS, NAS and BNR treatment configuration. The
results of the paired-t-test showed a statistical difference between the influent
and CAS, NAS and BNR effluents (p < 0.05). The BNR effluent values were
significantly lower than the CAS (p = 0.006) but not significantly lower than the
NAS effluent values. Further statistical analysis could not be employed to
discriminate between the means of the data set because of the relatively limited
number of available data. The removal efficiencies calculated as the percent
difference between the influent and effluent E2-Eq concentrations across each
treatment process were 84 ± 3 %, 89 ± 10 % and 98 ± 2 % for CAS, NAS and BNR
respectively. The results show that the BNR and the NAS performed better than
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the CAS in terms of estrogenicity removal. In general, the trend in the removal of
estrogenicity by the process configurations were consistent with the removal of
E1, NP and BPA that were previously presented.
50
45
40
35
E2-Eq (ng/L)
30
25
20
15
10
5
0
Influent CAS NAS BNR
Figure 3-11. Box plot comparing the E2-Eq responses of influent and effluents
from CAS, NAS and BNR (n = 4-5).
Conclusions
A comparative study of the performance of 3 different wastewater
treatment processes was conducted. The concentrations of 10 MPs including
acidic, basic and neutral compounds were determined in the influent and the
effluent of CAS, NAS and BNR treatment configurations. The estrogenic activity
of the influent and effluent from each of the treatment configuration was also
analyzed using the YES assay. The removal efficiency of TMP improved with the
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complexity of the three treatment processes configurations and SRTs. IBU, ADR,
SMX, NP, E1 and BPA had moderate to high removals (> 65%) while CBZ and
MEP remained recalcitrant in the three treatment process configurations. The
removal of GEM was better in the NAS than in the BNR and CAS treatment
configurations. The fact that only TMP showed an increase in removal as the
complexity of the treatment configuration increased from CAS to NAS and to
BNR suggests further study to assess the behavior of TMP in BNR systems. The
YES assay analyses showed an improvement in estrogenicity removal in the BNR
and NAS treatment configurations as compared to the CAS treatment
configuration. In general, similar trend was observed among the treatment
processes in terms of the removal of MPs and estrogenicity. However, it is
important to note that the similarity in the trends in MP and estrogenicity
removals does not imply that the reduction in estrogenicity across the treatment
trains was as a result of the MPs removed.
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Chapter 4 Assessment of the Removal of Estrogenicity in Biological
Nutrient Removal Wastewater Treatment Processes
An abridged version of this chapter was submitted for publication in the journal of Science of the
Total Environment on Dec. 14, 2014; resubmitted with revisions on Jan. 15, 2015 and accepted
for publication January 28, 2015.
Ogunlaja O. O., Parker W. J., Assessment of the removal of estrogenicity in
biological nutrient removal wastewater treatment processes, Sci. Total Environ
(2015), doi/10.1016/j.scitotenv.2015.01.100.
4.1 Introduction
Effluents from wastewater treatment plants (WWTPs) have been shown to
contain a mix of various endocrine disrupting compounds (EDCs) that could
induce physiological effects either individually or synergistically on aquatic
organisms (Desbrow et al., 1998; Sumpter, 1998; Nakada et al., 2004; Vajda et al.,
2011; Wojnarowicz, et al., 2013; Parker et al., 2014). The USEPA has defined EDCs
as exogenous agents that interfere with the synthesis, secretion, transport,
binding action, or elimination of natural hormones in the body that are
responsible for the maintenance of homeostasis, reproduction, development, and
/or behavior. Some of the effects of EDCs on aquatic organisms include reduced
reproductive capacity and vitellogenin production (precursor of egg yolk
protein) in male fish, decreased female fish fertility and survival of juveniles,
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reduced fish egg fertilization and thyroid hormone disruption in tadpoles
(Purdom et al., 1994; Jobling and Sumpter, 1993, Jobling et al., 1998; Andersen et
al., 2003; Vajda et al., 2011; Wojnarowicz, et al., 2013; Parker et al., 2014).
Synthetic and natural EDCs enter sewer systems through human and
animal excretions (Combalbert and Hernandez-Raquet, 2010). Stringent policies
could be formulated by regulatory agencies to attenuate the risks associated with
EDCs in the environment. However, the anthropogenic release of these
substances into the environment is difficult to control because some of these
compounds are naturally produced in the human or animal body. For example,
the natural estrogens, E1 and E2 excreted by pregnant women could be as high
as 600–940 and 170–330 μg/day/person respectively (Johnson et al., 2000). Hence,
the removal of EDCs in wastewater treatment processes will be required for
attenuating their release into the aquatic environment.
Biological nutrient removal (BNR) wastewater treatment processes are
advanced configurations that provide carbon, nitrogen and phosphorus removal.
The removal and biodegradation of macropollutants in BNR processes is well
documented but the fate of EDCs that are prevalent in wastewater in these
processes is less well understood. Previous studies that have investigated the
115
removal of EDCs in BNR wastewater treatment processes have reported greater
than 90% removal efficiencies of the compounds (Koh et al., 2009; Li et al., 2011).
However, it is still unclear whether the high removal of EDCs in BNR treatment
processes can be translated into a high reduction in estrogenic responses from
the systems. A recent study that compared the removal of estrogenicity in
conventional activated sludge (CAS), nitrifying activated sludge (NAS) and
biological nutrient removal (BNR) processes showed greater than 80%
estrogenicity removal in all processes with the highest removal in the BNR
treatment process (Ogunlaja et al. 2013). However, the impact of the different
stages of treatment on estrogenicity reduction was not examined in detail.
The quantification of the estrogenic potency of EDCs in WWTPs is not
trivial because EDCs exist as a cocktail in WWTPs influents and effluents. The
potential for synergistic action of the mixture of EDCs in wastewater has
challenged previous attempts to relate calculated EDC concentrations with
measured estrogenicity in WWTPs (Petrovic et al., 2004). Therefore, in order to
give a holistic assessment of the estrogenicity of a WWTP effluent, previous
studies have employed in vitro bioassays to augment chemical measurements
(Servos et al., 2005; Wu et al., 2011; Parker et al., 2014).
116
In general, there have been few studies that have employed bioassays to
investigate estrogen biodegradation in BNR activated sludge. Previous studies
that have investigated the biodegradation of EDCs in activated sludge systems
have monitored the disappearance of the compounds using chemical techniques
without an understanding of the estrogenicity associated with the disappearance
of the compounds (Joss et al., 2004; Dytczak et al., 2008). However, it has been
demonstrated in other process configurations that the disappearance of
estrogenic compounds does not necessarily eliminate estrogenicity. For example,
the transformation of 17β-estradiol (E2) in activated sludge processes was
reported to involve E2 oxidation to estrone (E1), another estrogenic compound
(Ternes et al., 1999; Shi et al., 2004; Dytczak et al., 2008). There is the potential for
differing conversions between estrogenic compounds in different redox
conditions (Joss et al., 2004; Czajka and Londry, 2006; Dytczak et al., 2008).
Hence, bioassay could be an important tool for characterizing the impact of
redox on estrogenicity.
This study employed the YES assay technique to investigate the removal
and biotransformation of EDCs in BNR activated sludge. In the current study, E1
and E2 were evaluated as target EDCs because several studies have shown that
117
E1 and E2 constitute a substantial fraction of the dominant estrogens found in the
effluents of WWTPs (Nakada et al., 2004; Aerni et al., 2004; Fernandez et al., 2007;
Muller et al., 2008). Specifically, this study employed the recombinant yeast
screen to 1) investigate the removal of estrogenicity in BNR processes operated
with both authentic and synthetic wastewater, 2) estimate the biotransformation
rate constants for E1 and E2 in aerobic, anoxic and anaerobic batch reactors, and
3) investigate the transformation kinetics between E2 and E1 under anaerobic,
anoxic and aerobic conditions.
4.2 Approach
A combination of pilot and bench scale tests, batch experiments and
mathematical modeling was employed to assess the removal and
biotransformation of EDCs in BNR activated sludge processes. The experimental
approach employed a pilot scale BNR process fed with authentic municipal
wastewater and a bench scale BNR process fed with synthetic wastewater that
was dosed with EDCs. The objective of operating the pilot scale BNR was to
investigate the removal and biotransformation of EDCs in each treatment zone of
a BNR treatment process. The goal of operating the bench scale BNR system was
to investigate the removal kinetics of the natural estrogens, E1 and E2 and their
118
associated estrogenicity under controlled conditions. In addition, a series of
batch experiments were conducted to investigate the transformation between E1
and E2 under aerobic, anoxic and anaerobic conditions in order to provide
further insight into the removal and biodegradation of the compounds in BNR
processes.
4.2.1 Pilot Scale BNR Wastewater Treatment Process
A detailed description of the UCT-BNR pilot plant was described
elsewhere (Ogunlaja et al, 2013). The operating and design conditions are
summarized in Table 4-1. The pilot UCT-BNR was operated on authentic
municipal wastewater that was augmented with sodium bicarbonate (22 g/L at
the rate of 14.4L/d) to provide alkalinity, di-potassium phosphate (11 g/L at the
rate of 14.4 L/d) as phosphorus source and sodium acetate (34.36 g/L at the rate
of 14.4 L/d) to enhance the proliferation of PAOs. The resultant influent
concentration of COD, alkalinity and total phosphorus were 367 ± 48 mg/L, 268 ±
21 mg/L and 11 ± 7 mg/L respectively. The pH range of the authentic wastewater
entering the bioreactor was 7.5 – 8.2. The bioreactor was partitioned into six 60L
cells to simulate pseudo plug flow (Figure 4-1). The first cell was operated as an
anaerobic zone, the next two cells were operated as anoxic zones and the last
119
three cells were operated as aerobic zones. Coarse bubble aerators were used for
aeration and mixing in the aerobic section of the bioreactor while mechanical
mixers were used in the non-aerated zones. The solids residence time (SRT) was
maintained by wasting mixed liquor from the last aerobic section of the
bioreactor. The temperature was controlled by an insulated water jacket that was
wrapped around the bioreactor, primary and final clarifier.
Table 4-1. Pilot BNR operating and design conditions

Unit Size/description Unit
Flow rate 1.3 m3/d
Primary clarifier Area = 0.46 m2
Depth = 1.56 m
Bioreactor Volume = 0.36 m3
Depth = 1.28 m
DO(aerobic) = 4-5 g/m3
DO(anoxic) = 1-2.5 g/m3
DO(anaerobic) = 0-0.2 g/m3
Final clarifier Area = 0.204 m2
Depth = 1.4 m
Recycle rate Aerobic = 2.6 m3/d
Anoxic = 1.3 m3/d
SRT 20 d
Aerobic SRT 10 d
RAS flow rate 0.9 m3/d
Waste rate 0.018 m3/d
HRT 7 h
Temperature 18 ± 2 oC
RAS-return activated sludge,SRT-solid residence time,HRT-hydraulic retension time
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Figure 4-1. Flow schematic of pilot BNR activated sludge treatment system.
4.2.2 Bench Scale BNR Wastewater Treatment Process
The bench scale UCT-BNR process consisted of 3 - 10 L coupled reactors
made of acrylic plastic and a 25 L final clarifier for solid –liquid separation
(Figure 4-2). The aerobic reactor was mixed and aerated with fine bubble aerators
while the anaerobic and anoxic reactors were mechanically mixed. The influent
flow to the system was maintained at 0.086 ± 0.01 m3/ day and the HRT was 5
hours. The return activated sludge was operated at 75% of the influent flow rate
and the internal recycle ratios were 200% and 100% of the influent flow rate for
the aerobic and anoxic recycles respectively. Approximately 0.9 ± 0.1 L/d of
sludge was wasted from the aerobic zone of the bioreactor to maintain a total
SRT of 20 days. The DO in the aerobic zone was maintained at 5-7 mg/L and the
121
temperature of the system was maintained at 20 ± 2oC. The operational
parameters of the bench scale BNR process were consistent with typical
operational parameters of commonly used BNR processes (WEF, 2005).
The bioreactors were inoculated with mixed liquor collected from the
return activated sludge stream of a full scale BNR WWTP in southern Ontario.
The system was fed with a synthetic wastewater containing carbon source,
nitrogen source, phosphate buffer and micronutrients. The constituents of the
synthetic wastewater and their target concentrations are presented in Table 4-2.
The system was initially maintained without addition of E1 and E2 into the
influent stream for one SRT to allow acclimatization of the biomass to the
synthetic feed. Subsequently, a volume of E2 and E1 dissolved in water was
dosed into the synthetic wastewater with the objective of achieving a target
concentration of 100 ng/L for E1 and E2 to acclimatize the biomass to the
estrogenic compounds. These dosed concentrations were higher than those
typically observed in Canadian wastewater (Servos et al., 2005). However, this
concentration enabled measurement of the estrogenicity in the samples,
considering the sample matrix. After two months of operation, steady state was
achieved and the aerobic bioreactor mixed liquor with an MLVSS concentration
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of 3817 ± 150 g MLVSS/ m3 was employed for the batch tests.
Figure 4-2. Flow schematic of the bench scale BNR system.
Table 4-2. Composition of influent synthetic wastewater

Molecular Target feed
Function Name Formula concentration Unit
Carbon source Sodium acetate NaCH3 CO2 475 g COD/ m3
Nitrogen source Ammonium chloride NH4 Cl 15 g N/ m3
Phosphorus Monopotassium
source phosphate KH2PO4 10 g P/ m3
Alkalinity Sodium bicarbonate NaHCO3 167 g/ m3
Calcium chloride CaCl2·2H2O 250 g/ m3
Magnesium sulfate MnSO4·H2O 180 mg/ m3
Copper sulfate CuSO4·5H2O 35 mg/ m3
Micronutrients
Zinc sulfate ZnSO4 ·7H2O 130 mg/ m3
Iron (II) sulfate FeSO4·7H2O 390 mg/ m3
Cobalt chloride CoCl3 ·6H2O 34 mg/ m3
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4.2.3 Monitoring and Sampling–Pilot and Bench Scale BNR
System
The pilot plant was operated for over 12 months with consistent
monitoring of the conventional and operational parameters for over 6 months.
The bench scale setup was operated for 3 months with consistent monitoring for
one month. Performance was assessed by monitoring chemical oxygen demand
(COD), Total Kjeldahl Nitrogen (TKN), ammonia (NH3), nitrite (NO2), nitrate
(NO3), phosphate (PO4), total suspended solid (TSS) and volatile suspended solid
(VSS).
Twenty four hour composite samples of the pilot plant influent and
effluent were collected in pre washed stainless steel containers, three times a
week for two weeks using a refrigerated autosampler for estrogenicity analysis.
Eight hour composite grab samples (sampled every 2 hours for eight hours and
mixed together) were also collected thrice a week for two weeks from the
anaerobic, anoxic and aerobic zones of the bioreactor. Five hour composite
samples were collected from the bench scale influent and effluent streams in
prewashed amber glass bottles thrice a week for 2 weeks for estrogenicity
analysis. In addition, grab samples with a volume of 40 mL were collected thrice
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a week for two weeks from the mixed liquors in the anaerobic, anoxic and
aerobic reactors. The samples were centrifuged at 4000 rpm for 5 minutes and the
supernatant filtered through 1.5 µm glass microfiber filters (Whatman, Toronto,
Ontario, Canada). The filtered samples for the pilot plant were adjusted to pH 8
with 0.1M NaOH and stored in amber bottles in preparation for solid phase
extraction while the bench scale samples were extracted immediately.
4.2.4 Batch Experiments
The mixed liquor from the aeration zone of the bench scale UCT-BNR was
employed as the innoculum in a series of batch tests that were conducted to
investigate the biotransformation of E2 and E1 under anaerobic, anoxic and
aerobic conditions using the YES assay method. The batch experiments were
conducted as described by Shi et al., 2004. The batch experiments were
performed using a 1L Erlenmeyer flask placed on a magnetic stirrer and
wrapped with aluminium foil to prevent photodegradation of the EDCs. In the
aerobic experiment, the mixed liquor was mechanically stirred and air was
supplied through air pumps to maintain a dissolved oxygen (DO) concentration
of approximately 2 – 7 mg/L. The mixed liquor was mechanically stirred in the
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anaerobic and anoxic experiments without aeration. The reactors were
maintained at a temperature of 20 ± 2oC and pH range of 7.2 - 8.5.
A solution of either E2 or E1 in ethanol solution was added to the
sterilized erlenmeyer flask to achieve a target concentration of 0.1g/m3. After
evaporation of the solvent, approximately 125 mL of mixed liquor from the BNR
process was diluted with an appropriate volume of synthetic wastewater to
achieve a mixed liqour volatile suspended solid (MLVSS) concentration of 255
gMLVSS/m3. The batch experiments were conducted once and duplicate samples
collected at every time interval The concentrations of the chemicals added into
the batch reactors to facilitate biomass growth throughout the duration of the
biotransformation tests are presented in Table 4-3.
Table 4-3. Feed composition for batch tests

Function Molecular Formula Anaerobic Anoxic Aerobic Unit
Carbon source NaCH3 CO2 305 330 375 gCOD/m3
Phosphorus source KH2PO4 20 15 20 g P/m3
Nitrogen source NH4 Cl n/a n/a 14 g N/m3
Nitrate source KNO3 n/a 72 n/a g N/m3
n/a-not added
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4.2.5 Batch Experiment Monitoring and Sampling
The concentrations of E2, E1 (as indicated by the YES test) and
conventional parameters in the mixed liquor in the batch reactors were analyzed
to assess the removal of estrogenicity and conventional pollutants. From each
batch reactor, 40 mL of mixed liquor samples were collected in prewashed amber
bottles before the addition of E1 and E2 to the reactors in order to quantify
background concentrations. Samples of the reactors’ mixed liquor were then
collected 15 minutes after dosing and subsequently every four hours for three
days. The collected samples were centrifuged at 4000 rpm for 5 minutes and the
supernatant liquids were filtered using 1.5 µm glass fiber filters (Whatman
934/AH). A volume of 10 mL was analyzed for conventional pollutants after
filtering and the remaining 30 mL was immediately analyzed using SPE analysis.
All parameters were analyzed in duplicates.
4.2.6 Sample Extraction
Solid phase extraction (SPE) is the most common extraction method used
to concentrate EDCs in environmental samples and hence the method of Li et al.,
(2010) was employed to extract the EDCs from the wastewater samples in this
study.
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4.2.6.1 Solid Phase Extraction
100 ml of the filtered samples were passed through 6cc/500 mg OASIS
HLB cartridges (Waters, USA) using Visiprep solid-phase extraction (SPE)
vacuum manifolds and a vacuum pump. The cartridges were initially
conditioned sequentially with 5 mL of methanol and 5 mL of Milli-Q water
before loading the samples onto the cartridge. After extraction, the cartridges
were rinsed with 5mL of water and 5 mL of hexane before being dried under
vacuum for 5 minutes. Two 5-mL aliquots of ethyl acetate at an approximate
flow rate of 1mL/min were subsequently used to elute the samples into 10 mL
borosilicate collection tubes. The eluate was dried under a gentle stream of
nitrogen gas and vortex-mixed with 500 µL of methanol. The final samples were
transferred into amber bottles and stored at 4oC until the YES assay procedure
was conducted.
4.2.6.2 Extraction Recovery
A volume of 100 mL of mixed liquor from the anaerobic, anoxic and
aerobic sections of the WWTP that was the source of the inoculum for the bench
scale BNR was dosed with 30 µg/L of E2 to determine the sample extraction
recovery. 100 mL of milli-Q water was also dosed with the same concentration of
128
E2 to investigate the possible effect of sample matrix on the extraction procedure.
The wastewater samples were centrifuged at 5000 rpm for 5 minutes and the
supernatant liquid were filtered using 1.5 µm glass fiber filter before solid phase
extraction and YES assays were conducted.
4.2.7 Chemical Analysis
Conventional wastewater parameters including chemical oxygen demand
(COD), total suspended solids (TSS), total Kjeldahl nitrogen (TKN), total
phosphorus, ammonia-nitrogen, nitrate-nitrogen, total suspended solid (TSS)
and volatile suspended solid (VSS) were analyzed according to Standard Methods
(Eaton., 2005).
4.2.8 YES Assay
One of the widely used in vitro bioassays for the determination of
estrogenicity in environmental samples is the recombinant yeast estrogen screen
(YES) (Fent et al., 2006; Leusch et al., 2010; Citulski and Farahbakhsh, 2012). The
recombinant yeast strain contains yeast cells that has been integrated with the
DNA of human estrogen receptors (hER). The YES assay provides a qualitative
and quantitative measure of the endocrine-disrupting potential of all EDCs
present in WWTP effluent (Leusch et al., 2010). Hence, this method has proved to
129
be a useful tool in determining the combined estrogenic effects of the mixture of
EDCs in complex environmental matrices such as WWTPs effluents.
A sample of concentrated human estrogen-receptor (hER) transfected
recombinant yeast stock was provided by Prof. C. Metcalfe, (Trent University,
Peterborough, ON). The YES assays were conducted as described by Routledge
and Sumpter (1996) with modifications as reported by Citulski and Farahbakhsh,
(2010). The bioassays were conducted in a sterilized laminar air flow cabinet to
minimize aerosol formation and contamination. Each 96 well optically flat-
bottom microtiter plate contained one row of 100 µL of serially diluted positive
control of E2 (10 nM to 5 x 10-3 nM) in triplicates, one row of ethanol as negative
control and three rows of serially diluted 80 µL extracted wastewater samples. A
volume of 200 µL of combined growth medium/CPRG/yeast mixture was added
to the dried wells of the plates. The plates were sealed with parafilm to prevent
drying of the well solution in the incubator and shaken at 150 rpm and 32oC for
72 hours. The yeast growth in the plates was measured using a micro titer plate
reader (Sunrise Basic TECAN) at an absorbance of 620 nm to measure turbidity
and 540 nm to detect color change of the assay medium.
130
4.2.8.1 Data Processing
The absorbance measurements for E2 standards and wastewater samples
were adjusted as per Fent et al (2006) (Equation 4-1) in order to adjust the
absorbance of chlorophenol red at 620 nm for the extent of yeast growth
(turbidity).
Adjusted Absorbance = AB540nm (sample) – [AB 620nm (sample) – AB 620nm
(blank, average)] 4-1
Where,
AB540nm (sample) = sample absorbance at 540 nm

AB 620nm (sample) = sample absorbance at 620 nm
AB 620nm (blank, average) = average of blank absorbance at 620 nm.
The dose response curve for the standards and environmental samples
were input to GraphPad Prism 6 (v. 6.02) that employs a four-parameter
sigmoidal Hill equation to calculate the EC-50. To obtain the estrogenicity values
for the extracted sample, the logarithm of the extraction dilution that yielded a
50% response was computed from the fitted standard curve. The estrogenicity of
the samples were expressed as 17β-estradiol equivalents (E2-Eq), or the
equivalent concentration of E2 that would have to be present in the wastewater
131
sample to achieve a response of the same magnitude. The E2-Eq was calculated
as the ratio between the amount of E2 in the incubation well at EC50 in the
standard curve and the equivalent volume at EC50 as follows:
𝑛𝑔 𝐸2 𝐸𝐶50( 𝑛𝑔⁄𝐿)
𝐸2 − 𝐸𝑞 ( 𝐿 ) = (𝐸𝐶50 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟∗0.4) 4-2
The value of 0.4 employed in equation 2 accounted for dilution when the air
dried 80µL wastewater sample extract applied to the titer well was reconstituted
with 200µL of the growth medium containing yeast cells (80/200 = 0.4). The
method detection limit for the assay was determined to be 1 ng/L.
4.2.8.2 Determination of Relative Potency between E1 and E2-

Eq (𝐲)
In order to determine the relative estrogenic response of E1 as E2-Eq, E1
stock solution was added to a volume of 100 mL of the mixed liquor used for the
batch tests to achieve concentrations in the range of 100-2000 ng/L. The dosed
mixed liquor samples were centrifuged at 5000 rpm for 5 minutes and the
supernatant liquids were filtered using 1.5 µm glass fiber filter before SPE and
YES assays were conducted.
132
Outliers in replicate measurements were detected using Grubb’s test. The
regressions used to construct the YES assay response curves for the samples were
compared to the response curves of estradiol (E2) standard using the F-test. To
check the yeast growth absorbance (AB620nm) for the presence of toxic effects
resulting from the wastewater extracts, the yeast growth was compared to the
average turbidity (±3 standard deviations) of 12 ethanol only negative-control
wells that were incubated in the same plate as the samples. Samples that had
yeast growth with turbidity values below the average minus 3 standard
deviations of the negative control were removed from the dose-response curve
analysis. The final conditioned data were employed to generate the dose-
response curve for the estimation of the EC50.
As previously described, this study integrated results from pilot and
bench scale BNR processes and batch tests to obtain an improved understanding
of the fate of EDCs in BNR processes. The UCT-BNR pilot and bench scale
systems were monitored with respect to the removal of conventional wastewater
pollutants and estrogenicity to establish the performance of the treatment
133
processes. Samples collected from the batch reactors were analyzed for
conventional pollutants to monitor the progress of typical metabolic processes
and to estimate the biodegradation and biotransformation kinetics of E1 and E2
in BNR activated sludge. The biotransformation and biodegradation rate
constants for E1 and E2 were estimated using pseudo first order kinetic
expressions.
4.3.1 Performance of Pilot and Bench Scale BNR System –

Conventional Pollutants
The conventional pollutants of the pilot and bench scale BNR processes
were analyzed to determine whether the processes were functioning within
typical operating ranges of a BNR process. The influent and effluent
concentrations of the conventional contaminants from the pilot and bench scale
BNR processes are presented in Table 4-4. As expected, low effluent
concentrations were observed for COD, TKN, NH4, NO3, and TP. The results
indicate that the treatment systems were effectively nitrifying, denitrifying and
biologically removing phosphorus during the sampling period. The performance
of both BNR systems was consistent with typical operation of BNR systems
(Jeyanayagam, 2005). Hence, it was assumed that the biomass contained
134
representative quantities of active ordinary heterotrophic organisms (OHOs),
nitrifiers and polyphosphate accumulating organisms (PAOs).
Table 4-4. Conventional pollutants responses in BNR processes (Mean ± SD.)

Pilot Scale (mg/L) Bench Scale (mg/L)
Parameter Influent Effluent Influent Effluent
COD 367 ± 48 33 ± 12 447 ± 150 23 ±13
TKN 25 ± 12 2.3 ± 0.2 20 ± 10 3.2 ± 0.1
NH4-N 19 ± 8 0.04 ± 0.03 15 ± 4 0.03 ± 0.01
NO3-N 0.54 ± 0.2 3±2 0.61 ± 0.24 4 ± 2
TP 11 ± 7 3±2 10 ± 2 1± 0.3
MLSS (gCOD/L) 6.8 ± 0.9 7.2 ± 0.3
4.3.2 Evaluation of Extraction Recovery
The extraction recoveries of the EDCs in the wastewater samples were
analyzed to assess the performance of the solid phase extraction procedure
employed in extracting the EDCs in the treated wastewater. The results of the
tests with known concentrations of E2 are presented in Table 4-5. The Table
shows that the extraction procedure gave a high recovery of E2 from the
wastewater samples and milli-Q water. As expected, the milli-Q water had the
highest recovery. The comparable recoveries observed in the mixed liquor
135
samples and the milli-Q sample were deemed to indicate that the mixed liquor
suspended solids did not adsorb the dosed E2 and the solid phase extraction
method extracted most of the dosed E2. Hence, the solid phase extraction
procedure used in this study was deemed appropriate to quantify the
estrogenicity in the treated wastewater samples.
Table 4-5. Solid phase extraction recovery of E2

Sample Recovery %
Anaerobic mixed liquor 90 ± 20
Anoxic mixed liquor 87.5 ± 11
Aerobic mixed liquor 86.4 ± 6
Milli Q water 98.8 ± 13
4.3.3 Estrogenicity Removal in Pilot and Bench Scale BNR
The estrogenicity was assessed at different points during the operation of
the pilot and bench scale BNR process in order to analyze and compare the
performance of the processes in terms of estrogenicity removal. The responses
reported as E2-Equivalent (E2-Eq) concentrations in the influent, effluent, and
the interstages of both systems are presented in Figure 4-3.The deviation bars in
the figure represent the standard deviation of the measurements (n = 6). It is
apparent from the figure that the estrogenicity concentration decreased through
each successive stage of both BNR processes. The overall estrogenicity removal
136
efficiency was calculated as the difference between the E2-Eq concentrations in
the influent stream into the bioreactor and that of the effluent exiting the
bioreactor, divided by the E2-Eq concentration in the influent stream into the
bioreactor. Thus, the overall estrogenicity removal efficiencies for the pilot and
bench scale BNR processes were 96 ± 5% and 95 ± 5% respectively. Hence,
significant estrogenicity removal was observed in the pilot and the bench scale
BNR processes. This observation was consistent with previous studies that
reported removal of estrogens in BNR wastewater treatment systems (Joss et al.,
2004; Wu et al., 2011, Li et al., 2011). The consistency of the high estrogenicity
removal in this study with the results of previous studies that have reported high
removal of EDCs based on chemical measurements suggests that a high removal
of EDCs in BNR process could be interpreted as a high removal of estrogenicity.
It was concluded that both pilot and bench scale BNR processes were performing
equivalently with respect to overall estrogenicity removal.
Mass balances were conducted to account for the effects of the recycle
streams on the observed estrogenicity removal efficiencies. The mass balances
around the bioreactors for both pilot and bench scale BNR are presented in
Figures 4-4 and 4-5 respectively. The degradation efficiency of EDCs in each zone
137
was calculated as the difference between mass flow entering and leaving the
zone, divided by the total mass flow entering the zone. The difference between
the mass inflow and outflow for each redox zone was assumed to be due to
biodegradation within the zone of the bioreactor. Previous studies have shown
that sorption typically accounts for less than 10% of the estrogen removal from
wastewater (Andersen et al., 2003; Joss et al., 2004; Ternes et al., 1999). Hence, the
degradation efficiencies in the anaerobic, anoxic and aerobic reactors were
calculated as 11± 9%, 18 ± 2%, 93 ± 10% and 8± 0.8%, 38 ± 4%, 85 ± 22% in the pilot
and bench scale reactors respectively. Generally, these results show that similar
degradation efficiencies were observed in corresponding redox zones of the pilot
and bench scale processes. Therefore, it was concluded that comparable
degradation of estrogenic compounds occurred in the individual zones of both
BNR processes, albeit at different percentages.
Similar estrogenicity degradation efficiencies were observed along the
bioreactors for both systems despite the fact that the pilot plant BNR was fed
with authentic wastewater with presumably a mix of estrogens and the bench
scale BNR was operated with synthetic wastewater with only two estrogens.
Previous studies have shown that approximately 95% of the estrogenic activity in
138
treated effluents was due to the presence of the natural estrogens while other
estrogenic compounds such as alkyl-phenol contributed less than 5% of the
estrogenicity in WWTPs effluents (Matsui et al., 2000; Aerni et al., 2004; Muller et
al., 2008). The results suggest that the majority of the estrogenic activity in the
authentic wastewater was contributed by estrogens similar to the ones added to
the synthetic wastewater.
4.3.4 Interstage Comparison of Estrogenicity Removal in Pilot and
Bench Scale BNR
The analysis of the mass balance in the BNR processes enabled an
assessment of the importance of the redox conditions towards the estrogenicity
removal. It also enabled a comparison between each redox zone in order to
assess which zone could be optimized to improve overall process performance.
The ratio of the degradation efficiencies in the aerobic, anoxic and anaerobic
zones for both pilot and bench scale BNR process were 8:2:1 and 11:5:1
respectively. The observed high degradation efficiencies in the aerobic zones of
the pilot and the bench scale BNR process was consistent with previous studies
that reported high degradation of EDCs as monitored by chemical concentrations
(Joss et al., 2004; Dytczak et al., 2008 ). Under aerobic conditions, heterotrophic
139
organisms have the capability to produce oxygenase enzymes that catalyze the
direct incorporation of oxygen molecule into the molecule of the organic
compounds. This oxygenase reaction weakens the ring structure in the synthetic
compounds which makes it accessible for subsequent oxidation steps and more
water soluble (Rittman and McCarty, 2001). Thus, the high reductions of
estrogenicity in the aerobic zone could be due to the combination of the redox
condition and the bacteria activity on the estrogenic compounds.
The performance of the anoxic zones was compared to that of the
respective aerobic zones of the BNR processes. The degradation efficiencies of
EDCs in the anoxic zones of the pilot and bench scale BNR processes were lower
than that of the aerobic zones. This result was consistent with previous studies
that reported biodegradation of EDCs or removal of estrogenic activities in the
anoxic zones of BNR processes (Jurgens et al., 2002 and Lee and Liu, 2002; Joss et
al., 2004; Wu et al., 2011). The lower biodegradation efficiency in the anoxic zones
as compared with the aerobic zones could be as a result of the lower energy
available to the biomass in the zones due to the utilization of nitrate as electron
acceptor instead of oxygen (Tchobanoglous et al., 2003). Hence, it was concluded
140
that estrogenicity removal can occur in anoxic zone of a BNR process, but at a
lower efficiency when compared to the estrogenicity removal in the aerobic zone.
The performance of the anaerobic zones were compared with the
respective aerobic and anoxic zones of the BNR processes. The degradation
efficiencies of EDCs in the anaerobic zones of the pilot and bench scale BNR
processes were the lowest out of the three zones. The lowest biodegradation
efficiency in the anaerobic zones as compared with the anoxic and aerobic zones
could be as a result of the low energy available to the biomass in the anaerobic
zones (Tchobanoglous et al., 2003). The comparable and low biodegradation
efficiencies in the anaerobic zones of both the pilot and bench scale BNR
processes suggest that only modest removals of estrogenicity occur in the
anaerobic zones of BNR processes.
141
180
160
140 Pilot Bench
E2-Equivalent (ng/L)
120
100
80
60
40
20
0
Influent AN AX AO Effluent
Figure 4-3. E2-Equivalent profiles along pilot and bench scale BNR bioreactors.
AN-Anaerobic, AX-Anoxic, AO-Aerobic.
Figure 4-4. Mass balances (µg/d) of estrogenicity around pilot BNR bioreactor.
142
Figure 4-5. Mass balances (µg/d) of estrogenicity around bench scale BNR
bioreactor.
4.3.5 Biotransformation of E2 and E1 in Batch Tests
A series of batch tests were conducted under similar redox conditions as
the stages of the BNR process in order to further understand the
biotransformation kinetics of E1 and E2 under the different redox conditions. The
profiles of E2 equivalents in each batch test dosed with either E1 or E2 are
presented in Figure 4-6. It is apparent from the Figure that the estrogenicity in all
the reactors decreased with time, although at different rates.
The E2-Eq in reactors dosed with either E1 or E2 decreased at the fastest
rate under aerobic conditions, followed by anoxic conditions and the slowest rate
was observed under anaerobic conditions. These results were consistent with the
previously described degradation efficiencies of the EDCs in the stages of the
143
pilot and bench scale BNR processes where the highest degradation occurred in
the aerobic zones, followed by the anoxic zones and lastly the anaerobic zones.
Therefore, it was concluded that biotransformation of E1 and E2 in the aerobic,
anoxic and anaerobic batch reactors leads to a reduction in estrogenic responses
in the reactors, albeit at different rates in the order of aerobic > anoxic> anaerobic.
Figure 4-6 shows that the E2 equivalence of the aerobic and anoxic
reactors dosed with E2 decreased quickly during the first 4.25 hours of the tests.
Previous studies have shown that the transformation or oxidation of E2 to E1 can
occur under both aerobic and anoxic conditions (Ternes et al., 1999; Shi et al.,
2004; Dytczak et al., 2008). This transformation reaction has also been previously
reported to be rapid and usually occur early in batch experiments (Onda et al.,
2002). It was not possible to separately monitor the biotransformation of E2 to E1
using the YES assay technique because the method measures the total estrogenic
response elicited by both E1 and E2 at every point interval. However, since the
relative estrogenic potency of E2 is significantly higher than that of E1 (Svenson
et al., 2003; Tanaka et al., 2001), a significant reduction in E2 in the reactors
would lead to a significant reduction in the total estrogenic response. Hence, the
observed sharp decline in estrogenicity within the first approximately 4.25 hours
144
of the tests suggested the transformation of E2 to E1 in the aerobic and anoxic
reactors.
From Figure 4-6 it can be seen that the E2-Eq of both E1 and E2 dosed
reactors declined at the same rate in the anoxic and aerobic reactors after
approximately 16 hours of reaction and this trend remained the same throughout
the duration of the experiment. The E2-Eq in the E2 dosed reactors were the
combined estrogenicity elicited by the residual E2 and the formed metabolite, E1
while the E2-Eq in the E1 dosed reactors only measured the estrogenicity due to
residual E1 during the experiment. Hence the results support the previous
observation that transformation of E2 to E1 occurred in the early stages of the
experiments with E2 dosed reactors and that after approximately 16 hours, the
majority of the estrogenicity was contributed by E1. Hence, these results suggest
an accumulation of E1 in the E2 dosed reactors before further degradation as the
experiment proceeded.
Previous studies have reported that biotransformation of E2 proceeded
through initial formation of E1 and subsequent formation of metabolites or
mineralization in anoxic and aerobic conditions (Ternes et al., 1999; Onda et al.,
2003; Shi et al., 2004; Dytczak et al., 2008). However, similar conclusions were not
145
reported for biotransformation of E1 and E2 under anaerobic conditions. The
reduction of E1 to E2 has been reported in anaerobic batch tests that were
conducted with activated sludge taken from an anaerobic/anoxic/aerobic MBR
pilot plant (Joss et al., 2004), an oxidation ditch (Mes et al., 2008) and a lab scale
anaerobic/aerobic SBR (Lust and Stensel, 2011). On the contrary, other studies
have reported oxidation of E2 to E1 under various anaerobic conditions ((Jürgens
et al., 2002; Lee and Liu, 2002; Czajka and Londry, 2006). Hence, this study used
bioassays to assess the biotransformation kinetics between E1 and E2 under
anaerobic conditions.
Based on the bioassay method employed in this study, it was initially
expected that the E2-Eq in the anaerobic reactor dosed with E2 would decrease to
confirm the oxidation of E2 to E1 and increase in the E1 dosed anaerobic reactor
to confirm the conversion of E1 to E2. This expectation was based on the fact that
the estrogenic potency ratio of E2 to E2-Eq was 1:1 and E1 to E2-Eq was 1: 0.56.
The results showed that the E2-Eq decreased in the anaerobic reactors dosed
with either E1 or E2. The decline in the E2-Eq in the E1 dosed anaerobic reactor
was faster than that of the E2 dosed reactor (Figure 4-6). This result implied that
E2 was converted to E1 in the E2 dosed reactor and that degradation of E1
146
produced other metabolites than E2 in the E1 dosed reactor. This result was
consistent with the previously observed reduction in estrogenicity in the
anaerobic zones of the bench scale BNR, which was the source of the mixed
liquor for the batch tests. Hence, it was concluded that the conversion of E1 and
E2 to less estrogenic metabolites occurred in the anaerobic batch tests, with E1
degrading at a faster rate than E2.
90
E2-O2 E2-AX
80
E2-AN E1-O2
70 E1-AX E1-AN
60
E2 Eq (ug/L)
50
40
30
20
10
0
0 10 20 30 40 50 60 70
Time(hr)
Figure 4-6. E2 equivalent profiles for batch tests dosed with E1 or E2. O2-
aerobic, AX- anoxic, AN-anaerobic.
147
4.3.6 Modeling of Biotransformation of E1 and E2 in Batch Tests
Mathematical modeling was employed to further understand the
biotransformation kinetics of E2 and E1 in terms of their estrogenicity response
under anaerobic, anoxic and aerobic conditions. The models commonly used to
predict biodegradation kinetics of EDCs in wastewater treatment typically
employ either first order (Zhao et al., 2008) or pseudo first order expressions
(Schwarzenbach et al., 2003; Joss et al., 2006). Therefore, a pseudo first order
expression was employed to characterize the biotransformation kinetics of E2
and E1 in the aerobic, anoxic and anaerobic batch tests (Equation 4-3).
𝑟𝑖 = −𝑘𝑏 𝑋𝑆𝑖 4-3
Where,
𝑟𝑖 = rate of biotransformation of compound i in batch reactor [µg L-1hr-1]
𝑘𝑏 = biotransformation rate constant [L gCOD-1hr-1]
𝑋= MLVSS concentration [gCOD L-1]
𝑆𝑖 = E2Eq due to E1 [µg L-1]
The use of the YES assay technique to describe the behavior of E1 and E2
in batch tests was novel to this study. However, to employ the results of this
study for modeling the biotransformation of E2 in the batch tests it was necessary
148
to address the degradation of E2 to E1, which is also an estrogenic compound,
and the subsequent degradation of E1 (Ternes et al., 1999; Shi et al., 2004;
Dytczak et al., 2008; Hashimoto and Murakami, 2009) (Equation 4-4). The
measured E2-Eq values in the E2 dosed batch tests were recognized as the total
estrogenic response elicited by E1 and E2 (Equation 4-5).
𝑘𝑏,1 𝑘𝑏,2
𝐸2 → 𝐸1 → 𝑢𝑛𝑘𝑛𝑜𝑤𝑛 4-4
Where,
𝑘𝑏,1 = biotransformation rate constant for the conversion of E2 to E1 [L gCOD-1hr-

1
]
𝑘𝑏,2 = biotransformation rate constant for the degradation of E1 [L gCOD-1hr-1]
𝑆′𝐸2,𝑡 = 𝑆𝐸2,𝑡 + 𝑦𝑆𝐸1,𝑡 @ t = 0, 𝑆𝐸1,𝑡 = 0 4-5
Where,
S′E2,t = total E2-Eq at time t [µg.L-1]
SE2,t = E2 concentration at time t [µg.L-1]
SE1,t = E1 concentration at time t [µg.L-1]
y = relative potency between E1 and E2-Eq
Equation 4-5 describes the E2-Eq concentrations in terms of summative
effects of E1 and E2 in the E2 dosed reactors. The relative potency (y) was
149
employed to convert the E1 concentration to E2-Eq. This conversion was not
required for E2 as the relative potency of E2 to E2-Eq is 1:1 (Svenson et al., 2003;
Rutishauser et al., 2004). The measured E2-Eq in the E2 dosed batch tests was
employed directly in the modelling of E2 in the reactors.
The mass balance on E1 in the E2 dosed batch reactors is presented in
Equation 4-6, and includes the production of E1 from E2 and the subsequent
biotransformation of E1
𝑑𝑆𝐸1
= 𝜇𝑘𝑏,1 𝑋𝑆𝐸2 − 𝑘𝑏,2 𝑋𝑆𝐸1 4-6
𝑑𝑡
Where,
μ = stoichiometric conversion between E2 and E1
𝑋= MLVSS concentration [g COD L-1]
𝑆𝐸1 =soluble concentration of E1 [µg.L-1]
𝑆𝐸2 =soluble concentration of E2 [µg.L-1]
𝑘b,1 = biotransformation rate constant for the conversion of E2 to E1 [L gCOD-1hr-

1
]
𝑘b,2 = biotransformation rate constant for the conversion of E1 to metabolites [L
gCOD-1hr-1]
Assuming a 1:1 mole ratio between E2 and E1, solving for E1 in equation 4-6 and
substituting into equation 4-5 yields equation 4-7.
150
𝑒 −𝑘1𝑋𝑡 𝑒 −𝑘2𝑋𝑡
𝑆′𝐸2,𝑡 = 𝑆𝐸2,𝑡 + 𝑦 ( 𝑆𝐸2,𝑡 𝑘1 ( + )) 4-7
𝑘2 −𝑘1 𝑘1 −𝑘2
The biotransformation rate constants k 2 and k1 were estimated by solving
equations 4-3 and 4-7 for each redox condition using an integral least square
method that minimized the sum of squares of the residuals between the
predicted and measured E2-Eq in the E1 and E2 dosed batch tests respectively.
4.3.7 Relative Potency between E1 and E2-Eq (𝐲)
The estrogenic potency of E1 relative to E2 was determined based on the
tests at known concentrations of E1 and measured E2 Eq. Figure 4-7 shows the
relationship between E1 concentrations and measured E2 Eq. It is apparent from
the plot that there was a linear relationship between the measured E2-Eq and the
E1 concentrations as indicated by the r2 value and residuals. The estrogenic
potency of E1 was obtained from the slope of the linear fit and was estimated to
be 0.56 ngE2/ngE1. This value was consistent with previously reported relative
potencies between E2-Eq and E1 as measured by YES assays (Rutishauser et al.,
2004; Muller et al, 2008). These previous studies reported relative potencies
between E1 and E2-Eq in the range of 0.35 - 0.7. Hence, the estimated value of
151
0.56 was assumed to be reasonable and was employed as the relative potency
between E1 and E2-Eq in this study.
2000
1800
Measured E2 Equivalent (ng/L)
y = 0.5633x
1600 R² = 0.995
1400
1200
1000
800
600
400
200
0
0 500 1000 1500 2000
E1 concentration (ng/L)
Figure 4-7. Correlation between E1 concentrations and E2 Equivalents as

measured by YES assay.
4.3.8 Modeling Results
The integral least squares method was employed to estimate the
biotransformation rate constants (kb) for E1 and E2 in the aerobic, anoxic and
anaerobic batch tests. Figure 4-8 presents the results of the model fitting for the
measured E2-Eq in each of the reactors dosed with E1 and E2 while Table 4-6
presents the estimated parameter values and model performance evaluators. The
152
model performance evaluators, r2 and NSE were deemed to be within a
satisfactory performance rating range and hence it was concluded that there was
a good fit between the predicted and measured E2-Eq in each of the reactors.
From Table 4-6, it can be seen that the kb values for both E2 and E1, decreased
from the aerobic, to anoxic and anaerobic reactors. The trend in the kb values
among the three redox conditions was consistent with the previously described
observations on the biodegradation efficiencies and removals from the different
redox zones in the BNR processes and batch reactors. Hence, it was concluded
that the biotransformation of E1 and E2 followed pseudo first order kinetics in
the anaerobic, anoxic and aerobic batch tests.
The confidence intervals of the estimated parameter values were
calculated in order to assess the uncertainty in the parameter values. The
estimated kb values along with their 95% confidence intervals are presented in
Table 4-6. The confidence intervals of the estimated parameter values were less
than 14%, 67% and 90% of the estimated values in the aerobic, anoxic and
anaerobic zones respectively. Considering the uncertainty associated with
analyzing estrogens in wastewater, this degree of uncertainty in the model
parameter values was deemed to be acceptable.
153
The biotransformation of E2 through the intermediate metabolite, E1 was
assumed to proceed as depicted in equation 4-4. Table 4-6 shows that the 𝑘1
values in the aerobic and anoxic reactors were approximately an order of
magnitude higher than the 𝑘2 values in the corresponding test conditions. By
contrast, the 𝑘1 and 𝑘2 values in the anaerobic reactor were not statistically
different. Thus, these results suggests that the biotransformation of E1 was the
slower step in the two step reaction occurring in the aerobic and anoxic batch
tests while under anaerobic conditions the rates of biotransformation of E2 to E1
and the biodegradation of E1 were similar.
Table 4-6. Model Performance and biotransformation rate constants with 95%
confidence intervals
Aerobic Anoxic Anaerobic
L.gCOD- L.gCOD- L.gCOD-
kb 1 -1 r2 NSE 1 -1 r2 NSE 1 -1 r2 NSE
d d d
k1 71 ± 1.5 0.901 0.998 31 ± 3.3 0.972 0.998 1 ± 0.9 0.998 0.998
k2 7.3 ± 1.0 0.959 0.998 3 ± 2.0 0.967 0.998 0.85 ± 0.6 0.998 0.998
154
Figure 4-8. Measured and predicted E2-Eq in E1 and E2 dosed batch tests
(aerobic (AO), anoxic (AX) and anaerobic (AN)).
4.3.9 Biotransformation Dynamics between E2 and E1 With

Respect to E2-Eq
The calibrated model was employed to simulate the behaviors of E1 and
E2 in the E2 dosed batch tests. This exercise was undertaken so as to elucidate
the dynamics of the transformation between E2 and E1 in each of the batch
reactors. Figures 4-9, 4-10 and 4-11 depict the results of the simulations of
aerobic, anoxic and anaerobic batch reactors respectively and include the
measured E2-Eq concentrations. It is apparent from the Figures that the
155
measured E2-Eq concentrations in all the reactors were described well by the
simulated E2 + yE1 curves. The simulated E1 values were greater than the E2 +
yE1 and E2-Eq responses in the aerobic and anoxic tests, but lower in the
anaerobic tests. However, the predicted E2 concentrations were consistently
lower than E2 + yE1 and E2-Eq in all three reactors. In the aerobic and anoxic
tests (Figure 4-9 and 4-10), there was a rapid decline in E2 which led to an
accumulation of E1 followed by subsequent degradation of E1. However, this
pattern was different in the anaerobic test due to the much slower rates of
transformation of E2 and degradation of E1 (Figure 4-11). The simulations
provided useful insight into the dynamic responses of E2 and E1 in the batch
tests that could not be directly elucidated from the YES responses.
156
100
E2 E1
80 E2Eq E2 + yE1
60
ug/L
40
20
0
0 10 20 30 40 50 60 70
Time (hr)
Figure 4-9. Simulated and observed estrogen concentration vs time in E2 dosed

aerobic batch tests.
90
80 E2 E1
70 E2Eq E2 + yE1
60
50
ug/L
40
30
20
10
0
0 10 20 30 40 50 60 70
Time (hr)
Figure 4-10. Simulated and measured estrogen concentrations vs time curve in

E2 dosed anoxic batch tests.
157
90
E2 E1
80
E2Eq E2 +yE1
70
60
50
ug/L
40
30
20
10
0
0 10 20 30 40 50 60 70
Time (hr)
Figure 4-11. Simulated and measured estrogen concentrations vs time in E2

dosed anaerobic batch tests.
4.3.10 Model Verification
The biotransformation rate constants estimated from the bench scale
testing were employed in a mass balance model to assess the behavior of E1 and
E2, measured as E2-Eq along the stages of the bench scale BNR bioreactor. The
model verification was conducted using the configuration and operating data
that were employed in the bench scale BNR process, which was the source of the
mixed liquor for the batch tests. Figure 4-12 shows the flows and concentrations
employed to develop the mass balance models of the bench scale BNR process.
158
Figure 4-12. Flow and mass balance schematic for prediction of E2-Eq in BNR
bioreactor.
The steady state mass balances for E2 and E1 in the anaerobic, anoxic and
aerobic reactors are presented in equations 4-10-4-15.
Anaerobic
𝑄𝑆𝐸2,0 − (𝑄 + 𝛼2 𝑄)𝑆𝐸2,1 + 𝛼2 𝑄𝑆𝐸2,2 − 𝑘1,𝑎𝑛 𝑆𝐸2,1 𝑋1 𝑉1 = 0 4-10
𝑄𝑆𝐸1,0 − (𝑄 + 𝛼2 𝑄)𝑆𝐸1,1 + 𝛼2 𝑄𝑆𝐸1,2 + 𝑘1,𝑎𝑛 𝑆𝐸2,1 𝑋1 𝑉1 − 𝑘2,𝑎𝑛 𝑆𝐸1,1 𝑋1 𝑉1 = 0 4-11
Anoxic
(𝑄 + ∝2 𝑄)𝑆𝐸2,1 − (𝑄 +∝3 𝑄 + 𝑄𝑅 )𝑆𝐸2,2 − ∝2 𝑄𝑆𝐸2,2 + ∝3 𝑄𝑆𝐸2,3 + 𝑄𝑅 𝑆𝐸2,3 −
𝑘1,𝑎𝑥 𝑆𝐸2,2 𝑋2 𝑉2 = 0 4-12
159
(𝑄 + ∝2 𝑄)𝑆𝐸1,1 − (𝑄 +∝3 𝑄 + 𝑄𝑅 )𝑆𝐸1,2 − ∝2 𝑄𝑆𝐸1,2 + ∝3 𝑄𝑆𝐸1,3 + 𝑄𝑅 𝑆𝐸1,3 +
𝑘1,𝑎𝑥 𝑆𝐸2,2 𝑋2 𝑉2 − 𝑘2,𝑎𝑥 𝑆𝐸1,2 𝑋2 𝑉2 = 0 4-13
Aerobic
(𝑄 + ∝3 𝑄 + 𝑄𝑅 )𝑆𝐸2,2 − (𝑄 + 𝑄𝑅 −𝑄𝑤 )𝑆𝐸2,3 −∝3 𝑄𝑆𝐸2,3 − 𝑄𝑤 𝑆𝐸2,3 −
𝑘1,𝑜2 𝑆𝐸2,3 𝑋3 𝑉3 = 0 4-14
(𝑄 + ∝3 𝑄 + 𝑄𝑅 )𝑆𝐸1,2 − (𝑄 + 𝑄𝑅 −𝑄𝑤 )𝑆𝐸1,3 −∝3 𝑄𝑆𝐸1,3 − 𝑄𝑤 𝑆𝐸1,3 + 𝑘1,𝑜2 𝑆𝐸2,3 𝑋3 𝑉3 −
𝑘2,𝑜2 𝑆𝐸1,3 𝑋3 𝑉3 = 0 4-15
Where,
𝑄 = influent flow rate
𝑆𝐸1,0 = influent E1 concentration
𝑆𝐸2,0 = influent E2 concentration
∝2 = anoxic recycle ratio with respect to influent flow rate
∝3 = nitrified recycle ratio with respect to influent flow rate
𝑘1,an = anaerobic zone biotransformation rate constant for E2 to E1
𝑘1,ax = anoxic zone biotransformation rate constant for E2 to E1
𝑘1,o2 = aerobic zone biotransformation rate constant for E2 to E1
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𝑘2,an = anaerobic zone biodegradation rate constant for E1
𝑘2,ax = anoxic zone biodegradation rate constant for E1
𝑘2,o2 = aerobic zone biodegradation rate constant for E1
𝑄𝑤 = wasted activated sludge flow rate
𝑄𝑅 = returned activated sludge flow rate
𝑉1 = anaerobic zone volume
𝑉2 = anoxic zone volume
𝑉3 = aerobic zone volume
𝑋1 = anaerobic zone MLVSS concentration
𝑋2 = anoxic zone MLVSS concentration
𝑋3 = aerobic zone MLVSS concentration
𝑦 = relative potency of E1 with respect to E2-Eq,
The equations incorporated the inflow and outflow of both E1 and E2 and
described the degradation of E2 through the intermediate metabolite E1 in each
of the redox zones. The six equations were solved simultaneously, using the
‘Inverse’ and ‘MMult’ matrices functions in Excel, to determine the 𝑆𝐸1 and 𝑆𝐸2 in
each of the redox zones. The relative potency of E1 that was previously estimated
as 0.56 was then employed to convert the predicted E1 concentrations to E2-Eq,
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to determine the E2-Eq in each of the redox zones (𝑆′𝐸2,1 , 𝑆′𝐸2,2 and 𝑆′𝐸2,3 ). Thus,
the predicted E2-Eq along with the measured E2-Eq are presented in Figure 4-13.
Figure 4-13 shows the plot of predicted E2, yE1, E2 +yE1 and the
measured E2-Eq in the BNR bench scale process. It is apparent from the plot that
the predicted E2 +yE1 values reasonably described the measured E2-Eq in the
BNR processes with slight deviation in the anoxic and aerobic zones. Figure 4-13
also shows that both the measured E2-Eq and the predicted E2 +yE1
concentrations decreased along the treatment zones of the BNR bioreactor. The
plot shows that majority of the predicted E2 degraded along the bioreactor
reaching a complete removal by the time the mixed liquor reached the aerobic
zone. In contrast, the E1 remained in the system with a slight decrease in the
anaerobic zone. The simulated behavior of E2 and yE1 in the bench scale BNR
process was consistent with the simulated behavior of E2 and yE1 in the batch
tests. In all, there was a good agreement between the measured E2-Eq and the
predicted E2 + yE1 concentrations along the stages of the bench scale BNR
process. The results were deemed to verify the model formulation describing the
interactions between E2 and E1 and the estimated biotransformation rate
constants reasonably estimated from the batch tests.
162
180
160
140
Concentration (ng/L)
120
100
80
60
40
20
0
E2 yE1 E2 + yE1 Measured E2Eq
Figure 4-13. Measured E2-Eq and predicted E2, yE1 and E2 + yE1 in the influent
and along the bioreactor in bench scale BNR process.
Conclusions
The estrogenicity in two BNR wastewater treatment processes was
measured using a recombinant yeast screen assay. The removal and
biodegradation of EDCs in a pilot BNR process fed with authentic wastewater
was investigated and 96 ± 5% of the estrogenicity exerted by the EDCs in the
wastewater was removed by the treatment process. The degradation efficiencies
in the anaerobic, anoxic and aerobic zones of the bioreactor were calculated as
11± 9%, 18 ± 2% and 93 ± 10% respectively. In order to further understand the

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performance of the BNR process in the removal of EDCs from wastewater, a
bench scale BNR process was operated with synthetic wastewater dosed with E1
and E2. The removal of estrogenicity in the bench scale system (95 ± 5%) was
comparable to the pilot BNR process and the degradation efficiencies were
estimated to be 8± 0.8%, 38 ± 4% and 85 ± 22% in the anaerobic, anoxic and
aerobic zones respectively. Both pilot and bench scale BNR processes showed
that the aerobic zone was key to the biodegradation of EDCs. The biodegradation
of EDCs in the bioreactors of the pilot and bench scale BNR processes followed
the trend of aerobic >anoxic > anaerobic. The results from the pilot and bench
scale BNR processes showed that BNR processes can effectively biotransform
EDCs in wastewater.
Batch experiments were conducted to estimate the biotransformation
kinetics of E1 and E2 in BNR activated sludge under different redox conditions.
The behavior of E1 and E2 in each of the batch tests was consistent with the
observed biodegradation efficiencies in the BNR process that was the source of
the sludge for the batch tests. The biotransformation rate constants for the
transformation of E2 to E1 were estimated as 71 ± 1.5, 31 ± 3.3 and 1 ± 0.9
L.gCOD-1d-1 for the aerobic, anoxic and anaerobic batch tests respectively while
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the biotransformation rate constants for the degradation of E1 were estimated to
be 7.3 ± 1.0, 3 ± 2.0, and 0.85 ± 0.6 L.gCOD-1d-1 for aerobic, anoxic and anaerobic
batch tests respectively. The biotransformation of E1 was the slower step in the
two step reaction occurring in the aerobic and anoxic batch tests while under
anaerobic conditions there was a balance between the biotransformation of E2 to
E1 and the biodegradation of E1.
A comparison between the simulated E2 + yE1 values and measured E2-
Eq along the bioreactor of the bench scale BNR process revealed that the
calibrated model parameters effectively predicted the steady state E2-Eq along
the bioreactor of the BNR process, which was the source of the sludge for the
batch tests. Hence, it was concluded that the calibrated model can be employed
to predict the E2-Eq concentrations in BNR treatment processes.
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Chapter 5 Biotransformation of Trimethoprim in Biological
Nutrient Removal Treatment System – The Role of Active
Microbial Groups
An abridged version of this chapter was submitted to the journal of Environmental
Science and Technology in manuscript form under the authorship of Ogunlaja O. O and
Parker W. J.
5.1 Introduction
The removal of trace organic compounds (TrOCs) in wastewater
treatment plants (WWTPs) has been an area of research for the past decade due
to the potential risks associated with the undesirable effects of TrOCs on the
ecosystem and human health (Heberer, 2002, Jones et al., 2004; Fent et al., 2006).
Many of these compounds are either recalcitrant or partially removed during
sewage treatment because the WWTPs were not designed to remove them
(Ternes et al., 2003; Joss et al., 2004; Clara et al., 2005a). Thus, effluents from
WWTPs have been identified as a primary source of TrOCs in the environment
(Kolpin et al., 2002; Ternes et al., 2004; Lishman et al., 2006). Consequently, the
optimization of WWTPs could play a critical role in attenuating the discharge of
TrOCs into the environment (Onesios et al., 2009; Pomies et al., 2013).
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WWTPs that are configured for biological nutrient removal (BNR) have
been reported to have higher removal efficiencies of TrOCs along with
macropollutants when compared to conventional activated sludge treatment
systems (Clara et al., 2005a, Kimura et al., 2007, Ogunlaja et al., 2013). These
previous studies have investigated the effects of WWTP design and operating
conditions on the removal of TrOCs from wastewater. Some of the parameters
that have been investigated included hydraulic retention time (HRT), solids
retention time (SRT), biomass type, type and presence of growth substrate,
temperature, pH, structure and physico-chemical properties of the TrOCs (Clara
et al., 2005a; Cirja et al., 2007; Helbling et al., 2012). Despite all these studies, it is
still unclear why BNR systems are more effective in removing TrOCs when
compared to other conventional activated sludge systems.
Biotransformation or biodegradation has been identified as a major
removal mechanism for attenuating the discharge of TrOCs. One of the plausible
biotransformation pathways for TrOCs in activated sludge systems is the
degradation of TrOCs by specialized microbial groups within the mixed liquor.
A few studies have attempted to identify these organisms in order to elucidate
the roles they play in the biotransformation of TrOCs (Shi et al., 2004; Batt et al.,
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2006; Khunjar et al., 2011). However, there is no agreement in the literature on
the type of active biomass (ammonia-oxidizing bacteria (AOB) and heterotrophic
organisms) that is responsible for the biodegradation of TrOCs in activated
sludge systems (Batt et al., 2006; Khunjar et al., 2011). Hence, further study is
required to investigate the role of active biomass groups in the biotransformation
of TrOCs.
Trimethoprim, one of the prevalent TrOCs found in wastewater, is a
hydrophilic synthetic antibiotic usually prescribed for treatment of chest or
urinary tract infections (Nolan et al., 1989; Batt et al., 2006). TMP is prevalent in
the effluent of wastewater treatment plants (Kolpin et al., 2002) due to its poor
biodegradability (Kummerer et al., 2000) and hydrophilic nature (Aga, 2008).
Trimethoprim concentrations as high as 0.53 µg/L has been reported in the
effluents of wastewater treatment plant in the United (Batt et al., 2005;
Glassmeyer et al., 2005). There are limited reports on the removal and
biodegradation of TMP in BNR treatment processes, hence the investigation of
the removal and biotransformation of TMP in a BNR activated sludge systems
was the focus of this paper.
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Models of TrOC fate can be usefully employed to analyze test data for the
purpose of obtaining an improved understanding of the processes involved in
TrOC removal. Biotransformation modeling of TrOCs in activated sludge
systems has usually been described by either first order (Byrns, 2001; Hashimoto
and Murakami, 2009) or pseudo first order kinetics (Maurer et al., 2007; Wick et
al., 2009). These kinetic expressions include one kinetic parameter, the dissolved
TrOC concentration and the biomass concentration. In previous biodegradation
studies, the biomass concentration has been approximated by the total or volatile
suspended solid concentration (Joss et al., 2005; Gaulke et al., 2009). A weakness
of this approach is that it does not consider the influence of growth substrates on
the biotransformation kinetics of the TrOCs. TrOCs are present in minute
concentrations in activated sludge systems and cannot sustain microbial growth.
Usually, co-substrates like soluble organics or ammonia are necessary to serve as
growth substrates (Pomies et al., 2013) for the active biomass that is presumably
responsible for the biotransformation of TrOCs. Hence, the integration of active
biomass fractions in biotransformation models may provide an improved
description of the biotransformation of TrOCs in wastewater.
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The objectives of this study were to 1) estimate the fractions of the active
biomass- (polyphosphate accumulating organism (PAO), ordinary heterotrophic
organism (OHO) and ammonia oxidizing bacteria (AOB)) in a BNR activated
sludge process, 2) investigate the removal of trimethoprim (TMP) in the BNR
process, 3) estimate the biotransformation rate constants of TMP with respect to
PAO, OHO and AOB in aerobic BNR activated sludge and 4) assess the
contributions of PAO, OHO and AOB towards the removal of TMP in aerobic
BNR activated sludge. The results of this study will improve the understanding
of the contributions of different biomass groups to TMP biotransformation,
developing a comprehensive modeling tool that can facilitate the quantification
of TMP removal in advanced wastewater treatment processes.
5.2 Approach
A BNR pilot plant was operated to investigate its performance with
respect to the removal of conventional pollutants and TMP. The conventional
pollutant data obtained from the BNR process operation were employed to
estimate the active biomass fractions in the BNR activated sludge. The mixed
liquor in the BNR pilot plant was employed as the source of the inoculum for
batch tests that investigated the biotransformation of TMP in aerobic batch tests.
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The estimated active biomass fractions and the batch experiment data were then
employed to calibrate developed pseudo first order model equations with
respect to each of the active biomass fractions. The TMP removal rates obtained
from the batch tests were then used to determine the contributions of each of the
active biomass fractions towards the overall TMP removal in the aerobic batch
test. Figure 5-1 summarizes the approach taken for the study.
Figure 5-1. Overview of study approach.
5.2.1 BNR Pilot Plant Description
The pilot scale UCT-BNR process consisted of a 0.718 m3 primary clarifier
tank, 0.025 m3 chemical dosing tank, 0.36 m3 bioreactor made of stainless steel,
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0.286 m3 final clarifier for solid–liquid separation and associated pumps for fluid
transfer. A schematic of the pilot BNR process is shown in Figure 5-2. A UCT-
BNR process was selected due to its ability to maintain a PAO population at low
influent carbon loading. The pilot BNR was operated on authentic municipal
wastewater that was augmented with sodium bicarbonate (22 g/L) to provide
alkalinity, di-potassium phosphate (11 g/L) as phosphorus source and sodium
acetate (34.36 g/L) to enhance the proliferation of PAOs. The bioreactor was
partitioned into six 0.06 m3 cells to simulate pseudo plug flow. The first cell was
operated as an anaerobic zone, the next two cells operated as anoxic zones and
the last three cells operated as aerobic zones. The aerobic section of the bioreactor
was mixed and aerated using coarse bubble aerators while the non-aerated
sections were mixed using mechanical mixers. The solids residence time (SRT)
was maintained by wasting mixed liquor from the last aerobic section of the
bioreactor. The operating and design conditions of the pilot BNR process are
summarized in Table 5-1. The temperature of the bioreactor was maintained at 18
± 2oC using a temperature controlled insulated water jacket that was wrapped
around the bioreactor while the pH was maintained within the range of 7.2-8.5.
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Figure 5-2. Flow schematic of pilot UCT-BNR activated sludge treatment
system.
Table 5-1. Pilot BNR operating and design conditions

Size/description Unit
Flow rate 1.3 m3/d
Primary clarifier Area = 0.46 m2
Depth = 1.56 m
Bioreactor Volume = 0.36 m3
Depth = 1.28 m
DO(aerobic) = 4 - 5 g/ m3
DO(anoxic) = 1-2.5 g/ m3
DO(anaerobic) = 0-0.2 g/m3
Final clarifier Area = 0.204 m2
Depth = 1.4 m
Recycle rate Aerobic = 2.6 m3/d
Anoxic = 1.3 m3/d
SRT 20 d
Aerobic SRT 10 d
RAS flow rate 0.9 m3/d
Waste rate 0.018 m3/d
HRT 7 Hrs.
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RAS-return activated sludge,SRT-solid residence time,HRT-hydraulic retension time
5.2.2 Pilot Monitoring and Sampling
The pilot plant was operated for over 12 months with regular monitoring
of conventional and operational parameters for 6 months. Stable plant
performance was ascertained by monitoring process operating conditions that
included, chemical oxygen demand (COD), Total Kjeldahl Nitrogen (TKN),
ammonia (NH3), nitrite (NO2), nitrate (NO3), phosphate (PO4), total suspended
solid (TSS) and volatile suspended solid (VSS). Twenty four hour composite
influent and effluent samples were collected thrice a week for three weeks (
except for the first week with 2 sampling campaigns) for TMP analysis. Eight
hour composite samples were also collected for TMP analysis from the anaerobic,
anoxic and aerobic zones of the bioreactor. For TMP analysis, the samples were
filtered using 1.5 µm glass micro fibre filters (GF/F Whatman), and extracted
using solid phase extraction.
5.2.3 Estimation of Active Biomass Fractions
The active biomass fractions in the BNR bioreactor were estimated by
simulating the biomass growth in the bioreactor based on growth substrate
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uptake as presented in detail elsewhere (Barker and Dold, 1997). The simulation
exercise was conducted using the activated sludge model no. 2d (ASM2d) within
the wastewater treatment modeling software BioWin 3.0 from Envirosim
(Hamilton, Ontario). Average influent wastewater parameters (Table 5-2) and the
pilot’s design and operating conditions in Table 5-1 were input into the simulator
prior to simulating steady state performance of the pilot BNR process. The
readily biodegradable fraction of the influent COD (fbs) was adjusted to reflect
the sodium acetate that was added to the influent stream. The readily
biodegradable fraction of the influent COD has been reported to affect the
biological phosphorus removal capability of BNR systems (Comeau et al., 1996;
Barnard and Steichen, 2006). Simulated and observed soluble effluent COD and
TKN were matched by adjusting the soluble unbiodegradable fractions of the
influent COD (fsu) and influent TKN (fSNI). Simulated and observed effluent solid
concentrations were matched by adjusting the final clarifier solids removal
efficiency. The simulated and observed bioreactor mixed liquor volatile
suspended solid concentrations were matched by adjusting the particulate
unbiodegradable fraction of influent COD (fup). Aside from these calibrations, the
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default values were used for the kinetic and stoichiometric parameters (Barker
and Dold, 1997).
Table 5-2. Average influent parameter values for BNR pilot plant simulation
Parameter Value Unit
Flow rate 1.3 ± 0.4 m3/d
COD 367 ± 48 gCOD/ m3
TKN 25 ±11 gN/ m3
TP 11 ± 6 gP/ m3
NO3-N 0.54 ± 0.2 gN/ m3
NO2-N 0.06 ± 0.03 gN/ m3
ISS 16 ± 10 gISS/ m3
pH 7.2 ± 0.2
Ca 169 ± 9 g/ m3
Mg 15 ± 4 g/ m3
Alkalinity 268 ± 21 g/ m3 as CaCO3
5.2.4 Batch Experiments
A series of batch tests were performed to investigate the
biotransformation of TMP in BNR activated sludge and to assess the
contributions of active biomass, PAO, OHO and AOB to the biotransformation of
TMP. The batch tests were designed using a combination of experimental
conditions to facilitate the growth of different biomass groups in the different
tests. Duplicate aerobic batch tests were conducted to asssess the role of PAO,
OHO and AOB on the biotransformation of TMP (Aerobic-1) while a third test
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(Aerobic-2) was conducted to assess the role of only PAO and OHO in the
biotransformation of TMP. A summary of the experimental conditions under
which the batch tests were performed is shown in Table 5-3.
The innocula for the batch tests were collected from the aerobic section of
the pilot BNR process. The batch reactors (Figure 5-3) had a working volume of
10 L that was filled with 6 L of activated diluted with 4 L of settled raw
wastwater to achieve a mixed liquor volatile suspended solid (MLVSS)
concentration of approximately 2050 mg/L. An aqueous stock solution of
ammonium chloride, di-potassium phosphate and sodium acetate was dosed
into the reactors at the same time as the TMP that was dissolved in methanol.
The initial TMP concentration in the batch reactors ranged from 0.2 to 1 µg/L.
The initial concentrations of the chemicals that were added to facilitate biomass
growth in the batch tests are shown in Table 5-3. The mixed liquor in the reactors
was mechanically stirred and aerated to maintain dissolved oxygen (DO)
concentrations of approximately 2 – 5 g/m3. The temperature of the reactors were
maintained at 18 ± 2oC and the pH was maintained in the ranged of 7.5 - 8.4.
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Figure 5-3. Schematic diagram of batch test.
Table 5-3. Initial concentrations of chemicals in batch reactors

Molecular AO- AO- AO-
Chemical added formula 1A 1B 2 Unit
Sodium acetate C2H3NaO2 COD 600 503 535 g COD/m3
Di-potassium phosphate K2HPO4 PO4-P 162 193 188 g P/m3
Ammonium Chloride NH4Cl NH3-N 114 98 n/a g N/m3
Allylthiourea C4H8N2S ATU N/A N/A 13 g/m3
OHO,
Target Active Biomass OHO,PAO,AOB PAO
n/a-not added to reactor; AO-Aerobic, Allylthiourea - Nitrification inhibitor
5.2.5 Batch Experiment Monitoring and Sampling
The time dependent changes in the concentration of TMP and
conventional parameters in the effluent samples were characterized to assess the
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reactors’ performance. From each batch reactor, 500 mL mixed liquor samples
were collected in prewashed amber bottles, just before and 15 minutes after the
addition of TMP to the reactors and subsequently every four hours for three
days. Each sample was centrifuged at 4000 rpm for 5 minutes and the centrate
was filtered using 1.5 µm glass fiber filter paper (Whatman 934/AH). A volume
of 60 ml of the filtrate was employed for analysis of COD, nitrogen species (NH4-
N, NO3-N, and NO2-N), soluble reactive phosphorus (PO4-P) and the unfiltered
samples were analyzed for TSS and VSS concentrations according to Standard
Methods (Eaton, 2005). A volume of 150 mL of the remaining filtered sample was
placed in a prewashed amber bottle and pH adjusted to pH 8 using 0.1M sodium
hydroxide. The samples were then spiked with 0.15 mL of a 0.5 ppm isotopically
labelled trimethoprim, dissolved in methanol, before solid phase extraction
(SPE). All parameters were analyzed in duplicate. The analysis of the
conventional parameters and SPE extraction were performed immediately after
filtering.
5.2.6 Biotransformation Model
A pseudo first order kinetic expression is commonly used to describe the
biodegradation of TrOCs in activated sludge systems (Cowan et al., 1993;
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Monteith et al., 1995; Schwarzenbach et al., 2003). Therefore, pseudo first order
kinetic expressions were used to estimate the biotransformation rate constants of
TMP in the batch experiments. Two different models were employed to
investigate the use of MLVSS concentration and estimated active biomass
concentration in determining the biotransformation rate constant of TMP in BNR
activated sludge. Both models assumed that the MLVSS and the active biomass
concentrations were constant throughout the duration of the batch tests. This
assumption is typically valid for short duration batch tests. Model 1 did not
differentiate between biomass species and hence the rate of biotransformation of
TMP was assumed to be a function of the MLVSS concentration in the reactors
(equation 5-1)
𝑟𝑖 = −𝑘𝑖 𝑋𝑚𝑙𝑣𝑠𝑠 𝑆𝑖 5-1
Where,
𝑟𝑖 = rate of biotransformation of compound i [µg L-1hr-1]
𝑘𝑖 = biotransformation rate constant for compound i [L gCOD-1hr-1]
𝑋𝑚𝑙𝑣𝑠𝑠 = mixed liquor volatile suspended solid concentration [gCOD L-1]
𝑆𝑖 =soluble concentration of compound i [µg.L-1]
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By contrast, model 2 incorporated three biotransformation rates where the rate of
biotransformation of TMP was assumed to be dependent on each of the active
biomass concentrations (equation 5-2).
𝑟𝑖 = − ∑𝑛=3
𝑗=1 𝑘𝑖,𝑗 𝑋𝑗 𝑆𝑖 5-2
Where,
𝑘𝑖,𝑗 = biotransformation constant for compound i [L gCOD-1hr-1] with respect to
biomass j
𝑋𝑗 = active biomass concentration [gCOD L-1]
𝑆𝑖 =soluble concentration of compound i [µg.L-1]

𝐽 = 𝑃𝐴𝑂, 𝑂𝐻𝑂, 𝐴𝑂𝐵
5.2.7 Chemical Analysis
The filtered samples collected from the pilot BNR and the batch
experiments were extracted for TMP analysis by solid phase extraction (SPE) (Li
et al., 2010) using Oasis HLB 60 mg cartridge from Waters. Prior to extraction, 0.1
mL of a mixture of an internal standard (500 ng/mL) containing the stable isotope
labeled surrogates of the analyte was added to the sample. The eluates were
evaporated to dryness under a gentle stream of nitrogen and reconstituted in 0.4
ml of methanol. An internal standard (Trimethoprim- 13C6 ) that was prepared by
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Prof. Chris Metcalfe’s lab in Trent Univerity, was added to the reconstituted
samples prior to instrumental analysis to improve the quantitative analysis. The
extracted samples were shipped on ice to be quantified at Trent University. The
TMP concentrations were measured using high performance liquid
chromatography interfaced to a tandem mass spectrometer with an electrospray
ionization source using an API 3000 instrument procured from AB SCIEX
(Concord Ontario Canada). A detailed description of the sample preparation and
LC-MS/MS procedure that was employed to quantify the TMP was described
elsewhere (Hoque et al., 2013). The limit of quantification and limit of detection
were calculated to be between 0.1 to 2 ng/L.
The conventional and chemical data were analyzed for outliers using the
Grubb’s test. The significant outliers (p > 0.05) were removed before conducting
descriptive statistics on the data. The averaged conventional data were
introduced into BioWin for the simulation exercise. The fit between the
simulated and measured TMP concentrations in the batch experiments was
assessed on the basis of r 2 values and the Nash-Sutcliffe Efficiency (NSE) metric
(Nash and Sutcliffe, 1970) (equation 5-3). NSE values ranges from–∞ to 1 but are
182
usually greater than 0. A negative value of NSE indicates that the mean of the
measured value is a better predictor than the model. Typically, NSE values
greater than 0.7 are considered to indicate a strong predictive characteristic of the
model (Moriasi et al., 2007).
𝜎2
𝑁𝑆𝐸 = (1 − 𝑒2) 5-3
𝜎 𝑜
Where, 𝜎𝑒 = variance of residuals
𝜎𝑒 = variance of observed values
The pilot BNR process was monitored with respect to the removal of
conventional wastewater pollutants. This data was used to ascertain the
performance of the BNR treatment plant in terms of conventional contaminants
removal and to provide an indication of the metabolisms of the different
microbial groups that were active in the bioreactor. The ASM2d model was used
to estimate the fractions of active biomass in the bioreactor on the basis of the
conventional data. A series of batch experiments was conducted to investigate
the biotransformation of TMP in BNR activated sludge. The biotransformation
constants were estimated using pseudo first order kinetic expressions. The
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estimated biotransformation constants were then used to calculate the
contributions of the active biomass fractions towards the biotransformation of
TMP.
5.3.1 Conventional Contaminants Removal
The measured responses of the conventional contaminants as presented in
Table 5-4 shows that the effluent from the pilot plant was relatively consistent
throughout the sampling campaign. This showed the efficient performance of the
pilot plant irrespective of the variability in the influent contaminants.
Carbonaceous (cBOD5) was consistently removed in the pilot with the effluent
concentration ranging from 2 to 9 mg/L. This result suggests a good removal of
biodegradable organic matter. In addition, the pilot was expected to have high
removal efficiencies for TKN, TAN, NO2-N and TP. These patterns were
consistently observed throughout the sampling period as shown in Table 5-4.
This suggests that the pilot plant was effectively nitrifying, denitrifying and
biologically removing phosphorus. Hence, it was concluded that the BNR pilot
plant was achieving levels of treatment that are typical of operations at technical
scale (Jeyanayagam, 2005; WEF, 2005).
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5.3.2 Pilot Plant Calibration and Active Biomass Estimation
The steady state effluent concentrations of the conventional pollutants and
the MLVSS concentration in the bioreactor were simulated using the BioWin
modeling software. To simulate the measured effluent and MLVSS
concentrations of the pilot BNR, the influent wastewater fractions were adjusted
to match the measured data. During the plant operation, the pilot plant influent
was augmented with sodium acetate to ensure the proliferation of PAOs for
biological phosphorus removal. Hence, the readily biodegradable fraction of the
total influent COD (0.55 g COD/ g of total COD) was adjusted in the simulator to
reflect the sodium acetate that was added to the influent stream. The simulated
and observed soluble effluent COD and TKN were then matched by adjusting
the soluble unbiodegradable fractions of the influent COD (0.04 gCOD/ g of total
COD) and influent TKN (0.035 gN/g TKN) in the simulator. The solids capture
rate in the final clarifier was adjusted to match the observed effluent suspended
solid concentration. The simulated and observed mixed liquor volatile
suspended solid (MLVSS) concentrations in the aerobic bioreactor were matched
by adjusting the particulate unbiodegradable fraction of influent COD (0.043 g
COD/ g of total COD). The measured and calibrated MLVSS concentrations in
185
the aerobic section of the bioreactor were 3600 ± 474 g COD/m3 and 3600 g
COD/m3 respectively. The simulated concentrations for the active biomass
groups- AOB, OHO and PAO in the aerobic section of the pilot BNR bioreactor
were 40, 780 and 2710 g COD/m3. The relative error between the measured and
steady state simulated values were below 20% which was deemed to signify
good agreement between the measured and simulated data (Table 5-4).
Table 5-4. Measured and predicted effluent concentrations (g/m3) from the
pilot BNR process
Response Primary Effluent Measured Calibrated Removal
(Influent) Effluent Effluent efficiency
Mean S.dev Mean S.dev %
COD 367 48 33 12 33 91
TKN 25 12 2.3 0.2 2.3 91
NH4-N 19 8 0.04 0.03 0.04 100
NO3-N 0.54 0.2 3 2 3 n/a
NO2-N 0.06 0.03 0.03 0.01 0.03 50
TP 11 7 3 1.7 3 73
TSS 32 2.5 9 1.6 10 72
n/a-not applicable
5.3.3 TMP Removal in BNR Pilot Plant
The concentrations of TMP in the pilot were measured to investigate the
performance of the pilot in terms of TMP removal. The influent TMP
concentration (n=8) was 78 ± 39 ng/L while the effluent TMP concentration (n=8)
186
was 28 ± 8 ng/L, hence the overall TMP removal efficiency was calculated as 59 ±
14%. The TMP removal efficiency obtained in this study was similar to a prior
study that investigated the removal of TMP in an MBR system (57 ±10 % )
(Radjenovic et al., 2009) but on average, 3-6 times higher than that reported for
conventional activated sludge systems (11 ± 31%) (Gobel et al., 2007; Radjenovic
et al., 2009; Ogunlaja et al., 2013). Thus, it was concluded that the performance of
the pilot BNR process in terms of TMP removal was comparable to the
performance of previously investigated advanced wastewater treatment
processes.
The bioreactor of the BNR process was divided into three different zones
operated at different redox conditions. Therefore, it was expected that the
different zones would contribute at different proportions towards the
biotransformation of TMP. This expectation was based on the fact that the
amount of energy that is usually captured by the microorganisms in aerobic
conditions is usually higher than the energy captured in anoxic and anaerobic
conditions (Tchobanoglous et al., 2003). This bioenergy could be instrumental to
the biotransformation of TMP in the BNR bioreactor. The TMP concentrations in
the influent, effluent and the interstage of the BNR bioreactor are presented in
187
Figure 5-4. It is apparent from Figure 5-4 that the TMP concentration decreased
along the interstage of the bioreactor, which suggested that each of the zones
contributed to the overall TMP removal.
However, during the operation of the pilot BNR process, mixed liquors
were recycled from the aerobic zone to the anoxic zone and from the anoxic zone
to the anaerobic zone. The returned activated sludge was also recycled from the
final clarifier to the anoxic zone of the bioreactor. These recycle flows can have
dilution effects on the pilot plant influent at different points of the treatment
system. Therefore, a set of mass balances were employed to characterize the fate
of TMP in the pilot BNR process (Figure 5-5).
In the mass balances, the biotransformation efficiency of TMP in each zone
was calculated as the difference between the mass flow entering and leaving the
zone, divided by the mass flow entering the zone. The difference between the
mass inflow and outflow in the aqueous phase across each zone of the bioreactor
was assumed to be due to microbial biotransformation within the zone of the
bioreactor. This assumption was based on the fact that previous studies had
shown negligible TMP removal by sorption in activated sludge systems
(Eichhorn et al., 2005; Perez et al., 2005; Batt et al., 2007; Aga, 2008). Therefore, the
188
TMP biotransformation efficiencies in the anaerobic, anoxic and aerobic sections
of the BNR bioreactor were calculated to be 13 ± 12%, 17 ± 10 % and 24 ± 4%
respectively. These results show that the biotransformation efficiency in the
bioreactor of the BNR pilot increased from the anaerobic to the anoxic and
aerobic zone. Hence, it was concluded that TMP was biotransformed in all the
redox zones of the BNR bioreactor, albeit at different percentages.
The TMP removals by each of the stages of the BNR process were
estimated to compare the contributions of each stage towards the overall TMP
removal in the BNR process. The contributions of the stages were calculated to be
22 ± 28%, 29 ± 24% and 41 ±10% for anaerobic, anoxic, aerobic zones respectively.
As expected, the results shows that the aerobic zone of the BNR process
contributed the highest removal, followed by the anoxic zone and lastly the
anaerobic zone. Hence it was concluded that the TMP removal in the BNR
process was related to the oxidation-reduction condition in each of the zones.
The energy that is captured by the microorganisms in aerobic conditions is
usually higher than the energy captured in anoxic conditions and even higher
than the energy captured in anaerobic conditions (Tchobanoglous et al, 2003).
This difference in bioenergy could explain why the aerobic zone had the highest
189
biodegradation efficiency among the three stages for the pilot BNR process. In
addition, aerobic heterotrophic organisms have the capability to produce
oxygenase enzymes that catalyze the direct incorporation of molecular oxygen
into organic compounds. This oxygenase reaction weakens the ring structure in
the TMP compounds that can make it accessible for subsequent oxidation steps
and more water soluble (Rittman and McCarty, 2001). Thus, the high
biotransformation efficiency of TMP in the aerobic zone could have been due to
the combination of the aerobic environment and the bacteria activity on the
compound. Hence, the aerobic zones as compared to the anoxic and aerobic
zones has the potential for significant biotransformation and removal of TMP in
a BNR process. However, further study is recommended to investigate methods
of improving the removal of TMP in anoxic and anaerobic environments.
190
140
120
TMP Concentration (ng/L)
100
80
60
40
20
0
Figure 5-4. Trimethoprim concentrations along BNR pilot plant. (deviation bar
represents standard deviation of measurements (n=8)).
Figure 5-5. Mass balances of Trimethoprim in BNR pilot plant (TMP mass
flow rate in µg/d).
191
5.3.4 Performance of Batch Reactors with respect to Conventional
Pollutants
Batch tests were conducted to obtain an improved understanding of the
kinetics associated with the removals that were observed in the BNR pilot plant.
The performances of the batch tests were initially ascertained by investigating
the behavior of the conventional pollutants through the duration of the
experiments. Figures 5-6 and 5-7 show the profiles of the conventional pollutants
in the batch tests conducted under aerobic conditions without nitrification
inhibition (Aerobic-1) and with nitrification inhibition (Aerobic-2) respectively. It
is apparent from Figure 5-6 that the AOB activity were active in Aerobic-1 batch
test as evidenced by the decrease in ammonia that occurred simultaneously with
an increase in nitrate. By contrast, there was no change in ammonia or nitrate
concentration throughout the duration of the experiment in Aerobic-2 batch
reactor, which indicated the inhibition of AOB activity by the added allylthiourea
(Figure 5-7).
Under aerobic conditions, PAOs oxidize intracellular poly-β-
hydroxybutyrates (PHBs) to obtain energy for growth and maintenance
requirements. The intracellular PHBs support PAO growth and soluble
phosphate (PO4) uptake from the bulk liquid in the reactor. The accumulated
192
polyphosphates provide the energy required for soluble VFA uptake in the
anaerobic zone. Figures 5-6 and 5-7 show a decline in the PO4 concentrations that
ceased after approximately 20-24 hours. The reduction in the PO4 concentration
in the reactors signified that the PAOs were actively taking up PO4 to form
intracellular polyphosphates. The cessation of PO4 uptake may have been due to
the depletion of the intracellular PHBs of the PAO. In the absence of PHBs, the
PAOs have no source of energy to carry out their metabolic activities under
aerobic conditions. Hence, it was concluded that the PAOs in aerobic 1 and 2
batch tests were active during the 20-24 hours of the tests and became inactive
for the rest of the duration of the batch tests.
Under aerobic conditions, ordinary heterotrophic organisms (OHO)
oxidize COD for cellular growth and maintenance. Figures 5-6 and 5-7 showed a
decline in the soluble COD concentrations, which effectively ceased after
approximately 20-24 hours. After the soluble COD concentration in aerobic 1 and
2 reached a minimum, the concentrations tended to gradually increase till the
end of the reaction in both aerobic 1 and 2 batch tests. During biomass growth,
decay and lysis also occur concurrently. The cell decay and lysis released soluble
substrates and particulate substrates into the bulk liquid in the reactor. The
193
particulate substrates can undergo hydrolysis to produce more soluble substrate
for cellular consumption (Grady et al., 1999). These results suggests that the rate
at which OHOs oxidized the soluble COD for growth was initially faster than cell
death and lysis at the beginning of the batch tests but after approximately 20-24
hours of reaction, the rate of COD consumption became slower than the rate of
release of soluble COD into the reactor by biomass death and lysis. This was
demonstrated by the slightly increasing COD concentration in both batch tests.
Hence, it was concluded that after 20-24 hours of reaction in the batch tests, the
biomass in the reactors entered a stationary phase as depicted by the constant
soluble COD concentrations.
180 700
160 PO4-P NH3-N
COD concentration (mgCOD/L)

600
NH4-N, NO3-N, NO2-N,PO4-P
140 NO3-N NO2-N

concentration (mg/L)
COD 500
120
100 400
80 300
60
200
40
20 100
0 0
0 10 20 30 40 50 60 70 80
Time(hrs)
Figure 5-6. Profile of conventional pollutant concentrations in batch

experiment without AOB inhibition (Aerobic-1).
194
200 600
180 PO4-P
NH3-N, NO3-N, PO4-P Concentration
500
COD Concentration (mgCOD/L)

160 NH3-N
NO3-N
140
COD 400
120
(mg/L)
100 300
80
200
60
40
100
20
0 0
0 20 40 60 80
Time (hrs)
Figure 5-7. Profile of conventional pollutant concentrations in batch
experiment with AOB inhibition (Aerobic-2).
5.3.5 Performance of Batch Reactors with respect to Trimethoprim

Removal
The TMP concentrations in the water phase were plotted against time
(Figure 5-8) so as to investigate the effect of AOB inhibition on TMP removal in
the batch reactors. Figure 5-8 shows that the TMP concentrations in the batch
experiments with and without AOB inhibition followed similar decreasing
trends. It is apparent from the plot that the biotransformation of TMP was faster
in aerobic-1 batch tests than in aerobic-2. In Figure 5-8, It can also be observed
195
that the result of the duplicate aerobic-1 batch tests were consistent with each
other, thereby implying good reproducibility of the results.
The previous analysis of the conventional parameters had shown that
PAO, OHO and AOB were active in aerobic-1 while PAO and OHO were active
in aerobic-2 batch tests. The difference in trends between aerobic-1 and aerobic-2
clearly suggests the impact of the AOB activity on the biotransformation of TMP.
The slower decline in TMP remaining in the aerobic reactor with nitrification
inhibition as compared to the batch reactor without nitrification inhibition
suggest that AOB plays a role in the biotransformation of TMP. Previous studies
have suggested the capability of AMO that is produced by AOBs to oxidize a
broad range of organic compounds along with the oxidation of ammonia (Batt et
al., 2006; Khunjar et al., 2011). The results obtained from the current batch tests
suggest that AOBs play a role in the biotransformation of TMP in the BNR
activated sludge. Hence, the incorporation of the concentrations of AOB in a
biotransformation model could provide a better description of the behavior of
micropollutants in activated sludge systems.
196
0.7
0.6
Aerobic-1A Aerobic -1B Aerobic-2
0.5
C (ug/L)
0.4
0.3
0.2
0.1
0
0 10 20 30 40 50 60 70 80
Time(h)
Figure 5-8. TMP Concentrations in the batch reactors: Aerobic 1A&B- without
AOB inhibition, Aerobic 2- with AOB inhibition.
5.3.6 Estimation of Biotransformation Rate Constants
The biotransformation of TMP in the batch tests was examined using two
pseudo first order kinetic models. In the application of the models, it was
assumed that the mixed liquor volatile suspended solids concentration was
constant in model 1 and that the estimated active biomass concentrations were
constant in model 2. For short duration batch tests, it was considered reasonable
to assume that the biomass concentration is constant. Hence, a non-linear integral
least square method (Chapra, 1997) that minimized the sum of squares of the
197
residuals between the predicted and measured TMP data was employed to
simultaneously fit the models to the TMP responses from the batch tests with
and without nitrification inhibition.
Figures 5-9 and 5-10 show plots of the measured and predicted
concentrations of TMP in the aerobic-1 and 2 batch tests respectively. The
goodness of fit of the two models were estimated using r2 and NSE. The results
(Table 5-5) show that the r2 and NSE values for both models 1 and 2 were larger
than 0.98 for the tests, indicating that both models could represent the behavior
of TMP in the batch tests. However, the higher residuals between the measured
TMP concentrations and model 1 predicted TMP concentrations (Figure 5-10) as
compared to model 2 predicted TMP concentrations indicated that model 2 was a
better fit than model 1 for the aerobic-2 batch test. Hence, it was concluded that
model 2 was able to better describe the behavior of TMP in the batch tests. An
additional feature of this model is the ability to estimate the contribution of each
active biomass group to the overall TMP removal in the BNR activated sludge.
The estimated biotransformation rate constants for TMP in model 1 and
model 2 are summarized in Table 5-5. From Table 5-5, it can be seen that the
model 2 biotransformation rate constants were inversely proportional to the
198
active biomass fractions in the BNR mixed liquor employed for the batch test. For
example, the AOB had the smallest proportion of the biomass concentration but
the highest rate constant while PAO had the highest proportion of the biomass
concentration but the lowest rate constant. The ratio of the estimated
biotransformation rate constants with respect to PAO, OHO and AOB was 1: 2:
48. The estimated biotransformation rate constant from model 1 was consistent
with a previous study that investigated the biotransformation of TMP under
aerobic condition using sludge from A2O-MBR process, two-phase fate model
and MLSS as the biomass concentration (Xue et al., 2010). However, the current
study was the first to estimate the biotransformation rate constant for TMP with
respect to active biomass groups in a BNR activated sludge, hence there was no
previous study for comparison.
199
0.8
0.7
0.6
0.5
C (ug/L)
Model-1
0.4
Model-2
0.3
Measured-A
0.2
Measured-B
0.1
0.0
0 10 20 30 40 50 60 70 80
Time (h)
Figure 5-9. Simulated and measured Trimethoprim concentrations in aerobic

batch reactors without AOB inhibition.
0.30
0.25
0.20
C (ug/L)
0.15 Model-1
Model-2
0.10
Measured
0.05
0.00
0 10 20 30 40 50 60 70 80
Time (h)
Figure 5-10. Simulated and measured Trimethoprim concentrations in aerobic

batch reactors with AOB inhibition.
200
Table 5-5. Estimated biotransformation rate constant for TMP with respect to
PAO, OHO and AOB
Model-1 Model-2
Aerobic-1 Aerobic-2 Aerobic-1 Aerobic-2
2
r 0.986 0.924 0.986 0.987
NSE 0.97 0.966 0.971 0.985

PAO OHO AOB
kb 0.58 ± 0.06 0.32 ± 0.06 0.58 ± 0.06 13.7 ± 0.06 L/gCOD/d
5.3.7 Contribution of Active Biomass Groups towards

Trimethoprim Biotransformation
To obtain an improved understanding of TMP removal rates by each
active biomass, the estimated biotransformation rate constants were employed in
model 2 to predict the species-specific rates. Figure 5-11 presents the
contributions of PAO, OHO and AOB towards the overall TMP removal rate in
the test reactors. It is apparent from Figure 5-11 that the observed TMP removal
rates in aerobic-1 were greater than that of aerobic-2. In aerobic-1, the AOB and
PAO contributed similarly while OHO contributed the least to the overall TMP
removal rate for the first 40 hours of the batch test. A similar trend was also
observed in the aerobic 2 batch test where PAO contributed a higher proportion
than OHO for the first 40 hours of the biotransformation reaction. Therefore, it
was concluded that each of the biomass groups in the BNR mixed liquor worked
201
collectively to achieve the observed TMP removal but contributed at different
proportions in the order AOB = PAO > OHO for aerobic-1 and PAO > OHO for
aerobic-2.
In all, these results suggests that both AOB and PAO are instrumental to
the significant removal of TMP in the BNR activated sludge. This means that
both nitrification and biological phosphorus removal may be beneficial for the
biotransformation of TMP in BNR activated sludge. This could explain why the
BNR performs better than other conventional activated sludge (CAS) treatment
systems for TMP removal. It is recommended that the approach employed in this
study for TMP should be extended to other prevalent micropollutants in
wastewater.
202
0.06
0.05
TMP removal rate (ug/L/d)
0.04
0.03
0.02
0.01
0.00
0 10 20 30 40 50 60 70 80
Time (hrs)
PAO-2 OHO-2 AOB-2 Predicted-2

PAO-1 OHO-1 AOB-1 Predicted-1
Figure 5-11. Contribution of PAO, OHO, and AOB to the overall TMP removal
rate.
Conclusion
A combination of pilot scale BNR activated sludge system, batch
experiments and a modeling exercise were employed to investigate the removal
and biodegradation kinetics of TMP in a BNR activated sludge. The results
showed that TMP can be effectively removed in a BNR activated sludge system
with each of the redox zones contributing different proportions. The TMP
removal efficiency was calculated as 59 ± 14% while the biotransformation
203
efficiencies in the anaerobic, anoxic and aerobic sections of the BNR bioreactor
were calculated to be 13 ± 12%, 17 ± 10 % and 24 ± 4% respectively. This result
indicated that TMP removal in the BNR process was related to the oxidation-
reduction condition in each of the zones. A comparison of TMP removal rates in
aerobic batch reactors with and without AOB inhibition showed a faster removal
rate in the reactor without AOB inhibition, suggesting that AOB plays a role in
TMP removal in BNR activated sludge. PAO, OHO and AOB were ascertained to
be actively undergoing metabolic activities in the batch tests as monitored by the
disappearance of the conventional pollutants associated with their growth and
cellular maintenance.
The biotransformation of TMP in BNR activated sludge under aerobic
condition obeyed a modified pseudo first order model that incorporated the
fractions of the active biomass groups. Biotransformation rate constants for TMP
with respect to PAO, OHO and AOB followed a pattern of kAOB > kOHO > kPAO.
This model showed that PAOs, OHOs and AOBs contributed different
proportions towards the biotransformation of TMP in BNR activated sludge with
the trend AOBs = PAOs > OHOs. Hence, the developed modeling tool have been
shown to provide enhanced quantification of TMP removal in BNR treatment
204
processes. The results of this study have improved the understanding of the
contributions of the active biomass groups in BNR activated sludge towards
TMP biotransformation in wastewater treatment. In all, the results of this study
indicated one of the possible reasons why BNR treatment processes deliver better
TMP removal as compared to CAS.
205
Chapter 6 Conclusions and Recommendations for Future Research
6.1 Conclusions
A comparison between the performances of three biological wastewater
treatment processes (CAS, NAS and BNR) in terms of their capability to remove
10 MPs that spanned a wide range of therapeutic classes indicated that the
removal efficiency of TMP improved with the complexity of the three treatment
processes configurations and SRTs. IBU, ADR, SMX, NP, E1 and BPA had
moderate to high removals (> 65%) while CBZ and MEP remained recalcitrant in
the three treatment process configurations. The removal of GEM was better in
the NAS than in the BNR and CAS treatment configurations. In order to assess
whether the observed chemical removal can be compared to the performance of
the processes in terms of estrogenicity removal, a comparison between the
performances of the three different process configurations in terms of their
estrogenicity removal was investigated. The YES assay analyses showed an
improvement in estrogenicity removal in the BNR and NAS treatment
configurations as compared to the CAS treatment configuration. Generally,
similar trend was observed between the treatment processes in terms of their
chemical removal and biological response reduction. The unique contribution of
206
this study was the side-by-side comparison of the wastewater treatment
configurations using both chemical and biological responses (YES assay).
In the comparative study that investigated the performances of the CAS,
NAS and BNR configurations in terms of their estrogenicity removal, the BNR
had the highest removal efficiency as compared to the other two processes.
However, the impact of the different stages of treatment on estrogenicity
reduction in the BNR process was not examined in detail. Therefore, the removal
of endocrine disrupting compounds in BNR wastewater treatment processes was
investigated. The results from the detailed BNR study indicated that the pilot
and bench scale BNR processes effectively removed EDCs in the authentic and
synthetic wastewater, with removal efficiencies as high as 95% for both systems.
The biodegradation of EDCs in the bioreactors of the two BNR processes
followed the trend of aerobic >anoxic > anaerobic. In order to understand the fate
of E1 and E2 under aerobic, anoxic and anaerobic conditions, a pseudo first order
model was calibrated with data generated from batch tests under the target
conditions. The biotransformation of E1 was estimated to be the slower step in
the two step reaction in the aerobic and anoxic batch tests while under anaerobic
conditions there was a balance between the biotransformation of E2 to E1 and the
207
biodegradation of E1. In order to further understand the transformation
dynamics between E1 and E2 under aerobic, anoxic and anaerobic conditions of
the BNR process, a mass balance model was used to verify the estimated
biotransformation rate constants using the operating conditions of the pilot
which was the source of the inoculum for the batch tests. The model effectively
predicted the fate of E1, E2, E2 + yE1 and E2-Eq in the different zones of the BNR
bioreactor. This study was the first to investigate, model, calibrate and validate
the biotransformation of E1 and E2 under different redox conditions in a BNR
activated sludge using biological responses.
The role of the active microbial groups on the biotransformation of
Trimethoprim in BNR processes was investigated. The results indicated that
AOB play a role in TMP removal in BNR activated sludge. TMP removal in the
BNR system was related to the oxidation-reduction conditions in each of the
zones. A combination of pilot scale BNR activated sludge system, batch tests and
modeling exercise were employed to evaluate the contributions of PAO, OHO
and AOB to the biotransformation of TMP in BNR activated sludge. Under
aerobic conditions, the biotransformation of TMP in BNR activated sludge
obeyed a modified pseudo first order model that incorporated the fractions of the
208
active biomass groups, where the biotransformation rate constants for TMP with
respect to PAO, OHO and AOB followed a pattern of kAOB > kOHO >kPAO. The
contributions of PAOs, OHOs and AOBs to the biotransformation of TMP in
BNR activated sludge followed the trend of AOBs = PAOs > OHOs. This study
have shown that both nitrification and biological phosphorus removal may be
beneficial for the biotransformation of TMP in BNR activated sludge. These
results could explain why the BNR performs better than other conventional
activated sludge (CAS) treatment systems for TMP removal. This study was the
first to investigate the role of PAO, OHO and AOB in the biotransformation of
TMP in BNR activated sludge.
6.2 Recommendations for Future Research
It is recommended that the biodegradation models that incorporated the
active biomass groups as applied to TMP in this study should be extended to
other prevailing MPs in wastewater in order to investigate the role of the active
biomass groups in their biotransformation. This approach will refine the current
practice of using the lumped parameter of MLSS or MLVSS as the biomass
concentration in biodegradation models and replace it with the specific active
biomass group directly related to the operating conditions of the treatment
209
system. This model can be applied in the current structure of activated sludge
models to effectively predict the concentrations of MPs in WWTP effluents in
order to assist MPs environmental risk analysis for policy formulation.
Additional research is also recommended to generate more data in order
to further validate the estimated biotransformation rate constants of TMP with
respect to the biomass groups and that of E1 and E2 under aerobic, anoxic and
anaerobic conditions. The set of parameters that were calibrated and verified in
this study are suitable for the operating conditions of the BNR process
investigated in this study and might not be applicable to other treatment
processes. Hence, further study is recommended to test the estimated
biotransformation rate constants for a wide range of operating conditions and
biological treatment processes in order to ascertain the robustness of the models.
It is recommended for future studies to investigate the effect of
temperature on the estimated biotransformation kinetics of the MPs in BNR
activated sludge. Biologically mediated reactions are usually dependent on
temperature because temperature affects microbial activities. Therefore, further
research is needed to estimate the temperature correction factor for the estimated
rate constants.
210
Studies should be performed to investigate the effects of varying
concentration of dissolved oxygen on the removal of MPs in BNR activated
sludge. The results of this study indicated that the degradation efficiencies of the
MPs and EDCs improved in the aerobic tests as compared to the anoxic and
anaerobic tests. Hence, further research is needed to quantify the effects of
different concentrations of dissolved oxygen on the removal of MPs in BNR
activated sludge.
Finally, further study of the metabolites formed during the
biotransformation of E1 in activated sludge systems is recommended. This
research investigated the estrogenic potency of this metabolite. However, it
would be beneficial to augment the biological analysis with a chemical
identification and quantification of the formed metabolites. This will enable the
formulation of a clearer biodegradation pathway for E2 and E1 in activated
sludge systems.
211
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Appendix A
Figure A 1. Sources and distribution of pharmaceuticals in the environment.

(Togunde, 2012).
237
Appendix B
Pilot plant diagrams and operational performance
Figure B 1. Pilot Plants (picture taken by Vince Pileggi).
238
600
500
400
COD (mg/L)
300
200
100
0
9-Feb-11 11-Mar-1110-Apr-1110-May-11 9-Jun-11 9-Jul-11 8-Aug-11 7-Sep-11 7-Oct-11
Date
Figure B 2. Daily pilot BNR influent and effluent COD.
250
200
CBOD5 (mg/L)
150
100
50
0
9-Feb-11 31-Mar-11 20-May-11 9-Jul-11 28-Aug-11 17-Oct-11
Date
Figure B 3. Daily pilot BNR influent and effluent CBOD5.
239
80
70
60
DOC (mg/L)
50
40
30
20
10
0
Date
Figure B 4. Daily pilot BNR influent and effluent DOC.
180
160
140
120
TOC (mg/L)
100
80
60
40
20
0
Date
Figure B 5. Daily pilot BNR influent and effluent TOC.
240
35
30
25
TAN (mgN/L)
20
15
10
0
Date
Figure B 6. Daily pilot BNR influent and effluent TAN.
35
30
25
NO3-N (mgN/L)
20
15
10
0
Date
Figure B 7. Daily pilot BNR influent and effluent NO3-N.
241
1
0.9
0.8
0.7
NO2-N (mgN/L)
0.6
0.5
0.4
0.3
0.2
0.1
0
Date
Figure B 8. Daily pilot BNR influent and effluent NO2-N.
50
45
40
35
TKN (mgN/L)
30
25
20
15
10
5
0
Date
Figure B 9. Daily pilot BNR influent and effluent TKN.
242
9
6
PO4-P (mgN/L)
0
Date
Figure B 10. Daily Pilot BNR influent and effluent PO4-P.

14
12
10
TP (mgN/L)
0
Date
Figure B 11. Daily Pilot BNR influent and effluent TP.
243
9
5
pH
0
Date
Anaerobic/C1 Anoxic/C3 Aerobic/C5 NAS CAS
Figure B 12. Daily pilot BNR influent, effluent and BNR stages pH.
244
400
350
300
mg/L CaCO3
250
200
150
100
50
0
Date
Influent NAS CAS BNR
Figure B 13. Daily pilot BNR influent and effluent alkalinity.
245
250
200
150
mgTSS/L
100
50
0
7-Jan-11 26-Feb-11 17-Apr-11 6-Jun-11 26-Jul-11 14-Sep-11 3-Nov-11
Date
Figure B 14. Daily pilot BNR influent and effluent TSS.
180
160
140
120
mgVSS/L
100
80
60
40
20
0
7-Jan-11 26-Feb-11 17-Apr-11 6-Jun-11 26-Jul-11 14-Sep-11 3-Nov-11
Date
Figure B 15. Daily pilot BNR influent and effluent VSS.
246
Table A1. QA/QC for Trimethoprim batch test
O2-2 O2-8 O2-11
Duplicate A (µg/L) 0.792 0.143 0.031
B (µg/L) 0.787 0.137 0.037
Relative % difference 0.68 4.77 18.04
in Milli Q Water
Spike Actual 1 µg/L
Spike Measured 1.53 µg/L
% Recovery 153 %
Blank ND
Background (Unspiked Mixed

Liquor) 0.049 µg/L
After Spiking
Spike Actual 1 µg/L
Average
Measured (n=3) 0.80 µg/L
StDev 0.12
% Recovery 75 %
247
Appendix C
UCT-BNR Simulation Environment
This section describes the modeling environment used to simulate the pilot BNR
process. The operation of the pilot plant was simulated using BioWin 3.1®
integrated model in the BioWin Platform. BioWin is a widely recognized
commercially available wastewater treatment process model and simulation
package that was developed by EnviroSim Associates Ltd, Canada. The
simulation platform includes activated sludge-anaerobic digestion models which
describes various biological processes in wastewater treatment, chemical
precipitation reactions, and gas-liquid mass transfer models using fifty state
variables and sixty process expressions. BioWin 3.1® modelling tool was chosen
because it was suitable for the study objective which requires effective modelling
of the different biological and physical processes that impact bacterial growth
rate under varying redox and operating conditions. Therefore, the selected
modeling environment was well equipped to effectively simulate the pilot UCT-
BNR process. Figure C1 shows the BioWin simulator configuration of the pilot
UCT- BNR process.
248
WAS
Figure C 1. BioWin Simulator flow diagram of UCT-BNR process.
249
Appendix D
YES Assay Preparatory Procedures and Solid Phase Extraction

The preparatory procedures in this section were adapted from Routledge and
Sumpter (1996) and the operating manual provided by Prof. C. Metcalfe as
presented by Citulski (2012).
Preparation of Stock solution

A) Estradiol (E2) Standard
 Weigh 55.6 mg of 98% pure 17-β-estradiol
 Transfer E2 to a 100mL volumetric flask
 Use Pasteur pipette to wash down any remaining E2 from the weighing
boat into the volumetric flask using absolute ethanol
 Add ethanol until the volumetric flask is filled; stopper the flask, seal with
Teflon tape, and hand-shake for ~30 seconds until the E2 is dissolved
 Charge a disposable 1mL glass pipette x3 with E2 stock solution; transfer

1mL to a second 100mL volumetric flask; reseal the stock solution with
stopper and Teflon tape, and place in freezer for storage
 Fill second 100mL volumetric flask with absolute ethanol, stopper, seal
with Teflon tape, and hand-shake for ~30 seconds
 Charge a disposable 1mL glass pipette x3 with intermediate E2 solution;

transfer 1mL to a third 100mL volumetric flask; dispose of the
intermediate solution after use
 Fill third 100mL volumetric flask with absolute ethanol, stopper, seal with
Teflon tape, and hand-shake for ~30 seconds, store in the -20oC freezer, but
return to room temperature before use (this is the working E2 standard for
the assay, and has a concentration of 54.48μg/L (2 x 10-7M) E2).
250
B) Minimal Medium
Add the following to 1000 mL of Milli-Q water and stir with magnetic stirrer on a
hot plate set to 75oC until all constituents are dissolved:
 13.61 g potassium phosphate monobasic (KH2PO4)
 1.98 g ammonium sulphate ((NH4)2SO4)
 4.2 g potassium hydroxide (KOH)
 0.2 g magnesium sulphate (MgSO4,)
 1mL of 40 mg/50 mL ferric sulphate (Fe2(SO4)3)
 50 mg L-leucine
 50 mg L-histidine
 50 mg adenine
 30 mg L-tyrosine
 30 mg L-isoleucine
 30 mg L-lysine hydrochloride
 25 mg L-phenylalanine
 20 mg L-arginine hydrochloride
 20 mg L-methionine
 100 mg L-glutamic acid
 150 mg L-valine
 375 mg L-serine
251
 Pipette 45mL aliquots into 100 mL Kimax bottles
 Autoclave at 121oC for 10 minutes
 Store sealed containers at room temperature
Glucose Solution
 Weigh out 20 g of (+)-D-Glucose, anhydrous, add to 250 mL Kimax bottle
 Measure 100 mL of Milli-Q water into the Kimax bottle
 Autoclave at 121oC for 10 minutes
 Store sealed container at room temperature
Vitamin Solution
 Add the following to 180 mL of Milli-Q water in a sterile beaker:

 8 mg of pyridoxine
 8 mg of thiamine hydrochloride
 8 mg (+)-pantothenic acid (D-calcium pantothenate)
 40 mg inositol
 20 mL of 2 mg/100 mL biotin in Milli-Q water
 Sterilize the solution by syringe-filtering (using a 0.2μm filter tip) into a

250 mL sterilized Kimax bottle
 Store at 4oC
L-Aspartic Acid Solution
 Add 100 mg of L-aspartic acid to 25 mL of Milli-Q water
 Sterilize the solution by autoclaving at 121oC for 10 minutes
252
 Store at room temperature
L-Threonine Solution
 Add 600 mg of L-threonine to 25 mL of Milli-Q water
 Sterilize the solution by autoclaving at 121oC for 10 minutes
 Store at 4oC
Cupric Sulphate Solution

 Add 320 mg of anhydrous cupric sulphate to 100 mL of Milli-Q water in a
sterile beaker
 Sterilize the solution by syringe-filtering (using a 0.2μm filter tip) into a

100mL sterilized Kimax bottle
 Store at room temperature
Chlorophenol Red-β-Galactopyranoside (CPRG) Solution

 Add 100 mg of chlorophenol red-β-D-galactopyranoside to 10mL of
absolute ethanol in an amber glass vial
 Store at 4oC
Preparation of HER Yeast Culture

Day One
 Retrieve 10x concentrated yeast stock from the -20oC freezer and thaw in a
beaker
 Prepare growth medium in laminar flow hood by pipetting the listed

solutions into a 45mL sterile aliquot of minimal medium:
 5 mL glucose solution
 1.25 mL aspartic acid solution
 0.5 mL vitamin solution
 0.4 mL threonine solution

253
 0.125 mL cupric sulphate solution
 Vortex mix thawed 10x concentrated yeast stock to suspend cells
 Add 250μL of 10x concentrated yeast stock to the growth medium, and
place in incubator/shaker set to 28oC and 250 rpm
Day Two (~ 24 hours after initiating incubation)
 Mix a second batch of growth medium, as per Day 1 instructions
 Using a sterile pipette, transfer 2 mL growth medium to a disposable

cuvette, and use this as a blank to measure the optical density of the yeast
solution; use a second sterile pipette to transfer 2 mL of the cultured yeast
solution to a disposable cuvette
 Measure the blank-corrected optical density of the yeast stock at 630nm

(Genesys 10 UV-Vis Scanning Spectrophotometer or equivalent)
 If OD630nm ~ 1.0, yeast stock is ready to be used (after a 24-hour incubation

period, the OD630nm has typically been 0.90 – 1.25)
 To the remaining growth solution, add cultured yeast in 0.5mL

increments, and check the OD630nm after each addition until an absorbance
of ~0.1 is achieved (about 2.5mL ± 0.5mL of yeast culture usually added)
 Prepare enough bottles of growth solution to fill the amount of microtiter

plates that will be used, and add sufficient yeast culture to each to achieve
OD630nm of ~0.1, and then add 0.5mL of CPRG solution to each bottle; this
mixture can now be used to fill the microtiter plates
3.6.2 Preparation of the Microplates
 Use 300 μL flat-bottomed microtiter plates (Whatman)
 Prepare a ― ”dummy row” (sufficient to create a quadruplicate set of

standard curves) as follows:
254
 Pipette 100μL of absolute ethanol into wells C2-C12 of a plate
 Pipette 200μL of E2 working standard into well C1; then withdraw

100μL from this well and add to well C2, fill and discharge the
pipette x5 to mix the contents (this will yield a 2x dilution in well
C2)
 Withdraw 100μL of solution from well C2, and add to well C3,
charging the pipette x5 to mix; repeat this process across the
remaining wells, and discard the final excess 100μL from well C12
 Cover the ― ”dummy row” with lab or autoclave tape, and then –
working from the lowest concentration well, C12 – pipette four
10μL aliquots of diluted E2 standard into wells E/F/G/H 12; repeat
this process, successively uncovering wells C11 - C1 and adding the
solution to the corresponding E/F/G/H wells
 Pipette 100μL of absolute ethanol into each of wells A1 - A12 to create a

negative blank
 Allow ethanol/E2 solution to evaporate by placing the microplate in the

laminar flow hood
 When all solvent has evaporated from individual wells, add 200μL of the
combined growth medium/CPRG/yeast mixture to each well in the
sample assay microplate
 After addition of yeast culture and incubation, the standard

curve/negative control plate should resemble Figure D2.
Environmental Sample Extracts
 Add 80μL of cleaned solvent extract from an environmental sample to

well 1 of the row; add another 80μL of extract to well 2, and as with the E2
curve procedure, serially-dilute the contents of well 2 across the remaining
wells; repeat as desired to obtain replicate curves
 Allow solvent to evaporate by placing the microplate in the laminar flow

hood
255
 When all solvent has evaporated from individual wells, add 200μL of the
combined growth medium/CPRG/yeast mixture to each well in the
sample assay microplate
 Seal the plate(s) with autoclave tape
 Place plates in the incubator/shaker and tape to the shaker plate; incubate
at 32oC with the shaker tray set to 150 rpm, for 72 hours
3.6.3 Reading the Microplates

 A microplate reader (TECAN) was used to determine the absorbance of
the samples at both 620nm (to evaluate sample turbidity) and 540nm (to
evaluate colour development)
 The corrected absorbance values were calculated as follows:

 Corrected Absorbance = AB540 nm – (AB620nm – Control620nm, average)
3.6.4 Preparation and storage of 10X concentrated yeast stock

Day One
 Make growth medium (minimal medium + vitamin solution/etc.) and add
125μL of concentrated yeast stock (stored in the -80oC freezer) thawed in a
beaker of ice in the laminar flow hood
 Place in incubator at 28oC with the orbital shaker set to 250 rpm for
approximately 24 hours
Day Two
 Make more growth medium and add 1mL of yeast from ― ” Day 1”
culture per ~50mL (several can be made at one time)
 Incubate at 28oC with the orbital shaker set to 250rpm for approximately
24 hours
Day Three
 Transfer each culture made on Day Two to a sterile 50mL centrifuge tube
(with closure)
256
 Centrifuge at 4oC for 10 minutes at 2000g
 In laminar flow hood, decant supernatant and re-suspend centrifuged

yeast pellet in 5mL of a minimal medium/glycerol mixture (45mL minimal
medium + 5.5mL glycerol, previously autoclaved at 121 oC/10 minutes)
 Transfer 0.5 mL aliquots to sterile GC vials (previously autoclaved at 121

o
C/10 minutes), cap and crimp, and store at -20 oC
 The prepared yeast stocks are good for 4 months
Figure D-1. Sample Extraction Process (Multi-Residue Extraction Technique).
257
Figure D-2. Example of Ethanol Negative Control Row (blank) and E2
Standard Curve.
Figure D-3. Example of YES-Assay Dose Response Curve for Blank and E2
Standard Curve.
258
Appendix E
Chemical Structure of Selected Chemical Compounds
Ibuprofen (C13H18O2)
Meprobamate (C9H18N2O4)
Carbamazepine (C15H12N2O)
259
Gemfibrozil (C15H22O3)
Trimethoprim (C14H18N4O3)
Sulfamethoxazole (C10H11N3O3S)
Androstenedione (C19H26O2)
260
Estrone (C18H22O2)
17-β-Estradiol (C18H24O2)
Nonyl-phenol (C15H24O)
261
Bisphenol-A (C15H16O2)
Ethinylestradiol (C20H24O2)
262
Appendix F
Micropollutant concentrations in treatment trains
IBU MEP GEM CBZ TMP SMX ADR E1

ng/L July
Sewage C1 7/7/2011 1427 16 24 242 134 82 72 40
Influent C2 7/7/2011 1377 9 21 233 107 80 66 24
CAS - N C3 7/7/2011 5 8 4 260 71 16 ND ND
CAS C4 7/7/2011 141 9 31 280 86 32 7 3
CAS -
BNR C5 7/7/2011 ND 11 9 287 32 42 ND 2
Sewage D1 7/19/2011 1247 8 22 209 118 107 50 24
Influent D2 7/19/2011 1267 5 20 194 104 111 36 22
CAS - N D3 7/19/2011 ND 5 ND 227 92 21 ND ND
CAS D4 7/19/2011 440 5 37 223 97 42 8 6
AN D5 7/19/2011 1327 5 21 198 44 50 24 18
03AX D6 7/19/2011 657 2 10 146 26 14 ND 7
C5O2 D7 7/19/2011 2 6 9 218 33 22 ND ND
CAS BNR D8 7/19/2011 ND 5 9 234 30 16 ND ND

Average Sewage 1337 12 23 226 126 94 61 32
Influent 1322 7 21 214 105 96 51 23
CAS - N 5 7 4 244 82 18 ND ND
CAS 291 7 34 251 92 37 7 4
CAS - BNR ND 8 9 260 31 29 ND 2
Stdev IBU MEP GEM CBZ TMP SMX ADR E1

Sewage 127 5 2 23 11 18 16 11
Influent 78 3 1 28 2 22 21 1
CAS - N 0 1 0 12 7 2 ND ND
CAS 106 2 2 20 4 4 1 1
CAS - BNR ND 4 0 37 1 19 ND ND
ng/L August IBU MEP GEM CBZ TMP SMX ADR E1

Sewage E1 8/3/2011 1257 9 23 175 112 148 59 2
Influent E2 8/3/2011 1413 9 24 196 116 150 51 16
CAS - N E3 8/3/2011 nd 14 nd 234 72 34 24 24
CAS E4 8/3/2011 48 13 30 219 82 94 nd 2
CAS -
BNR E5 8/3/2011 nd 15 8 223 44 121 nd nd
Sewage F8 8/24/2011 530 9 33 229 91 196 42 4
Influent F1 8/24/2011 427 14 26 246 101 20 43 2
CAS F2 8/24/2011 nd 22 nd 316 46 4 nd 2
263
CAS - N F3 8/24/2011 194 11 40 229 32 9 nd nd
CAS -
BNR F4 8/24/2011 nd 15 nd 274 68 2 54 42
AN F5 8/24/2011 254 14 17 252 68 17 6 37
AX F6 8/24/2011 224 11 20 232 116 47 nd 4
O2 F7 8/24/2011 480 11 12 247 94 19 39 3
Sewage G8 8/30/2011 25 19 9 195 105 36 10 8
Influent G2 8/30/2011 27 21 11 219 101 34 10 9
CAS G1 8/30/2011 nd 20 nd 293 97 nd nd 6
CAS - N G3 8/30/2011 2 19 nd 258 96 41 nd 2
CAS -
BNR G4 8/30/2011 nd 10 nd 176 18 nd nd nd
AN G5 8/30/2011 nd 22 nd 280 71 nd 3 8
AX G6 8/30/2011 3 25 nd 308 75 5 nd 1
O2 G7 8/30/2011 4 24 9 297 86 40 nd 2

Average Sewage 604 12 22 200 103 127 37 5
Influent 622 14 20 221 106 68 34 9
CAS - N nd 15 40 240 67 28 24 13
CAS 81 18 30 276 75 49 nd 3
CAS - BNR nd 13 8 224 44 62 54 42

Sewage 619 6 12 27 11 82 25 3
Influent 661 6 8 25 9 71 22 7
CAS - N nd 4 ND 15 32 17 ND 16
CAS 100 5 ND 51 26 64 ND 3
CAS - BNR nd 3 ND 49 25 84 ND ND
RSD Sewage 103 47 56 14 11 65 67 71

Influent 106 41 39 11 8 105 64 75
CAS - N ND 29 ND 6 49 60 ND 120
CAS 123 27 ND 18 35 129 ND 81
CAS - BNR ND 22 ND 22 58 136 ND ND
ng/mL September IBU MEP GEM CBZ TMP SMX ADR E1

Influent H1 Sept-14-2011 ND 0.017 ND 0.216 0.034 0.009 ND 0.004
CAS H2 Sept-14-2011 ND 0.024 ND 0.226 0.033 0.004 ND 0.004
CAS-N H3 Sept-14-2011 ND 0.027 ND 0.284 0.042 0.002 ND ND
CAS-BNR H4 Sept-14-2011 ND 0.022 ND 0.254 0.025 ND ND 0.003
AN H5 Sept-14-2011 ND 0.021 ND 0.262 0.030 ND ND 0.009
AX H6 Sept-14-2011 ND 0.023 ND 0.270 0.025 ND ND 0.013
O2 H7 Sept-14-2011 ND 0.021 ND 0.258 0.028 ND ND 0.009
Sewage H8 Sept-14-2011 ND 0.017 ND 0.208 0.043 0.009 ND 0.005
Influent I1 Sept-16-2011 1.607 0.008 0.035 0.168 0.031 0.130 ND ND
264
CAS I2 Sept-16-2011 0.034 0.008 0.043 0.193 0.037 0.077 ND ND
CAS-N I3 Sept-16-2011 ND ND ND 0.002 0.001 ND ND ND
CAS-BNR I4 Sept-16-2011 0.023 0.010 0.036 0.210 0.024 0.086 ND ND
AN I5 Sept-16-2011 ND ND ND ND ND ND ND ND
AX I6 Sept-16-2011 ND ND ND ND ND ND ND ND
O2 I7 Sept-16-2011 ND ND ND ND ND ND ND ND
Sewage I8 Sept-16-2011 1.529 0.011 0.049 0.225 0.041 0.185 ND ND
Influent J1 Sept-20-2011 1.367 0.009 0.026 0.128 0.027 0.103 ND ND
CAS J2 Sept-20-2011 0.035 0.008 0.031 0.209 0.031 0.119 ND ND
CAS-N J3 Sept-20-2011 0.041 0.009 0.007 0.238 0.033 0.079 ND ND
CAS-BNR J4 Sept-20-2011 0.019 0.009 0.031 0.218 0.026 0.116 ND ND
AN J5 Sept-20-2011 0.022 ND ND 0.002 0.001 0.003 ND ND
AX J6 Sept-20-2011 0.590 0.009 0.031 0.201 0.022 0.176 ND ND
O2 J7 Sept-20-2011 0.022 0.009 0.029 0.187 0.022 0.162 ND ND
Sewage J8 Sept-20-2011 1.437 0.008 0.024 0.146 0.035 0.164 ND ND
Average IBU MEP GEM CBZ TMP SMX ADR E1

Sewage 1483 12 36 193 39 119 ND 4
Influent 1487 11 30 171 30 81 ND 4
CAS - N 41 12 7 175 26 41 ND ND
CAS 35 13 37 209 34 67 ND ND
CAS - BNR 21 14 33 227 25 101 ND ND

Sewage 65 5 17 42 4 96
Influent 170 5 6 44 3 64
CAS - N 0 12 0 151 22 55
CAS 1 9 8 17 3 58
CAS - BNR 3 8 4 23 1 21
265
Appendix G
Trimethoprim batch test data
Aerobic-1B ug/L mg/L mg/L mg/L mg/L mg/L

hr TRM-2 std-2 C/Co COD PO4-P NH3-N NO3-N NO2-N
0 0.529 0.124 1 503.00 193.76 98.27 4.03 0.42
0.25 0.643 0.012 0.940469 451.00 175.52 94.73 2.83 0.37
4.25 0.362 0.011 0.818182 231.00 148.04 69.54 26.67 0.93
16.07 0.161 0.004 0.174487 166.00 82.54 31.30 65.67 0.06
20.07 0.137 0.032 0.101026 154.00 79.62 18.59 78.33 0.17
24.65 0.103 0.021 0.083284 154.00 76.17 10.78 88.69 0.01
39.65 0.079 0.023 0.017889 168.33 80.44 5.56 91.67 0.02
43.73 0.073 0.022 0.01654 172.33 84.13 0.04 97.33 0.01
47.9 0.028 0.002 0.016129 186.00 85.03 0.01 98.67 0.01
64.07 0.022 0.002 0.016129 208.67 89.01 0.05 100.33 0.02
68.07 0.019 0.003 0.016129 216.00 92.19 0.02 101.00 0.01
73.07 0.018 0.002 0.016129 229.00 95.28 0.01 102.33 0.01
266
Aerobic-1 (ug/L)
hr TRM-1-1 C/Co Std COD SD PO4-P SD NH3-N SD NO3-N SD NO2-N SD
0.00 0.656 0.179 600.33 28.22 162.40 3.81 114.67 0.99 0.40 0.00 0.02 0.00
0.25 0.549 1.000 0.022 599.67 22.14 152.00 4.35 113.40 2.09 0.20 0.00 0.01 0.00
4.25 0.489 0.892 0.018 286.67 51.64 118.00 3.35 81.23 1.24 1.70 0.00 0.73 0.00
16.07 0.160 0.292 0.001 171.30 18.88 40.46 0.65 39.60 1.18 30.00 1.00 0.76 0.01
20.07 0.122 0.222 0.021 144.67 4.04 32.39 0.21 25.40 11.88 47.00 1.00 0.01 0.01
24.65 0.094 0.171 0.013 128.33 4.62 22.30 0.59 19.10 0.50 52.00 1.00 0.69 0.01
39.65 0.058 0.106 0.030 135.33 2.08 22.20 0.21 7.73 0.21 76.00 1.00 0.05 0.02
43.73 0.054 0.098 0.027 138.67 4.51 22.30 0.20 0.40 0.20 86.33 0.58 0.21 0.01
47.90 0.024 0.043 0.006 142.67 1.53 23.20 0.05 0.33 0.06 88.33 0.58 0.10 0.01
64.07 0.020 0.036 0.004 146.30 0.00 23.20 0.00 0.17 0.03 89.00 1.00 0.04 0.00
68.07 0.018 0.032 0.002 152.33 0.58 23.10 0.05 0.06 0.01 93.00 1.00 0.03 0.00
73.07 0.017 0.031 0.002 166.33 1.53 30.20 0.10 0.07 0.01 95.00 1.00 0.02 0.00
Aerobic-2 ug/L
hr TRM-2 Std-1 C/Co COD SD NH3-N SD NO3-N SD PO4-P SD
0 0.218 0.18 535.00 19.47 15.20 0.99 3.50 0.00 187.52 1.73
0.25 0.214 0.02 1.000 462.67 22.03 15.20 2.09 3.40 0.00 170.32 3.49
4.25 0.165 0.02 0.771 231.33 19.35 16.00 1.24 3.50 0.00 36.68 0.62
16.07 0.095 0.00 0.446 172.33 2.52 15.80 1.18 3.60 1.00 16.68 0.19
20.07 0.086 0.02 0.400 164.67 3.79 16.70 1.80 3.80 1.00 12.42 0.04
24.65 0.072 0.01 0.337 169.67 6.03 15.90 0.50 3.90 1.00 12.44 0.46
39.65 0.039 0.03 0.182 174.33 1.53 16.80 0.21 3.60 1.00 13.61 0.10
43.73 0.038 0.03 0.178 188.33 2.52 15.20 0.20 4.20 0.58 14.35 0.05
47.9 0.026 0.01 0.121 194.00 13.00 15.30 0.06 3.50 0.58 13.58 0.03
64.07 0.021 0.00 0.098 202.00 12.00 14.50 0.03 3.50 1.00 12.87 0.03
68.07 0.014 0.00 0.065 217.50 6.36 15.70 0.01 3.50 1.00 14.39 0.11
73.07 0.009 0.00 0.041 219.00 11.00 15.20 0.01 3.40 1.00 20.07 0.05
267

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Impact of Biological Nutrient Removal Process

Operating and Design Conditions on the Removal

of Micropollutants from Wastewater

Olumuyiwa Omotola Ogunlaja

presented to the University of Waterloo

thesis requirement for the degree of

Waterloo, Ontario, Canada, 2015

©Olumuyiwa Omotola Ogunlaja 2015

including any required final revisions, as accepted by my examiners.

I understand that my thesis may be made electronically available to the public.

The efficacy of three different wastewater treatment configurations, conventional

assay. The selected micropollutants (MPs) covered a broad spectrum of therapeutic

classes, i.e non-prescription analgesic (Ibuprofen (IBU)), anti-convulsant/epileptic

(meprobamate (MEP) and carbamazepine (CBZ), lipid lowering drug (Gemfibrozil

(GEM)), antibiotic (trimethoprim (TMP) and sulfamethoxazole (SMX)), steroid hormone

(androstenedione (ADR)), estrogen (estrone (E1)) and estrogenic compounds (nonyl

an improvement in estrogenicity removal in the BNR and NAS treatment

configurations as compared to the CAS treatment configuration. Comparing the

process, 96 ± 5% of the estrogenicity exerted by the EDCs in the wastewater was

efficiencies were estimated to be 8± 0.8%, 38 ± 4% and 85 ± 22% in the anaerobic, anoxic

of E1 and E2 in the BNR process.

biotransformation of trimethoprim (TMP) in a BNR activated sludge. The

concentrations of the active microbial groups- ammonia oxidizing bacteria (AOB),

ordinary heterotrophic organism (OHO) and polyphosphate accumulating organism

activated sludge with the trend AOBs = PAOs > OHOs.

assistance of my supervisor, Prof. Wayne J. Parker. I count it as a rare privilege to have

have made a contribution to his research group.

I would like to appreciate the support of my advisory committee members, Profs.

Servos for his advice on analysis of estrogens in wastewater and granting me

unhindered access to his laboratory. I am also grateful to my external examiner, Prof.

introduced me to the world of wastewater treatment processes when I joined WTC.

wastewater samples collected during this study respectively.

I would like to appreciate the friendship and support of some of my colleagues

the lab technicians of the department of Civil and Environmental Engineering,

wastewater treatment plant.

University of Waterloo, Thomas Sullivan.

Canadian Water Network (CWN), EnviroSim, Civil and Environmental Engineering

Department-University of Waterloo, Center for Control of Emerging Contaminant

(CCEC)-University of Waterloo and Graduate Student Office (GSO) - University of

rest of our lives.

To my wife Sileola Ogunlaja (Abebi) and my children, Daniel Ireoluwa Ogunlaja

supported and encouraged me throughout my program.

AUTHOR’S DECLARATION ...................................................................................... ii

Abstract ..................................................................................................................... iii

Table of Contents ........................................................................................................xi

List of Figures ...........................................................................................................xvi

Nomenclature ......................................................................................................... xxii

Chapter 1 Introduction ................................................................................................ 1

1.1 BACKGROUND ...................................................................................................................1

1.2 SCOPE AND OBJECTIVES ....................................................................................................8

1.3 THESIS OUTLINE................................................................................................................9

Chapter 2 Literature Review...................................................................................... 10

2.1 MICROPOLLUTANTS ........................................................................................................10

2.2 BIOLOGICAL NUTRIENT REMOVAL PROCESSES IN WASTEWATER TREATMENT ...........11

2.2.1 Summary of BNR Processes in Wastewater Treatment ......................................19

2.3 BNR PROCESS CONFIGURATIONS ..................................................................................19

2.3.1 Summary on BNR Process Configurations ...........................................................27

2.4 BIOLOGICAL PHOSPHORUS REMOVAL IN BNR PROCESS ..............................................28

2.4.1 VFA Potential of the Influent Stream.....................................................................30