Applied Science, Faculty of
Engineering, School of (Okanagan)
DSpace
UBCO
Rahman, Nahian
Master of Applied Science - MASc
University of British Columbia
Wastewater treatment plants (WWTP) generate mixed sludge (MS) containing high volumes of organic and inorganic matter, posing treatment and disposal challenges. Hydrothermal liquefaction (HTL) is a potential thermo-chemical technology for converting sludge to fuel (biocrude oil) offering a smaller footprint alternative to typical sludge digestion methods such as anaerobic digestion (AD) for treating high-volume sludge. However, the large amount of aqueous by-product (HTLaq) from the HTL poses challenges for industrial-scale use. Onsite biological treatment can be a low-cost and effective solution for HTLaq. Whereas, complex substances and highly soluble organic compounds in HTLaq inhibit downstream biological treatment processes, making pretreatment of HTLaq before biological treatment a necessity for commercializing HTL in sludge management. This study used hydrogen peroxide (H₂O₂) to pretreat HTLaq, derived from municipal sludge cake at HTL conditions of 350ºC for 15 min residence time, and the biodegradability efficiency of pretreated HTLaq was evaluated. Previous studies used HTLaq from other feedstocks. Quenchers, such as sodium carbonate (Na₂CO₃) and sodium sulphite (Na₂SO₃), were used to remove the residual H₂O₂ from the pretreated HTLaq, which caused discrepancies in total chemical oxygen demand (tCOD) results and additional inhibition in downstream biological treatment. Pretreatment of aqueous for 2 h at dosages of 0.25, 0.50, and 0.75 g H₂O₂/g COD of HTLaq, followed by a quencher addition (H₂O₂: quencher at 1:1 (w/w)) and 3 h waiting period, achieved the highest efficiency in tCOD removal from HTLaq. Approximately 18% of tCOD removal was achieved from pretreated HTLaq at 0.75 g H₂O₂/g COD with sodium carbonate as a quencher. Additionally, pretreated HTL at 0.5 g H₂O₂/g COD with Na₂CO₃ as a quencher achieved a reduction of phenolics by 50% compared to unpretreated HTLaq. Further biological degradability assessments using mesophilic and thermophilic biochemical methane potential (BMP) assays yielded 16% and 63% higher specific cumulative methane yield, respectively, for pretreated HTLaq at 0.25 g H₂O₂/g COD with Na₂CO₃ quencher. Additionally, the aerobic biodegradability index, estimated by the ultimate biochemical oxygen demand (BOD)/tCOD ratio, increased from 0.75 to 0.85 for HTLaq pretreated assays. Results indicate that low-dosage H₂O₂ can be suitable for pretreating HTLaq before downstream biological treatment.
PRETREATMENT OF HYDROTHERMAL LIQUEFACTION AQUEOUS FROM MUNICIPAL SLUDGE BY HYDROGEN PEROXIDE FOR ENHANCED BIOLOGICAL TREATMENT by Nahian Rahman B.Sc., Islamic University of Technology, Bangladesh, 2020 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Civil Engineering) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) October 2024 © Nahian Rahman, 2024 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis entitled: Pretreatment of Hydrothermal Liquefaction Aqueous from Municipal Sludge by Hydrogen Peroxide for Enhanced Biological Treatment submitted by Nahian Rahman in partial fulfilment of the requirements for the degree of Master of Applied Science Examining Committee: Dr. Cigdem Eskicioglu, School of Engineering Supervisor Dr. Nicolas Peleato, School of Engineering Supervisory Committee Member Dr. Alyse Kiesser, School of Engineering Supervisory Committee Member Dr. Joshua Brinkerhoff, School of Engineering University Examiner iii Abstract Wastewater treatment plants (WWTP) generate mixed sludge (MS) containing high volumes of organic and inorganic matter, posing treatment and disposal challenges. Hydrothermal liquefaction (HTL) is a potential thermo-chemical technology for converting sludge to fuel (biocrude oil) offering a smaller footprint alternative to typical sludge digestion methods such as anaerobic digestion (AD) for treating high-volume sludge. However, the large amount of aqueous by-product (HTLaq) from the HTL poses challenges for industrial-scale use. Onsite biological treatment can be a low-cost and effective solution for HTLaq. Whereas, complex substances and highly soluble organic compounds in HTLaq inhibit downstream biological treatment processes, making pretreatment of HTLaq before biological treatment a necessity for commercializing HTL in sludge management. This study used hydrogen peroxide (H2O2) to pretreat HTLaq, derived from municipal sludge cake at HTL conditions of 350ºC for 15 min residence time, and the biodegradability efficiency of pretreated HTLaq was evaluated. Previous studies used HTLaq from other feedstocks. Quenchers, such as sodium carbonate (Na2CO3) and sodium sulphite (Na2SO3), were used to remove the residual H2O2 from the pretreated HTLaq, which caused discrepancies in total chemical oxygen demand (tCOD) results and additional inhibition in downstream biological treatment. Pretreatment of aqueous for 2 h at dosages of 0.25, 0.50, and 0.75 g H2O2/g COD of HTLaq, followed by a quencher addition (H2O2: quencher at 1:1 (w/w)) and 3 h waiting period, achieved the highest efficiency in tCOD removal from HTLaq. Approximately 18% of tCOD removal was achieved from pretreated HTLaq at 0.75 g H2O2/g COD with sodium carbonate as a quencher. Additionally, pretreated HTL at 0.5 g H2O2/g COD with Na2CO3 as a quencher achieved a reduction of phenolics by 50% compared to unpretreated HTLaq. Further biological iv degradability assessments using mesophilic and thermophilic biochemical methane potential (BMP) assays yielded 16% and 63% higher specific cumulative methane yield, respectively, for pretreated HTLaq at 0.25 g H2O2/g COD with Na2CO3 quencher. Additionally, the aerobic biodegradability index, estimated by the ultimate biochemical oxygen demand (BOD)/tCOD ratio, increased from 0.75 to 0.85 for HTLaq pretreated assays. Results indicate that low-dosage H2O2 can be suitable for pretreating HTLaq before downstream biological treatment. v Lay Summary Disposal of untreated mixed sludge generated from wastewater treatment plants has adverse environmental effects. Hydrothermal liquefaction technology can be an alternative to conventional treatment processes for sewage sludge treatment. This method treats sludge at a high temperature (350ºC) in a pressurized container, and valuable products, such as biofuels and nutrients are recovered. However, a large amount of liquid by-product generated from this process creates a bottleneck in commercializing the technology as it contains many pollutants. This thesis evaluates adding hydrogen peroxide (H2O2), as a chemical pretreatment, to the liquid by-product before treating it in existing biological processes at sewage treatment plants. In particular, the study focuses on finding a suitable dosage of H2O2 and optimum conditions for the pretreatment process. Additionally, the efficiency of the pretreated hydrothermal liquefaction aqueous phase in increasing methane recovery from this liquid stream by various microbial cultures in enclosed mixed/heated bioreactors is investigated. vi Preface The following research was completed by the author at the University of British Columbia School of Engineering, under the guidance and supervision of Dr. Cigdem Eskicioglu, principal investigator of the Bioreactor Technology Group (BTG). This work includes the conceptualization, laboratory experimentation of hydrothermal liquefaction (HTL) of dewatered municipal sludge, separation of liquid by-product of HTL (HTLaq), H2O2 pretreatment and biodegradability assessment of HTLaq through anaerobic and aerobic batch assays, analyzing results and writing of the thesis. The preliminary design of the H2O2 pretreatment experiment was done by Dr. Parmila Devi (former research engineer at BTG). Dr. Huan Liu, Dr. Ibrahim Alper Basar (previous Ph.D students at the BTG lab), and Ronald Kizza, (Ph.D student at the BTG), helped in running the bench-scale high-temperature/pressure HTL reactor to generate the HTLaq by-product. Farid Alizad Oghyanous, a Ph.D. student at BTG contributed to the set-up of the aerobic biological assessment of HTLaq assays. Jacky-Takeuchi, the research technician at BTG, assisted with the analysis of organic compounds using gas chromatography-mass spectrometry (GC-MS). Milad Goodarzi, a Ph.D student at the BTG helped with proofreading the thesis. Artificial intelligence was used to improve the writing quality of the thesis. A version of the Methodology and Results and Discussion chapters will be submitted for journal publication soon. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ................................................................................................................................ v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................. x List of Figures ............................................................................................................................. xiii List of Abbreviations ................................................................................................................ xvii Acknowledgements .................................................................................................................... xix Dedication ................................................................................................................................... xxi Chapter 1: Introduction ........................................................................................................... 1 1.1 Background ................................................................................................................. 1 1.2 Research motivation.................................................................................................... 2 1.3 Objectives ................................................................................................................... 4 Chapter 2: Literature review ................................................................................................... 5 2.1 Wastewater treatment processes ................................................................................. 5 2.2 Sludge treatment processes ......................................................................................... 9 2.2.3.1 Hydrothermal carbonization ................................................................................. 15 2.3 HTL product streams ................................................................................................ 17 2.4 Pretreatment options of HTLaq ................................................................................ 21 2.5 Pretreatment using H2O2 ........................................................................................... 23 viii 2.6 Summary ................................................................................................................... 24 Chapter 3: Materials and Methods ....................................................................................... 26 3.1 Equipment ................................................................................................................. 26 3.2 HTL operation for HTL aqueous generation from dewatered mixed sludge............ 27 3.3 Preliminary experiments for HTLaq pretreatment with H2O2 .................................. 28 3.4 Optimized HTLaq pretreatment with H2O2 .............................................................. 31 3.5 Analytical characterization methods ......................................................................... 32 3.6 Biodegradability assessment of HTLaq assays before and after pretreatment ......... 38 Chapter 4: Results and discussion ......................................................................................... 44 4.1 Part 1: H2O2 dosage optimization for pretreatment of HTLaq with/without quenchers 44 4.2 Aerobic and anaerobic biodegradability assessments of HTLaq .............................. 63 Chapter 5: Conclusions .......................................................................................................... 81 5.1 Summary part 1 ......................................................................................................... 81 5.2 Summary part 2 ......................................................................................................... 82 5.3 Overall summary and conclusions ............................................................................ 83 5.4 Limitations and recommendations ............................................................................ 84 Bibliography ................................................................................................................................ 86 Appendices ................................................................................................................................... 96 Appendix A ........................................................................................................................... 96 Appendix B ........................................................................................................................... 97 Appendix C ......................................................................................................................... 102 ix Appendix D ......................................................................................................................... 103 x List of Tables Table 4-1: HTL mixed sludge (MS) feedstock and HTLaq characterization. .............................. 44 Table 4-2 Characterization of non-pretreated and pretreated HTLaq-1 samples at different times. 0.75H+NC and 0.75H+NS represent pretreated samples at the dose of 0.75 g H2O2/g COD of HTLaq-1 followed by sodium carbonate and sodium sulphite quenchers (H2O2: quencher of 1:1 by wt.), respectively. H2O2 and quencher additions were at room temperature (22C) and 602C, respectively. .................................................................................................................................. 53 Table 4-3 N-heterocyclic compounds removal from pretreated HTLaq-1 samples at different times. 0.75H+NC and 0.75H+NS represent pretreated samples at the dose of 0.75 g H2O2/g COD of HTLaq-1 followed by sodium carbonate and sodium sulphite quenchers (H2O2: quencher of 1:1 by wt.), respectively. H2O2 and quencher additions were at room temperature (22C) and 602C, respectively. .................................................................................................................................. 54 Table 4-4 ANOVA summary table for analysis of the significant mean difference between different....................................................................................................................................................... 56 Table 4-5 Characterization of non-pretreated and pretreated HTLaq samples versus time. 0.75H+NC and 0.75H+NS represent pretreated samples at the dose of 0.75 g H2O2/g COD of HTLaq followed by sodium carbonate and sodium sulphite quenchers (H2O2: quencher of 1:1 by wt.), respectively. H2O2 addition was at 602C for 2 h, followed by quencher addition/cooling to room temperature (22C) in 2-5 h................................................................................................. 57 Table 4-6 N-heterocyclic compounds removal from pretreated HTLaq-1 samples versus times. 0.75H+NC and 0.75H+NS represent pretreated samples at the dose of 0.75 g H2O2/g COD of HTLaq followed by sodium carbonate and sodium sulphite quenchers (H2O2: quencher of 1:1 by xi wt.), respectively. H2O2 addition was at 602C for 2 h, followed by quencher addition/cooling to room temperature (22C) in 2-5 h................................................................................................. 58 Table 4-7 Parameter characterizations for pretreated HTLaq with and without quencher for different dosages. H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C)............................................................................................................................................ 60 Table 4-8 N-heterocyclic compound removal % for pretreated HTLaq-1 and HTLaq-2 with and without quencher for different dosages. H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). ......................................................................................................... 62 Table 4-9 Inoculum characterization for aerobic and anaerobic biodegradability assessment..... 63 Table 4-10 Kinetic parameters for mesophilic (35°C) and thermophilic (55°C) BMP assays for HTLaq samples. ............................................................................................................................ 72 Table 4-11: ANOVA summary table for analysis of the significant mean difference between different ......................................................................................................................................... 73 Table 4-12: Equality probability between different pretreatment conditions for mesophilic BMP assays. ........................................................................................................................................... 74 Table 4-13 Equality probability between different pretreatment conditions for thermophilic BMP assays. ........................................................................................................................................... 75 Table 4-14 Biodegradability assessment of raw and pretreated HTLaq under aerobic conditions........................................................................................................................................................ 78 Table 4-15 Kinetic parameters for ultimate BOD test for HTLaq samples. ................................. 79 xii Table 4-16 ANOVA summary table for analysis of the significant mean difference between different ......................................................................................................................................... 80 Table 4-17: Equality probability between different pretreatment conditions for aerobic biodegradability index of HTLaq samples. ................................................................................... 80 xiii List of Figures Figure 2.1 Schematic diagram of Annacis Island wastewater treatment plant (BC, Canada). ....... 7 Figure 2.2 Biochemical pathways and products in AD (adapted from Appels et al., 2008; Ostrem, 2004). ............................................................................................................................................ 12 Figure 3.1 Hydrothermal liquefaction process for mixed sludge at BTG laboratory. .................. 27 Figure 3.2 Lab-scale set up for H2O2 and quencher pretreatment of HTLaq samples.................. 30 Figure 3.3. Images of (a) New Brunswick Innova®44 shaking incubator, (b) batch of BMP assay bottles, and (c) Agilent 7820A gas chromatography - thermal conductivity detector. ................. 40 Figure 3.4 Image of (a) batch of BOD assays and (b) online respirometer for aerobic biodegradability assessment.......................................................................................................... 42 Figure 4.1 (a) tCOD, (b) pH and (c) color of pretreated HTLaq-1 at room temperature (23C) at different pretreatment times for 0.25H, 0.5H and 0.75H. ............................................................. 45 Figure 4.2 Mesophilic (35ºC) specific cumulative methane yield over time for positive control (glucose), pretreated (0.5H) and unpretreated HTLaq-1. Sample of 0.5H corresponds to 0.50 g H2O2/g COD of HTLaq. ................................................................................................................ 47 Figure 4.3 VFA concentration during mesophilic (35ºC) biochemical methane potential for (a) unpretreated HTLaq-1 and (b) pretreated (0.5H). Sample of 0.5H corresponds to 0.50 g H2O2/g COD of HTLaq. ............................................................................................................................ 48 Figure 4.4 pH during mesophilic (35ºC) biochemical methane potential assays for pretreated (0.5H) and unpretreated HTLaq-1. Sample of 0.5H corresponds to 0.50 g H2O2/g COD of HTLaq........................................................................................................................................................ 49 Figure 4.5 (a) The impact of sodium sulphite addition as quencher on tCOD of HTLaq-1 and pretreated HTLaq-1 at different dosages, (b) theoretical and experimental tCOD comparison of xiv sodium sulphite at different dosages, (c) the impact of sodium carbonate addition as quencher on tCOD of HTLaq and pretreated HTLaq at different dosages, (d) theoretical and experimental tCOD comparison of sodium carbonate at different dosages. Both H2O2 pretreatment and quencher addition was at room temperature (22ºC). NS: sodium sulphite, NC: sodium carbonate............. 50 Figure 4.6 tCOD concentration of pretreated HTLaq-1 samples at different times (1-4 h). 0.75H+NC and 0.75H+NS represent pretreated samples at the dose of 0.75 g H2O2/g COD of HTLaq-1 followed by sodium carbonate and sodium sulphite quenchers (H2O2: quencher of 1:1 by wt.), respectively. H2O2 and quencher additions were at room temperature (22C) and 60  2C, respectively, total pretreatment+quencher addition time is reported. At t=0, no pretreatment was conducted. ..................................................................................................................................... 52 Figure 4.7 tCOD concentration of pretreated HTLaq-1 samples versus time. 0.75H+NC and 0.75H+NS represent pretreated samples at the dose of 0.75 g H2O2/g COD of HTLaq-1 followed by sodium carbonate and sodium sulphite quenchers (H2O2: quencher of 1:1 by wt.), respectively. H2O2 addition was at 60  2C for 2 h, followed by quencher addition/cooling to room temperature (22C) in 2-5 h, total pretreatment time is reported. At t=0, no pretreatment was conducted...... 55 Figure 4.8 (a) tCOD concentration for pretreated HTLaq-1 with and without quencher for different dosages, (b) tCOD concentration for pretreated HTLaq-2 with and without quencher for different dosages. H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). 59 Figure 4.9 Mesophilic (35°C) BMP results for pretreated HTLaq for different dosage of H2O2 (a) cumulative specific methane yield of glucose, (b) cumulative specific methane yield of H2O2 pretreated HTLaq-2 without quencher, (c) cumulative specific methane yield for H2O2 pretreated HTLaq-2 with sodium carbonate as quencher, (d) cumulative specific methane yield for H2O2 xv pretreated HTLaq-2 with sodium sulphite as quencher. H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). H: hydrogen peroxide, NC: sodium carbonate, NS: sodium sulphite. ..................................................................................................................... 65 Figure 4.10 Thermophilic (55°C) BMP results for pretreated HTLaq for different dosage of H2O2 (a) cumulative specific methane yield of glucose, (b) cumulative specific methane yield of H2O2 pretreated HTLaq-2 without quencher, (c) cumulative specific methane yield for H2O2 pretreated HTLaq-2 with sodium carbonate as quencher, (d) cumulative specific methane yield for H2O2 pretreated HTLaq-2 with sodium sulphite as quencher. H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). H: hydrogen peroxide, NC: sodium carbonate, NS: sodium sulphite. ..................................................................................................................... 66 Figure 4.11 VFA concentration throughout mesophilic BMP assays for pretreated and unpretreated HTLaq-2. H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). H: H2O2, NC: Sodium carbonate, NS: Sodium sulphite. ................................................................... 69 Figure 4.12 VFA concentration throughout thermophilic BMP assays for pretreated and unpretreated HTLaq-2. H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). H: H2O2, NC: Sodium carbonate, NS: Sodium sulphite. ............................. 70 Figure 4.13 N-heterocyclic compounds distribution at the termination of thermophilic BMP assays utilizing pretreated HTLaq. For pretreatment, H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both xvi pretreatments at room temperature (23C). H: hydrogen peroxide, NC: sodium carbonate, NS: sodium sulphite. ............................................................................................................................ 71 Figure 4.14 Respirometric biochemical oxygen demand (BOD) concentration of unpretreated HTLaq and H2O2 pretreated HTLaq, For pretreatment, H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). H: hydrogen peroxide, NC: sodium carbonate, NS: sodium sulphite. ..................................................................................................................... 76 Figure 4.15 Oxygen uptake rate of unpretreated and pretreated HTLaq with time. Shaded area represents standard deviation. For pretreatment, H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). H: hydrogen peroxide, NC: sodium carbonate, NS: sodium sulphite. ............................................................................................................................ 77 xvii List of Abbreviations AD Anaerobic digestion AOP Advanced oxidation process BC British Columbia BTG Bioreactor technology group BMP Biochemical methane potential BOD Biochemical oxygen demand COD Chemical oxygen demand EMA Environmental Management Act GAC Granular activated carbon GAE Gallic acid equivalence GC Gas chromatograph GC-FID Gas chromatography with flame ionization detector GC-MS Gas chromatography with mass spectrometry GC-TCD Gas chromatography with thermal conductivity detector HTL Hydrothermal liquefaction HTLaq Hydrothermal liquefaction aqueous waste product HTC Hydrothermal carbonization MS Mixed sludge NC Sodium carbonate NS Sodium sulphite NSERC Natural Sciences and Engineering Research Council of Canada PS Primary sludge xviii STP Standard temperature and pressure TAN Total ammonium nitrogen tCOD Total chemical oxygen demand TS Total solids TSS Total suspended solids UV Ultraviolet VFAs Volatile fatty acids VS Volatile solids VSS Volatile suspended solids WAS Waste activated sludge Wt% Percent by weight WWTP wastewater treatment plant xix Acknowledgements First and foremost, I would like to thank Dr. Cigdem Eskicioglu for supervising, mentoring, supporting, and providing knowledge throughout my research and academic journey. During my program, her feedback and advice have been invaluable. She taught me how to think out of the box and implement new ideas in my work. Additionally, I want to extend my gratitude to my committee members, Dr. Nicolas Peleato and Dr. Alyse Kiesser. I would like to extend my sincere appreciation to everyone at the BTL lab, whose support as both colleagues and friends has been invaluable throughout my journey. Special thanks Alison Cox, Dr. Ibrahim Alper Basar, Ronald Kizza and Dr. Huan Liu for training me on different BTG laboratory procedures. I am deeply grateful to Jacky Takeuchi and Dr. Parmila Devi for their guidance and assistance in different lab setups. I wholeheartedly appreciate the support and help from my BTG fellow lab mates Milad Goodarzi, Farid Alizad Oghyanous, Kemal Aktas and Maryam Hosseinikhah. Your help and encouragements made my experience more rewarding. On a personal note, I would like to express my heartfelt gratitude to family and friends who have been my pillars of strength throughout my academic journey. To my parents Ms. Nazma Sultana and Mr. Matiur Rahman, thank you for giving me the confidence and motivation to pursue my dreams. I am deeply grateful to my sisters Nahid Rahman and Nafis Rahman for being my lifelong role models. A special thank you Satabdi and Anmol for always standing by my side, boosting up my confidence, and believing in me when I needed most. Thanks to Sonia apu and Kanon bhai for giving me fresh perspective about life and future. Sohan, Eranga, Omang, Afra, and Parinda, thank you for always been there when I needed the support and friendship. I would like to thank my cousin Nazia and friend Sristy, Ilay and Akanksha for their constant motivation and the invaluable life lessons they shared, helping me navigate every challenging situation. And xx lastly special thanks to my teacher Samia Syeoti Ramim, for her unwavering guidance, from my undergraduate studies to my successful application to the master’s program at UBC. xxi Dedication To my beloved family and friends who stood by me throughout my journey in this distant land. 1 Chapter 1: Introduction 1.1 Background Wastewater from different sources such as municipalities, textile industries, agriculture, etc., have different types of pollutants that threaten the ecosystem and human health. With the increasing population, municipal wastewater production is increasing, which results in more semi-solid (slurry) waste production from wastewater treatment plants (WWTP). The semi-solid wastes from WWTP are called “sludge” and contains mainly organics (excess microbial cells from biological treatment), along with inorganic matter. Converting wastewater organics into bioenergy can play an important role in decreasing carbon emissions and environmental sustainability in wastewater treatment. Conversion of organics to bioenergy also prevents these pollutants from entering our water and soil ecosystem. Anaerobic digestion (AD) is the most frequently used process by larger municipalities to treat wastewater sludge by reducing pathogens and organic matter along with biogas recovery. Biogas is a renewable energy produced through breakdown of organic matter. Although anaerobic digestion (AD) has demonstrated potential advantages such as biogas production, pathogen removal, and odor reduction, it has some limitations. These include slow and low organic removal efficiency (50-60%), the need for large reactor volumes, and the presence of residual pollutants (micropollutants, fecal coliforms, and heavy metals) in biosolids that are applied to land as soil amendments. For this reason, there is a dire need for other sludge treatment processes, such as thermochemical and advanced biochemical methods. In this context, hydrothermal liquefaction (HTL) known as a thermochemical technology that effectively converts organic waste into valuable products, such as bio-oil and biogas, is of great importance (Lu et al., 2022). HTL has been explored as a potential alternative to AD to overcome 2 some of its limitations. In HTL process, sludge is treated under high pressure (4-22 MPa) and temperature (200-400◦C), which converts organics into four different streams, including liquid and gaseous streams (Watson et al., 2020). The liquid product from the HTL process is called hydrothermal liquefaction aqueous phase (HTLaq). However, the large amount (80% of the HTL by products) of produced HTLaq stream is creating a bottleneck for incorporating HTL in industrial use. HTLaq contains high concentrations of organic carbon as chemical oxygen demand (COD), ammonium, nitrogenous organics, and harmful aromatics which inhibits the downstream biological treatment processes in the WWTP (Aktas et al., 2024a; Watson et al., 2020). This research focuses on pretreating HTLaq using hydrogen peroxide (H2O2) and determining the biodegradability efficiency of the pretreated HTLaq for downstream processing within the vicinity of WWTP, with the goal of minimizing the costs associated with building new treatment units. 1.2 Research motivation As discussed, in section 1.1, the treatment and safe disposal of municipal sludge is a worldwide challenge. In the Province of British Columbia (BC), Canada, Metro Vancouver is one of the most rapidly growing areas. It currently has five WWTPs producing 55,000 tons of biosolids (treated sludge) annually. Within the next three decades, the population of Metro Vancouver is expected to grow from 2,767,000 to 3,836,800 (Liu, 2024). This expansion in population is expected to affect biosolids production significantly. For example, sludge production is projected to rise to 150,000 tons per year by 2050 (Liu, 2024). For this waste stream, AD has been the most frequently used municipal sludge treatment process for medium- to large-scale WWTPs, including the plants operated by Metro Vancouver. However, in AD no nutrient e.g. nitrogen (N) and phosphorus (P) recovery occurs as a commercial revenue stream, and it only captures 50-60% of 3 carbon (Basar et al., 2023). Furthermore, the disposal of treated municipal sludge can take up to 50% of the operational cost of WWTPs (Appels et al., 2008). To solve this issue, Metro Vancouver is planning to construct a pilot-scale HTL unit in Annacis Island WWTP. In a large-scale process, effectively managing HTLaq on-site, which is a byproduct of HTL, is critical due to the large volume (80% of process output) and soluble organic and inorganic compounds in HTLaq with the potential to inhibit downstream biological treatment processes. This necessitates detoxifying HTL aqueous by a pretreatment process which does not diminish biodegradation potential. Chemical treatment, such as the advanced oxidation process (AOP) or oxidation with H2O2, can be a feasible solution in pretreating HTLaq. Pretreatment with H2O2 is comparatively inexpensive than other AOPs as it will not add any additional treatment chamber or system as well as H2O2 is comparatively cheaper than other oxidants and readily available. Furthermore, a mild dose (2-8 L per 100 mL of sludge) of H2O2 has shown effectiveness in treating mixed sludge for bacterial disintegration since OH• radicals are very reactive even in low concentrations (Preethi et al., 2022). During H2O2 oxidation process OH• radicals reacts with complex compounds and increases the biodegradability of those compounds (Siciliano et al., 2014). However, there is still a research gap in determining the effectiveness of H2O2 pretreatment of HTLaq derived from municipal sludge, as HTLaq is a significantly different waste stream than the municipal sludge itself. The valuable findings from this study fill the research gap and provide a comprehensive knowledge of the H2O2 treatment process for the incorporation of HTL process into municipal WWTPs. Although the laboratory testing utilizes municipal sludge from Annacis Island WWTP, the results can be applied to other municipal WWTPs with similar processes. On a broader scale, this study is set to significantly impact the future wastewater treatment industry by helping them to upgrade their sludge treatment processes with a more resource recovery focus. 4 1.3 Objectives The objectives of this research are defined as follows: 1. Determining optimum chemical dose and time for H2O2 pretreatment of HTLaq, derived from municipal sludge, 2. Determining removal efficiencies of the H2O2 pretreatment process for a large group of organic and inorganic pollution parameters in HTLaq, 3. Determining efficiencies of various quenchers, utilized to remove residual H2O2 from pretreated samples, in eliminating interference in pollutant parameter quantification and biodegradability assessment, 4. Determining anaerobic biodegradability of H2O2 pretreated HTLaq using the optimum dosage identified in objective 1, 5. Determining aerobic biodegradability of H2O2 pretreated HTLaq using the optimum dosage identified in objective 1. 5 Chapter 2: Literature review This chapter focuses on the existing technologies in the wastewater treatment process, sludge treatment as well as the HTL process. A brief review of HTLaq pretreatment methods is discussed. And lastly, a summary is given for the overall HTL process and HTLaq pretreatment alternatives. 2.1 Wastewater treatment processes With the increasing usage of water, water pollution and wastewater volume are increasing. The sources of wastewater could be domestic, industrial, agricultural etc. (Crini, 2019). Typically, municipal wastewater includes streams from residential areas, hospitals, educational institutions, industrial buildings, parks, hotels, restaurants, airports, etc. (Metcalf, 2014). Depending on the source, municipal wastewater contains organic and inorganic pollutants such as pathogenic microorganisms, pharmaceuticals, pesticides, heavy metals and micropollutants that are hazardous to the environment. Thus, an adequate treatment process is needed for public health safety and aquatic life. Generally, a WWTP includes primary/preliminary, secondary, and tertiary treatment processes. Figure 2-1 shows a schematical view of a WWTP. While primary treatment includes physical treatment units and processes such as screens and bars, secondary treatment includes biological treatment units, such as trickling filters or rotating biological contractors. Lastly, tertiary treatment can be a chemical, physical, or biological process, such as the disinfection chamber in Figure 2-1, depending on the discharge regulations of the receiving water body (Metcalf, 2014). Sludge is mainly produced from primary and secondary treatment processes and later, it goes to sludge treatment processes. 6 2.1.1 Preliminary treatment In preliminary treatment, screens and bars are used to remove coarse debris from influent wastewater. To remove the grits, a grit removal process is introduced after the screening chamber. The preliminary treatment process prevents damages that might occur in the downstream unit processes by removing rags, sticks, floatables, grease, and grits (Metcalf, 2014). Screens are typically of three types: coarse, fine and micro screens. Rags, plastics, bottles, sanitary products, sticks and leaves etc. are removed through a coarse screen because of its opening size > 6 mm. Based on the wastewater characteristics, additional fine screens with opening sizes of 0.6 to 6 mm are used to remove remaining papers, rags, razor blades, and plastics for the influent water (Metcalf, 2014). Grit chambers are used following the screens where solids are separated using gravity. Solids such as coffee grinds, eggshells, and sands are settled and removed in the grit chamber. Grits and coarse materials can damage the downstream units as well as clog the pipelines. Therefore, preliminary treatment is required for the removal of unwanted larger particles and grits which are transported to the landfill after preliminary treatment chambers. 2.1.2 Primary treatment processes Primary treatment includes sedimentation and/or floatation to remove suspended organic and inorganic matter. The primary treatment unit uses mainly physical processes such as equalization tanks followed by the primary clarifiers. Equalization tanks allow uniform flow of the wastewater to the primary clarifiers (Metcalf, 2014). In primary clarifier, settleable solids are removed from the influent. This unit aims to prevent the clogging of biological units in the WWTP by reducing inorganic matter. According to Metcalf (2014), primary sedimentation tanks can remove 50-70% of total suspended solids (TSS) and 25-40% of biochemical oxygen demand (BOD). Settled sludge 7 at the bottom of the primary clarifier is called primary sludge (PS), and it is sent to the gravity thickener for further stabilization, as seen in Figure 2-1. The effluent wastewater from primary treatment goes to the secondary treatment unit for the biological treatment process. Figure 2.1 Schematic diagram of Annacis Island wastewater treatment plant (BC, Canada). 2.1.3 Secondary treatment processes Secondary treatment includes single or multiple biological units to remove most of the biodegradable organic matter (85%) as well as small, suspended solids from the effluent of the primary clarifier (Metcalf, 2014). The secondary treatment process reduces the pathogens from wastewater as well as degrades dissolved organics using biological treatment methods. Microorganisms (bacteria and fungi) transform organic matter into water, CO2, and other byproducts. In this process, nutrients such as N and P are utilized for the new cell growth of microorganisms (Metcalf, 2014). Biological units, such as conventional activated sludge and trickling filters, are used in the secondary treatment. Trickling filters are used to remove carbonaceous BOD and ammonia (via nitrification/denitrification) (Metcalf, 2014). In a trickling filter, aerobic biological treatment 8 happens where wastewater flows through a bed of filter media where microorganisms are attached. Due to its higher surface area, bacteria can grow and multiply more. Thus, it degrades the organic matter while influent water passes through the media. Some examples of filter media are rocks, gravel, ceramic, and plastic media. Conventional trickling filters used rocks with lower depth (4-6 ft) as a medium. However, modern trickling filters can be taller, and the depth varies from 16 to 33 ft. Plastic packing media is used due to its high void space (90 to 95%) (Metcalf, 2014). The popularity of trickling filters decreased with time, and in the 1990s, conventional activated sludge systems started to gain popularity. In the conventional activated sludge process, the effluent of primary treatment enters the aeration tank. Aerobic bacteria and wastewater are mixed with pressurized air for a certain duration (4-8 h) in the aeration tank (Davis & Cornwell, 2013). During this stage, organic matters are converted into inorganic byproducts. The secondary clarifier is installed after the aeration tank where treated wastewater, including microorganisms (biomass), enters, and with the help of gravitational force, treated wastewater is separated from the solids, which settle in the bottom of the clarifier. This solid stream contains aerobic microorganisms from the aeration tank and is called waste-activated sludge (WAS) (Metcalf, 2014). Effluent wastewater from the secondary clarifier enters tertiary treatment processes. Notably, solids from the bottom of the secondary clarifier, WAS, are partly recycled back to the aeration tank and the rest is sent to a dissolved air floatation or gravity thickener for further sludge treatment. Typically, the conventional activated sludge process removes almost 95% TSS (by wt.) and 92% BOD (Metcalf, 2014) 2.1.4 Tertiary treatment processes Pollutants that were not treated in the secondary treatment processes, such as trace metals, remaining pathogens, and nutrients, are the main targets in the tertiary treatment phase (Metcalf, 9 2014). Physical techniques and chemical methods are used as tertiary treatment. Oxidation, membrane filtration, ion exchange, reverse osmosis, disinfection, etc., are some examples of tertiary treatment processes (Crini, 2019). Depending on the effluent quality of secondary treatment and the discharge requirement of the receiving water body, the tertiary treatment processes vary. The studied WWTP (Annacis Island) uses disinfection with sodium hypochlorite (NaOCl), known as chlorination, for tertiary treatment (Figure 2-1). Chlorination is the most frequently used process and can remove almost 99% of pathogens before discharging the effluent into water bodies (Metcalf, 2014). However, chlorination can be harmful to aquatic life due to the disinfection by-products (DBPs) (Davis & Cornwell, 2013). DBPs can be formed when chlorine reacts with the organic matter in the water bodies (Davis & Cornwell, 2013). Alternatives such as advanced oxidation (different combinations of ultraviolet (UV) light, H2O2, and ozone) can be used for disinfection (Droste, 1997). Biological nutrient removal (BNR) is also a recognized tertiary treatment process to eliminate nutrients such as N and P (Metcalf, 2014). If the effluent contains higher N and P, it can increase biological growth, resulting in algal bloom in the lakes. Activated sludge with BNR as a tertiary treatment can remove 96% of BOD and after the tertiary treatment, total phosphorus decreases to 0.5 - 2 mg P/L from 4 - 12 mg P/L (Metcalf, 2014). 2.2 Sludge treatment processes Sludge is the solid or semi-solid by-products generated from the wastewater treatment processes mentioned above. Sludge contains different types of pollutants in large volumes, such as heavy metals, pharmaceuticals, nutrients, pathogens, etc. Thus, sludge treatment is essential for environmental sustainability. Sludge treatment includes sludge thickening and sludge stabilization (Metcalf, 2014). In the sludge thickening, the sludge is concentrated to reduce the excess PS and SS volume. The process where the pollutants are removed from sludge is called sludge 10 stabilization. Different kinds of sludge stabilization processes are chemical treatment, biochemical treatment, and physical treatment, along with more advanced versions of these processes, such as thermochemical treatment (Metcalf, 2014). After the sludge stabilization and final dewatering process, biosolids are generated and disposed of in the environment. Land application, composting, incineration, etc., are some examples of sludge disposal methods. Different countries have different regulations for biosolids disposal. For example, British Columbia has regulatory frameworks through its Environmental Management Act (EMA) and its Organic Matter Recycling regulations (British Columbia, 2018). 2.2.1 Chemical Treatment processes Alkaline treatment, such as the addition of lime (calcium oxide), is a common chemical sludge stabilization process. In the chemical stabilization method, the pH level is increased by an alkaline reagent leading to reduced odour and pathogens. Among the established alkaline reagents, sodium hydroxide (NaOH) has shown more stabilization efficiency than calcium hydroxide [Ca(OH)2] (Li et al., 2015). The alkaline stabilization process is comparatively cheaper than other sludge stabilization methods. However, from this process dust and ammonia odour might be produced as well as the resulting product has a larger volume due to the addition of chemicals and might not be utilizable as a soil amendment (Metcalf, 2014). 2.2.2 Biochemical treatment processes In general, suspended growth reactors are used for biochemical sludge treatment. In a suspended growth system, microorganisms responsible for treating sludge are maintained in the liquid suspension by mixing (Metcalf, 2014). Aerobic and anaerobic digesters are the main examples of suspended growth reactors designed for biochemical sludge treatment. These processes are more related to the scope of this thesis and are explained below. 11 2.2.2.1 Aerobic digestion Aerobic digestion occurs in the presence of oxygen with microbial degradation of organic materials in primary and secondary sludge streams. Having higher microbial growth in aerobic sludge digesters facilitates a shorter retention time requirement. Aerobic digesters can reduce almost 38-50% of VS (Metcalf, 2014). However, high aeration cost is a major drawback for aerobic digesters, and the byproduct of aerobic digesters (mainly CO2) with further environmental concerns (Shabbir et al., 2021). 2.2.2.2 Anaerobic digestion Currently, at medium- to large-scale WWTPs, AD is playing an important role by converting organic substances in primary and secondary sludge streams into biogas, energy sources, and nutrient-rich digestate, land applied as fertilizer (Appels et al., 2008). In AD, degradation of organic compounds occurs in the absence of oxygen through a mixed culture of anaerobic microorganisms (Metcalf, 2014). AD takes place in four different stages (Figure 2-2): hydrolysis, acidogenesis, acetogenesis, and methanogenesis (Metcalf, 2014). A conventional AD digester is a continuously stirred tank reactor (CSTR) where these four steps occur sequentially over a specific retention period. During hydrolysis, high molecular weight compounds and insoluble organics (soluble monomers), such as proteins, complex carbohydrates, and fats are converted into amino acids, simple sugars, and fatty acids, respectively (Ostrem, 2004). Afterwards, in the acidogenesis step, these soluble organic matter are converted into volatile fatty acids (VFAs) with the help of fermentative bacteria. The third step is acetogenesis, where alcohols and higher organic acids (e.g., propionic and butyric acids) are converted into acetic acid (acetate). In the final stage, biogas with 60-70% methane (CH4) is produced from the product of the previous stages with the help of methanogenic archaea, 12 and the remaining 30-40% of biogas contains mostly carbon dioxide and trace gases, e.g., hydrogen sulphide (H2S), ammonia (NH3) and moisture (H2O) (Appels et al., 2008). The production of biogas is the most remarkable advantage of AD over other sludge treatment processes. Notably, 70-80% of the incorporated energy from the total organics is converted into recovered energy through generated methane (Ostrem, 2004). Additionally, the growth of anaerobic biomass is much lower than aerobic digestion, as a result, it reduces the volume and mass of the final biosolids disposed into the environment (Ostrem, 2004). Despite all the aforementioned advantages, AD has some disadvantages, e.g., lengthy process, production of Figure 2.2 Biochemical pathways and products in AD (adapted from Appels et al., 2008; Ostrem, 2004). 13 undesired gases (H2S) aside methane, and production of harmful substances which might negatively affect the downstream biogas utilization infrastructures (Appels et al., 2008). The biochemical reaction rates and microbial cultures in AD depend on factors such as temperature and pH. Temperature plays a crucial role in maintaining reaction rates in AD. Two types of temperature regimes are common for AD: Mesophilic (30-35◦C) and thermophilic (50-70◦C). The reaction rate increases with temperature up to a certain extent. This is why mesophilic digesters have lower biogas yield compared to thermophilic digesters (Meegoda et al., 2018), but the yield also depends on the sludge retention time. It is very important to maintain the temperature in industrial digesters because fluctuations in temperature can inhibit biogas production (Zhang et al., 2015). Digester pH is also an important factor affecting the microbial community in AD. Methanogens can be inhibited in both acidic and basic conditions, and the optimum pH for AD ranges between 6.8 - 7.2 (Cioabla et al., 2012). During acidogenesis, pH can decrease by forming organic acids, imposing possible pH instabilities. For example, methanogenic activity is affected significantly when pH goes below 6.3 and above 7.8. Therefore, maintaining the pH levels throughout the operation is required (Ostrem, 2004; Zhang et al., 2015). According to Goodarzi et al. (2024), elevated pH to 9.0 can increase the lag time (250%) of anaerobic digestion process while reducing microbial activity and syntropy between microorganisms in the system. There have been some reports on the addition of conventional buffers like lime to maintain the pH, while, it is not recommended as a common practice due to operational cost (Verma, 2002). There are also other operational paramaters, e.g., solid content, VFAs, nutrient content, organic loading rate, retention time, and mixing speed (Verma, 2002; Zhang et al., 2015). For instance, if the feedstock has more lignocellulosic substances, thermal or biological pretreatment before AD 14 is recommended to increase the biogas production by enhancing the hydrolysis. Excess amount of VFAs can cause toxicity in the digester, thus monitoring VFAs is crucial for reactor performance assessment (Zhang et al., 2015). Retention time determines the size of the digester in a way that longer retention time necessitates larger digester. Reducing the retention time leads to a small reactor size, saving capital and operational costs (Ostrem, 2004). However, longer solid retention time (SRT) is preferred for slow-growing microorganisms, in particular acetoclastic methanogenic archaea (Azarmina, 2024). For preliminary lab-scale AD performance assessment, biochemical methane potential (BMP) test is used as a screening tool. BMP assay is a batch assessment where the biodegradability of organic waste, as well as methane-production rate and extent, are measured. To determine if the organic waste (substrate) can cause acute and/or chronic inhibition in different AD cultures, a BMP assay can be a fast and cost-effective method before more investments are made in larger-scale continuous-flow AD digesters. In a BMP assay, substrate and anaerobic inoculum are added to a serum bottle at a certain substrate-to-inoculum ratio (SIR) based on an optimum value reported in the literature for similar AD conditions/ substrate. Based on the characterization of the substrate and inoculum, alkalinity and the C/N/P ratio are adjusted by adding external buffer and nutrient solutions. After adding the necessary elements, the bottles are purged with ultra-pure N gas (99.99%) to eliminate oxygen, sealed, and placed in an incubator/shaker until they finish producing biogas. The incubation temperatures are typically mesophilic (30 to 38ºC) or thermophilic (50 to 70ºC) (Metcalf, 2014). Generally, inoculum is starved for a few days (4-5 days) before setting up the BMPs to reduce its VFAs and other readily biodegradable substrates. As a result, inoculum itself refrains from producing biogas and most of the biogas/methane originates from the substrate in BMP assay. In this assay methane, carbon dioxide, oxygen and nitrogen percentages are 15 measured by collecting biogas from the BMP bottles. Through BMP assessment, specific methane is calculated by measuring cumulative methane volume divided by the COD content (for soluble waste) or volatile solids (VS) of the substrate (for particulate/slurry type of waste) added to the BMP bottle initially. 2.2.3 Thermochemical treatment In the thermochemical treatment, organic feedstock, such as municipal sludge, lignocellulosic material, and microalgae, is converted into biofuels through thermal conversion. In the thermochemical processes of sludge, the physical, biological, and chemical characteristics of municipal sludge are changed to produce fuel (solid, liquid, or gas) and other by-products (Das et al., 2021). Liquefaction, wet oxidation, hydrothermal carbonization, hydrothermal liquefaction, hydrothermal gasification, combustion, and pyrolysis are some examples of thermochemical treatment processes (Jiang et al., 2021). Thermochemical treatments aim to valorize the sludge energy content while minimizing the negative impacts on the environment (Manara & Zabaniotou, 2012). 2.2.3.1 Hydrothermal carbonization Hydrothermal carbonization (HTC) is an exothermic process where various feedstocks such as lignocellulosic biomass waste, municipal waste, and sewage sludge are converted into solid phase (hydrochar), liquid (bio-oil) and gas phases (biogas). In this process, water is utilized as a solvent in subcritical conditions (Akbari et al., 2023). Subcritical water stays in the liquid state under high pressure at a temperature between 100-370°C with lower dielectric constants (Ko et al., 2020). Different studies have mentioned different operational conditions for carrying out this process. Studies have mentioned that mild temperature and high pressure are needed. According to Akbari et al. (2023), the temperature ranges between 150-350°C. However maximum biomass 16 production yield was reported at 168°C with licorice root pulp as feedstock. Son Le et al. (2022) mentioned that the HTC process converts wet feedstocks in a temperature ranging between 170–250°C in a short processing time (5-240 min). Compared to other thermochemical techniques, HTC requires less temperature and energy consumption. Furthermore, the HTC process does not need any feedstock pre-drying process (Akbari et al., 2023). HTC can be a potential replacement for pyrolysis because of the stability and rich carbon content in the byproduct (Son Le et al., 2022). 2.2.3.2 Hydrothermal gasification Gasification is a potential biomass-to-gas process in which combustible syngas is produced from waste. Steam reforming, auto-thermal reforming, steam gasification, oxidative gasification, hydrothermal gasification (HG), etc., are some notable gasification processes. HG is the most promising method among these processes due to its low energy consumption and cost efficiency (Sarker et al., 2022). HG can be divided into supercritical water gasification (SCWG) and subcritical gasification. The water phase at a pressure higher than the critical pressure (> 22.1 MPa) and temperature (> 374°C) of pure water, is called supercritical water (Sarker et al., 2022). To achieve higher efficiency, Chen et al. (2021) reported that a temperature higher than 1000°C might be needed while doing SCWG of coal. Efficient solubilization of biomass happens because a homogenous reaction medium builds up due to the solubility, diffusion, and heat transfer capacity of supercritical water. Different types of feedstocks can be valorized in this process, such as fruit waste, municipal sludge, chicken manure, and plastics (Sarker et al., 2022). 2.2.3.3 Hydrothermal liquefaction In hydrothermal liquefaction (HTL) treatment, wet urban residues, such as municipal sludge, are converted into valuable resources or solvents (liquid and gaseous) while the process is 17 conducted at a high temperature (280-400˚C) and pressure (10-35 MPa) (Li et al., 2022, Gollakota et al., 2018). Because of these features, HTL is an innovative technology for converting municipal sludge feedstock into useful products. HTL has the potential to replace AD for municipal sludge treatment due to its net positive energy recovery, reduction of management costs of the biosolids, and effective sterilization of pathogens and micropollutants due to high pressure/temperature used (Liu et al., 2023). Also, it is more cost-efficient than gasification (Basar et al., 2023). Previous studies indicated that the HTL process of municipal sludge leads to high energy and nutrient recovery. For example, more than 64% of energy can be recovered as biocrude from dewatered mixed sludge cake at an HTL temperature and pressure of 350˚C 170 bar, respectively (Basar et al., 2023). Furthermore, the HTL process is able to concentrate more than 80% of phosphorus from dewatered mixed sludge cake into hydrochar (Liu et al., 2021). From lab-scale testing, studies indicated that HTL treatment using PS at 325-350˚C for < 20 min can lead to energy recovery of up to 69% (Silva Thomsen et al., 2020). Nevertheless, the lack of literature on the HTL aqueous treatment process is a drawback for incorporating it in WWTPs. 2.3 HTL product streams Four product streams are derived from the HTL processing of feedstock, including crude, hydrochar, aqueous, and gas phases. 2.3.1 Biocrude Biocrude is produced in the HTL process through the Maillard reaction in the recombination pathways (Basar et al., 2021). Biocrude is a dark brown, hydrocarbon-rich, free-flowing liquid resulting from thermal breakage and reformation of biomass macromolecules (Chand et al., 2022). Biocude is the primary fuel product of HTL, known as an environment-friendly alternative to crude oil, that can be refined into transportation fuel. Depending on the initial feedstock characteristics, 18 biocrude contains saturated fatty acids, aromatic hydrocarbons, N- and O- heterocyclic compounds, alcohols, ketones, amines, esters and amides at different concentrations. The concentration of these compounds also depends on the HTL operating conditions (temperature, pressure, operating time, and feedstock) and the biocrude separation method (Liu et al., 2023). A high number of heavy metals including sulphur (53%) and arsenic (55%) were found in biocrude generated from HTL of mixed sludge at 350C and 15 min residence time. These heavy metals may cause challenges in the downstream processing of biocrude (Liu et al., 2023). Increasing reaction time in HTL operation can be an option to improve the biocrude quality while higher residence time might decrease the energy recovery (Liu, 2024). Additionally, Marrone et al. (2018) suggested that hydrotreated PS-derived biocrude after upgrading might have comparable quality to petroleum. 2.3.2 Hydrochar Hydrochar is the solid, ash-like content derived from the HTL process. However, the yield depends on the treatment conditions of the HTL along with the feedstock (Liu et al., 2021). A study by Marrone et al. (2018) reported that hydrochar yield from PS, SS and digested sludge (DS) were 9.5, 20.5, and 36.4% (by wt.), respectively in a continuous plug-flow HTL system with an operating condition at 358˚C for 18-30 min of residence time. Hydrochar may contain high amounts of nutrients (such as P), as well as pollutants, such as heavy metals, polycyclic aromatic hydrocarbons (PAHs), etc. (Liu et al., 2023). Despite containing a high amount of nutrients, it is not advisable to use hydrochar as fertilizer due to its toxic elements, such as Cr and Ni. Studies reported that heavy metals, such as Cr, Ni, Cu, Hg, Zn, and Pb, can be found in hydrochar derived from municipal sludge (Liu et al., 2023). Acidic extraction for P recovery from hydrochar is 19 reported to be a sustainable solution for fertilizer use instead of land applying hydrochar containing heavy metals (Liu, et al., 2023). 2.3.3 Gas phase The gaseous phase of the HTL process contains mainly, carbon dioxide, and hydrogen, with carbon dioxide taking 90% of the gas phase (Azarmina, 2024). Decarboxylation, decarbonylation, and deamination are the main mechanisms behind gas production in HTL (Basar et al., 2021). At high operational temperatures, gas yield increases due to the production of secondary tar (Jiang et al., 2021). 2.3.4 HTL aqueous phase The aqueous phase of the HTL process (HTLaq) constitutes the highest volume (80-81% by wt.) of reactor output among the 4 products of the HTL process when municipal sludge is treated at 20% of total solids by wt. (Liu et al, 2023). Therefore, finding a safe disposal method for HTLaq within the WWTP is crucial for waste utilities to agree to replace AD with HTL for sludge treatment. The characteristics of HTLaq depend on the type of feedstock (manure, algae, lignocellulose, sludge) and HTL operational parameters, such as temperature/retention time (Basar et al., 2023; Watson et al., 2020). Basar et al. (2024) studied how different municipal sludge composition (PS to SS ratio) changes HTLaq characteristics. They showcased that higher SS content increased N-heterocyclic compounds, total alkalinity, tCOD, orthophosphate, and VFAs, while high PS led to an increase in phenolic compounds, and dissolved protein in HTLaq (Basar et al., 2024). According to Basar et al. (2021), HTLaq derived from municipal sludge mainly contains ketones, carboxylic acids, amides, and amines, in addition to other organic and inorganic compounds. HTLaq also contains various complex molecules, such as N-heterocyclic compounds. Studies showed that only 33% of organic carbon in the feedstock is transferred into HTLaq. Also, 20 organic acids and most of the nitrogenous compounds accumulate in the aqueous phase (SundarRajan et al., 2021; Watson et al., 2020). High carbon contents can be detected in HTLaq derived at low-temperature HTL operation. On the other hand, high temperature and reaction time result in a high content of nitrogen in the HTLaq (Watson et al., 2020). Organic compounds in HTLaq could be used as value-added products if recovered. Such as ethanol as transportation fuel, acetic acid in vinyl acetate production, phenol in resins and dyes production. In the case of inorganics, N, P, and potassium (K) could be used as a component in commercial fertilizers. Thus, valorization of value-added products from HTLaq is a preferred method. Furthermore, for valorizing HTLaq, biological conversion of organics, in particular to bioenergy (methane, hydrogen), is also an option, as most WWTPs have biological treatment processes on-site (Watson et al., 2020). Due to high COD, VFAs, and alkalinity, studies recommended the anaerobic biological treatment of HTLaq (Liu et al., 2023). However, HTLaq contains a significant number of inhibitory compounds for AD, such as phenols, N-heterocyclic compounds, furans, and ammonia, that can affect the performance of the biological treatment processes. Also, recirculation of HTLaq in HTL has shown a positive impact on the biocrude yield. Also, it showed a declining trend in generating HTLaq (SundarRajan et al., 2021). However, recirculating HTLaq to HTL reactor might change the solid content ratio of feedstock and optimal conditions of the HTL process need to be adjusted. A study reported that AD represents a low-energy-cost HTLaq treatment (SundarRajan et al., 2021). However, due to having complex inhibitory compounds in HTLaq, it becomes challenging to treat HTLaq through biological processes like AD. From a recent study by Azarmina (2024), it is known that co-digestion of HTLaq, derived from municipal sludge cake at HTL conditions of 350°C and 170 bar, under thermophilic (55°C) AD shows inhibition on methane 21 generation. The study was conducted by semi-continuous flow anaerobic co-digesters of HTLaq with mixed sludge along with dewatering centrate and reported that increasing HTLaq tCOD contribution percentage from 12% to 23.1% and then to 43.3% showed inhibition on biogas production after 42, 29 and 15 days of reactor feeding, respectively, at a digester SRT of 20 days, corresponding to an organic loading rate (OLR) of 2.125, 1.677 and 1.211 kg COD/m3.day respectively (Azarmina, 2024). Another study showed that phenols, N-heterocyclic compounds and furans are considered primary inhibitors in the HTLaq derived from cornstalk at HTL temperature of 260˚C, which reduces the methane yield in upflow anaerobic sludge and packed bed reactors (Si et al., 2018). Thus, pretreatment of HTLaq is needed before incorporation into AD. Also, Basar et al. (2024) reported that in an HTL temperature and retention time ranges of 290˚C to 360˚C and 0 to 30 min, respectively, the operational parameters of HTL impact the anaerobic biodegradability of HTLaq derived from mixed sludge cake (20% TS). The highest biodegradability levels of mesophilic and thermophilic BMP assays with HTLaq as substrate were 66% and 49%, respectively, which indicated the more susceptibility of the thermophilic BMPs to HTLaq whereas aerobic biodegradability was 74% in the same study (Basar et al., 2024). For aerobic biodegradability, HTLaq obtained at temperatures less than 340˚C was reported to be easily biodegradable, and on the contrary, inhibition was detected for HTLaq obtained at higher temperatures than 360˚C which was also reported to be unsuitable for AD (Basar et al., 2024). 2.4 Pretreatment options of HTLaq Due to the inhibitory effects of HTLaq on biological treatment when used as a substrate, pretreatment of HTLaq is a dire need for incorporating the HTL process in the full-scale WWTP for sludge treatment. HTLaq can be pretreated using chemical, biochemical, and physical methods for enhanced downstream biological processes. 22 Physical pretreatment methods like adsorption of recalcitrant pollutants using activated carbon could be an option for increase methane yield. Hydrochar produced from HTL itself is also found as a strong adsorbent (SundarRajan et al., 2021). However, the adsorption capacity of biochar and hydrochar is significantly lower than granular activated carbon (GAC) (Aktas et al., 2024a). Also, the regeneration of adsorbent is another concern in this process (Aktas et al., 2024a). Recent studies indicated that GAC demonstrated higher adsorption capability by removing 66% of tCOD, 94% of phenolics, and 95% of cyclic ketones from HTLaq, derived from municipal sludge cake at 350°C, 15 min HTL conditions. In the biodegradability assessment of GAC adsorbed HTLaq, with a GAC range of 5-100 g/L, methane yield ranged from 9-97% and 10-83% of theoretical methane yield at standard temperature and pressure (0°C, 1 atm) under mesophilic and thermophilic conditions. Furthermore, an improvement was detected in aerobic biodegradation with a higher concentration of GAC (5 – 100 g/L), resulting in an ultimate BOD/tCOD ratio range of 0.81-0.93, defined as the biodegradability index (Aktas et al., 2024b). In addition to physical pretreatments described above, various chemical and thermochemical methods such as ozonation, oxidation with various chemicals such as H2O2, ammonia stripping/recovery, and ultraviolet (UV) oxidation can be options for HTLaq pretreatment (Aktas et al., 2024a; Cox & Eskicioglu, 2024; Watson et al., 2020). Cox & Eskicioglu (2024) worked on thermochemical (high temperature/pH) ammonia stripping and recovery from HTLaq derived from municipal sludge cake at 350°C, 15 min residence time. They also assessed anaerobic co-digestion of pretreated HTLaq with mixed sludge and dewatering centrate at an SRT of 20 days, corresponding to an OLR range of 1.8 to 2.5 kg COD/m3.d. The authors reported that under thermophilic (55°C) conditions, pretreated HTLaq showed less inhibition than untreated HTLaq and under mesophilic (35°C) conditions neither pretreated nor untreated HTLaq showed 23 any inhibition. This trend indicates a low tolerance of thermophilic conditions toward HTLaq co-digestion and supports other AD studies utilizing HTLaq as a substrate (Cox & Eskicioglu, 2024). Among oxidation pretreatments of HTLaq, ozonation requires expensive equipment (Javeed et al., 2023) and additional energy costs. Furthermore, the process requires the replacement or renewal of equipment to maintain the functionality of the treatment in the long run. Therefore, the following section will focus on a lower-cost oxidation alternative using H2O2, which is also the main topic of this thesis. 2.5 Pretreatment using H2O2 Oxidation is a widely recognized process to treat WWTP effluent with toxic compounds. For the pretreatment of HTLaq derived from sludge prior to biological treatment, H2O2 oxidation can be tested as a potential process. H2O2 is known as a powerful oxidizing agent and can eliminate, inhibitory and complex organic compounds from HTLaq. H2O2 breaks into highly reactive hydroxyl radicals (OH•) which can oxidize recalcitrant organics into easily biodegradable compounds with low molecular weights (Siciliano et al., 2014). These recalcitrant compounds can be converted into simple compounds or might be mineralized to CO2 and H2O. An increase in H2O2 dosage enhances the OH• formation. However, several studies have shown discrepancies in COD values while treating wastewater with H2O2 due to the residual OH• interfering with the analytical method for COD quantification (Bezsenyi et al., 2021; Lee et al., 2011). Therefore, it can be challenging to determine the optimum H2O2 dosage for the pretreatment process if COD removal is used as the only response parameter. Quenching excess H2O2 with chemicals (sodium-sulfite, sodium-bisulfite, sodium carbonate, and catalase, etc.) can be a solution to this issue (Chen et al., 2023; Liu et al., 2003; Wang et al., 2019). Sodium sulphite and sodium 24 carbonate are more affordable compared to other quenching chemicals. Also, sodium carbonate has shown efficiencies in promoting the formation of OH• from H2O2 (Acero & Gunten, 2000). So far, H2O2 has been tested for the HTLaq derived from feedstocks different than municipal sludge. Pretreatment with a low-medium dosage of H2O2 (0.25-0.625 g H2O2/g COD) has shown increasing methane yield in mesophilic BMP assays utilizing HTLaq derived from Spirulina biomass, a type of blue-green algae (Quispe-Arpasi, 2018). The author reported COD reduction and color change during the H2O2 pretreatment of HTLaq derived from Spirulina as feedstock at HTL of 300˚C for 30 min. The COD and color reduction efficiencies increased with the addition of H2O2 from 9.9% to 15.3% and 79.6% to 93.8%, respectively (Quispe-Arpasi, 2018). However, there might be negative impact on AD due to excessive use of H2O2 (10 – 60 L per 100 mL of sludge) (Preethi et al., 2022; Watson et al., 2020). H2O2 pretreatment of HTLaq derived from municipal sludge still needs to be studied to determine the optimal H2O2 dosage toward biodegradability assessment. 2.6 Summary Overall, HTL can be a better solution than AD for municipal sludge treatment, because of the more efficient energy output and nutrient recovery possibilities. For implementing HTL as part of WWTP operation, handling HTLaq can be a great challenge. Furthermore, to achieve a sustainable sludge treatment process that incorporates carbon and nutrient recovery from HTLaq, a downstream biological treatment is preferred as WWTPs have biological treatment processes already in place. Although technologies for valorizing HTLaq within WWTP have many advantages, they encounter some drawbacks (Watson et al., 2020). The inhibitory compounds in HTLaq require an on-site, cost-effective, and efficient pretreatment for detoxification. Pretreatment with H2O2 can be a possible solution considering these aspects. However, high 25 dosages of H2O2 can leave residual hydroxyl radicals, which can inhibit the microorganism in downstream treatment processes by reducing the VFA. A suitable quencher and optimum H2O2 dose selection can solve this issue. The next chapters of this thesis focus on a lab-scale testing methodology and results on H2O2 pretreatment of HTLaq derived from municipal sludge, the effectiveness of various quenchers for removing residual hydroxyl radicals, as well as the biodegradability assessment of pretreated HTLaq under anaerobic and aerobic conditions. 26 Chapter 3: Materials and Methods The experiments done in the thesis were divided into two parts: (1) Preliminary experiments for optimizing H2O2 dosage for pretreatment of HTLaq with/without quenchers, and (2) Biodegradability assessment of H2O2 pretreated HTLaq under two conditions: in the absence (anaerobic) and presence of dissolved oxygen (aerobic). In this chapter, the materials and methodology for both parts are explained briefly. 3.1 Equipment This research was conducted in the University of British Columbia Okanagan Campus’s Bioreactor Technology Group’s (BTG) laboratory. The lab-scale equipment used to conduct this research is tabulated in Table 3-1. Table 3-1. Equipment list of all experiments performed. Equipment Model Manufacturer Analytical balance XA105DU Mettler Toledo Centrifuge (with large rotor) Sorvall Legend XT Thermo Thermo Fisher Scientific Centrifuge (with small rotor) Legend micro-21 Thermo Fisher Scientific Parr reactor 4570 HT/HP Parr Instrument Company Oven Isotemp 625G Thermo Fisher Scientific Muffle furnace W-13 Paragon Industries Hot plate CIMAREC Thermo Fisher Scientific Dual channel pH/ion meter XL25 Thermo Fisher Scientific Microplate reader SynergyTM HT Multi -Detection Bio Tek Instruments Spectrophotometer GENESYS 10S UV-VIS Thermo Fisher Scientific Gas chromatograph (GC/FID) 7890A GC with Flame Ionization Detector Agilent Technologies Benchtop homogenizer mixer PT 10-35 GT Kinematica Polytron Gas chromatograph (GC/MS) 8860 GC with 5977B Mass Spectrometer Agilent Technologies Shaker incubator Innova 44 Rotary Shaking Incubator New Brunswick Scientific Gas flowmeter M-0.5SCCM Alicat Gas chromatograph (GC/TCD) 7820A GC with Thermal Conductivity Detector Agilent Technologies Respirometer PF-8000 Pulse Flow Respirometer Respirometer Systems and Applications, LLC 27 3.2 HTL operation for HTL aqueous generation from dewatered mixed sludge The experiments needed a sufficient volume of HTLaq before the H2O2 pretreatment processes. HTL processing was done two times for: (1) dosage optimization of pretreatment, and (2) biological degradation assessment of pretreated and un-pretreated HTLaq. For HTL operation, mixed sludge (MS) was sampled twice from a local WWTP in Vancouver, BC, on the months of May and November of 2023. PS (gravity-thickened) and SS (thickened through dissolved air floatation) were pre-mixed at the WWTP at 50:50 (vol.%) to make the MS before being shipped to the BTG laboratory. Figure 3.1 shows different steps of the generating HTLaq from MS. Figure 3.1 Hydrothermal liquefaction process for mixed sludge at BTG laboratory. At the BTG lab, to achieve 20% (by wt.) of total solids (TS), MS was centrifuged at 4,000 g for 10 min in 750 mL centrifuge bottles. After removing the supernatant, the remaining solids were centrifuged at 20,000 g for 8 min using 50 mL centrifuge tubes. Ultimately, the collected solids were characterized for the solid content and preserved in a container. The collected solid 28 portion was named dewatered sludge cake. For HTL runs, 500 g of the dewatered sludge cake was transferred to a 1-L Parr batch reactor (Parr 4570) and treated at 350C for 15 min residence time. The selection of the HTL temperature (350C) was influenced by research conducted by the Pacific Northwest National Laboratory and the U.S. Department of Energy (Snowden-Swan et al., 2022). Similarly, the residence timeframe of 15 min was determined through an earlier investigation carried out by the BTG (Liu et al., 2022). This chosen time doesn’t account for the ramping or cooling duration for the Parr batch reactor. Details on the HTL treatment of dewatered sludge cake are detailed in theses by previous BTG members (Basar, 2024; Liu, 2024). After the HTL operation, the set-up was allowed to cool, and the reactor was opened. To separate the HTLaq from hydrochar and biochar, the effluent was filtered through a 0.45 m filter. Biochar and hydrochar were outside the scope of this thesis, therefore only the HTLaq was retained. 3.3 Preliminary experiments for HTLaq pretreatment with H2O2 To determine the optimum dosage of H2O2 for pretreatment, different preliminary experiments were conducted. In line with the literature that studied H2O2 pretreatment of HTLaq from Spirulina biomass (Quispe-Arpasi, 2018), three dosages of 0.25, 0.50 and 0.75 g H2O2/g COD of HTLaq were tested. For pretreatment, the 3 target dosages of H2O2 was added to HTLaq in 3 amber septum bottles (30 mL) at room temperature (22C) initially and mixed using a magnetic stirrer (60 rpm). tCOD, pH, and color in HTLaq was analyzed after different pretreatment reaction times by removing a 2 mL samples from the amber septum bottles with a syringe. However, tCOD values of these samples showed discrepancies which are discussed later in Chapter 4. This issue occurred due to the unreacted H2O2 remaining in the pretreated assays and interfering with the tCOD method as an additional oxidant. 29 In previous literature (Acero & Gunten, 2000; Chen et al., 2023; Liu et al., 2003; Wang et al., 2019), sodium carbonate and sodium sulphite were used as quenchers to remove the residual H2O2 after the pretreatment processes at different quenchers to H2O2 ratios (w/w) of 1:1, 1:2, 1:2.5, 1:3, 1:3.5, and 1:4. The molar calculation for the quencher dosage can be found in Appendix section A . The highest dosage of H2O2 was chosen to select the optimized quencher ratios due to having higher residual H2O2 after the pretreatment process. To determine if the quenchers had additional tCOD added to the system, tCOD was also measured for quenchers themselves along with H2O2 pretreated assays (HTLaq + quencher). Based on the results from preliminary experiments, an optimal quencher type and dosage was selected. To determine the optimum pretreatment time and to assess if the temperature had any effects on the pretreatment process, H2O2 pretreatment was also tested at elevated temperature in addition to the room temperature at different pretreatment time durations. For this experiment the highest dosage of H2O2 (0.75 g H2O2/g COD) was selected due to having higher H2O2 residuals. Two sets of experiments were conducted. From the first set of experiments, pretreatment time duration was optimized where pretreatment was conducted using 0.75 g H2O2/g COD dose at room temperature (C) for different time durations (1 h, 2 h, 3 h, 4 h). 4 amber bottles (total volume of 10 mL) with pretreated HTLaq samples at 4 different time durations were set-up. At the end of each pretreatment duration, quenchers were added to each bottle. After addition of quencher, temperature was elevated to 60  C using a water bath and the experiment was continued for additional 3 h. In the end, pretreated HTLaq samples were characterized for various parameters (tCOD, TAN, VFA, total phenolics, pH and N-hetorocyclic compounds), detailed in section 3.5. From the second set of experiments, optimum waiting time after quencher addition was determined. In this step, pretreatment of HTLaq with 0.75 g H2O2/g COD dose at 60  C using 30 a water bath was conducted at fixed time duration of 2 h. These experiments were conducted in 4 amber bottles (total volume of 10 mL) (Figure 3.2) with 5 mL of HTLaq samples and then a quencher providing a 1:1 (quencher:H2O2 dosage) (w/w) ratio was added to each bottle and experiments were continued for different quencher addition waiting times (2 h, 3 h, 4 h, and 5 h). At the end of the experiments, different parameters (tCOD, TAN, VFA, total phenolics, pH and N-hetorocyclic compounds) were measured for pretreated HTLaq samples. Two sets of analysis of variance test (ANOVA) tests were conducted using SPSS software to determine the mean difference in COD results while optimizing quencher addition time. Figure 3.2 Lab-scale set up for H2O2 and quencher pretreatment of HTLaq samples. 31 3.4 Optimized HTLaq pretreatment with H2O2 Using the preliminary screening of pretreatment time, temperature and quencher type, the optimized pretreatment was performed at different H2O2 concentrations. In 9 amber glass bottles (20 mL) with septa, 15 mL of HTLaq and H2O2 were mixed at three different dosages of 0.25, 0.50, and 0.75 g H2O2/g COD of HTLaq at room temperature (22C) for 5 h. Among those 9 bottles (assays), 3 assays pretreated HTLaq at 3 different H2O2 dosages but without quenchers, and the remaining 6 assays pretreated HTLaq at similar H2O2 dosages but also with a quencher of sodium carbonate or sodium sulphite using a 1:1 (w/w) H2O2: quencher ratio. While preparing 6 of the pretreatment assays with quenchers, H2O2 pretreatment was conducted for 2 h at room temperature (22°C), and after that, quenchers were added, and the assays were mixed at 60 rpm for another 3 h (total of 5 h for entire pretreatment) at room temperature (22C). Table 3-2 compiles the pretreatment assay labels based on the aforementioned properties of pretreatment assays. The labels are defined including the H2O2 dosage at the beginning. The later alphabets stand for the chemicals used in the pretreatment process; for example, H stands for assays only pretreated with H2O2 without any quencher, whereas NC and NS mean quenchers of sodium carbonate and sodium sulphite, respectively, added after pretreatment with H2O2. Table 3-2 Names of the pretreated HTLaq assay. Assay name H2O2 dosage (g H2O2/g COD of HTLaq) Quencher used Quencher: H2O2 ratio (w/w) 0.25Ha 0.25 None N/A 0.50H 0.50 None N/A 0.75H 0.75 None N/A 0.25H+NCb 0.25 Sodium carbonate 1:1 0.50H+NC 0.50 Sodium carbonate 1:1 0.75H+NC 0.75 Sodium carbonate 1:1 0.25H+NSc 0.25 Sodium sulphite 1:1 0.50H+NS 0.50 Sodium sulphite 1:1 0.75H+NS 0.75 Sodium sulphite 1:1 ahydrogen peroxide, bsodium carbonate, csodium sulphite. 32 3.5 Analytical characterization methods To determine the effect of the H2O2 and quencher pretreatments on HTLaq samples, characterization techniques included total chemical oxygen demand (tCOD), pH, color analysis, VFAs, total ammonium ammonium-nitrogen (TAN), total alkalinity, and total phenolics. The same characterization methods were also performed on MS samples before HTL procedure as well as anaerobic and aerobic inocula. Additional characterization of TS/VS, TSS, VSS were performed on MS, as well as anaerobic and aerobic inocula before biodegradability assessments. 3.5.1 Total chemical oxygen demand (tCOD) To measure tCOD, a colorimetric method based on Standard method 5220D was employed (American Public Health Association, 2005). Various samples (mixed sludge, HTLaq, pretreated HTLaq etc.) were diluted with reverse osmosis (RO) water to ensure the tCOD concentration was within the calibration range (0-700 mg/L). Subsequently, 2.5 mL of either the diluted sample or the standard, along with 3.5 mL of digestion solution and 1.5 mL of tCOD catalyst solution, were mixed in securely capped test tubes. The digestion solution contained mercury sulphate (HgSO4) and potassium chromate (K2Cr2O7), while the tCOD catalyst solution contained silver sulphate (Ag2SO4). Both solutions included a high concentration of sulfuric acid (H2SO4), thus the process was conducted under a fume hood with necessary personal protective equipment. After preparing the samples, test tubes were subjected to heating at 150C for two hours in a heating block, and for the next hour, the test tubes were cooled down. Afterward, using a Kimwipe, the exterior of the test tubes was wiped, and assessed using the GENESYS 10S UV-VIS spectrophotometer, at absorbance of 600 nm. The tCOD value was calculated after a calibration curve was created using the calibration standard’s absorbance (y-axis) versus the calibration concentration (x-axis). The 33 absorbance values for the samples were inputted into the formula determined from the calibration curve to calculate the concentration of tCOD in the sample. In Appendix B, a calibration curve is provided as an example. 3.5.2 pH To measure pH, a dual-channel AccumetTM pH meter XL25 was used. pH was measured by submerging a pH probe in various assays. 3.5.3 Color analysis by UV/Visible range absorbance The color in HTLaq assay was analyzed using a GENESYS 10S UV-VIS spectrophotometer. To determine the best absorbance to use, a UV/visible spectrum analysis was done in the 200-700 nm range. After reviewing the spectral absorbance data Appendix B and referring to the literature, absorbance at 538 nm was chosen as the wavelength for color assessment to replicate and compare the results from the literature (Quispe-Arpasi, 2018). This decision was made based on the maximum absorbance of organic compounds at this wavelength. For the analysis, samples were transferred to quartz cells/cuvettes and data was collected at aforementioned wavelength. 3.5.4 Volatile fatty acids (VFAs) Volatile fatty acids (VFA) concentration was measured utilizing an Agilent 7890 gas chromatograph equipped with a flame ionizer detector (GC/FID) with iso-butyric acid as the internal standard (Ackman, 1972). To prepare the sample, 500 uL of the sample plus 500 uL of iso-butyric acid for internal standardization were added to autosampler vials. The instrument was calibrated using the internal standard method where calibration standards were prepared to match the internal standard concentration in each sample (for each calibration standard, 500 uL of calibrant plus 500 uL of the internal standard were analyzed). Additionally, a standard sample with 34 a known concentration of VFAs: 2000 mg/L of acetic acid, propionic acid, and butyric acids was tested with every batch of samples. During sample preparation, samples with solids (MS, inoculum etc.) underwent centrifugation (12000 g, 15 minutes) and were filtered through 0.45 m syringe filters. Samples with higher VFA content (HTLaq) were diluted 10 times with RO water. Subsequently, 0.5 mL of prepared samples/calibration check standard, and 0.5 mL of iso-butyric acid were added to glass autosampler vials and capped. Afterward, these vials were loaded into the GC/FID autosampler, and using the Chemstation software, the VFA concentration in each vial was determined. Chemstation software used a calibration table incorporated with internal standard quantification. 3.5.5 Total ammonium nitrogen (TAN) TAN concentration in different samples was measured following the colorimetric method based on Environmental Protection Agency method 350.1 (Cimon et al., 2020). Similar to VFA sample preparation, samples with higher solids were centrifuged and filtered (12000 g, 15 minutes, and 0.45 m syringe filter). For HTLaq assays, these steps were not required. Next, the samples were diluted to a certain concentration using Type-1 water in volumetric flasks where the TAN concentration was within the calibration standards range (0.1 to 7.5 mg/L). In a 10 mL test tube, by combining 0.61 mL of diluted sample or calibration standard, 2.0 mL of complexing solution, 0.86 mL of alkaline phenol, 0.01 mL of sodium hypochlorite and 0.36 mL of a 500 mg/L nitroferricyanide solution, a mixture was prepared. Then, the test tubes were placed in the dark for 1 h while the indophenol, a blue-colored compound, was formed depending on the TAN concentration in the sample. GENESYS 10S UV-VIS spectrophotometer was used to measure absorbance at 640 nm to detect the indophenol’s blue color. Finally, the measured 35 absorbance values were compared to the prepared calibration curve from the standard solution’s absorbance values. In Appendix B, a calibration curve is provided as an example. 3.5.6 Total phenolic compounds To determine total phenolic compounds, short in total phenolics, a modified Folin-Ciocalteau method was used (Singleton et al., 1999). A Synergy HT microplate multi-detector reader was used to detect total phenolics concentration as Gallic Acid Equivalence (GAE) through a colorimetric process. Gallic acid was used as the calibrant for this method. Samples were centrifuged (12000 g, 15 minutes) and the supernatant was filtered using a 0.45 m syringe filter. Filtered samples were diluted to ensure that the concentration of total phenolics stayed within the calibration ranges from 0 to 500 mg GAE/L. For samples that contained HTLaq, the dilution was 10-fold. In 2 mL microcentrifuge tubes, 0.1 mL diluted sample or standard, 0.5 mL of 10 times diluted Follin-Ciocalteu reagent and 0.4 mL of 75 g/L sodium carbonate, were mixed. The entire mixing process was required to be done under the fume hood for safety purposes. After mixing the solution in a vortex mix, 0.2 mL of solution was transferred to a well in a microplate. Lastly, the microplate was inserted into the microplate reader where, after five minutes of heating at 40C and five seconds of shaking, absorbance at 735 nm was measured. Follin-Ciocalteu reagent converted phenolics to different oxides with a dark blue color with a peak absorbance between 725 to 750 nm. The absorbance reading of the samples from the microplate reader was later compared to the absorbance of the standard calibration curve to determine the sample's total phenolics concentration as mg GAE/L. In Appendix B, a calibration curve is provided as an example. 36 3.5.7 Total alkalinity To measure total alkalinity, the samples were titrated with 0.1 N sulfuric acid. From a 25 mL burette, 0.1 N sulfuric acid was added drop by drop into the preweighed sample in a beaker. Using a pH probe, the pH was measured continuously, and acid was added until the pH reached to 4.6. The volume of acid consumed was recorded from the burette reading. Total alkalinity was calculated using the equation 3.1. Total alkalinity (mg CaCO3L) =50,000 × Acid volume (mL) × Normality of the acid usedSample volume (mL ) Equation 3.1 3.5.8 Total solids and volatile solids (TS and VS) To measure TS/VS, first, the mass of the crucible (used to hold samples) was measured, and a certain amount of sample was added, and the mass of the crucible with the sample was measured. After that, the crucible with the sample was dried in the oven at 105C for a minimum of 12 h. After this step, the mass was recorded. Ultimately, the sample was burnt in a muffle furnace at 550C for 30 minutes, cooled to room temperature and weighed. For calculating TS and VS, the following equations of 3.2 and 3.3 were used, respectively: TS (% gg ) =dry weight plus crucible (g) - crucible weight (g)sample weight plus crucible (g) - crucible weight (g)×100 Equation 3.2 VS ( % gg ) =dry weight plus crucible (g) - burnt weight plus crucible (g)sample weight plus crucible (g) - crucible weight (g)×100 Equation 3.3 3.5.9 Total suspended solids and volatile suspended solids (TSS and VSS) For characterizing aerobic inoculum, TSS and VSS test was conducted. The percentage of TSS and VSS were measured by drying and burning aerobic inoculum with a filter that caught the suspended solids from the samples. In this experiment, an aluminum weighing dish was weighed. 37 Later, a 5 mL sample was vacuum filtered through the prepared glass fibre filter and placed on the same aluminum dish. For measuring TSS, the filter with the aluminum dish was dried at 105C for 12 h. After cooling down to room temperature, the weight of the dried filter with the aluminum dish was noted. Later, it was placed into a muffle furnace to burn the organic material on the filter at 550C for 30 minutes. After cooling the dish and filter, the weight of the burnt sample filter with the aluminum dish was recorded. Following equations 3.4 and 3.5, TSS and VSS were quantified, respectively. TSS (%gmL) =(dry weight of filter + dish) (g)-(filter weight + dish) (g)sample volume (mL)×100 Equation 3.4 VSS (%gmL) =(dry weight of filter + dish) (g)-(burnt weight of filter + dish) (g)sample volume (mL)×100 Equation 3.5 3.5.10 N-heterocyclic compound analysis HTLaq (pretreated and unpretreated) assays were filtered through 0.45 m syringe filters to remove precipitated solids during storage. For each sample, 1 mL of the HTLaq was transferred into a glass test tube, then 2 mL of dichloromethane (DCM) was added. A vortex mixer was used to mix the DCM with the sample two times. 1 mL of the bottom DCM layer was transferred into an autosampler vial with an added internal standard. In autosampler vials, dilutions were conducted 10 and 50 times with DCM and internal standards. The analysis also included samples taken from biodegradability assays. The extraction procedure for the samples from the BMP assays was done as follows: at the end of the experiment the sample from the BMP bottle was centrifuged for 15 minutes at 9,000 ×g. Around 15 mL of aqueous portion was collected after the centrifugation of each sample. To extract the samples, 5 mL was measured into large glass test tubes, and 2 mL of DCM was added, and was vortexed 38 twice. Autosampler vials were prepared by labeling and adding 40 uL of the 50 ug/mL internal standard. 1 mL of the extracted sample (lower layer) was transferred into the autosampler vial, and the samples were injected in duplicate to an Agilent 8860 GC/5977B-Mass Spectrometer (GC/MS) equipped with an Agilent 30 m HP5MS column. Additionally, a standard vial was prepared at 10 times dilution with DCM and internal standard. The injector temperature of the GC/MS was 250C, the oven program was used, and the MSD full scan mode was set to 40 to 400 amu range. The instrument was calibrated using stock standards and internal standard techniques. Using the calibration and multiple dilutions of the sample extraction sample results were determined. 3.6 Biodegradability assessment of HTLaq assays before and after pretreatment For the biodegradability assessment of HTLaq samples before and after pretreatment, BMP and biochemical oxygen demand (BOD) assessment were conducted to evaluate anaerobic and aerobic biodegradability, respectively. 3.6.1 Biochemical methane potential (BMP) assays for anaerobic biodegradability assessment In total, three batches of BMP assays were performed. As a preliminary experiment for Part 1, a batch of BMP assays under mesophilic conditions (35C) was conducted over the course of 38 days. In Part 1, each BMP bottle received 50 mL of anaerobic inoculum (bacterial culture) and different volumes of substrate (HTLaq-1, and pretreated sample of 0.5H in Table 3-2, no quencher) to maintain a substrate-to-inoculum ratio (SIR) of 0.25 mg COD of substrate/mg VS of inoculum. This set of BMPs was performed to assess the inhibitory effect of residual H2O2 on the anaerobic biodegradability of pretreated HTLaq-1. Later for Part 2, one thermophilic (55C) and one mesophilic (35C) batch of BMP assays were set up with various substrates (pretreated and unpretreated HTLaq-2 in Table 3-2, with/without quencher) at similar SIR ratio as part one (0.25 39 mg COD of substrate/mg VS of inoculum). This SIR ratio was selected as an optimized SIR ratio for similar conditions with HTLaq as substrate done previously in BTG (Basar, 2024). Anaerobic mesophilic and thermophilic inocula for the BMP assays, was harvested from two lab-scale digesters operating under semi-continuous flow conditions at 35C and 55C, with SRTs of 20 days. MS from Annacis Island WWTP was used to feed the digesters. Before setting up the BMP assays, bacterial cultures were starved for three to five days to minimize the VFA and methane production from the inoculum itself. After the acclimatization and starvation, inoculum was characterized for different parameters (pH, TS, VS, VFA, TAN, total phenolics, total alkalinity and tCOD). The BMP assays were conducted using 162 mL serum bottles with a liquid volume 70 mL of total liquid leaving 92 mL for gas headspace (Figure 3.3). The set up for the assays followed the method developed by BTG lab (Başar et al., 2022; Kumar et al., 2023). For each BMP assays four replicates were set up, and one set of positive controls (glucose as substrate) and blanks (inoculum only) were included. Among the four BMP bottles, one (sacrificial) bottle was for measuring pH and VFA and three bottles for biogas/methane production measurements. Substrate volume was determined based on tCOD of unpretreated and pretreated HTLaq samples, while the inoculum (50 mL) and total liquid volume (70 mL) were consistent for all the BMP bottles in the same batch of testing. After addition of substrate and inoculum, RO water was used to make up 70 mL of liquid volume. No external buffer or nutrients were needed, as HTLaq contains sufficient amount due to the polar and nonpolar compounds in HTLaq (Basar, 2024). However, for the BMP assays for Part 2, 1 M hydrochloric acid solution to adjust the initial pH to neutral range (7.5 - 8) before sealing the bottles. This decision was made to neutralize the high pH imposed by the quencher. At the end, the bottles were purged with ultra-pure nitrogen gas (99.99 %) for two 40 minutes to remove dissolved oxygen and sealed with air-tight rubber septa and aluminum caps. Later, the bottles were incubated under mesophilic (35C) or thermophilic (55C) conditions with a mixing speed of 95 rpm in the rotary shaking incubators (Figure 3.3). Figure 3.3. Images of (a) New Brunswick Innova®44 shaking incubator, (b) batch of BMP assay bottles, and (c) Agilent 7820A gas chromatography - thermal conductivity detector. After the setup, a handheld manometer was used to periodically check the pressure inside the bottles until the specific (mL/g CODfed) cumulative methane yield reached a plateau. An Agilent 7820A GC equipped with thermal conductivity detector (TCD) (Figure 3.3) was used to analyze the biogas composition, which quantified methane, carbon dioxide, oxygen and nitrogen content. To calibrate the GC/TCD, a standard gas mixture of 7% nitrogen, 20% carbon dioxide, and 73% methane was used. Specific cumulative methane yield of substrate was determined by deducting methane production from blank BMP bottles (inoculum only) from methane production from other assays (substrate + inoculum), and results were reported as mL CH4/mg COD of HTLaq at standard temperature and pressure (0C, 1 atm). Alongside gas measurements, pH and VFA 41 samples were taken at the measurement points to evaluate the VFA/pH changes throughout the BMP assay. Further details regarding BMP assays are included in Appendix B. Tables B-1, B-2 and B-3 show examples of set-up calculation for each BMP assay from Part 1 mesophilic BMP, and Part 2 mesophilic and thermophilic BMPs, respectively. 3.6.2 Biochemical oxygen demand (BOD) assays for aerobic biodegradability assessment To determine the aerobic biodegradability of HTLaq samples before and after pretreatment, BOD assays were conducted using an online PF-800 Pulse Flow Respirometer. The methodology was based on Standard Methods 5210 D (American Public Health Association, 2005). In short, aerobic inoculum was obtained from a solid contact tank in a local WWTP in Vancouver, BC. Before the BOD test, the inoculum was pre-aerated and acclimatized to HTLaq in a separate reactor. This acclimatizing process was done by supplying 0.3% of the acclimation reactor volume (4 L) with HTLaq every day for almost 2 weeks. A total of 12 sets of BOD assays were set up in duplicates, which includes 10 sets of unpretreated and pretreated HTLaq, 1 set of blank (only aerobic inoculum without any substrate), and 1 set of positive control (glucose as substrate). The online respirometer had borosilicate bottle reactors with 600 mL volume, which were used as BOD assays. The SIR ratio for the BOD assays was 10 g COD of substrate/g VSS of inoculum. This SIR ratio was chosen based on the optimized BOD method on the same substrate performed previously in BTG. BOD bottles were filled up with acclimatized aerobic inoculum (approximately 75% of bottle volume determined by SIR), RO water (500 mL), mineral base, nutrient base, nitrification inhibitor (10 mg/L of allythiourea), and HTLaq sample as substrate or glucose (5 mL). The details of the nutrient and mineral base 42 chemicals are in Appendix section C. In each BOD bottle, a tube with 5 mL of 30% potassium hydroxide solution was placed above the assays to absorb the generated CO2 gas. After the set-up, BOD assays were sealed, submerged in the water bath (20C), and connected to a gas counter with ultra-pure oxygen (99.9%)-feeding from a pressured cylinder. Each reactor bottle was constantly mixed at 600 rpm with a magnetic stirrer. Once the cumulative oxygen uptake reached a plateau (26 days), the respirometer analysis was terminated. The respirometer recorded the oxygen uptake rate versus time (Figure 3.5). BOD of the samples was calculated after subtracting the oxygen uptake of the blank (aerobic inoculum only) from the oxygen uptake of other bottles (substrate + inoculum), in units of mg cumulative oxygen uptake per L of the substrate. Figure 3.4 Image of (a) batch of BOD assays and (b) online respirometer for aerobic biodegradability assessment. 3.6.3 Data analysis of biodegradability assessments To fit the cumulative methane production (BMPt) and cumulative oxygen uptake curves (BODt) versus time to the modified Gompertz model, the following equation 3.6 was used by OriginPro software. 43 BMPt or BODt=P×exp{-exp[Rm×eP×(λ-t)+1]} Equation 3.6 where: P = maximum specific methane production (mL CH4/g COD) or oxygen consumption (mg O2/L) Rm = maximum rate (mL CH4/g COD/d or mg O2/L/d)  = lag phase duration (d) t = incubation time (d). Along with all these parameters, the co-efficient of determination (R2) was calculated to observe the accuracy of the model’s fit. If the R2 was close to 1, the fit was more accurate. To account for the uncertainties, experimental error during BMP and BOD assay, standard deviation and standard error was considered. To determine the significance between the results of biodegradation rate and extent obtained for each substrate, an ANOVA and Tukey HSD (honestly significant difference) post hoc comparison test was conducted for different incubation times. SPSS software was used in this analysis. Later p-value was compared to determine the significance difference between the assays. 44 Chapter 4: Results and discussion This chapter includes the results and discussion of the laboratory experiments conducted. The chapter is divided into two parts: Part 1: H2O2 dosage optimization for pretreatment of HTLaq with/without quenchers, and Part 2: Biodegradability assessment of HTLaq samples before and after various H2O2 pretreatments. 4.1 Part 1: H2O2 dosage optimization for pretreatment of HTLaq with/without quenchers 4.1.1 Characterization of HTL feedstock (MS) and HTLaq streams As mentioned in Chapter 3, two batches of HTL processing were conducted to generate two different HTLaq samples (HTLaq-1 and HTLaq-2) from mixed sludge sampled from the WWTP at different seasons (MS-1 and MS-2). It is worth mentioning that the MS characteristics change based on seasonality. Table 4-1 presents HTL feedstock and HTLaq characterization results for HTLaq-1 and HTLaq-2 and their corresponding sludges (MS-1 and MS-2, respectively). The pH of HTLaq-1 was found at 5.7, which was much lower than that of HTLaq-2 (pH of 8.1), which was also produced under the same HTL operating conditions but from a different MS feedstock. This could be a result of the more acidic nature of MS-1 compared to MS-2. Table 4-1: HTL mixed sludge (MS) feedstock and HTLaq characterization. Parameter MS-1 HTLaq-1 MS-2 HTLaq-2 tCODa (g/L) 49.7 ±g 0.6 64.0 ± 0.76 49.0 ± 0.3 63.0 ± 0.2 Total VFAb (g/L) 3.9 ± 0.01 14.0 ± 0.04 3.8 ± 0.14 13.0 ± 0.07 TANc (g NH4-N/L) 0.7 ± 0.02 2.4 ± 0.05 0.7 ± 0.01 2.7 ± 0.07 Total phenolics (g GAEd/L) 0.1 ± 0.01 2.2 ± 0.03 0.1 ± 0.01 1.2 ± 0.01 Total alkalinity (g CaCO3/L) 2.1 ± 0.02 5.1 ± 0.02 2.1 ± 0.01 7.1 ± 0.02 TSe % (w/w) 3.2 ± 0.6 N/A 3.0 ± 0.7 N/A VSf % (w/w) 2.8 ± 0.6 N/A 2.5 ± 0.6 N/A pH 5.85 ± 0.01 5.67 ± 0.01 6.5 ± 0.01 8.1 ± 0.02 aTotal chemical oxygen demand, btotal volatile fatty acids as summation of acetic, propionic and butyric acids, ctotal ammonium nitrogen, dgallic acid equivalent, etotal solids, fvolatile solids, gdata represents the average ± standard deviation of triplicates, N/A: not available. 45 4.1.2 H2O2 pretreatment of HTLaq without quenchers In the initial stage of the pretreatment process, only H2O2 was added to HTLaq-1 at dosages of 0.25, 0.50, and 0.75 g H2O2/g COD of HTLaq (corresponding to sample names of 0.25H, 0.5H and 0.75H, respectively), and tCOD, pH and color in HTLaq-1 samples were measured over different pretreatment times. According to Figure 4.1(a), tCOD levels in HTLaq-1 samples did not show a trend, and there were large discrepancies and high standard deviation among results, even for replicates from the same sample. This may be due to residual H2O2 in the pretreated samples, which interferes with the tCOD method. Quispe-Arpasi (2018) reported that with an increase of H2O2 dosage (0.25 to 0.75 g H2O2/g COD) in pretreated HTLaq (Spirulina biomass), COD was reduced, and the COD removal percentage increased from 9.9% 1.6 to 15.3%  1.2 with time. However, this doesn’t align with the findings in current study due to the interference of residual H2O2 with tCOD method. Figure 4.1 (a) tCOD, (b) pH and (c) color of pretreated HTLaq-1 at room temperature (23C) at different pretreatment times for 0.25H, 0.5H and 0.75H. Figure 4.1(b) presents pH evolution over pretreatment time. As seen from the graph, all H2O2 dosages followed a decreasing trend, while after 5 h pretreatment, it reached to a plateau in all dosages. Among pretreated samples, 0.75H showed the highest pH decrement (from 5.70 to 46 5.40), followed by 0.5H (from 5.67 to 5.43) and 0.25H (from 5.67 to 5.48). Therefore, with a higher dosage of H2O2, a higher pH reduction in HTLaq-1 occurred. Color change assessment in HTLaq-1 also showed a similar trend to pH reduction (Figure 4.1(c)). With the increased H2O2 dosage, the color reduction was more visible. However, even after 24 h of pretreatment, the color intensity continued to decline due to the remaining H2O2 residual in the pretreated sample. 4.1.2.1 Anaerobic biodegradability assessment of pretreated HTLaq-1 for possible inhibition from residual H2O2 To determine the anaerobic biodegradability of the pretreated HTLaq-1, BMP assays were conducted with 3 substrates (HTLaq-1, 0.5H, and glucose) under mesophilic conditions as shown in Figure 4.2. For pretreatment dose 0.5H was considered based on the results derived from Quispe-Arpasi, (2018) where the highest ultimate cumulative methane yield improvement (13%) during mesophilic BMP assays was achieved while HTLaq (Spirulina biomass) pretreated with 0.5 g H2O2/g COD was used as a substrate. Therefore, 0.5 g H2O2/g COD was considered to see if residual H2O2 can be inhibitory for this optimal dosage. Figure 4.2 shows cumulative specific methane yield over incubation time for 38 days. Methane production results showed acute inhibition for the BMP assay using pretreated HTLaq-1 at dosage of 0.5H (no quencher) for the first 12 days of the incubation. In the figure it is visible that positive control (glucose) had an ultimate specific methane potential of 340 mL CH4/g COD, which is 97% of the ultimate theoretical methane yield of glucose at STP. This result shows that the inoculum (microbial culture) was working properly during the BMP test. 47 Figure 4.2 Mesophilic (35ºC) specific cumulative methane yield over time for positive control (glucose), pretreated (0.5H) and unpretreated HTLaq-1. Sample of 0.5H corresponds to 0.50 g H2O2/g COD of HTLaq. While HTLaq-1 was used as the substrate, the assay resulted in an ultimate specific methane production of 160 mL CH4/g COD over 37 days of incubation and showed no inhibition during the process. However, for the 0.5H pretreated sample (at 0.5 g H2O2/g COD) as substrate, acute inhibition was exhibited during the first 12 days of the experiment. After that, the inoculum started to recover, and the ultimate specific cumulative methane yield reached 160 mL CH4/g COD after 37 days of incubation which is the same as the un-pretreated HTLaq-1’s ultimate specific methane yield. The acute inhibition in early incubation time for 0.5H assay was due to the H2O2 residuals in 0.5H causing oxidative stress on the anaerobic cultures. BMP assay VFA results are shown in Figure 4.3. For both HTLaq-1 and 0.5H assays, the VFA concentration reached maximum at day 1. At day 1, unpretreated HTLaq-1 assays reached to 690 mg/L of total VFA and pretreated HTLaq (0.5H) resulted in 850 mg/L of VFA. Later, 48 unpretreated HTLaq-1 VFA dropped at day 2 and showed a gradual increase until day 12; later, on, day 17, VFA dropped to 120 mg/L, and all the VFAs were consumed after day 17. For pretreated HTLaq (0.5H), a different trend was visible where after the initial increase at day 1 it started to gradually decrease and reached zero at day 6. For both assays, VFAs dropping to 0 mg/L indicates successful completion of batch digestion as the anaerobic cultures had consumed all VFAs available. Unpretreated HTLaq-1 took longer to utilize the VFA content for methane generation, whereas pretreated HTLaq exhausted VFAs in 6 days. However, higher VFA content on days 1 and 2 of 0.5H BMP assays could be correlated to acute inhibition from residual H2O2. Figure 4.3 VFA concentration during mesophilic (35ºC) biochemical methane potential for (a) unpretreated HTLaq-1 and (b) pretreated (0.5H). Sample of 0.5H corresponds to 0.50 g H2O2/g COD of HTLaq. Throughout the test, the pH of the BMP assay’s content was monitored, and the results are shown in Figure 4.4. In the assays, the pH ranged from 8.1 to 7.7, which was in the optimized range for anaerobic cultures. The initial pH was slightly high due to the high alkalinity present in HTLaq-1. 49 Figure 4.4 pH during mesophilic (35ºC) biochemical methane potential assays for pretreated (0.5H) and unpretreated HTLaq-1. Sample of 0.5H corresponds to 0.50 g H2O2/g COD of HTLaq. 4.1.3 Optimization of the quenchers toward removal of residual peroxide Based on the results showing acute inhibition in Figure 4.2, finding an appropriate quencher became necessary for removing the residual H2O2 from the pretreated HTLaq. Sodium carbonate and sodium sulphite were used as quenchers. The HTLaq-1 sample was used in all the experiments in this section. 4.1.3.1 Finding the optimum dosage of quencher For determining the optimal quencher, the main consideration was that the quenchers needed to have the minimum interference with the tCOD method. Firstly, to determine if the quenchers brought additional tCOD to samples, tCOD tests were conducted on quenchers as well as HTLaq-1 and H2O2 pretreated HTLaq-1 with added quenchers at different dosages at room temperature. The results were compared with the theoretical tCOD contribution based on the quencher’s chemical formula. 50 When sodium sulphite was added to HTLaq-1 at different dosages (Figure 4.5 (a)), it increased the tCOD of HTLaq-1 from 64,000 mg/L to a maximum of 115,000 mg/L at 1:4 level. On the other hand, adding sodium sulphite to pretreated HTLaq-1 lowered the tCOD at lower dosages of sodium sulphite (quencher: H2O2 ratios of 1:1, 1:2 by wt.), but started to increase tCOD with an increase of sodium sulphite dosage. Sodium sulphite itself also showed tCOD contribution Figure 4.5 (a) The impact of sodium sulphite addition as quencher on tCOD of HTLaq-1 and pretreated HTLaq-1 at different dosages, (b) theoretical and experimental tCOD comparison of sodium sulphite at different dosages, (c) the impact of sodium carbonate addition as quencher on tCOD of HTLaq and pretreated HTLaq at different dosages, (d) theoretical and experimental tCOD comparison of sodium carbonate at different dosages. Both H2O2 pretreatment and quencher addition was at room temperature (22ºC). NS: sodium sulphite, NC: sodium carbonate. 51 in a range of 50-400 mg/L at different concentrations, which were approximately 15% lower than the theoretical tCOD of sodium sulphite in all concentrations (Figure 4.5 (b)). When sodium carbonate was added to unpretreated HTLaq-1 as a quencher (Figure 4.5(c)), it increased the tCOD by ≈ 10%. Oppositely, the impact of sodium carbonate addition to pretreated HTLaq-1 was not discernable compared to sodium sulphite’s effect on tCOD. For both HTLaq samples (untreated and pretreated), tCOD stayed in the range of 72,000-78,000 mg/L. Also, testing tCOD for sodium carbonate itself proved that this quencher did not bring additional tCOD to samples (Figure 4.5 (d)). The measured tCOD of sodium carbonate was negligible (2-12 mg/L) compared to 64,000 mg COD/L of HTLaq. Ultimately, based on these results, the lowest dosage of quencher (H2O2: quencher of 1:1 (by wt.)) was selected as it showed the minimum effect on tCOD of HTL samples for the next experimental steps for both sodium sulphite and sodium carbonate quenchers. 4.1.4 Optimizing pretreatment temperature and time in the presence of quencher As mentioned in Chapter 3, in this part, two sets of experiments were conducted. In the first set, H2O2 pretreatment was performed on HTLaq-1 at room temperature (22C), and quenchers were added to pretreated samples at different pretreatment times (1 h, 2 h, 3 h, and 4 h). For each sample, at the end of pretreatment, the temperature was increased to 60  2C upon quencher addition and maintained for an additional 3 h. This 3 h additional time was provided to provide enough time for the reaction of quencher with the residual H2O2. Considering the additional 3 h, the total pretreatment time was 4, 5, 6, and 7 h. 52 Figure 4.6 shows time vs. tCOD for pretreated HTLaq-1 at different time duration. The results show that between 4-7 h of pretreatment, the tCOD values were similar for both quenchers (60,000-62,000 mg/L). Therefore, 2 h of pretreatment before the addition of quencher (totaling 5 h of pretreatment + quencher) was selected to reduce the total time requirement for the treatment process. Figure 4.6 tCOD concentration of pretreated HTLaq-1 samples at different times (1-4 h). 0.75H+NC and 0.75H+NS represent pretreated samples at the dose of 0.75 g H2O2/g COD of HTLaq-1 followed by sodium carbonate and sodium sulphite quenchers (H2O2: quencher of 1:1 by wt.), respectively. H2O2 and quencher additions were at room temperature (22C) and 60  2C, respectively, total pretreatment+quencher addition time is reported. At t=0, no pretreatment was conducted. Table 4-2 reports other characteristics of non-pretreated and pretreated HTLaq-1 samples in the same experiment. While sodium sulphite was used as a quencher (0.75H+NS), TAN increased from 2680 to around 3300-3800 mg NH4-N/L. On the other hand, TAN reduction occurred when sodium carbonate was used (0.75H+NC with 1600 mg NH4-N/L). For both 53 quenchers, throughout the pretreatment process, total VFA decreased slightly and stayed in the range of 10,000-12,000 mg/L. Total phenolics were also reduced by 15 and 50%, for 0.75H+NS and 0.75H+NC samples, respectively. However, sodium carbonate showed a higher effect on pH by increasing it from 8.10 to 10.39 due it its buffering capacity, which is concerning for the biodegradability assessment of 0.75H+NC. Table 4-2 Characterization of non-pretreated and pretreated HTLaq-1 samples at different times. 0.75H+NC and 0.75H+NS represent pretreated samples at the dose of 0.75 g H2O2/g COD of HTLaq-1 followed by sodium carbonate and sodium sulphite quenchers (H2O2: quencher of 1:1 by wt.), respectively. H2O2 and quencher additions were at room temperature (22C) and 602C, respectively. Sample Time (h) Total pretreatment time (h) TANa (mg NH4-N/L) Total phenolics (mg GAEb/L) Total VFAc (mg/L) pH HTLaq-1 2680 ±f 65 1190 ± 12 13000 ± 73 8.10 0.75H+NS 1 4 3800 ± 49 966 ± 23 10200 ± 82 8.18 2 5 3600 ± 42 953 ± 23 11200 ± 51 8.30 3 6 3800 ± 41 1000 ± 6 11500 ± 22 8.30 4 7 3300 ± 16 1010 ± 25 10800 ± 26 8.17 0.7H+NC 1 4 1600 ± 22 638 ± 12 11000 ± 138 10.31 2 5 2100 ± 9 577 ± 74 11200 ± 107 10.33 3 6 2300 ± 59 638 ± 18 11700 ± 50 10.39 4 7 1600 ± 12 631 ± 10 11800 ± 15 10.32 aTotal ammonium nitrogen, bgallic acid equivalent, ctotal volatile fatty acids as summation of acetic, propionic and butyric acids, fdata represents the average ± standard deviation of triplicates. In Table 4-3, the concentration of the selected group of N-heterocyclic compounds in HTLaq-1 and their percent removals after pretreatment is tabulated. 2-methyl pyrazine was found to be the highest concentrated N-heterocyclic compound in HTLaq (1198 mg/L). For 0.75H+NS, the highest % removal for the N-heterocyclic compounds was after 2 h pretreatment (totaling 5 hr H2O2 + quencher). For 0.75H+NC, the removal percentage of the N-heterocyclic compounds 54 increased with time. However, for both samples, the pretreatment lowered the N-heterocyclic compound concentrations in the HTLaq-1. Table 4-3 N-heterocyclic compounds removal from pretreated HTLaq-1 samples at different times. 0.75H+NC and 0.75H+NS represent pretreated samples at the dose of 0.75 g H2O2/g COD of HTLaq-1 followed by sodium carbonate and sodium sulphite quenchers (H2O2: quencher of 1:1 by wt.), respectively. H2O2 and quencher additions were at room temperature (22C) and 602C, respectively. aData represents the average ± standard deviation of triplicates, bundetected components, total pretreatment duration is reported. In the second part of the experiments, H2O2 pretreatment was conducted at higher temperature (602C for 2 h) followed by a quencher addition. 2 h pretreatment time was selected based on the first part of the experiment. The samples were, then, left at room temperature (22C) for different times (2 h, 3 h, 4 h, and 5 h). Figure 4.7 shows time vs. tCOD for pretreated HTLaq-1 assays for different quencher addition waiting time. Results reveal that the quencher reaction time does not impact the tCOD. For example, for 0.75H+NC, 3 h after quencher addition, the tCOD concentration is similar to tCODs after 4 h and 5 h. Also, for 0.75H+NC, there is an overall tCOD removal of 15% after 5 h. Compound name Concentration in HTLaq-1 (mg/L) 0.75H+NS 0.75H+NC 4 h 5 h 6 h 7 h 4 h 5 h 6 h 7 h 2-methyl pyridine 27.7 ±a 0.08 51% 65% 52% 46% 59% 58% 61% 73% 2-methyl pyrazine 1198 ± 3.42 14% 44% 10% 29% 46% 17% 43% 35% 2,6-dimethylpyridine 2.05 ± 0.14 41% 65% 50% 45% -b 60% 63% 80% 2-ethyl-pyridine 0.89 ± 0.08 21% 58% 49% 33% - - - - Ethyl-pyrazine 228 ± 0.72 39% 45% 32% 32% 30% 32% 33% 49% 2,3-eimethyl pyrazine 153 ± 0.05 37% 38% 25% 27% 20% 27% 27% 39% 2,3,5-trimethylpyrazine 108 ± 5.27 43% 48% 27% 32% 16% 33% 32% 51% 5-ethyl-2-methyl- pyridine 3.05 ± 0.29 78% 87% 83% 80% 66% 69% 69% 82% 55 Figure 4.7 tCOD concentration of pretreated HTLaq-1 samples versus time. 0.75H+NC and 0.75H+NS represent pretreated samples at the dose of 0.75 g H2O2/g COD of HTLaq-1 followed by sodium carbonate and sodium sulphite quenchers (H2O2: quencher of 1:1 by wt.), respectively. H2O2 addition was at 60  2C for 2 h, followed by quencher addition/cooling to room temperature (22C) in 2-5 h, total pretreatment time is reported. At t=0, no pretreatment was conducted. Two sets (for two quenchers) of one-way ANOVA test were conducted to determine the statistical difference between means in the COD results for the last three waiting periods (5-7 h overall pretreatment time). For the assays where sodium carbonate was used as quencher, probability of the similarity of the assays with different quencher reaction times (p-value) was 0.075>0.05. Similarly, in case of COD results for assays with sodium sulphite as quencher, p-value was 0.063>0.05. Consequently, for both cases the results do not show statistically significant difference. This observation means that based on COD results, quencher addition waiting period between 3-5 h are similar. 56 Table 4-4 ANOVA summary table for analysis of the significant mean difference between different COD results during quencher optimizing waiting period (5-7 h overall pretreatment time), at a 95% confidence interval (α=0.05). aResults between different time duration, bresults for same assays, cdegree of freedom, dsignificance or p-value. For parameters other than tCOD, Table 4-5 shows a similar trend as Table 4-3. For 0.75H+NS, TAN concentration changed from 2680 to around 3000-3300 mg NH4-N/L. On the contrary, TAN was almost reduced to half for 0.75H+NC compared to unpretreated HTLaq-1. For both quenchers, total phenolics were reduced where 0.75H+NC showed more reduction compared to 0.75H+NS. Total VFA showed an almost similar trend for both cases. VFA was reduced by 7-14% for both quenchers. Similar to the part-I, pH was also considerably high for 0.75H+NC which was around 10.3 due to extra buffering capacity from sodium carbonate. ANOVA test results: Sodium carbonate as quencher Sum of Squares dfc Mean Square F Sig.d Between Groupsa 46055501.930 2 23027750.965 4.103 0.075 Within Groupsb 33677057.005 6 5612842.834 Total 797332558.935 8 ANOVA test results: Sodium sulphite as quencher Sum of Squares df Mean Square F Sig. Between Groups 165401329.89 2 82700664.943 4.555 0.063 Within Groups 108940014.42 6 18156669.069 Total 274341344.30 8 57 Table 4-5 Characterization of non-pretreated and pretreated HTLaq samples versus time. 0.75H+NC and 0.75H+NS represent pretreated samples at the dose of 0.75 g H2O2/g COD of HTLaq followed by sodium carbonate and sodium sulphite quenchers (H2O2: quencher of 1:1 by wt.), respectively. H2O2 addition was at 602C for 2 h, followed by quencher addition/cooling to room temperature (22C) in 2-5 h. Sample Time (h) Total pretreatment time (h) TANa (mg NH4-N/L) Total phenolics (mg GAEb /L) Total VFAc (mg/L) pH HTLaq-1 2680 ±f 65 1190 ± 12 13000 ± 73 8.10 0.75H+NS 2 4 3140 ± 370 966 ± 23 11100 ± 107 8.12 3 5 3300 ± 110 953 ± 23 11700 ± 100 8.08 4 6 3020 ± 500 1024 ± 6 17600 ± 234 8.05 5 7 3300 ± 160 1034 ± 25 12049 ± 174 8.10 0.75H+NC 2 4 416 ± 18 759 ± 14 12200 ± 85 10.22 3 5 1900 ± 10 692 ± 7 11900 ± 102 10.31 4 6 1400 ± 27 671 ± 10 11200 ± 33 10.31 5 7 1830 ± 32 673 ± 7 11900 ± 117 10.33 aTotal ammonium nitrogen, bgallic acid equivalent, ctotal volatile fatty acids as a summation of acetic, propionic and butyric acids, fdata represents the average ± standard deviation of triplicates. Similar to the first set of experiments, a clear reduction of N-heterocyclic compounds was detected in this experiment (Table 4-6). 0.75H+NS sample yielded the highest compound removal after 3 h of treatment. Similar to Table 4-3, with longer treatment time, N-heterocyclic compound removal % was higher for the 0.75H+NC sample. From the results in section 4.1.4, it was determined that temperature changes did not affect the parameters of HTLaq-1 for the pretreatment process. In both sets of testing, the results showed a similar trend. Also, 2 h after H2O2 addition and 3 h after quencher addition, HTLaq-1 showed a stable tCOD value, therefore these pretreatment conditions were implemented for the next steps. Also, the effect of temperature on the pretreatment process was negligible and changing temperature during the overall pretreatment is not energy efficient. Which is why the room temperature (22C) was selected for the next experiments. 58 Table 4-6 N-heterocyclic compounds removal from pretreated HTLaq-1 samples versus times. 0.75H+NC and 0.75H+NS represent pretreated samples at the dose of 0.75 g H2O2/g COD of HTLaq followed by sodium carbonate and sodium sulphite quenchers (H2O2: quencher of 1:1 by wt.), respectively. H2O2 addition was at 602C for 2 h, followed by quencher addition/cooling to room temperature (22C) in 2-5 h. aData represents the average ± standard deviation of triplicates, total pretreatment time is reported, b“-“ depicts undetected compound. 4.1.5 Pretreatment of HTLaq under optimized conditions Based on the optimization results presented so far, for this section, pretreatment was conducted for both HTLaq-1 and HTLaq-2 samples at dosages of 0.25, 0.50, and 0.75 g H2O2/g COD of HTLaq for 2 h at room temperature, followed by a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h) at room temperature (23C). Figures 4.8 (a) and (b) show the tCOD concentrations for HTLaq-1 and HTLaq-2 samples, respectively. For HTLaq-1, tCOD was reduced from 67,000 to almost 55,000 mg/L for the highest dosage of 0.75 g H2O2/g COD of HTLaq for both quencher scenarios (0.75H+NC and 0.75H+NS). Compound name Concentration in HTLaq-1 (mg/L) 0.75H+NS 0.75H+NC 4 h 5 h 6 h 7 h 4 h 5 h 6 h 7 h 2-methyl pyridine 27.7 ± a 0.08 51% 65% 52% 46% 59% 58% 61% 73% 2-methyl pyrazine 1198 ± 3.42 14% 44% 10% 29% 46% 17% 43% 35% 2,6-dimethylpyridine 2.05 ± 0.14 41% 65% 50% 45% - b 60% 63% 80% 2-ethyl-pyridine 0.89 ± 0.08 21% 58% 49% 33% - - - - Ethyl-pyrazine 228 ± 0.72 39% 45% 32% 32% 30% 32% 33% 49% 2,3-dimethyl pyrazine 153 ± 0.05 37% 38% 25% 27% 20% 27% 27% 39% 2,3,5-trimethylpyrazine 108 ± 5.27 43% 48% 27% 32% 16% 33% 32% 51% 5-ethyl-2-methyl- pyridine 3.05 ± 0.29 78% 87% 83% 80% 66% 69% 69% 82% 59 For the other two dosages (0.25, 0.50 g H2O2/g COD), tCOD values were still lower than untreated HTLaq. Figure 4.8(b) shows the tCOD results for the HTLaq-2 sample pretreated under similar conditions as the HTLaq-1. tCOD was reduced by 18% with the highest dosage of H2O2 with a quencher. In contrast, samples pretreated with only H2O2 exhibited a substantial increase (67,000 to almost 86,000 mg/L) in tCOD due to the residual H2O2 in the samples. Figure 4.8 (b) confirms that the increase of H2O2 leads to increasing residual H2O2 resulting in higher false tCOD. Thus, it can be concluded that samples with quencher result in accurate tCOD by removing the residual H2O2 from the sample. In Tables 4-7, other parameter concentrations are reported for the pretreatment process of HTLaq-1 and HTLaq-2. For HTLaq-1, more phenolics and VFA removal occurred when quenchers were used. However, TAN and pH increased after using a quencher in the sample. When the treatment was conducted without any quencher (0.5H and 0.75H) TAN was more than half of Figure 4.8 (a) tCOD concentration for pretreated HTLaq-1 with and without quencher for different dosages, (b) tCOD concentration for pretreated HTLaq-2 with and without quencher for different dosages. H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). 60 what was in the HTLaq. But for the samples with the quencher, TAN increased with the H2O2 dosages increase. For samples with sodium carbonate, pH was higher ranging between 9.98 and 10.35, compared to other samples. Table 4-7 Parameter characterizations for pretreated HTLaq with and without quencher for different dosages. H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). Sample TANa (mg NH4-N/L) Total phenolics (mg GAE b /L) Total VFAc (mg/L) pH HTLaq-1 HTLaq-1 2680 ± 65 1190 ± 12 13000 ± 73 8.10 0.25H 2500 ± 240 870 ± 27 10300 ± 300 8.10 0.5H 1200 ± 140 1200 ± 30 9800 ± 180 8.10 0.75H 1000 ± 27 575 ± 2 7900 ± 240 7.99 0.25H+NSe 6300 ± 70 742 ± 66 7420 ± 120 8.40 0.5H+NS 7530 ± 50 612 ± 90 6900 ± 110 8.45 0.75H+NS 7580 ± 170 980 ± 17 11200 ± 17 7.96 0.25H+NCf 5120 ± 50 570 ± 170 8080 ± 170 9.98 0.5H+NC 5840 ± 100 450 ± 25 6600 ± 280 10.25 0.75H+NC 5990 ± 73 640 ± 17 12000 ± 180 10.35 HTLaq-2 HTLaq-2 2460 ± 57 1900 ± 41 8090 ± 100 7.83 0.25H 1400 ± 350 710 ± 14 8840 ± 80 7.26 0.5H 1150 ± 21 705 ± 11 7700 ± 66 7.11 0.75H 470 ± 160 926 ± 13 6280 ± 44 7.04 0.25H+NS 2380 ± 30 1060 ± 10 7430 ± 9 7.86 0.5H+NS 2070 ± 24 940 ± 2 7500 ± 48 7.65 0.75H+NS 2230 ± 5 440 ± 40 7500 ± 34 8.69 0.25H+NC 1850 ± 9 800 ± 4 8300 ± 73 10.01 0.5H+NC 1270 ± 26 550 ± 5 7200 ± 37 10.31 0.75H+NC 1260 ± 80 620 ± 10 8090 ± 40 10.44 aTotal ammonium nitrogen, bgallic acid equivalent, ctotal volatile fatty acids as summation of acetic, propionic and butyric acids, ddata represents the average ± standard deviation of triplicates, esodium sulphite, fsodium carbonate. 61 In the case of HTLaq-2, TAN and total phenolics decreased with the pretreatment. Samples with Na2CO3 as a quencher resulted in higher TAN and phenolic removal compared to those with Na2SO3. Despite the pretreatment process, VFA levels remained similar in the pretreated samples, which have the potential for valorization and can produce methane during AD. However, the pH values were higher in the samples mixed with Na2CO3 as a quencher. GC-MS results for pretreated HTLaq-1 and HTLaq-2 are shown in Table 4-8. Overall, both HTLaq samples exhibited higher pyrazine removal. For HTLaq-1, pretreatment with the highest H2O2 dosage without quenchers (0.75H) resulted in the highest percentage of N-heterocyclic compound removal. For samples with quenchers, 0.25H+NS and 0.5H+NC achieved the maximum reduction of 2-methyl-pyridine, 2-methyl-pyrazine, 2-ethyl-pyridine, 2,3-dimethyl-pyrazine, and 5-ethyl-2-methyl-pyridine. For HTLaq-2, the results showed fluctuations in N-heterocyclic compound levels with pre-treatment. Pyrazines increased with a higher dosage of H2O2, except for the 0.75H+NC sample, which showed a lower value. No clear removal trend was observed for pyridines. 62 Table 4-8 N-heterocyclic compound removal % for pretreated HTLaq-1 and HTLaq-2 with and without quencher for different dosages. H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). aData represents average ± standard deviation of triplicates, bconcentration in HTLaq cnegative value represents % increase, H: hydrogen peroxide, NS: sodium sulphite, NC: sodium carbonate.Compound name HTLaq b (mg/L) 0.25H 0.5H 0.75H 0.25H+NS 0.5H+NS 0.75H+NS 0.25H+NC 0.5H+NC 0.7H+NC HTLaq-1 2-methyl pyridine 27.7 ± a 0.08 8% 15% 34% 41% 33% 26% 43% 48% 23% 2-methyl pyrazine 1198 ± 3.42 20% 22% 34% 9% 24% 13% 25% 51% 12% 2,6-dimethylpyridine 2.05 ± 0.14 8% 13% 21% 28% 28% 2% 34% 37% 37% 2-ethyl-pyridine 0.89 ± 0.08 13% 21% 22% 8% 15% 27% 16% 19% 24% Ethyl-pyrazine 228 ± 0.72 14% 17% 29% 29% 14% 9% 35% 43% 9% 2,3-dimethyl pyrazine 153 ± 0.05 13% 15% 27% 25% 9% 8% 36% 44% 1% 2,3,5-trimethylpyrazine 108 ± 5.27 18% 12% 33% 29% 12% 6% 37% 42% 4% 5-ethyl-2-methyl- pyridine 3.05 ± 0.29 11% 13% 31% 72% 66% 59% 31% 36% 9% HTLaq-2 2-methyl pyridine 43.4 ± 1.1 -4%c 7% 39% -17% -9% -31% -8% -25% 0% 2-methyl pyrazine 933 ± 15.1 5% -3% -17% 0% -8% -11% -1% -11% 0% 2,6-dimethylpyridine 29 ± 2.1 23% 32% 18% 23% 25% 16% 27% 17% 35% 2-ethyl-pyridine 14.3 ± 0.0 16% 26% 22% 19% 17% 17% 27% 19% 25% Ethyl-pyrazine 10.7 ± 0.2 23% 22% 50% 23% 24% 21% 43% 41% 18% 2,3-dimethyl pyrazine 5.9 ± 0.1 24% 33% 19% 22% 24% 8% 17% 8% 21% 2,3,5-trimethylpyrazine 174 ± 2.2 1% -9% -17% -5% -11% -36% -6% -15% -7% 5-ethyl-2-methyl- pyridine 80.7 ± 0.9 2% -8% -23% -4% -11% -35% -4% -14% -6% 63 4.2 Aerobic and anaerobic biodegradability assessments of HTLaq 4.2.1 Inoculum characterization Characterizations of anaerobic (mesophilic and thermophilic) and aerobic inoculum are provided in Table 4-10. Based on the results, all the parameter measurements align well with previous research done by BTG using inocula from similar WWTPs (Aktas, et al., 2024b). Table 4-9 Inoculum characterization for aerobic and anaerobic biodegradability assessment. Parameter Mesophilic anaerobic inoculum Thermophilic anaerobic inoculumb Aerobic inoculumc pH 7.4 ±a 0.02 8.1 ± 0.13 6.4 ± 0.05 TSb % (w/w) 1.17 ± 0.04 1.56 ± 0.01 - VSc % (w/w) 0.86 ± 0.00 1.01 ± 0.00 - Total suspended solids (mg/L) -h - 1228 ± 30.20 Volatile suspended solids (mg/L) - - 1093 ± 27.03 Total VFAd (mg/L) 38.41 ± 8.34 223.5 ± 10.67 454 ± 53.12 TANe (mg NH4-N/L) 1490 ± 17.80 1155 ± 17.70 40.1 ± 0.25 Total phenolics (mg GAEf/L) 16.2 ± 0.60 39.7 ± 2.50 4.1 ± 1.23 Total alkalinity (mg CaCO3/L) 6201 ± 129 5381 ± 615 190 ± 24 tCODg (mg/L) 19800 ± 640 14500 ± 750 2100 ± 53 aData represents the average ± standard deviation of triplicates, btotal solids, cvolatile solid, dtotal volatile fatty acids as summation of acetic, propionic and butyric acids, etotal ammonium nitrogen, fgallic acid equivalent, gtotal chemical oxygen demand, hnot measured. 64 4.2.2 Biodegradability assessment under anaerobic conditions Figures 4.9 and 4.10 illustrate specific cumulative methane yield (mL CH4/mg COD) for H2O2 pretreated HTLaq-2 as substrate at mesophilic (35C) and thermophilic (55C) temperatures over incubation time, respectively. BMP assays with glucose were used as positive control in both mesophilic and thermophilic conditions. The glucose sample achieved an ultimate specific cumulative methane yield of 0.33 mL CH4/mg COD at both temperatures, equaling to ≈94% of the ultimate theoretical methane yield from glucose at STP in Figures 4.9(a) and 4.10(a). Under mesophilic temperature, untreated HTLaq-2 resulted in an ultimate methane production of 0.18 mL CH4/mg COD over 38 days. This ultimate methane yield was significantly lower than the ultimate methane yields positive control (glucose as substrate) achieved under a similar condition (Figures 4.9(a/b)). This shows that non-pretreated HTLaq contains refractory COD, not amenable to methane production. For HTLaq-2 pretreated with only H2O2 at a higher dosage (0.75H), there was acute inhibition in the first days (1-3 d) of the incubation. However, at lower H2O2 dosages, 0.25H did not show acute inhibition. 0.25H also exhibited an increase in ultimate specific cumulative methane yield to 0.19 mL CH4/mg COD when compared to other H2O2 dosages (0.75H and 0.5H). Notable, when quencher was used, 0.25H+NC and 0.5H+NC assays resulted in higher ultimate specific cumulative methane yield than unpretreated HTLaq-2, (16% and 6% improvement, respectively) (Figure 4.9c). On the contrary, assays with sodium sulphite were as quencher showed no improvement in the ultimate specific methane yield compared to unpretreated HTLaq. Based on these results, it can be said that lower dosages of H2O2 with sodium carbonate as quencher had higher ultimate specific methane yield. Quispe-Arpasi (2018), studied the effect of pretreated H2O2 HTLaq (Spirulina biomass) on cumulative methane production under mesophilic conditions and reported the highest ultimate cumulative methane 65 production. Pretreated HTLaq with 0.5g H2O2/g COD dosage (no quencher), achieved 15% increase in mesophilic specific methane production, compared to unpretreated HTLaq (Quispe-Arpasi, 2018). Figure 4.9 Mesophilic (35°C) BMP results for pretreated HTLaq for different dosage of H2O2 (a) cumulative specific methane yield of glucose, (b) cumulative specific methane yield of H2O2 pretreated HTLaq-2 without quencher, (c) cumulative specific methane yield for H2O2 pretreated HTLaq-2 with sodium carbonate as quencher, (d) cumulative specific methane yield for H2O2 pretreated HTLaq-2 with sodium sulphite as quencher. H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). H: hydrogen peroxide, NC: sodium carbonate, NS: sodium sulphite. 66 Under thermophilic conditions, the overall ultimate specific cumulative methane yield for all the substrates were lower compared to BMP assays at mesophilic temperatures. These results agree with previous studies that compared mesophilic and thermophilic cultures for HTLaq treatment and are attributed to much lower microbial diversity at thermophilic temperatures Figure 4.10 Thermophilic (55°C) BMP results for pretreated HTLaq for different dosage of H2O2 (a) cumulative specific methane yield of glucose, (b) cumulative specific methane yield of H2O2 pretreated HTLaq-2 without quencher, (c) cumulative specific methane yield for H2O2 pretreated HTLaq-2 with sodium carbonate as quencher, (d) cumulative specific methane yield for H2O2 pretreated HTLaq-2 with sodium sulphite as quencher. H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). H: hydrogen peroxide, NC: sodium carbonate, NS: sodium sulphite. 67 (Aktas, et al., 2024b). The unpretreated HTLaq substrate resulted in an ultimate specific cumulative yield of 0.11 mL CH4/mg COD, which was 67% lower than the ultimate specific methane yield of glucose under similar conditions (Figures 4.10(a/b)). BMP samples treated only with H2O2 at thermophilic temperature showed more severe inhibition compared to mesophilic BMP assays, resulting in no improvement in ultimate specific methane yield compared to unpretreated HTLaq (Figures 4.10b). However, substrates pretreated with sodium carbonate (0.25H+NC and 0.5H+NC) as a quencher exhibited an improvement of 63.6% and 36.4% in the ultimate methane yield, respectively, compared to unpretreated HTLaq. The 0.75H+NC BMP assay yielded ultimate specific cumulative methane yield at almost the same level as the unpretreated HTLaq (Figures 4.10c). The ultimate specific cumulative methane yields for 0.25H+NS and 0.5H+NS samples, on the other hand, were slightly higher than the yield of HTLaq, but 0.75H+NS resulted in a significantly lower ultimate specific cumulative methane yield (Figures 4.10d). This indicates an inhibition stemmed from NS quencher itself on methanogens. Higher ultimate specific cumulative methane yield levels in assays using pretreated HTLaq-2 with sodium carbonate quencher as substrate indicates that the pretreatment with H2O2 can reduce the recalcitrant effect of HTLaq. The compounds that are difficult to degrade during biological treatment are converted into easily biodegradable compounds due to partial oxidation from H2O2 pretreatment (Quispe-Arpasi, 2018). Due to the oxidation phase during H2O2 treatment, N-heterocyclic compounds are decreased, which leads to higher methane production during biological treatment (Siddique, 2014). Figures 4.11 and 4.12 show different types of VFAs over incubation time of 38 days for mesophilic and 35 days for thermophilic BMP assays, respectively. Results revealed that total VFA was exhausted after 3 days in the mesophilic BMP assays, except for 0.75H+NS (Figure 68 4.11) where the total VFA was exhausted after 10 days of incubation. In Figure 4.12, the total VFA in pretreated substrates were exhausted over 25 days of thermophilic incubation. Similar to the mesophilic condition, 0.75H+NS also resulted in higher total VFAs under thermophilic condition (Figure 4.12). Inhibition due to high sodium sulphite dosage could be a reason behind the increasing VFA results. During the reaction of sodium sulphite and H2O2, sodium sulphate is generated. Studies showed sulphate (SO42-) can cause inhibition in AD of organic matter and decrease methane yield (Chen et al., 2019; Yuan et al., 2020). Higher VFAs show that 0.75H+NS had delayed hydrolysis and acidogenesis phase during AD. Figures 4.11 and 4.12 indicate that thermophilic archaea (culture) took longer to convert VFAs into methane. In case of different type VFA concentration, it is visible that butyric acid concentration was less than acetic acid and propionic acid during BMP assays (mesophilic and thermophilic). During mesophilic BMP assays (Figure 4.11), at day zero acetic acid concentration was highest with low amount of propionic acid for almost all the samples. However, for 0.75H+NS, all the propionic acid was used after 3 days of mesophilic incubation and acetic acid concentration was increased to ≈325 mg/L at day 3. Compared to mesophilic BMP assays, thermophilic BMP assays (Figure 4.12) had higher VFA (acetic and propionic acid) throughout the 35 days incubation period. Similar to mesophilic assays almost all the butyric acid was exhausted. The substrates which showed inhibition in methane yield results resulted in higher propionic acids compared to other substrates during the last day of the incubation period. 69 Figure 4.11 VFA concentration throughout mesophilic BMP assays for pretreated and unpretreated HTLaq-2. H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). H: H2O2, NC: Sodium carbonate, NS: Sodium sulphite. 70 Figure 4.12 VFA concentration throughout thermophilic BMP assays for pretreated and unpretreated HTLaq-2. H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). H: H2O2, NC: Sodium carbonate, NS: Sodium sulphite. 71 More pyridines were utilized than pyrazines during thermophilic AD (Figure 4.13). Almost 99% of the pyridines were degraded through thermophilic BMPs during the 35 days. Samples with 0.5H+NC and 0.75H+NC showed lower N-heterocycles concentration. The results indicate that pretreated samples with sodium carbonate as quencher reduced the inhibition due to reduced N-heterocyclic compounds. Figure 4.13 N-heterocyclic compounds distribution at the termination of thermophilic BMP assays utilizing pretreated HTLaq. For pretreatment, H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). H: hydrogen peroxide, NC: sodium carbonate, NS: sodium sulphite. Apart from VFA and N-heterocyclic compounds, pH was also monitored throughout the BMP for both mesophilic (Appendix Table D1) and thermophilic (Appendix Table D2) incubation. During mesophilic incubation, the pH ranged from 7.07 to 8.04 over 38 days, while for thermophilic BMP assays, the pH ranged from 7.42 to 8.24 over 35 days. Monitoring the pH helped determine whether the anaerobic inoculum was in optimal condition. The Modified Gompertz equation was used to estimate kinetic parameters from specific cumulative methane curves (Table 4-10). It was observed that the reaction rate (Rm) was higher in 72 mesophilic conditions compared to thermophilic counterparts, confirming that the thermophilic inocula are more sensitive to HTLaq substrates. In both mesophilic and thermophilic BMP assays, 0.25H+NC had the highest maximum (ultimate) cumulative methane production (P). Substrates that exhibited acute inhibition in the first few days of the mesophilic BMP experienced a lag phase, whereas other substrates’ lag phase result was 0. Additionally, the kinetic rate (Rm) decreased with increasing H2O2 dosage under thermophilic conditions. On the contrary, no such trend was observed for pretreated HTLaq substrates in mesophilic BMP assays. Table 4-10 Kinetic parameters for mesophilic (35°C) and thermophilic (55°C) BMP assays for HTLaq samples. Mesophilic biochemical methane potential Kinetic parameters HTLaq 0.25H 0.5H 0.75H 0.25H+NC 0.5H+NC 0.75H+NC 0.25H+NS 0.5H+NS 0.75H+NS P 167 182 160 101 197 183 145 163 123 122 Rm 49.9 48.92 31.57 49.31 47.87 46.83 45.47 55.99 77.02 5.00  0 0 0 1 0 0 0 0 0 2 R2 0.8902 0.9143 0.9381 0.9583 0.9277 0.9267 0.9892 0.9235 0.9895 0.9870 Adj-R2 0.8588 0.8897 0.9204 0.9463 0.9071 0.9058 0.9861 0.9016 0.9865 0.9833 Thermophilic biochemical methane potential Kinetic parameters HTLaq 0.25H 0.5H 0.75H 0.25H+NC 0.5H+NC 0.75H+NC 0.25H+NS 0.5H+NS 0.75H+NS P 104 30 - - 164 145 111 112 121 12 Rm 28.14 9.81 - - 23.20 25.98 12.27 27.54 27.40 0.86  0 8 - - 0 0 0 0 0 0 R2 0.9407 0.9954 - - 0.9689 0.9856 0.9938 0.9885 0.9923 0.8436 Adj-R2 0.9170 0.9936 - - 0.9565 0.9798 0.9914 0.9839 0.9893 0.7810 P = maximum specific methane production (mL CH4/g COD), Rm = maximum reaction rate (mL CH4/g COD/d),  = lag phase duration (d), Adj-R2= adjusted R2, HTLaq= hydrothermal liquefaction aqueous phase, NC: sodium carbonate, NS: sodium sulphite. ANOVA and Tukey HSD post hoc tests were conducted to compare the significance of the aforementioned discussion on BMP assays. One intermediate day before the specific cumulative methane yield reached a plateau (day 9 for mesophilic and day 8 for thermophilic) as well as the last day of BMP assays were chosen to analyze the significant difference between all BMPs. For mesophilic BMP assays, day 9 and the final day of the experiment were chosen, while for thermophilic BMP assays, day 8 and the final day were chosen for statistical analysis. Overall, 73 there was a significant difference between the substrates (Table 4-11), because the p-value was <0.001, less than 0.05 for all the analyzed days. Table 4-11: ANOVA summary table for analysis of the significant mean difference between different pretreatment dosages in anaerobic BMP assays, at a 95% confidence interval (α=0.05). aresults between different pretreated HTLaq as substrates, bresults for same pretreated HTLaq, cdegree of freedom, dsignificance or p-value. In Table 4-12, a pairwise comparison between different pretreatment conditions is shown for mesophilic BMP assays. There is a significant difference between most of the assay pairs indicated with green cells. Throughout the mesophilic BMP assays, 0.25H+NC exhibited a significant difference with almost all the pretreatment conditions except 0.5H+NC and 0.25H. Comparing unpretreated HTLaq with pretreated dosages, such as 0.5H+NC, 0.75H+NC, and 0.25H+NS, resulted in p-values higher than 0.05, meaning these samples aren’t significantly ANOVA test results: mesophilic BMP (day 9) Sum of Squares dfc Mean Square F Sig.d Between Groupsa 0.066 9 0.007 218.667 <0.001 Within Groupsb 0.001 20 0.000 Total 0.066 29 ANOVA test results: mesophilic BMP (day 38) Sum of Squares df Mean Square F Sig. Between Groups 0.032 9 0.004 95.919 <0.001 Within Groups 0.001 20 0.000 Total 0.032 29 ANOVA test results: thermophilic BMP (day 8) Sum of Squares df Mean Square F Sig. Between Groups 0.296 9 0.033 757.983 <0.001 Within Groups 0.001 20 0.000 Total 0.296 29 ANOVA test results: thermophilic BMP (day 35) Sum of Squares df Mean Square F Sig. Between Groups 0.418 9 0.046 435.264 <0.001 Within Groups 0.002 20 0.000 Total 0.420 29 74 different during the initial days of mesophilic BMP. During the last day, no difference was exhibited for samples such as 0.25H, 0.5H, 0.5H+NC, and 0.25H+NS with unpretreated HTLaq. Table 4-12: Equality probability between different pretreatment conditions for mesophilic BMP assays. Mesophilic BMP (day 9) Mesophilic BMP (day 38) HTLaq 0.25H 0.5H 0.75H 0.25H+NC 0.5 H+NC 0.75H+NC 0.25H+NS 0.5H+NS 0.75H+NS HTL 0.25H 0.5H 0.75H 0.25H+NC 0.5 H+NC 0.75H+NC 0.25H+NS 0.5H+NS 0.75H+NS HTLaq 0.25H 0.5H 0.75H 0.25H+NC 0.25H+NC 0.25H+NC 0.25H+NC 0.25H+NC 0.25H+NC HTLaq: hydrothermal liquefaction aqueous phase, H: hydrogen peroxide, NC: sodium carbonate, NS: sodium sulphite. Red depicts no significant difference, green depicts a significant difference between the two pretreatment conditions. For pretreatment, H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). In the case of thermophilic BMPs (Table 4-13), for the initial days, almost all the assays were found to be different compared to unpretreated HTLaq except 0.75H+NC. However, after 35 days of incubation, 0.75H+NC, 0.5H+NS and 0.75H+NS assays resulted in similar yields. Assays with the highest methane yield, which was 0.25H+NC, showed equality with 0.5H+NC.0.25H+NS and 0.5H+NS for the initial days of thermophilic BMPs. At the end of the thermophilic BMP assay, due to the higher pretreatment efficiency, only 0.5H+NC stayed similar to 0.25H+NC. 75 Table 4-13 Equality probability between different pretreatment conditions for thermophilic BMP assays. Thermophilic BMP (day 8) Thermophilic BMP (day 35) HTLaq 0.25H 0.5H 0.75H 0.25H+NC 0.5 H+NC 0.75H+NC 0.25H+NS 0.5H+NS 0.75H+NS HTL 0.25H 0.5H 0.75H 0.25H+NC 0.5 H+NC 0.75H+NC 0.25H+NS 0.5H+NS 0.75H+NS HTLaq 0.25H 0.5H 0.75H 0.25H+NC 0.25H+NC 0.25H+NC 0.25H+NC 0.25H+NC 0.25H+NC HTLaq: hydrothermal liquefaction aqueous phase, H: hydrogen peroxide, NC: sodium carbonate, NS: sodium sulphite. Red depicts no significant difference, green depicts a significant difference between the two pretreatment conditions. For pretreatment, H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). Overall, the similarity between different pretreatments was greater in the mesophilic BMP assays, whereas in thermophilic BMP assays, assays resulted in more significant differences. This shows that impact of pretreatment of HTLaq on thermophilic AD cultures were greater, compared to its corresponding control. 4.2.3 Biodegradability assessment under aerobic conditions To assess the biodegradability of HTLaq substrates under aerobic conditions, BOD test was conducted using an online respirometer. Figure 4.14 shows the BOD results over the course of 26 days (BOD26). The 0.75H+NS sample achieved the highest BOD26 resulting in a 3.1% 76 increase from the BOD26 of unpretreated HTLaq. The highest BOD removal of 19.15% was achieved for 0.25H+NC. Figure 4.14 Respirometric biochemical oxygen demand (BOD) concentration of unpretreated HTLaq and H2O2 pretreated HTLaq, For pretreatment, H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). H: hydrogen peroxide, NC: sodium carbonate, NS: sodium sulphite. Figure 4-15 exhibits the oxygen uptake rate for different pretreatment conditions of H2O2 with/without quencher. From the graphs, it can be said that, with time the oxygen uptake rate reduced to  50 mg O2 L-1 h-1 over 26 days for all the samples.77 Figure 4.15 Oxygen uptake rate of unpretreated and pretreated HTLaq with time. Shaded area represents standard deviation. For pretreatment, H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). H: hydrogen peroxide, NC: sodium carbonate, NS: sodium sulphite. 78 A similar trend was also observed in terms of the biodegradability index, calculated by ultimate BOD (BOD26) divided by tCOD for each HTLaq sample (Table 4-14). The sample of 0.75H+NS resulted in the highest biodegradability index of 0.85, while it was 0.75 for untreated HTLaq. For BOD5, 0.75H+NC reached the lowest BOD of 27.45 ± 5.45 g/L. Table 4-14 Biodegradability assessment of raw and pretreated HTLaq under aerobic conditions. Sample BOD5a (g/L) Ultimate BOD26 (g/L) tCODb (g/L) Ultimate BOD26/tCOD ratio (biodegradability index) HTLaq 36.92 ±f 0.88 47.42 ± 0.47 63.00 ± 1.06 0.75 0.25Hc 33.65 ± 1.86 44.34 ± 1.58 67.00 ± 0.90 0.76 0.5H 32.31 ± 0.44 43.70 ± 0.69 68.11 ± 1.19 0.78 0.75H 33.02 ± 0.36 43.30 ± 2.16 84.47 ± 3.39 0.68 0.25H+NCd 27.96 ± 6.14 38.34 ± 2.50 67.16 ± 0.45 0.63 0.5H+NC 33.34 ± 1.05 42.85 ± 1.43 59.85 ± 0.45 0.71 0.75H+NC 27.45 ± 5.45 40.30 ± 3.5 56.90 ± 1.19 0.71 0.25H+NSe 34.46 ± 1.42 46.61 ± 1.9 62.97 ± 2.52 0.74 0.5H+NS 30.49 ± 5.35 37.63 ± 8.7 58.39 ± 1.02 0.64 0.75H+NS 39.07 ± 1.78 48.90 ± 2.9 57.44 ± 0.39 0.85 aBiochemical oxygen demand, btotal chemical oxygen demand, chydrogen peroxide, dsodium carbonate, esodium sulphite, fdata represents average ± standard deviation of triplicates. For pretreatment, H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). Non-biodegradable compounds in HTLaq are complex and has strong bonds. These types of molecules are not common in the natural environment and to break down such molecular bonds, microorganisms require different enzymes. The aerobic microorganisms lack of such enzymes to breakdown complex HTLaq molecules and because of that a substantial percentage (up to 37% in certain cases of Table 4.14) of tCOD from HTLaq samples could not be utilized during the aerobic biodegradation. 79 The rate of aerobic degradation was also assessed using kinetic modelling through the Modified Gompertz equation (Table 4-15). The maximum oxygen consumption (P) displayed a decreasing trend with the increasing dosage of H2O2. The pretreated sample of 0.25H+NC resulted in the highest oxygen consumption rate (Rm), which exhibits faster conversion of organics to CO2. The 0.75H+NS sample resulted in the highest maximum oxygen consumption. No lag phase was observed for any substrates. This result suggests that pretreatment with lower dosages of H2O2 removed inhibitory compounds to aerobic cultures making the HTLaq compounds faster and more amenable to biodegradation. Table 4-15 Kinetic parameters for ultimate BOD test for HTLaq samples. Aerobic degradability assessment Kinetic parameters HTLaq 0.25H 0.5H 0.75H 0.25H+NC 0.5H+NC 0.75H+NC 0.25H+NS 0.5H+NS 0.75H+NS P 43719 40974 40173 39981 45139 38789 36717 42234 34583 45565 Rm 8840 7665 6915 7230 10971 8265 5160 8141 9909 10273  0 0 0 0 0 0 0 0 0 0 R2 0.9010 0.8945 0.8901 0.9102 0.9239 0.8833 0.8023 0.8533 0.8677 0.9027 Adj-R2 0.9009 0.8945 0.9000 0.9102 0.9239 0.8832 0.8022 0.8532 0.8676 0.9026 P = ultimate biochemical oxygen demand (mg O2/L), Rm = maximum oxygen consumption rate (mg O2/L /d),  = lag phase duration (d), Adj-R2= adjusted R2, HTLaq= hydrothermal liquefaction aqueous phase, NC: sodium carbonate, NS: sodium sulphite. For pretreatment, H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). Similar to anaerobic BMP tests, ANOVA and Tukey HSD post hoc were conducted to compare the mean effect of different pretreatment conditions of HTLaq on aerobic biodegradability index and the results are given in Table 4-15 and Table 4-16, respectively. The results indicated an overall significant difference (p-value < 0.05) of pretreatment conditions of HTLaq in the ANOVA test result (Table 4-16). While comparing pairwise between two pretreatment conditions, almost all the HTLaq samples exhibited statistically significant differences in their aerobic biodegradability index (Table 4-17, p-value < 0.05, green cells). 80 However, similarity was also observed (p-value > 0.05, red cells) between some of the pairs, such as 0.5H vs 0.5H+NS, 0.5H+NC vs 0.75H+NC, and 0.25H+NS vs 0.25H. Table 4-16 ANOVA summary table for analysis of the significant mean difference between different pretreatment conditions in aerobic biodegradability index of HTLaq samples, at a 95% confidence interval (α=0.05). aresults between different pretreated HTLaq as substrates, bresults for same pretreated HTLaq, cdegree of freedom, dsignificance or p-value. Table 4-17: Equality probability between different pretreatment conditions for aerobic biodegradability index of HTLaq samples. Aerobic biodegradability index (BOD/tCOD) HTLaq 0.25H 0.5H 0.75H 0.25H+NC 0.5 H+NC 0.75H+NC 0.25H+NS 0.5H+NS 0.75H+NS HTLaq 0.25H 0.5H 0.75H 0.25H+NC 0.25H+NC 0.25H+NC 0.25H+NC 0.25H+NC 0.25H+NC HTLaq: hydrothermal liquefaction aqueous phase, H: hydrogen peroxide, NC: sodium carbonate, NS: sodium sulphite. Red depicts no significant difference and green depicts a significant difference between the two pretreatment conditions. For pretreatment, H2O2 addition was for 2 h, followed a quencher addition (quencher: H2O2 ratio of 1:1 by wt.) for another 3 h waiting time (total of 5 h), both pretreatments at room temperature (23C). ANOVA test results: aerobic biodegradability index Sum of Squares dfc Mean Square F Sig.d Between Groupsa 0.219 9 0.024 2616.774 <0.001 Within Groupsb 0.000 20 0.000 Total 0.220 29 81 Chapter 5: Conclusions 5.1 Summary part 1 Part 1 of this study determined the optimum dosage and assessed the efficiency of the H2O2 pretreatment process with/without quenchers for HTLaq samples derived from municipal sludge cake at 350oC, 15 min HTL conditions. The main findings of part 1 are as follows: • Treating HTLaq only with H2O2 leaves H2O2 residual, especially at high dosages, which interferes with the tCOD analysis leading to erroneous increment in tCOD concentrations in the pretreated sample. However, using quenchers, such as sodium carbonate, can eliminate the interference lowering the tCOD gradually with an increase dosage. • Residual H2O2 in the pretreated sample also react with the anaerobic inoculum and inhibit mainly the archaea. This leads to acute inhibition of BMP assays during the early stage of incubation, especially for HTLaq samples pretreated at high H2O2 dosages. • HTLaq pretreatment optimization results indicate that 2 h and 3 h waiting times after H2O2 and quencher additions (total pretreatment time of 5 h) provides the optimum efficiency for pollutant removal. Furthermore, moving from room temperature to elevated temperature is not necessary for increasing pollutant removal efficiency. These findings fulfil the first and third objectives of the thesis. • The second objective of this thesis was to determine removal efficiencies of the H2O2 pretreatment process for organic/inorganic pollutants. The results indicated that the overall pretreatment (H2O2 with quencher) had effects on reducing tCOD, phenolics, and TAN etc. Based on the tCOD results of HTLaq, sodium carbonate as a quencher has higher efficiency compared to sodium sulphite. Up to 20% tCOD was removed from HTLaq when H2O2 pretreatment was combined with sodium carbonate. Similarly, more than 55% of total 82 phenolic removal was achieved in the samples pretreated with H2O2/sodium carbonate. However, due to additional buffering capacity provided by sodium carbonate, the pH of pretreated HTLaq was higher, which led to a need for readjustment of pH for downstream biodegradability assessment. 5.2 Summary part 2 Part 2 of the study mainly focused on meeting the last two objectives of the thesis, which was biodegradability assessment of H2O2 pretreated HTLaq. This part assessed the efficiency of H2O2 pretreated HTLaq in downstream anaerobic and aerobic biological treatment processes. The main findings of part 2 are: • The specific methane yield from HTLaq samples under thermophilic temperature was comparatively lower than mesophilic temperature, confirming that thermophilic cultures are more sensitive toward complex compounds in HTLaq. Under mesophilic conditions, the lowest dosage of H2O2 with sodium carbonate quencher (0.25H+NC) had the highest ultimate specific methane yield, which was only 16% higher compared to control. Moreover, with an increase in H2O2 dosage, ultimate specific methane yield from HTLaq decreased. On the other hand, under thermophilic conditions, the same sample (0.25H+NC) achieved almost 55% improvement in ultimate specific cumulative methane yield compared to unpretreated HTLaq. This result shows that improvements coming from pretreatments are more pronounced under thermophilic conditions since thermophilic control BMPs are challenged more than mesophilic control BMPs due to narrower microbial diversity at elevated temperatures. • Addition of quenchers, in particular sodium carbonate, to HTLaq after H2O2 pretreatment were able to eliminate acute inhibition observed in BMP assays. However, using sodium 83 sulphite as quencher caused additional inhibition due to generation of SO42- while reacting with H2O2. • Pretreated samples with sodium carbonate as quencher reduced higher N-heterocyclic compounds under thermophilic digestion. • In aerobic biodegradability assays, the highest biodegradability index (0.85) was achieved at the highest dosage (0.75H+NS). However, the 0.25H+NC sample achieved the highest BOD removal of 19.15% with an aerobic biodegradability index of 0.63. • Higher oxygen uptake rate was observed for HTLaq samples of 0.25H+NC exhibiting faster conversion of organics to CO2 at lower H2O2 dosages with sodium carbonate. • Overall, samples with lower dosages of H2O2 with sodium carbonate as quencher exhibited the highest aerobic and anaerobic biodegradability. This is also advantageous from the operational cost point of view. 5.3 Overall summary and conclusions This thesis addresses challenges in incorporating the HTL process for municipal sludge treatment to WWTPs. It proposed a pretreatment method of HTLaq through using H2O2 for mainly removing potential inhibitory compounds from HTLaq for enhanced biological treatment within the vicinity of WWTP. It also contributed to resource recovery and circular economy by facilitating simultaneous production of biocrude oil and biogas (methane) from municipal sludge via HTL. The following main conclusions can be summarized from this research: • Treating HTLaq with only H2O2 might not be a feasible solution due to negative effect of residual H2O2 on beneficial cultures in downstream biological treatment. To reduce the H2O2 residual in the HTLaq, sodium carbonate can be used as a quencher. 84 • Anaerobic treatment of HTLaq under mesophilic conditions is more efficient and robust than under thermophilic conditions, due to higher sensitivity of thermophilic cultures to inhibitory compounds. However, if thermophilic AD has to be the choice due to WWTP operational restrictions, H2O2 pretreatment combined with sodium carbonate can enhance the methane recovery from HTLaq. • In a dose range of 0.25-0.75 g H2O2/g COD of HTLaq, the lowest dosage of H2O2 combined with sodium carbonate quencher (H2O2: quencher at 1:1 (w/w)) is an optimum choice for both anaerobic and aerobic downstream treatment from process and operational cost point of view. 5.4 Limitations and recommendations One limitation of this study is that only 3 levels of H2O2 dosage were tested to observe the effects on HTLaq characterization and tCOD removal was the main criteria for dosage optimization. For avoiding errors and challenges related to measuring very low amount of H2O2 in the lab, the minimum dosage was set to 0.25 g H2O2/g COD, which also helped to compare the results with existing literature on other HTL feedstock. However, not being able to test lower dosages than 0.25 g H2O2/g COD is a limitation of this study. Another limitation is that the study was conducted on two batches of mixed sludge were used in generating HTLaq stream which does not stimulate a full-scale HTL operation in WWTP. Lastly, no long-term continuous-flow AD operation was conducted for anaerobic biodegradability assessment of pretreated HTLaq, since only batch BMP assays were conducted as preliminary screening of the treatment conditions. 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The molar mass of Na₂CO₃ is 105.99 g/mol, Mass of Na₂CO₃ = 0.00979 mol ×105.99 g/mol =1.04 g Na₂CO₃ For 0.25 g H₂O₂/g COD dosage, in 1L of HTLaq, Required H₂O₂ = 101 mL or 112.4 g So, required Na₂CO₃ = 105.04 g So, 105.04 g Na₂CO₃ required for quenching 1L of H₂O₂ pretreated HTLaq where the peroxide dosage was 0.25 g H₂O₂/g COD dosage. 97 Appendix B Figure B.2 tCOD calibration curve. Figure B.1 Colorimetric analysis for tCOD. 98 ` Figure B.4 Colorimetric analysis for TAN. Figure B.3 TAN calibration curve. 99 Figure 0.5 Colorimetric analysis for phenolics. Figure B.6 Phenol calibration curve. 100 Figure B.7 Spectrum analysis for HTLaq. BMP setup conditions Table 0-1 Preliminary mesophilic BMP setup parameters for part 1. Substate Substate tCOD (mg) Inoculum VS (mg) Volume of inoculum (mL) SIR (mg COD/mg VS) Total volume (mL) Sample 133.34 13.33 40 0.25 70 Table 0-2 Mesophilic BMP setup parameter for part 2. Substate Substate tCOD Inoculum VS Volume of inoculum SIR (mg COD/mg VS) Total volume 101 (mg) (mg) (mL) (mL) Sample 85.91 8.59 40 0.25 70 Table 0-3 Thermophilic BMP setup parameter for part 2. Substate Substate tCOD (mg) Inoculum VS (mg) Volume of inoculum (mL) SIR (mg COD/mg VS) Total volume (mL) Sample 108.69 10.87 40 0.25 70 102 Appendix C Respirometer test details Mineral Base I Add the following to 800 mL of type 1 water. Dilute to 1L. • CoCl2.6H2O 0.25 g • Na2MoO4.2H2O 0.005 g • FeCl2.4H2O 4 g • H3BO3 0.025 g • NiCl2.6H2O 0.025 g • CuCl2.2H2O 0.007 g • MnCl2.4H2O 0.05 g • ZnCl2 0.025 g • Na2SeO4 0.025 g Mineral Base II Dissolve the following in 800 mL type 1 water. Dilute to 1 L. • CaCl2 15 g • MgCl2.6H2O 20g Nutrient Base Dissolve the following in 800 mL type 1 water. Neutralize pH using 50% NaOH. Dilute to 1 L. • NH4Cl 53 g • Na2SO4 15 g • KH2PO4 12 103 Appendix D Change in pH over time in BMP assays Table 0-1 Change in pH during mesophilic BMP assays. Time (d) HTLaq 0.25H 0.5H 0.75H 0.25H+NC 0.5H+NC 0.75H+NC 0.25H+NS 0.5H+NS 0.75H+NS 0 7.47 7.43 7.55 7.57 7.07 7.24 7.63 7.47 7.45 7.54 1 7.77 7.72 7.67 7.69 7.51 7.76 8.14 7.78 7.73 7.89 2 7.71 7.69 7.75 7.76 7.52 7.74 8.13 7.79 7.79 7.82 3 7.65 7.66 7.69 7.67 7.40 7.67 8.04 7.76 7.65 7.75 5 7.66 7.63 7.63 7.61 7.39 7.65 7.99 7.81 7.62 7.70 9 7.57 7.52 7.61 7.58 7.32 7.57 7.90 7.57 7.53 7.68 15 7.51 7.48 7.59 7.50 7.39 7.54 7.77 7.55 7.58 7.70 20 7.53 7.49 7.51 7.48 7.37 7.60 7.80 7.57 7.61 7.73 30 7.51 7.49 7.5 7.49 7.37 7.59 7.80 7.57 7.62 7.72 38 7.52 7.48 7.49 7.48 7.40 7.61 7.81 7.60 7.62 7.73 Table 0-2 Change in pH during thermophilic BMP assays. Time (d) HTLaq 0.25H 0.5H 0.75H 0.25H+NC 0.5H+NC 0.75H+NC 0.25H+NS 0.5H+NS 0.75H+NS 0 8.02 8.04 8.09 8.10 8.07 7.84 8.10 8.12 8.10 8.11 1 8.04 8.03 8.13 8.11 8.04 8.01 8.22 8.13 8.11 8.17 2 8.10 8.06 8.07 8.07 8.14 8.03 8.24 8.13 8.11 8.18 3 8.14 8.08 8.03 7.97 8.12 7.96 8.19 8.05 8.09 8.16 8 7.93 7.97 7.90 7.87 8.00 7.89 8.09 7.96 7.96 8.09 13 7.74 7.66 7.66 7.66 7.75 7.61 7.83 7.75 7.70 7.86 23 7.61 7.64 7.60 7.54 7.60 7.42 7.70 7.57 7.65 7.72 35 7.57 7.58 7.62 7.68 7.63 7.57 7.72 7.63 7.67 7.70
Thesis/Dissertation
eng
Civil Engineering
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Eskicioglu, Cigdem
Pretreatment of hydrothermal liquefaction aqueous from municipal sludge by hydrogen peroxide for enhanced biological treatment
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