Impact of Ozone Pre-treatment on Aerobic and Anaerobic Biodegradability of Aqueous Phase from Hydrothermal Liquefaction of Municipal Sludge

Impact of Ozone Pre-treatment on Aerobic and Anaerobic Biodegradability of Aqueous Phase from Hydrothermal Liquefaction of Municipal Sludge A. Parajulia and C. Eskicioglua,b,c* aUBC Bioreactor Technology Group, School of Engineering, University of British Columbia, Kelowna, BC V1V 1V7 Canada bICREA – Catalan Institution for Research and Advanced Studies, Pg. Lluís Companys 23, Barcelona, Spain cGEMMA – Group of Environmental Engineering and Microbiology, Department of Civil and Environmental Engineering, Universitat Politecnica de Catalunya – Barcelona Tech., c/Jordi Girona 1-3, Building D1, E-08034 Barcelona, Spain *Corresponding author: Cigdem Eskicioglu (cigdem.[email protected], [email protected]) Abstract Hydrothermal liquefaction (HTL) of municipal sludge is a promising alternative to anaerobic digestion due to resource recovery into biocrude oil (refined to transportation fuel), nutrient-rich hydrochar, smaller footprint, enhanced micropollutant destruction and substantially reduced solids for final disposal. For integration of HTL to wastewater treatment plants (WWTPs), its highest volume aqueous product (HTLaq) requires treatment on-site. However, HTLaq contains soluble refractory organics inhibitory to downstream biological processes. In this study, the impact of ozone (O3) pre-treatment on characteristics and subsequent aerobic and anaerobic biodegradability of HTLaq were studied. HTLaq was obtained at 350oC, 15 min from sludge cake. Within a pretreatment dose range of 0.03-0.18 g (dissolved) O3/g chemical oxygen demand (COD) of HTLaq, the highest dose of 0.18 g O₃/g COD achieved a 43% COD removal and 90% aerobic biodegradability improvement compared to control (no pre-treatment) at 20oC. For anaerobic biodegradability, the optimum pre-treatment dose was 0.14 g O₃/g COD with 98% and 89% improvements in specific methane yields from HTLaq under mesophilic (35oC) and thermophilic (55oC) temperatures, compared to controls. Removal of inhibitory N-heterocyclics and phenolics from HTLaq via ozonation was the main reason for enhanced biodegradation and biogas recovery, improving overall carbon recovery from municipal sludge. Keywords Biological treatment; hydrothermal liquefaction aqueous; municipal sludge; ozone; N-heterocyclics. INTRODUCTION Wastewater treatment plants (WWTPs) are shifting their focus towards resource recovery, particularly from sludge which contains valuable inorganic nutrients and organic compounds. While anaerobic digestion (AD) is widely adopted for sludge treatment, its limitations such as long retention time, high volume of final biosolids, and persistence of macroand micro-pollutants have led to exploration of hydrothermal liquefaction (HTL). HTL is a faster, more compact process that achieves enhanced pollutant destruction while producing value-added products (biocrude oil, biogas, nutrients) from product streams. HTL processes wet organic feedstock under high temperatures (200-374°C) and pressures (15-220 bars) within 1 to 60 minutes (Basar et al., 2021). However, the HTL process produces wastewater with high levels of organic and inorganic inhibitory compounds including chemical oxygen demand (COD), phenolics, ammonium, nitrogen-containing organics, and toxic aromatic compounds, which requires treatment to enable large-scale HTL implementation for municipal sludge (Watson et al., 2020). The strong oxidizing ability of ozone (O3) makes it a promising pre-treatment method for eliminating or converting refractory to more biodegradable organics (Yang et al., 2018). This study evaluated how O3 pre-treatment affects the properties of sludge-derived HTL aqueous (HTLaq) as well as its downstream biodegradability under aerobic and anaerobic conditions. MATERIALS AND METHODS The HTL of dewatered mixed sludge (20% total solids by weight) was conducted using a 1-L Parr® 4570 reactor at 350°C and under 168 bar for 15 minutes. These conditions were adopted to maximize biocrude oil production from mixed sludge but resulted in high concentrations of inhibitory compounds in the wastewater byproduct (Liu et al., 2023). The mixed sludge was a combination of primary and secondary municipal sludges (obtained from a local WWTP in BC, Canada) at 50:50% by volume. For secondary treatment, the plant uses tricking filters followed by solids contact tank process. Aerobic and anaerobic inocula were obtained from the solids contact tanks and sludge digesters, respectively, from the same plant. After HTL reactor cooled down, the HTLaq was obtained and filtered via a 0.45 µm membrane. Compared to previous research that used 0-0.12 g O3/g COD for HTLaq from swine manure, our study explored a wider range of O3 doses for municipal sludgederived HTLaq (Yang et al., 2018). The ozonation was performed at 1.4 L/min (corresponding to 7.8 g O3/hr), where 100 mL of 10-times diluted HTLaq (dilution necessitated due to foaming at benchscale) was treated in a glass contact vessel. The characterizations of pH, COD, total phenolics, total volatile fatty acids (VFAs), and a selected group of N-heterocyclics of HTLaq were made. For anaerobic biodegradability, mesophilic (35°C) and thermophilic (55°C) biochemical methane potential (BMP) assays utilizing HTLaq before and after various O3 pretreatments were set-up at a food-to-microorganism ratio (F/M) of 0.25 g COD of aqueous/g volatile solids (VS) of inocula. The F/M ratio was optimized by preliminary BMP assays, and no external buffer/nutrient addition was necessary. The BMP assays (125 mL) had triplicates for each pretreatment and an additional bottle for pH and VFAs monitoring during incubation, while blank reactors (containing only inoculum) were used to calculate the net methane yield from each substrate by subtraction. Positive controls with glucose were also included and the assays were monitored until biogas production ceased. For aerobic biodegradability, HTLaq before and after various O3 pretreatments was evaluated using a PF8000 online respirometer at 20°C according to Standard Procedures 5210D. The HTLaq samples (40 mL) were mixed with acclimated aerobic inoculum (F/M ratio of 5 mg COD/mg volatile suspended solids (VSS)), nutrients, buffer (pH of 7), and nitrification inhibitor in borosilicate reactors equipped with KOH-filled CO2 absorption tubes. Oxygen uptake was monitored every 10 min while mixing at 600 rpm, with blank reactors (inoculum only) used to calculate oxygen consumption from substrate only as 5-day biochemical oxygen demand (BOD5) and ultimate (28-day) BOD (UBOD28). Further details about the methodology and results are available elsewhere (Parajuli & Eskicioglu, 2025). RESULTS AND DISCUSSION Materials characteristics The pH of diluted HTLaq decreased sharply from 8.0 to 6.2 at low O₃ doses (0-0.03 g O₃/g COD), then gradually decreased to 5.3 at 0.12 g O₃/g COD before stabilizing around 5.2-5.3 at higher doses (0.18 g O₃/g COD). The formation of carboxylic acids and other acidic compounds during ozonation of organic matter results in decreased pH levels (Yang et al., 2018). The initial COD of 10-times diluted HTLaq was 7.1 g/L. The COD removal was initially fast (32% at 0.12 g O₃/g COD), followed by 41% at 0.14 g O₃/g COD and 43% at 0.18 g O₃/g COD, suggesting that the initial degradation of easily oxidizable compounds were followed by more recalcitrant organics at higher doses. Total phenolics removal increased from 35% at 0.03 g O₃/g COD to 71% at 0.12 g O₃/g COD and remained almost the same, indicating ozone’s high effectiveness in degrading oxidizable phenolic compounds until reaching either mass transfer limitations or depletion of degradable phenolics. Ozonation increased total VFAs from 8 to 9.5 g/L, primarily through formation of acetic and propionic acids from the breakdown of larger organic molecules, which explains the observed pH reduction, while butyric acid levels remained stable. N-heterocyclic compounds of HTLaq ranged from 2ethylpyridine (0.43 mg/L) to 2-methylpyrazine (60.30 mg/L) in un-pretreated diluted HTLaq, with pyridine derivatives (2-methylpyridine, 2-ethylpyridine, and 5-ethyl-2-methylpyridine) showing increased removal at higher doses, reaching 69% removal for 2-methylpyridine and 2,6-lutidine at 0.18 g O₃/g COD. The concentration of pyrazine derivatives (2-methylpyrazine and ethylpyrazine) initially increased before degrading at higher O3 doses. Biochemical methane potential (BMP) assays As shown in Figure 1, the mesophilic inoculum's activity was validated by methane yield of glucose (0.32 mL CH4/mg COD, 91% of theoretical yield at 0°C, 1 atm), while HTLaq (before pre-treatment) yielded only 0.116 mL CH4/mg COD due to recalcitrant organics. After the optimum pre-treatment dose at 0.14 g O3/g COD, HTLaq achieved 0.229 mL CH4/mg COD (98% improvement), because of removal of inhibitory organics while preserving VFAs. The pH range for BMP assays remained between 7.25 to 7.9, which is considered ideal for mesophilic AD. Under thermophilic conditions, positive control (glucose) achieved 0.33 mL CH4/mg COD (94% of theoretical yield), while HTLaq (before pre-treatment) yielded only 0.119 mL CH4/mg COD. Although 0.14 g O3/g COD remained as the optimal pre-treatment dose, thermophilic BMPs showed a more gradual improvement with increasing O3 dose with lower overall yields (0.226 mL CH4/mg COD, 89% improvement) compared to mesophilic conditions, demonstrating thermophiles’ higher sensitivity to HTLaq, like adsorption pretreatment studies (Aktas et al., 2024). This was attributed to narrower microbial diversity in AD processes at elevated (thermophilic) temperatures. VFAs degraded more slowly in thermophilic BMPs than in mesophilic conditions, particularly in O3-pretreated samples. (a) (b) Figure 1. Specific cumulative methane yields of glucose, HTLaq and O3-pre-treated HTLaq samples at varying doses under (a) mesophilic (35°C) and (b) thermophilic (55°C) anaerobic conditions. Biochemical oxygen demand (BOD) assays Table 1 shows the pre-treatment impact on BOD and COD of (diluted) HTLaq samples derived from municipal sludge. When O3 dose increased, COD steadily dropped from 7,135 mg/L to 3,930 mg/L, showing that O3 effectively broke down organics in the solution. The 5-day and 28-day BOD5 and UBOD28 first went up as more O3 was added, peaking at a dose of 0.12 g O3/g COD, before declining at higher doses. The aerobic biodegradability of the HTLaq improved significantly with O3 treatment. The 5-day biodegradability index (BOD5/COD) nearly doubled from 0.42 to 0.80, while the 28-day index (UBOD28/COD) increased from 0.54 to 0.95. This shows that O3 treatment successfully converted hard-to-degrade organic matter into more easily biodegradable forms. The optimal O3 dose denoting highest biodegradability index for aerobic treatment was found to be 0.18 g O3/g COD (unlike anaerobic treatment). For comparison, a previous study on similar HTLaq showed more modest improvements in biodegradability (from 0.31 to 0.41) using a lower O3 dose (Yang et al., 2018). The extent of HTLaq biodegradation was correlated with the extent of microbial diversity, such as aerobic, mesophilic anaerobic followed by thermophilic anaerobic processes. Table 1. Aerobic biodegradability assessment of HTLaq derived from municipal sludge, before and after O3 pretreatment. Data are reported for 10× diluted HTLaq. Dissolved O3 dose BOD5 UBOD28 COD BOD5/COD UBOD28/COD g O3/g COD mg/L mg/L mg/L - - 0 3001±45 3869±98 7135±350 0.42 0.54 0.03 3228±70 4169±145 5543±98 0.58 0.75 0.12 3219±64 4222±171 4958±408 0.65 0.85 0.14 2843±51 3714±78 4215±166 0.67 0.88 0.16 2916±72 3744±100 4073±72 0.71 0.92 0.18 3167±94 3745±138 3930±27 0.80 0.95 CONCLUSION • At the optimal dissolved dose of 0.14 g O3/g COD for HTLaq derived from municipal mixed sludge, pre-treatment reduced COD by 41%, total phenolics by 71%, and nitrogen-containing compounds (61% of 2-methylpyridine and 67% of 2,6-lutidine), and retaining VFAs, beyond which minimal changes were observed in the samples. • The pre-treatment dose of 0.14 g O3/g COD achieved the highest mesophilic and thermophilic methane production, that were 98% and 89% higher than controls, respectively. Utilizing pretreatment doses above 0.14 g O3/g COD resulted in slightly lower methane production. • The highest O3 dose tested (0.18 g O3/g COD) greatly improved how well bacteria could break down the HTLaq under aerobic conditions, with the ultimate biodegradability index rising from 0.54 to 0.95. This suggests that O3-treated HTLaq could potentially be returned to WWTPs’ aerobic solids contact tank for downstream treatment. FUNDING INFORMATION The authors acknowledge the support of Metro Vancouver and NSERC’s Industrial Research Chair Program in Advanced Resource Recovery from Wastewater (IRCPJ 548816-18). REFERENCES Aktas, K., Liu, H., Basar, I.A. and Eskicioglu, C., 2024. Adsorption enhanced biological treatment of hydrothermal liquefaction aqueous phase derived from municipal sludge. Bioresource Technology, 407, 131093. Basar, I.A., Liu, H., Carrere, H., Trably, E. and Eskicioglu, C., 2021. 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