Impact of polystyrene nanoplastics on primary sludge fermentation under acidic and alkaline conditions: Significance of antibiotic resistance genes.

As a part of industrial or commercial discharge, the influx of nanoplastics (NPs) to the wastewater treatment plants is inevitable. Consequently, it has become a must to understand the effects of these NPs on different unit processes. This study aimed to investigate the impact of three different concentrations of polystyrene nanoplastics (PsNPs) on the fermentation of primary sludge (PrS), implemented in batch anaerobic bioreactors, at pH 5 and 10, considering the pH-dependent nature of the fermentation process. The results showed that PsNPs stimulated hydrogen gas production at a lower dose (50 µg/L), while a significant gas suppression was denoted at higher concentrations (150 µg/L, 250 µg/L). In both acidic and alkaline conditions, propionic and acetic acid predominated, respectively, followed by n-butyric acid. Under both acidic and alkaline conditions, exposure to PsNPs boosted the propagation of various antibiotic resistance genes (ARGs), including tetracycline, macrolide, ß-lactam and sulfonamide resistance genes, and integrons. Notably, under alkaline condition, the abundance of sul2 gene in the 250 µg PsNPs/L batch exhibited a 2.4-fold decrease compared to the control batch. The response of the microbial community to PsNPs exposure exhibited variations at different pH values. Bacteroidetes prevailed at both pH conditions, with their relative abundance increasing after PsNPs exposure, indicating a positive impact of PsNPs on PrS solubilization. Adverse impacts, however, were detected in Firmicutes, Chloroflexi and Actinobacteria. The observed variations in the survival rates of various microbes stipulate that they do not have the same tolerance levels under different pH conditions.

Impact of aging of primary and secondary polystyrene nanoplastics on the transmission of antibiotic resistance genes in anaerobic digestion.

Sewage sludge is a significant reservoir of nano/microplastics (NPs/MPs) and antibiotic resistance genes (ARGs). Research has revealed that NPs/MPs may exert an inhibitory effect on anaerobic digestion (AD) of sludge. Moreover, NPs/MPs can influence microbial community diversity and composition, potentially increasing ARGs dissemination. The morphological changes to NPs/MPs surface due to aging contribute to modifying hydrophobic properties. To date, there is limited comprehension regarding how various surface properties of NPs influence ARGs dissemination during AD. This study investigated the impact of primary aged/non-aged and secondary aged/non-aged polystyrene nanoplastics (PSNPs) on ARGs and mobile genetic elements (MGEs) propagation during AD. The findings indicated that the UV-aging process resulted in surface oxidation and distinct morphological characteristics in both primary and secondary PSNPs, while the surface oxidation effect was more pronounced in the secondary aged PSNPs. High concentrations (150 µg/L) of primary and secondary PSNPs inhibited methane production, with secondary PSNPs causing greater inhibition by 16 to 20 % compared to control. In contrast, low concentration (25 µg/L) had negligible or slightly positive effects on methane production. PSNPs at 150 µg/L reduced total VFA concentration, indicating an inhibitory effect on the fermentation step in the AD process. Primary and secondary PSNPs exhibited changes in EPS characteristics. ARGs abundance was enriched in reactors amended with PSNPs, with the highest abundance of 8.54 × 105 copies/g sludge observed in the secondary aged PSNPs (150 µg/L) reactor. Reactors exposed to aged PSNPs exhibited a relatively higher abundance of ARGs compared to reactors exposed to non-aged PSNPs. Exposure to PSNPs increased the microbial community diversity within the digesters and triggered the enrichment of Comamonadaceae and Syntrophaceae, belonging to Proteobacteria phylum. On the other hand, archaeal communities tended to shift towards hydrogenotrophic methanogens in PSNPs reactors. The correlation analysis showed that Comamonadaceae were positively correlated with the majority of ARGs and intl1. A positive correlation was observed between MGEs and most ARGs, suggesting that the increased proliferation of ARGs under PSNPs exposure may be linked to the abundance of MGEs, which in turn promotes the growth of hosts carrying ARGs. These findings suggest that aged and non-aged NPs could substantially impact the spread of ARGs and MGEs, which also led to notable alterations in the composition of the microbial community. Overall, this study provides valuable insights into the multifaceted impacts of PSNPs with various characteristics on AD processes, microbial communities, and ARGs proliferation, highlighting the urgent need for comprehensive assessments of NPs pollutants in the environment.

Comprehensive hydrothermal pretreatment of municipal sewage sludge: A systematic approach.

This study provides a comprehensive analysis of the potential impact of hydrothermal pretreatment (HTP) on municipal thickened waste-activated sludge (TWAS) and its integration with anaerobic digestion (AD). The research demonstrates that HTP conditions (170 °C, 3 bars for 30 min) can increase the solubilization of macromolecular organic compounds by 41%, which enhances biodegradability in semicontinuous bioreactors. This treatment also results in a 50% reduction in chemical oxygen demand (COD) and a 63% increase in the destruction of volatile solids (VS). The combination of HTP with AD significantly boosts methane yields by 51%, reaching 176 ml/g COD, and improves the digestate dewaterability, doubling the solid content in the dewatered cake. However, a higher polymer dose is required compared to conventional AD. Microbial community analysis correlates the observed performance and alterations; it indicates that HTP enhances resilience to stress conditions such as ammonia toxicity. This comprehensive study provides valuable insights into the transition from wastewater treatment plants (WWTPs) to resource recovery facilities (RRF) in line with circular economy principles.

Coupling hydrothermal liquefaction and anaerobic digestion for waste biomass valorization: A review in context of circular economy.

In light of the substantial global production of biomass waste, effective waste management and energy recovery solutions are of paramount importance. Hydrothermal liquefaction (HTL) and anaerobic digestion (AD) have emerged as innovative techniques for converting biomass waste into valuable resources. Their integration creates a synergistic framework that mitigates inherent limitations, leading to improved efficiency, enhanced product quality, and the comprehensive utilization of biomass. This review paper investigates the integration of HTL and AD, highlighting its significance and potential benefits as well as the optimal sequencing (HTL followed by AD and AD followed by HTL). The review encompasses experimental procedures, factors influencing both sequencing options, energy recovery characterizations, final product outcomes, as well as toxicological assessments and discussions on reduction. Additionally, it delves into the transition towards a circular bioeconomy and discusses the challenges and opportunities intrinsic to these processes. The findings presented in this review offer valuable insights to shape future research in this evolving field.

Can low-temperature thermal hydrolysis mitigate the oxidative stress of polystyrene nanoplastics on anaerobic digestion?

The presence of micro/nanoplastics (MPs/NPs) in sewage sludge has sparked considerable apprehensions over their potential negative effects on anaerobic digestion (AD) performance. The occurrence of MPs/NPs can trigger oxidative stress on the anaerobic microbiome, leading to potential inhibition of the AD process. While the thermal hydrolysis process (THP) is an extensively utilized sludge pretreatment method for AD, its impact on stress induced by MPs/NPs during AD remains poorly understood. In this study, we assessed the impacts of low-temperature THP (90 °C, 90 min) on AD of sewage sludge in the presence of 150 µg/L of polystyrene nanoplastics (PsNPs) under different solid retention times (SRTs) of 20, 15, and 10 d. The presence of PsNPs resulted in a higher reactive oxygen species (ROS) production and a higher abundance of antibiotic resistance genes (ARGs). Additionally, their presence caused a significant inhibition of methane production by 28.2%, 29.3%, and 38.8% for SRTs of 20, 15, and 10 d, respectively. Introducing low-temperature THP prior to the AD could partially recover methane production by mitigating ROS-induced stress and curbing the propagation of ARGs during the AD process. These results shed light on the potential benefits of THP and further optimization opportunities in alleviating the adverse effects of MPs/NPs-induced stress during sewage sludge AD.

Utilizing waste eggshells as a calcium precursor for contact precipitation of phosphorus from digested sludge centrate.

Phosphorus (P) recovery from wastewater is an essential component of the global P cycle. A contact precipitation process using chicken eggshells as a calcium (Ca) precursor was used to recover P from synthetic wastewater and real digested sludge centrate. Up to 96.4 % of P could be recovered from the digested sludge centrate after three repeated cycles of the contact precipitation process. In addition, 36.1 % of total chemical oxygen demand and 37.6 % of total ammonia nitrogen were removed from the centrate. Finally, most of the precipitates could be collected by a simple washing step. Scanning electron microscopy-energy dispersive spectroscopy and x-ray diffraction results indicated that the eggshells played three roles in this process: Ca source, precipitation substrate, and filter medium. Precipitates were mainly brushite. This research provides a new perspective on P recovery from wastewater using waste eggshells, and if further optimized, has a potential for practical future applications.

Low-temperature thermal hydrolysis for enhancing sludge anaerobic digestion and antibiotic resistance management: Significance of digester solids retention time.

Recently, there has been a growing inclination towards utilizing primary sludge (PS) fermentation prior to anaerobic digestion (AD) in water resource recovery facilities (WRRFs), where sludge liquor containing volatile fatty acids is used for biological nutrient removal. Nevertheless, using a low-temperature thermal hydrolysis process (THP) to improve AD in WRRFs adopting PS fermentation remains an area that has received limited research attention. Here, we studied the impact of THP (90 °C, 90 min) on anaerobic co-digestion of thickened waste activated sludge (TWAS) and fermented primary sludge (FPS) under varying solids retention times (SRTs) in semi-continuous mode. The study involved two THP schemes: scheme 1, where THP was done for both TWAS and FPS, and scheme 2, where THP was applied to TWAS only. The results demonstrated that reducing SRT from 20 to 15 and 10 d leads to decreased methane yield in both schemes. However, THP significantly enhances methane production, showing improvements of up to 37.9 % (scheme 1) and 31.2 % (scheme 2) under a 15-d SRT. Furthermore, while decreasing SRT increased the proliferation of antibiotic resistance genes (ARGs), thermal hydrolysis could effectively reduce most ARGs, indicating its potential to mitigate antibiotic resistance in the AD process. Overall, these results provide useful perceptions regarding the potential adoption of low-temperature THP in WRRFs with PS fermentation.

Boosting thermophilic anaerobic digestion with conductive materials: Current outlook and future prospects.

Thermophilic anaerobic digestion (TAD) can provide superior process kinetics, higher methane yields, and more pathogen destruction than mesophilic anaerobic digestion (MAD). However, the broader application of TAD is still very limited, mainly due to process instabilities such as the accumulation of volatile fatty acids and ammonia inhibition in the digesters. An emerging technique to overcome the process disturbances in TAD and enhance the methane production rate is to add conductive materials (CMs) to the digester. Recent studies have revealed that CMs can promote direct interspecies electron transfer (DIET) among the microbial community, increasing the TAD performance. CMs exhibited a high potential for alleviating the accumulation of volatile fatty acids and inhibition caused by high ammonia levels. However, the types, properties, sources, and dosage of CMs can influence the process outcomes significantly, along with other process parameters such as the organic loading rates and the type of feedstocks. Therefore, it is imperative to critically review the recent research to understand the impacts of using different CMs in TAD. This review paper discusses the types and properties of CMs applied in TAD and the mechanisms of how they influence methanogenesis, digester start-up time, process disturbances, microbial community, and biogas desulfurization. The engineering challenges for industrial-scale applications and environmental risks were also discussed. Finally, critical research gaps have been identified to provide a framework for future research.

Responses of syntrophic microbial communities and their interactions with polystyrene nanoplastics in a microbial electrolysis cell.

Microbial electrochemical technologies are promising for simultaneous energy recovery and wastewater treatment. Although the inhibitory effects of emerging pollutants, particularly micro/nanoplastics (MPs/NPs), on conventional wastewater systems have been extensively studied, the current understanding of their impact on microbial electrochemical systems is still quite limited. Microplastics are plastic particles ranging from 1 µm to 5 mm. However, nanoplastics are smaller plastic particles ranging from 1 to 100 nm. Due to their smaller size and greater surface area, they can penetrate deeper into biofilm structures and cell membranes, potentially disrupting their integrity and leading to changes in biofilm composition and function. This study first reports the impact of polystyrene nanoplastics (PsNPs) on syntrophic anode microbial communities in a microbial electrolysis cell. Low concentrations of PsNPs (50 and 250 µg/L) had a minimal impact on current density and hydrogen production. However, 500 µg/L of PsNPs decreased the maximum current density and specific hydrogen production rate by ∼43 % and ∼48 %, respectively. Exposure to PsNPs increased extracellular polymeric substance (EPS) levels, with a higher ratio of carbohydrates to proteins, suggesting a potential defense mechanism through EPS secretion. The downregulation of genes associated with extracellular electron transfer was observed at 500 µg/L of PsNPs. Furthermore, the detrimental impact of 500 µg/L PsNPs on the microbiome was evident from the decrease in 16S rRNA gene copies, microbial diversity, richness, and relative abundances of key electroactive and fermentative bacteria. For the first time, this study presents the inhibitory threshold of any NPs on syntrophic electroactive biofilms within a microbial electrochemical system.

Boosting resilience of microbial electrolysis cell-assisted anaerobic digestion of blackwater with granular activated carbon amendment.

Poor hydrolysis and methanogenesis efficiencies remain the main challenges for blackwater anaerobic digestion. This study investigated the performance of a granular activated carbon (GAC) amended microbial electrolysis cell-assisted anaerobic digester (MEC-AD) treating blackwater. Due to hydrolysis limitation, both MEC-AD and control reactors experienced performance declines as the organic loading rate increased from 3.0 to 4.5 g COD/L-d. Then, adding GAC without mixing formed GAC-sludge aggregates that improved methane yield to 38.3% and 32.3% in the MEC-AD and control reactor, respectively, and enhanced hydrolysis efficiency. The amended MEC-AD also successfully overcame the performance deterioration due to a temperature drop. Biomarker identification revealed the crucial roles of GAC biofilms and settled sludge in promoting methanogenesis and hydrolysis, respectively. This study demonstrated the GAC addition and the electrochemical environment could have a reciprocal influence, leading to more robust syntrophic microbial interactions, which could guide the future application of conductive materials in MEC-AD systems.

Nano and microplastics occurrence in wastewater treatment plants: A comprehensive understanding of microplastics fragmentation and their removal.

Nano/microplastic (NP/MP) pollution is a growing concern for the water environment. Wastewater treatment plants (WWTPs) are considered the major recipients of MP before discharging into local waterbodies. MPs enter WWTPs mainly from synthetic fibers through washing activities and personal care products. To control and prevent NP/MP pollution, it is essential to have a comprehensive understanding of their characteristics, fragmentation mechanisms, and the effectiveness of the current treatment processes used in WWTPs for NP/MP removal. Therefore, the objectives of this study are to (i) understand the detailed mapping of NP/MP in the WWTP, (ii) understand the fragmentation mechanisms of MP into NP, and (iii) investigate the removal efficiency of NP/MP by existing processes in the WWTP. This study found that fiber is the dominant shape of MP, and polyethylene, polypropylene, polyethylene terephthalate, and polystyrene are the major polymer type of MP in wastewater samples. Crack propagation and mechanical breakdown of MP due to water shear forces induced by treatment facilities (e.g., pumping, mixing, and bubbling) could be the major causes for NP generation in the WWTP. Conventional wastewater treatment processes are ineffective for the complete removal of MPs. Although these processes are capable of removing ∼95% of MPs, they tend to accumulate in sludge. Thus, a significant number of MPs may still be released into the environment from WWTPs on a daily basis. Therefore, this study suggested that using DAF process in the primary treatment unit can be an effective strategy to control MP in the initial stage before it goes to the secondary and tertiary stage.

Phosphorus recovery from wastewater via calcium phosphate precipitation: A critical review of methods, progress, and insights.

Phosphorus (P) is one of the important elements for human, animal, and plant life. Due to the development of the circular economy in recent years, the recovery of P from wastewater has received more attention. Recovery of P from domestic, industrial, and agricultural wastewater in the form of calcium phosphate (CaP) by precipitation/crystallization process presents a low-cost and effective method. Recovered CaP could be used as P fertilizer and for other industrial applications. This review summarizes the effects of supersaturation, pH, seed materials, calcium (Ca) source, and wastewater composition, on the precipitation/crystallization process. The recovery efficiency and value proposition of recovered CaP were assessed. This in-depth analysis of the literature reports identified the process parameters that are worth further optimization. The review also provides perspectives on future research needs on expanding the application field of recovered CaP and finding other more economical and environmentally friendly Ca sources.

Ensemble machine learning approach for examining critical process parameters and scale-up opportunities of microbial electrochemical systems for hydrogen peroxide production.

Hydrogen peroxide (H2O2) production in microbial electrochemical systems (MESs) is an attractive option for enabling a circular economy in the water/wastewater sector. Here, a machine learning algorithm was developed, using a meta-learning approach, to predict the H2O2 production rates in MES based on the seven input variables, including various design and operating parameters. The developed models were trained and cross-validated using the experimental data collected from 25 published reports. The final ensemble meta-learner model (combining 60 models) demonstrated a high prediction accuracy with very high R2 (0.983) and low root-mean-square error (RMSE) (0.647 kg H2O2 m-3 d-1) values. The model identified the carbon felt anode, GDE cathode, and cathode-to-anode volume ratio as the top three most important input features. Further scale-up analysis for small-scale wastewater treatment plants indicated that proper design and operating conditions could increase the H2O2 production rate to as high as 9 kg m-3 d-1.

Fate of intracellular, extracellular polymeric substances-associated, and cell-free antibiotic resistance genes in anaerobic digestion of thermally hydrolyzed sludge.

Thermal hydrolysis of sludge is a promising approach to mitigate antibiotic resistance genes (ARGs) propagation in anaerobic digestion (AD). Although ARGs in sludge may be fractioned into intracellular, extracellular polymeric substance (EPS)-associated, and cell-free ARGs, the fate of these different fractions in AD has never been investigated. This study presents a detailed characterization of intracellular and extracellular ARGs in AD of sludge thermally hydrolyzed at 90 °C and 140 °C. EPS-associated ARGs represented the major fraction of the total extracellular ARGs in all samples, while its lowest abundance was observed for thermal hydrolysis at 140 °C along with the lowest EPS levels. The results suggested a positive correlation between EPS-associated ARGs with intracellular and cell-free ARGs. Furthermore, various EPS components, such as proteins and e-DNA, were positively correlated with ß-lactam resistance genes. sul1 dominated all samples as an EPS-associated resistance gene. These results provide new insights into the significance of different ARGs fractions in their overall dissemination in AD integrated with thermal hydrolysis.

Mitigating methane emission from oil sands tailings using enzymatic and lime treatments.

Engineering strategies to reduce greenhouse gases (GHGs) emissions by inhibiting methanogenesis in oil sands tailings have rarely been examined. In this study, we explored the potential impact of chemical treatment (lime) and biological treatment using enzymes (lysozyme and protease) on inhibiting methane emissions from tailings. Overall, treatment with protease 3%, lysozyme 3%, and lime 5000 ppm reduced CH4 production (by 52%, 28%, and 25%, respectively) and were weakly associated with the archaeal abundance. Enzymes treatment resulted in a higher reduction in CH4 production compared with lime treatment. A 3% lysozyme treatment suppressed CH4 production (the change in methane was 0.48 mmol) and reduced the degradation of hexane throughout the experiment. Similarly, 3% protease suppressed CH4 production throughout the experiment (the change in methane was 0.78 mmol), which could be attributed to the pH reduction to pH 4.9 at week 23 resulting from the formation of volatile fatty acids. Another possible mechanism could be the formation of toxic compounds, such as high nitrogen content, after protease treatment that inhibited the microbial community. The toxicity effect to Vibrio fischeri was greater with lysozyme 3% and protease 3% treatment than with lime treatment (124 TU and 76 TU, respectively). Lime treatment resulted in the highest reduction in 16S rRNA gene copies from 5.7 × 106 cells g-1 (control) to 2.7 × 105, 1.71 × 105, and 1.4 × 105 cells g-1 for 1600, 3500, and 5000 ppm treatments, respectively. This study supports further work to examine and determine the optimum conditions (e.g., enzyme and lime dosages) for CH4 inhibition.

Carbon cloth amendment for boosting high-solids anaerobic digestion with percolate recirculation: Spatial patterns of microbial communities.

The addition of conductive materials in anaerobic digestion (AD) is a promising method for boosting biomethane recovery from organic waste. However, conductive additives have rarely been investigated for the high-solids anaerobic digestion (HSAD). Here, the impact of adding carbon cloth in the solid phase of an HSAD system with percolate recirculation was investigated. Furthermore, spatial patterns of microbial communities in suspended biomass, percolate, and carbon cloth attached biofilm were assessed. Carbon cloth increased biomethane yield from source-separated organics (SSO) by 20% more than the unamended control by shortening the lag phase (by 15%) and marginally improving the methanogenesis rate constant (by ∼8%) under a batch operation for 50 days. Microbial community analysis demonstrated higher relative abundances of the archaeal population in the carbon cloth amended reactor than in unamended control (12%-21% vs. 5%-15%). Compared to percolate and suspension, carbon cloth attached microbial community showed higher enrichment of known electroactive Pseudomonas species along with Methanosarcina and Methanobacterium species, indicating the possibility of DIET-based syntrophy among these species.

Exploration of machine learning algorithms for predicting the changes in abundance of antibiotic resistance genes in anaerobic digestion.

The land application of digestate from anaerobic digestion (AD) is considered a significant route for transmitting antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) to ecosystems. To date, efforts towards understanding complex non-linear interactions between AD operating parameters with ARG/MGE abundances rely on experimental investigations due to a lack of mechanistic models. Herein, three different machine learning (ML) algorithms, Random Forest (RF), eXtreme Gradient Boosting (XGBoost), and Artificial Neural Network (ANN), were compared for their predictive capacities in simulating ARG/MGE abundance changes during AD. The models were trained and cross-validated using experimental data collected from 33 published literature. The comparison of model performance using coefficients of determination (R2) and root mean squared errors (RMSE) indicated that ANN was more reliable than RF and XGBoost. The mode of operation (batch/semi-continuous), co-digestion of food waste and sewage sludge, and residence time were identified as the three most critical features in predicting ARG/MGE abundance changes. Moreover, the trained ANN model could simulate non-linear interactions between operational parameters and ARG/MGE abundance changes that could be interpreted intuitively based on existing knowledge. Overall, this study demonstrates that machine learning can enable a reliable predictive model that can provide a holistic optimization tool for mitigating the ARG/MGE transmission potential of AD.

Enhancing quorum sensing in biofilm anode to improve biosensing of naphthenic acids.

The feasibility of enhancing quorum sensing (QS) in anode biofilm to improve the quantifications of commercial naphthenic acid concentrations (9.4-94 mg/L) in a microbial electrochemical cell (MXC) based biosensor was demonstrated in this study. First, three calibration methods were systematically compared, and the charging-discharging operation was selected for further experiments due to its 71-227 folds higher electrical signal outputs than the continuous closed-circuit operation and cyclic voltammetry modes. Then, the addition of acylase (5 µg/L) as an exogenous QS autoinducer (acylase) was investigated, which further improved the biosensor's electrical signal output by ∼70%, as compared to the control (without acylase). The addition of acylase increased the relative expression of QS-associated genes (lasR, lasI, rhlR, rhlI, lasA, and luxR) by 7-100%, along with increased abundances of known electroactive bacterial genera, such as Geobacter (from 42% to 47%) and Desulfovibrio (from 6% to 11%). Furthermore, toxicities of different NAs concentrations measured with the Microtox bioassay test were correlated with corresponding electrical signals, indicating that MXC-biosensor can provide a dual platform for rapid assessment of both NA concentrations and NA-associated toxicity.

A critical review of process parameters influencing the fate of antibiotic resistance genes in the anaerobic digestion of organic waste.

The overuse and inappropriate disposal of antibiotics raised severe public health risks worldwide. Specifically, the incomplete antibiotics metabolism in human and animal bodies contributes to the significant release of antibiotics into the natural ecosystems and the proliferation of antibiotic-resistant bacteria carrying antibiotic-resistant genes. Moreover, the organic feedstocks used for anaerobic digestion are often highly-rich in residual antibiotics and antibiotic-resistant genes. Hence, understanding their fate during anaerobic digestion has become a significant research focus recently. Previous studies demonstrated that various process parameters could considerably influence the propagation of the antibiotic-resistant genes during anaerobic digestion and their transmission via land application of digestate. This review article scrutinizes the influences of process parameters on antibiotic-resistant genes propagation in anaerobic digestion and the inherent fundamentals behind their effects. Based on the literature review, critical research gaps and challenges are summarized to guide the prospects for future studies.

Propagation of antibiotic resistance genes during anaerobic digestion of thermally hydrolyzed sludge and their correlation with extracellular polymeric substances.

The positive impact of the thermal hydrolysis process (THP) of sewage sludge on antibiotic resistance genes (ARGs) removal during anaerobic digestion (AD) has been reported in the literature. However, little information is available on how changes in different extracellular polymeric substances (EPS) due to THP can influence ARG propagation during AD. This study focused on systematically correlating EPS components and ARG abundance in AD of sewage sludge pretreated with THP (80 °C, 110 °C, 140 °C, 170 °C). THP under different conditions improved sludge solubilization followed by improved methane yields in the biochemical methane potential (BMP) test. The highest methane yield of 275 ± 11.5 ml CH4/g COD was observed for THP-140 °C, which was 40.5 ± 2.5% higher than the control. Increasing THP operating temperatures showed a non-linear response of ARG propagation in AD due to the rebound effect. The highest ARGs removal in AD was achieved with THP at 140 °C. The multivariate analysis showed that EPS polysaccharides positively correlated with most ARGs and integrons, except for macrolides resistance genes. In contrast, EPS protein was only strongly correlated with ß-lactam resistance genes. These results suggest that manipulating THP operating conditions targeting specific EPS components will be critical to effectively mitigating the dissemination of particular ARG types in AD.