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.

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.

Pilot-scale investigation of conductive carbon cloth amendment for enhancing high-solids anaerobic digestion and mitigating antibiotic resistance.

This study examined the effectiveness of introducing conductive carbon cloth into a pilot-scale high-solids anaerobic digestion (HSAD) system. Adding carbon cloth increased methane production by 22 % and improved the maximum methane production rate by 39 %. Microbial community characterization indicated a possible direct interspecies electron transfer-based syntrophic association among microbes. Using carbon cloth also enhanced microbial richness, diversity, and evenness. Carbon cloth effectively reduced the total abundance of antibiotic resistance genes (ARGs) by 44.6 %, mainly by inhibiting horizontal gene transfer, as shown by the significant decrease in the relative abundance of integron genes (particularly intl1). The multivariate analysis further demonstrated strong correlations of intl1 with most of the targeted ARGs. These findings suggest that carbon cloth amendment can promote efficient methane production and attenuate the spread of ARGs in HSAD systems.

Antibiotic resistance genes proliferation under anaerobic degradation of polylactic acid and polyhydroxy butyrate bioplastics.

As the global concern over plastic pollution grows, efforts are underway to find environmentally friendly alternatives to traditional plastics. Bioplastics are being extensively researched and developed as a possible solution. This study compared the impact of two bioplastics, polylactic acid (PLA) and polyhydroxy butyrate (PHB), on the proliferation of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) during anaerobic digestion (AD). Both bioplastics (250-500 particles) could be degraded to a certain extent over 79 days, as indicated by higher methane production than the control without bioplastic particles. The PHB 500 reactor showed the highest methane yield along with the highest biodegradation efficiency (91 %) than other reactors amended with PHB and PLA particles. The highest ARG and MGE abundances were also observed in PLA 500, and the lowest ARG abundance was in PLA 250. Conversely, PHB reactors showed a relatively lower ARG abundance than the control. The correlation analysis suggested that most ARGs were positively correlated with PLA and negatively correlated with PHB (except for tetA, tetB, and tetX). Moreover, a correlation between MGEs and ARGs in PLA and PHB reactors was revealed by correlation analysis. These results show that AD responds differently to the different types/levels of bioplastics, which can ultimately influence the behavior of ARG proliferation. Thus, bioplastics may also pose a potential risk for spreading antibiotic resistance. These findings can be used as a basis for setting environmental standards for bioplastics and creating monitoring and control measures to prevent potential negative impacts on public health.

Granular activated carbon remediates antibiotic resistance propagation and methanogenic inhibition induced by polystyrene nanoplastics in sludge anaerobic digestion.

Nano/microplastics (NPs/MPs) in sewage sludge can induce oxidative stress to the anaerobic digestion (AD) and also proliferate antibiotic resistance genes (ARGs). Recently, granular activated carbon (GAC) has been used as an additive to enhance methane production in AD via direct interspecies electron transfer (DIET); however, its impact on AD exposed to NPs/MPs is yet to be studied. This study examined the effect of GAC (5 and 15 g/L) on sludge AD exposed to 150 µg/L of polystyrene nanoplastics (PsNPs). PsNPs decreased methane yield by 32.3% due to elevated levels of reactive oxygen species. However, GAC addition counteracted this adverse effect and improved methane production, attributed to the potential enrichment of DIET-active microbes and the adsorption of PsNPs by GAC. Moreover, GAC reduced the total abundance of ARGs, which was increased by PsNPs exposure. Thus, GAC can provide dual benefits in mitigating methanogenic inhibition caused by PsNPs and ARG spread.

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.

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.

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.

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.

Molecular biology and modeling analysis reveal functional roles of propionate to acetate ratios on microbial syntrophy and competition in electro-assisted anaerobic digestion.

This study examined the significance of propionate to acetate (HPr/HAc) ratios on microbial syntrophy and competition in microbial electrolysis cell-assisted anaerobic digestion (MEC-AD). In addition to molecular biology and phylogenetic analysis, a numerical MEC-AD model was developed by modifying Anaerobic Digestion Model No.1 to predict the effects of different HPr/HAc ratios (0.5, 1.5, 2.5, and 5). The HPr/HAc ratios of 0.5 and 1.5 maintained efficient syntrophy among electroactive bacteria, hydrogenotrophic methanogens, and homoacetogens, leading to higher methane yields. In contrast, higher HPr/HAc ratios of 2.5 and 5 were detrimental to methanogenesis. Both microbial community analysis and numerical modeling results suggested that higher propionate levels could promote the enrichment of H2-utilizing acetogens, thereby triggering their competition with hydrogenotrophic methanogens. Moreover, protein fraction in extracellular polymeric substances and the relative expression of genes associated with extracellular electron transfer in both anode and cathode biofilms were markedly decreased with increasing HPr/HAc ratios, indicating partial inhibition of microbial electroactivity. Overall, these results illuminate deep insight into anaerobic syntrophy, contributing to the process kinetics and methane yields in MEC-AD systems. Furthermore, from a practical viewpoint, the results can also be helpful in effective control of MEC-AD operation without propionate accumulation.

Significance of different mixing conditions on performance and microbial communities in anaerobic digester amended with granular and powdered activated carbon.

Conductive materials amendment in anaerobic digestion (AD) is a promising strategy for boosting the methanogenesis process. Despite mixing is a critical parameter, the behavior of digesters amended with conductive additives upon different mixing conditions has rarely been investigated. This study investigated continuous mixing, intermittent mixing (10 min in every 12 h), and non-mixing conditions for digesters amended with granular activated carbon (GAC) and powdered activated carbon (PAC). The non-mixed GAC digester provided the highest methane yield (318 ± 28 mL/g COD) from synthetic blackwater, while intermittently mixed GAC and control exhibited similar methane yields (290-294 mL/g COD). For non-mixed systems, microbial richness and diversity increased with GAC and PAC amendment. In contrast, continuous and intermittent mixing increased microbial diversity and richness in control reactors while reduced the same in GAC and PAC amended reactors. Overall, various mixing conditions distinctly changed the degree of enrichment/retention of microbes and consequently influenced methane recovery.

High-rate blackwater anaerobic digestion under septic tank conditions with the amendment of biosolids-derived biochar synthesized at different temperatures.

Septic tanks have been widely used for blackwater treatment in developing countries, while high-rate septic tanks with improved methane recovery are yet to be achieved. This study investigated biosolids-derived biochar (synthesized at 300℃, 425℃, and 550℃) as an additive for developing high-rate septic tanks. The experiments were conducted with anaerobic bioreactors operated with synthetic blackwater under septic tank conditions. All biochar amended reactors demonstrated a steady increase in daily methane production for increasing OLR from 0.08 to 3 g COD/L/d. The control reactor showed significant process disturbances at OLRs ≥ 2 g COD/L/d with an accumulation of volatile fatty acids followed by pH drop. At OLR of 3 g COD/L/d, the daily methane production from biochar amended reactors was ~ 4.3 times higher than the control (300 vs. 70 mL per day). Biochar addition established a robust microbiome consisted of a higher abundance of hydrogenotrophic and acetoclastic methanogens and hydrogen-producing fermentative bacteria.

Characterization and significance of extracellular polymeric substances, reactive oxygen species, and extracellular electron transfer in methanogenic biocathode.

The microbial electrolysis cell assisted anaerobic digestion holds great promises over conventional anaerobic digestion. This article reports an experimental investigation of extracellular polymeric substances (EPS), reactive oxygen species (ROS), and the expression of genes associated with extracellular electron transfer (EET) in methanogenic biocathodes. The MEC-AD systems were examined using two cathode materials: carbon fibers and stainless-steel mesh. A higher abundance of hydrogenotrophic Methanobacterium sp. and homoacetogenic Acetobacterium sp. appeared to play a major role in superior methanogenesis from stainless steel biocathode than carbon fibers. Moreover, the higher secretion of EPS accompanied by the lower ROS level in stainless steel biocathode indicated that higher EPS perhaps protected cells from harsh metabolic conditions (possibly unfavorable local pH) induced by faster catalysis of hydrogen evolution reaction. In contrast, EET-associated gene expression patterns were comparable in both biocathodes. Thus, these results indicated hydrogenotrophic methanogenesis is the key mechanism, while cathodic EET has a trivial role in distinguishing performances between two cathode electrodes. These results provide new insights into the efficient methanogenic biocathode development.

An intermittent power supply scheme to minimize electrical energy input in a microbial electrolysis cell assisted anaerobic digester.

From the perspective of energy saving in the operation of microbial electrolysis cell assisted anaerobic digester (MEC-AD), this study focused on developing an intermittent power supply scheme. The applied potential was switched off for 12 and 6 hours/day during the operation of a laboratory-scale MEC-AD system fed with glucose. The results from the operation under continuous applied potential served as the control. The overall biomethane generation and net energy income from the process were unaffected when the applied potential turned off for 6 hours/day. Both quantitative and qualitative analyses of microbial communities suggested that a balanced microbiome could be maintained under short-term switching-off the applied potential. However, performance substantially deteriorated when the applied potential turned off for 12 hours/day. Overall, the results of this study suggest that MEC-AD operation does not need a continuous power supply, and higher energy efficiency can be effectively achieved by intermittently powering the reactor.

A high-rate anaerobic biofilm reactor for biomethane recovery from source-separated blackwater at ambient temperature.

Anaerobic bioreactors for source-separated blackwater are mostly operated at low organic loading rates (OLRs) due to low biodegradability and the potential of ammonia inhibition. In this study, an anaerobic biofilm reactor having conductive carbon fibers as the media was investigated for the high-rate treatment of blackwater collected from vacuum toilets. The bioreactor was operated at different OLRs ranged from 0.77 to 3.01 g COD/L-d in four stages for a total operating period of ~ 250 days. With the increase of OLRs, the specific methane production rate increased from 105.3 to 304.6 ml/L-d with high methane content in biogas (75.5%-83%). The maximum methane yield was achieved at hydraulic retention time (HRT) of 15 days. Highest organics and suspended solids removal (80%-83%) were achieved at 20-days HRT, while increased OLRs resulted in diminished removal efficiencies. The state variables, including pH, total ammonia nitrogen, short-chain volatile fatty acids, and soluble chemical oxygen demand, indicated the system had a great capability to withstand the high OLRs. Microbial community analysis revealed that the high performance might be attributed to direct interspecies electron transfer (DIET) facilitated by potentially electroactive bacteria (e.g., Syntrophomonas, Clostridium) and electrotrophic archaea (e.g., Methanosaeta and Methanosarcina species) enriched on the carbon fibers. PRACTITIONER POINTS: An anaerobic biofilm reactor was investigated for biomethane recovery from source-separated blackwater. Conductive carbon fibers were utilized as the media to stimulate enrichment of potentially electroactive methanogenic communities. The bioreactor was operated at ambient temperature for over 250 days. High methane production rate and high-quality biogas were achieved at OLRs ranged from 0.77 to 3.01 g COD/L-d. Microbial community analysis suggested direct interspecies electron transfer (DIET) between specific electroactive bacteria and electrotrophic archaea.

Changes in syntrophic microbial communities, EPS matrix, and gene-expression patterns in biofilm anode in response to silver nanoparticles exposure.

Understanding the toxic effect of silver nanoparticles (AgNPs) on various biological wastewater treatment systems is of significant interest to researchers. In recent years, microbial electrochemical technologies have opened up new opportunities for bioenergy and chemicals production from organic wastewater. However, the effects of AgNPs on microbial electrochemical systems are yet to be understood fully. Notably, no studies have investigated the impact of AgNPs on a microbial electrochemical system fed with a complex fermentable substrate. Here, we investigated the impact of AgNPs (50 mg/L) exposure to a biofilm anode in a microbial electrolysis cell (MEC) fed with glucose. The volumetric current density was 29 ± 2.0 A/m3 before the AgNPs exposure, which decreased to 20 ± 2.2 A/m3 after AgNPs exposure. The biofilms produced more extracellular polymeric substances (EPS) to cope with the AgNPs exposure, while carbohydrate to protein ratio in EPS considerably increased from 0.4 to 0.7. Scanning electron microscope (SEM) imaging also confirmed the marked excretion of EPS, forming a thick layer covering the anode biofilms after AgNPs injection. Transmission electron microscope (TEM) imaging showed that AgNPs still penetrated some microbial cells, which could explain the deterioration of MEC performance after AgNPs exposure. The relative expression level of the quorum signalling gene (LuxR) increased by 30%. Microbial community analyses suggested that various fermentative bacterial species (e.g., Bacteroides, Synergistaceae_vadinCA02, Dysgonomonas, etc.) were susceptible to AgNPs toxicity, which led to the disruption of their syntrophic partnership with electroactive bacteria. The abundance of some specific electroactive bacteria (e.g., Geobacter species) also decreased. Moreover, decreased relative expressions of various extracellular electron transfer associated genes (omcB, omcC, omcE, omcZ, omcS, and pilA) were observed. However, the members of family Enterobacteriaceae, known to perform a dual function of fermentation and anodic respiration, became dominant after biofilm anode exposed to AgNPs. Thus, EPS extraction provided partial protection against AgNPs exposure.