ACS ES&T Water

The 2020 SARS-CoV-2 pandemic renewed global interest in wastewater-based epidemiology (WBE) as a tool for monitoring public health. Molecular analyses of wastewater are often limited by the small volumes of wastewater that can be processed, due to column clogging, handling constraints, and processing time. Additionally, inhibitors in the complex wastewater matrix reduce the sensitivity of downstream assays such as RT-PCR and sequencing. To address these limitations, we developed a novel column by incorporating a hydrophobic pre-filtration layer and sequential glass fiber filters. This enhanced column design, PureBioX Xpurify Column, enables processing of 1.58 times more wastewater (a 58% increase in throughput) while reducing RT-PCR inhibitors and maintaining compatibility with existing workflows. Despite a modest reduction in nucleic acid yield, the modified column consistently improved viral RNA detection sensitivity, including for SARS-CoV-2. This accessible, scalable upgrade strengthens the utility of direct capture methods in WBE-based public health surveillance.

Rare earth elements (REEs) are critical components of green technologies, but current mining and purification methods remain environmentally unsustainable due to their high energy consumption and intensive chemical requirements. Bio-hydrometallurgical processes have the potential to concentrate and recover REEs at a circumneutral pH. Work presented here uses bacteria at neutral pH to concentrate REEs from solution and subsequently recover those REEs using sodium citrate. Shewanella oneidensis MR-1 was incubated anaerobically in a culture media solution spiked with 14 REEs and yttrium for one to six days. REE concentrations remaining in solution were then compared to REE concentrations on cell pellets. For these same timepoints, the loosely bound extracellular polymeric substance (LB-EPS) was removed from cells prior to quantifying REEs on pellets to narrow down the location of REE binding. Moreover, cell pellets collected after 5 days in REE spiked solution were subjected to a time series desorption assay using sodium citrate. Shewanella oneidensis at a starting OD600 of 0.6 adsorbed 1.18mg/g of REE after 3 days. 80% of these REEs were located in the LB-EPS. In 10 minutes, 0.5 M sodium citrate desorbed about 75% of REEs from cells and over 95% after 24 hours. This method was also applied to Alaskan coal and showed that 68-86% of REEs were desorbed form S. oneidensis. This study elucidates the REE binding location and capacity of S. oneidensi, REE removal efficiency of sodium citrate overtime, and the application of this sustainable biotechnology for REE recovery at a circumneutral pH from Alaskan coal.

Hospital-based wastewater surveillance may complement community and clinical surveillance data in important ways, and may be useful in jurisdictions without community-based wastewater surveillance. From May 2024-April 2026, we analyzed weekly samples (n=190) from three hospitals in New York City using digital PCR to evaluate the sensitivity, specificity, and positive predictive value (PPV) of wastewater viral detection against facility SARS-CoV-2 and influenza A/B inpatient caseloads. Sensitivity was 38-42% for SARS-CoV-2 and 36-49% for influenza A/B, while specificity exceeded 72% for all pathogens. During respiratory seasons, sensitivity reached 81% for SARS-CoV-2 and 81% for influenza A; both had 100% sensitivity during peak case weeks. Notably, off-peak influenza detections occurred in hospital wastewater at all three hospitals in summer 2024 without corresponding hospital case detection, suggesting the presence of undiagnosed cases. These findings underscore the potential utility of hospital-based wastewater monitoring for tracking respiratory virus activity.

Since the COVID-19 pandemic, wastewater-based surveillance (WBS) has emerged as a key approach to assess community-level health and the evolution of pathogens. To date, most established WBS systems focus on polymerase chain reaction (PCR) based detection and targeted sequencing of known pathogens because these approaches are well-accepted and include amplification of pathogen target sequences of interest thereby enabling lower limits of detection. Metagenomic next-generation sequencing (mNGS) is a promising approach to enable pathogen detection and surveillance beyond predefined pathogen lists, but its regular application to WBS has not been yet widely adopted because many key performance characteristics are not well-understood, including limit of detection (LOD) and false positive/negative rates. This paper describes a computational analysis to estimate the operational LOD of various sequencing approaches using a simplified model of a local wastewater (WW) system involving a military base. This paper also presents findings from two types of experiments: 1) laboratory-spiked, those for which Atlantibacter subterraneus (Asub) is introduced into real-world WW samples in a laboratory setting, and 2) system-spiked, those for which Asub is introduced at a source location of a real-world WW system. Findings indicate that mNGS detection performance varies with sequencing method and the data analysis process. In addition, findings indicate that site-specific method characterization should be used when implementing mNGS for WBS because sites can have different WW system configurations, background organisms and sequencing inhibitors.

Nutrient-rich food-processing wastewaters represent valuable yet under-utilised side streams for sustainable protein production in the form of microbial biomass. Here we present an integrated dual-loop bioprocess that converts soybean-processing wastewater into microbial single-cell protein (SCP) while achieving substantial nutrient removal and product refinement. In the first loop, previously enriched microbial consortia were inoculated and cultivated in four parallel sequencing batch reactors (SBRs) for 44days at a hydraulic retention time (HRT) of 3days. This bioprocess configuration demonstrated features that support future scale-up while maintaining process stability, achieving a protein content of 33.3{+/-}3.2%, doubling the protein yield (15.32{+/-}3.49g dry weight per g soluble TKN) and quadrupling the production rate (0.29{+/-}0.06g dry weight L-1 d-1) compared to operating reactors without inoculation (HRT: 7.2days). Effluent treatment was stable, with 84% carbon and 78% nitrogen removal efficiencies, demonstrating efficient nutrient recovery. The SCP biomass was enriched in functional taxa, including Acidipropionibacterium, Lactococcus, Megasphaera, and Azospirillum, suggesting that reactor conditions and inoculum selection promoted a stable, protein-productive microbial community with potential probiotic benefits. In the second loop, bioreactor effluent was reused as aqueous matrix for heat treatment (60{degrees}C) of the SCP biomass, reducing the RNA content from 8.6% to 2.6%, with a 39% biomass loss accompanied by a 30% increase in total amino acid concentration. Hence, our valorisation approach integrates microbial biomass production, effluent reuse, and product refinement within a circular framework. The system provides a resource-efficient pathway for converting food-sector side streams into high-quality microbial community-based SCP, highlighting its potential scalability for sustainable nutrient and water management.

Wastewater-based epidemiology (WBE) has the potential to fill gaps in clinical surveillance for respiratory syncytial virus (RSV) burden. Previous studies have shown RSV to be detectable in wastewater, but few have linked detections to clinical data because, in many locations, RSV is not a reportable disease. Further, studies that have linked RSV in wastewater to clinical data have not distinguished between pediatric and adult infections, as is common in studies of RSV. Using 2,662 influent wastewater samples collected from twenty-four treatment plants in four New York counties, we measured the RSV concentration in wastewater and compared levels detected to RSV hospitalizations from September 2022 to July 2024. RSV concentrations in wastewater correlated well with RSV hospitalizations ({rho} between 0.52 and 0.85, P <0.001). RSV concentrations in wastewater lagged hospitalizations for under 10-year-olds by an average of three weeks but were a leading indicator for hospitalizations in patients over 50 by up to two weeks. In predictive models, wastewater explained 88 percent of the variance in RSV hospital admissions among people over 50. Wastewater-based epidemiology of RSV is therefore a reliable surveillance method and could provide early warning for increases in RSV hospitalizations among older populations.

Disagreement between molecular and culture-based assays for Legionella pneumophila detection is widely reported, yet comparisons have largely been based on direct assay-derived concentrations or binary positive/negative outcomes. However, it remains unclear whether molecular-culture disagreement reflects concentration-level incompatibility or unaccounted methodological and physiological differences related to DNA recovery and cell culturability. In this study, we observed substantial disagreement between molecular and Legiolert assays in source and finished drinking water samples collected from eight full-scale drinking water systems across the United States. Molecular thresholds adjusted for DNA recovery and cell culturability only partially resolved these discrepancies. We therefore developed a probabilistic Monte Carlo framework that incorporates sample-specific DNA recovery and cell culturability to evaluate the quantitative consistency of culturable L. pneumophila concentrations estimated by molecular and Legiolert assays. Quantitatively consistent and inconsistent samples occurred across both binary concordant and discordant classifications, demonstrating that positive/negative agreement poorly reflects concentration-level comparability. Overall, molecular and Legiolert assays showed strong quantitative consistency once sample-specific DNA recovery and cell culturability were considered. A small proportion of persistent inconsistencies at specific sampling sites, coupled with atypical microbial indicators, suggest that sample heterogeneity likely contributed to the remaining discrepancies. These findings demonstrate that integrating DNA recovery and cell culturability enhanced quantitative consistency between molecular and Legiolert assays and supports the use of molecular methods as rapid quantitative tools to complement culture-based L. pneumophila monitoring.

Surface waters in urban watersheds receive episodic inputs of wastewater, runoff, and road-salt residues during spring, yet the contribution of these short hydrological windows to antibiotic resistance gene (ARG) loading remains poorly resolved. Weekly samples were collected from offshore and nearshore sites in the Detroit River, a Great Lakes transboundary connecting channel, from February to December 2025. Five clinically relevant ARGs encoding resistance to carbapenems, methicillin, and colistin, alongside the fecal marker pepper mild mottle virus (PMMoV), were quantified by qPCR and paired with ten conventional water-quality variables. blaNDM, mcr-1, and blaVIM-7 were not detected while blaKPC occurred as discrete pulses. One week (5 May) accounted for 37.2% of annual offshore blaKPC loading, and three weeks in late April to early May accounted for 72.8%, with peak concentrations reaching 5.4 x 10E3 and 7.2 x 10E3 copies/L. mecA was detected in nearly all samples without a dominant pulse. PMMoV normalization showed blaKPC did not vary seasonally (Kruskal-Wallis p = 0.198), consistent with diluted wastewater during spring precipitation events rather than an emergent source. mecA/PMMoV varied seasonally (p = 0.003) and was lowest in spring, implicating non-wastewater inputs in summer and autumn. Seven water-quality variables were significantly elevated during blaKPC pulse weeks. PCA distinguished pulse from background conditions, explaining 81.3% of variance. A random forest classifier achieved leave-one-out AUC of 0.917; ROC AUC reached 0.943 for total phosphorus, 0.924 for chloride, and 0.974 for the multivariate model. These results demonstrate that blaKPC and mecA operate through distinct source pathways and that routine water-quality monitoring can flag elevated blaKPC risk without additional sampling infrastructure.

Wastewater surveillance (WS) has been widely adopted as a cost-effective and population-representative infectious disease monitoring tool and is increasingly being applied to bacterial and antimicrobial resistance gene (ARG) targets. However, some of these targets may persist in pipe biofilms and detach into wastewater, complicating accurate WS interpretation. To investigate biofilm contributions to wastewater pathogen and ARG signals, paired sink-drain biofilm, branch-drain-plumbing biofilm (sewer biofilm), and wastewater were collected from five hospital sites over a four-month period and analyzed using 16S rRNA gene amplicon sequencing and probe-capture metagenomics. Overall, sewer biofilm bacterial communities were as diverse as wastewater. Across sites, a mean of 9% (0.9 to 23.3%) of wastewater bacterial communities could be attributed to sewer biofilm communities. Many clinically relevant pathogens were consistently detected both in sewer biofilm and wastewater, including environmentally persistent and/or biofilm-associated taxa (e.g., Pseudomonas aeruginosa, Klebsiella pneumoniae). While many ARGs overlapped between wastewater and biofilms (e.g., tetA, sul1, blaCTX-M, vanA), others were significantly enriched in sewer biofilms (e.g., qacL, van-operon and OXA genes). Together, these findings confirm that wastewater pathogen and resistome profiles integrate inputs from both human shedding and pipe-resident communities and therefore need to be considered when selecting WS targets and interpreting signal.

In response to the growing need to recycle E-waste, specifically that of lithium-ion batteries (LIBs), the development of sustainable recycling methodologies will be vital. As one of the most sustainable and low-cost options, biohydrometallurgy (BioHM) uses biological organisms to facilitate the recovery of critical metals from spent LIBs. Despite the advantages of BioHM, its slow kinetics, due to the reliance on the metabolic activity of microorganisms, limit its large-scale application for the closed-loop recycling of LIBs. In this work, we investigate the correlation between the incubation time of Acidithiobacillus ferrooxidans (Atf) and its leaching efficiency of four common elements in LIBs: Li, Ni, Mn, and Co. We assess how specific incubation times along the biological growth curve affect the leaching efficiency of Li[Ni0.6Mn0.2Co0.2]O2 (NMC622), a model electrode material. Our results show that pH alone is not an accurate descriptor of the leaching efficiency of Atf cultures used at different growth stages. The addition of NMC622 during the bacterial lag phase reaches similar or even improved extraction compared to cultures used after reaching the exponential or stationary phases. A simple model of leaching dynamics shows predictions consistent with our experimental observations under the condition that the inhibition of bacterial growth by NMC is not severe. Our findings indicate that early-stage cultures can alleviate the kinetic bottleneck and improve the throughput of recovering critical materials from spent batteries.

Bioelectrochemical reduction of dilute nitrate (NO-; sub-mM to low-mM range) to ammonium (NH) offers a promising route toward circular nitrogen management from contaminated groundwater and environmental waters. However, on-site application of bioelectrochemical systems remains challenging due to low reduction rates and poor electron transfer efficiency of naturally formed biofilm electrodes. Here, we constructed a hydrogel biocathode by applying a carbon black/riboflavin/sodium alginate/cellulose hydrogel incorporating Shewanella oneidensis MR-1 cells to a graphite felt electrode via brush coating. The hydrogel electrode achieved NH production rates of 0.16-0.19 mol m-3 h-{superscript 1} without NO2- accumulation, and these rates were maintained without significant performance loss across three consecutive cycles with medium exchange over 1.5 days of total operation. The hydrogel electrode increased the current density by more than 5-fold compared with a conventional S. oneidensis biofilm electrode, indicating enhanced electron transfer efficiency per unit biomass, which directly contributed to the high NH production rates. The electricity consumption for NH production of 1.68-2.39 x 10{superscript 2} kJ g-N-{superscript 1} was substantially lower than that of metal catalyst systems at comparable NO- concentrations (typically, >104 kJ g-N-{superscript 1}). These findings demonstrate that the hydrogel electrode design represents an energy-efficient, and readily fabricated platform for bioelectrochemical NH production from dilute NO-.

Mass gatherings pose significant public health risks by facilitating the spread of infectious diseases. While wastewater-based surveillance (WBS) has been widely used to monitor pathogens in high-income settings, its use as a practical, multi-pathogen surveillance tool during mass gatherings in low- and middle-income countries remains limited. This study aimed to assess the operational feasibility, epidemiological significance, and public health utility of multi-pathogen WBS during the African Nations Championship (CHAN) football tournament in Uganda. Wastewater surveillance was conducted at Mandela National Stadium during eight match days in August 2025. Moore swabs were deployed at 38 manholes receiving wastewater from different toilet facilities across the stadium to capture representative wastewater samples. Samples were processed using Nanotrap(R) microbiome virus particles to concentrate pathogens, followed by nucleic acid extraction. Samples were analyzed for multiple enteric and respiratory pathogens, including Mpox, using quantitative PCR (qPCR). Descriptive analyses were performed to characterize pathogen detection patterns, positivity rates, and temporal distribution across surveillance sites. A total of 304 wastewater samples were collected and analyzed, of which 259 (85.2%) tested positive for at least one pathogen. Multiple pathogens were consistently detected across sampling days, with enteric pathogens predominating, particularly Shigella spp. (53.6%), Rotavirus A (35.9%) and Enterovirus (32.2%). The mpox virus was also detected in a notable proportion of samples (28.6%) across several sampling days. Respiratory pathogens, including SARS-CoV-2 (11.8%) and Influenza B (8.2%), were identified intermittently at lower frequencies. Pathogen diversity varied over time, with up to eight pathogens detected on a single day, and co-detection of multiple pathogens observed in the majority of positive samples. Cq value distributions further demonstrated variability in detected signal patterns across pathogens. Surveillance findings informed real-time public health interventions, including sanitation reinforcement, intensified hygiene promotion, environmental disinfection, and targeted risk communication, strengthened syndromic surveillance with on-site triage, and targeted environmental health assessments of food handling and wastewater infrastructure. These findings demonstrate the operational feasibility and public health utility of integrating multi-pathogen wastewater-based surveillance into mass-gathering preparedness and response frameworks in low-resource settings. By capturing diverse pathogen signals and informing targeted interventions during the CHAN football tournament, WBS can provide actionable population-level insights that can support outbreak preparedness and response. Scaling WBS within national preparedness systems could strengthen epidemic intelligence, enhance early warning capacity, and support data-driven public health decision-making during future mass gatherings and emerging infectious disease threats.

While exposure to volatile organic compounds such as ethylene dichloride and vinyl chloride monomer is a well-established cause of liver disease, particularly hepatic hemangiosarcoma, characterizing real-world exposure profiles in communities surrounding industrial centers remains challenging. Calvert City, Kentucky (population ~2,500), provides a unique setting characterized by both active industrial emissions and legacy sources of air toxics. To address these complexities, this method paper describes the framework for the Biomonitoring and Environmental Assessment for Community Outreach and Neighborhood Safety (BEACON) study. By utilizing a novel, multi-dimensional exposure assessment strategy, BEACON aims to characterize air toxic exposures and provide actionable data for community health and safety. For the BEACON study, we will leverage Kentucky Department of Air Quality measures of air toxics, analyze urine samples in a small cohort of community volunteers, analyze community urine via wastewater in an adjacent community, geocode citizen odor reporting, assess blood markers in wildlife, survey small and large animal veterinarians in the area for anomalies in morbidity and mortality, and work with the regional health system to enhance vigilance for health issues associated with toxicants present in the area. In addition, blood samples will be collected at three time points and biobanked for future analyses. Efforts will be made to link this study to additional large-scale long-term cohorts where possible. Throughout the project, community engagement will play a critical role by raising awareness, fostering collaboration, and ensuring that the voices of affected residents are heard.

Wastewater-based epidemiology (WBE) has emerged as a powerful population-level surveillance tool, but its coverage is structurally concentrated in in-network urban areas, potentially leaving rural populations underrepresented. Routine human movement between sewered (in-network) and unsewered (off-network) areas may, however, cause wastewater treatment plant (WWTP) measurements to reflect infectious disease dynamics beyond sewer boundaries. We evaluated this hypothesis using daily clinical COVID-19 testing data (January 2021-April 2022) across New York State excluding New York City (NYC). We disaggregated weekly cases and tests into in-network (WWTP catchment area) and off-network (outside WWTP catchment area) components applied to two geographic frameworks: administrative counties (N = 53 mixed-coverage) and mobility-defined communities identified through Walktrap community detection applied to census tract-level movement networks (N = 32 mixed-coverage). In/off-network COVID-19 trends were strongly correlated under both frameworks. County-level statewide aggregate correlations were high (incidence r = 0.994, positivity r = 0.996), as were individual county correlations (median r = 0.909 and 0.932, respectively). Mobility-defined community-level statewide correlations were similarly strong (r = 0.990 and 0.992), with comparable unit-level medians (r = 0.877 and 0.894). The mobility-defined community framework provided better population balance between in-network and off-network strata (87.5% vs. 69.8% in balanced range) and a higher floor on representativeness (minimum r = 0.440 vs. 0.177). Population size was the dominant predictor of in-network/off-network alignment at both scales; wastewater infrastructure density and off-network signal variability provided additional explanatory power at the mobility-defined community level. WWTPs broadly represent COVID-19 dynamics in surrounding off-network populations, supporting their use as sentinel surveillance sites. Representativeness weakens in smaller, more rural communities, and mobility-defined communities provide a complementary framework for identifying where this occurs.

Trace elements, such as iron, nickel, and cobalt are known to regulate methanogenic activity in anaerobic digestors used for waste valorisation, but the potential role of rare earth elements remains poorly understood. This study investigated the effects of lanthanum (La) supplementation on biogas production, methane generation, volatile fatty acid (VFA) formation, and carbohydrate utilisation in anaerobic digestion (AD). Biomethane potential (BMP) assays conducted under mesophilic conditions (37C) using methanogenic sludge granules, and glucose as substrate, were supplemented with 0.1, 1, 10, and 100 mg/L lanthanum chloride (LaCl3). Biogas production and composition was monitored over a 96-h incubation, while sacrificial, batch bioreactors were used to evaluate temporal VFA and carbohydrate profiles. La supplementation significantly enhanced biogas and methane production in a concentration-dependent manner. The highest cumulative biogas yield (478.9 mL, corresponding to 179.5 mL biogas/g COD) and methane production (285.7 mL, corresponding to 107.1 mL CH4/g COD) were observed with 100 mg/L LaCl3, corresponding to increases of 88.7% and 186%, respectively, compared with La-free controls. CO2 production also increased with La concentration, whereas hydrogen production remained comparatively low. Acetic and butyric acids represented the dominant fermentation products (80-88% of total VFAs), but profiles of accumulated VFA in the bioreactors diversified with La addition, including showing caproate production, indicating changed biodegradation dynamics in the methanogenic microbiome. These findings demonstrate that lanthanum can stimulate anaerobic digestion performance and methane generation, highlighting the potential as a novel trace element additive to enhance biogas production. Research is now required to elucidate the underlying microbial and biochemical mechanisms, and establish optimal dosing strategies for large-scale applications.

Surface water quality is a significant public health concern because contaminated rivers and lakes can spread waterborne diseases and expose communities to harmful pollutants, particularly in areas where water treatment and regulation are limited. This study assesses the anthropogenic impact on surface water quality in the Ahafo Ano Southwest District, Ghana. Water samples were collected from three locations within the Mankran watershed during both wet and dry seasons and analyzed for key parameters. Pollutant concentrations exhibited marked spatial and seasonal variability, with nitrate ranging from 1.09 to 13.77 mg/L, phosphate from 1.58 to 10.55 mg/L, and total suspended solids from 140.0 to 1680.0 mg/L. Heavy metals frequently exceeded acceptable limits, with zinc ranging from below detection to 0.11 mg/L, copper from below detection to 0.84 mg/L, and lead from below detection to 0.46 mg/L. Seasonal Water Quality Index (WQI) analysis indicated degraded water quality during the wet season in Baniekrom and Kunsu, due to increased runoff carrying agricultural residues, sediments, and mining contaminants. In contrast, Mmobrem showed improved wet-season quality, attributed to rainfall dilution. The Piper diagram further revealed that the water samples predominantly fall within the Ca-Cl hydrochemical facies, reflecting strong geogenic control through basaltic rock weathering, coupled with chloride enrichment likely intensified by agricultural and domestic inputs. Principal component analysis indicated that multiple anthropogenic and natural factors drive water quality variations across the catchment. Ecological risk assessment identified potential risks to algae, resulting from elevated levels of nutrients and metals. Overall, the combined WQI and hydrochemical evidence indicate an intensified nutrient influx during the wet season and increased suspended particulate matter during the dry season, primarily driven by agriculture, deforestation, and mining. The findings underscore the urgent need for sustainable water management strategies to mitigate the impacts of human activities on surface water quality in the region.

Newly established freshwater aquaria rely on development of biofilter nitrifying populations to prevent ammonia and nitrite accumulation that can negatively impact fish health. Although initial fish loads impact water chemistry of new aquaria, little is known about the corresponding impact on microbial community succession within freshwater aquarium biofilters. To address this gap, fourteen home aquarium systems were established, stocked with a range of fish loads, and maintained for eight months. Aquaria were sampled regularly to monitor nitrogen species, microbial community composition (16S rRNA gene sequencing), and the abundance of nitrifiers (qPCR). Aquaria with higher fish loads developed microbial communities that were compositionally distinct from those with lower fish loads, and were dominated by Pseudomonas, Rhodobacter, and Planctomycetes. These patterns are consistent with increased nutrient availability supporting biofilm development, whereas lower fish loads may delay biofilm maturation. Increasing the number of fish in an aquarium significantly increased maximum ammonia and nitrite concentrations, although both were ultimately depleted within similar timeframes across treatments. Comammox Nitrospira were among the most abundant biofilter nitrifiers and were present in all biofilter samples regardless of fish load. Ammonia-oxidizing bacteria were detected at relatively low abundance but showed increases in relative abundance within high fish load aquarium filters. Ammonia-oxidizing archaea were below sequencing detection limits and detected only at low levels by qPCR, suggesting that their establishment in aquarium biofilters may require higher initial inoculation or longer timeframes. Overall, these results demonstrate that fish load shapes microbial community development in newly established aquarium biofilters, and that comammox Nitrospira dominate among nitrifiers during early biofilter establishment.

Simultaneous removal of organic waste and industrial gypsum was assessed in continuous flow-through bioreactors that treat sulfate-rich sewage sludge. Metabolic fluxes, the composition of microbial communities, and profiles of organic matter in the presence of different organic loads were tracked over [~]190 days. The addition of a pre-enriched microbial community enhanced the rates of sulfate reduction during the establishment of the sludge blanket, but microbial diversity in established reactors depended primarily on organic loading. Organic removal rates were comparable to those in standard anaerobic digesters, but methane production accounted for [~]1% of electron flow compared to >70% in traditional systems. Stoichiometric analyses revealed that molar COD: sulfate ratios below [~]1 favored complete oxidation of acetate by sulfate-reducing bacteria (SRB) and those above [~]2.1 permitted either complete or incomplete oxidation, allowing sulfate reduction and methanogenesis to co-occur. Sequencing of the 16S rRNA confirmed these trends by revealing that the faster-growing SRB that do not oxidize acetate were more abundant at higher organic loads and during the establishment of the sludge blanket, whereas complete oxidizers became more abundant when the molar COD: sulfate ratio was [&le;]3.2. In reactors that had been seeded with the pre-enriched communities, acetate-oxidizing SRB became prevalent over the incomplete oxidizers 25-50 days earlier. These results enable targeted design and control of microbial processes and bioreactors that remove waste organics and gypsum while producing less methane due to the competition for acetate between methanogenic archaea and SRB that oxidize acetate.

Conventional anaerobic digestion emits methane from organic waste. Here, we investigate a sulfate-based alternative that suppresses methane production and generates alkaline solutions that may sequester carbon by carbonate precipitation. Although methanogenesis is known to occur when reduced organic carbon is replete and sulfate is limiting, it remains unclear whether methane emissions during microbial conversion of waste gypsum are primarily driven by community composition or organic availability. By comparing fluxes of electrons from organic matter toward sulfate or methane in microbial communities grown on different organic loads, we show that community composition, microbial growth, and organic availability collectively determine sulfide and methane fluxes. Lower organic loads increase the importance of syntrophic interactions with fermenters and competition between sulfate reducing bacteria and methanogens due to scarcity of substrates. Microbes present in the original sewage sludge reduce less sulfate, produce more methane, and generate less alkalinity compared to the communities enriched by multiple cycles of growth in the presence of sulfate and sewage sludge. The inoculation of communities enriched at low organic loadings in the presence of sulfate decreases the production of methane by enabling the growth of sulfate reducing bacteria from the order Desulfobacterales that can oxidize acetate to CO2 and compete with methanogens for acetate. The use of such enrichments in sludge treatment systems can stimulate the removal of organic substrates and waste gypsum, while suppressing methane production, over timescales comparable to those in the current sludge treatment systems that do not contain sulfate.

Combined sewer overflows (CSOs) are a major pathway for untreated wastewater into urban streams, yet their role in shaping antimicrobial resistance (AMR) dynamics remains poorly understood. Here, we used high-frequency, time-resolved sampling during two storm-triggered CSO events across two monitoring locations and one stormwater-only control site in an urban stream to quantify how these disturbances affect microbial communities, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) in an urban stream. CSO events caused rapid, up to two orders of magnitude, increases in bacterial, pathogen, and ARG abundance, with multiple transient peaks occurring within single overflow episodes. However, these increases were largely proportional to the total bacterial load, and most ARGs and MGEs did not change in relative abundance, indicating that CSOs primarily act as mass-transfer events rather than drivers of in situ selection. Downstream attenuation was governed by hydrological dilution despite additional CSO inputs: Both microbial and resistance signals largely returned to baseline within short time frames. This demonstrates that CSOs function as hydrologically driven pulse disturbances that generate acute but transient AMR exposure. Because CSO events lack the sustained pressure associated with continuous wastewater discharges, rapid washout prevents the long-term establishment of sewage-derived resistance. These findings highlight that AMR risk in CSO-impacted systems is driven primarily by short-term exposure rather than by persistent ecological transformation, with important implications for urban water management under increasingly extreme rainfall conditions.