The recovery of C, N, and P elements by sludge biorefinery potentially reduces operation costs and increases the extra benefits. Herein, we analyzed the elemental stoichiometry of C, N, and P and functional microbiome involved in enzymatic anaerobic fermentation. Enzymatic hydrolysis was observed to increase the release of C, N, and P into the sludge supernatants by 21.8 %–26.3 %. Metatranscriptome analysis indicated that enzymatic pretreatment enhanced the metabolism of the organic carbon degradation, ammonium conversion, and P solubilization in subsequent fermentation. Specifically, enzymatic pretreatment enhanced endogenous carbon hydrolase activity by 48.4 %–72.7 % and upregulated intra-C metabolic pathways, such as glycolysis and pyruvate metabolism. Ammonium transport and conversion were significantly increased by 4–6 fold, stimulating the synthesis of glutamine and endogenous amino acids. Additionally, enzymatic hydrolysis promoted phosphatase secretion and enhanced bacterial P uptake. These effects improved the recovery of C, N, and P as dentification carbon source and struvite by 13.7 %–41.8 % and the dry sludge production was reduced by 24.3 %–28.1 %. Life cycle assessment (LCA) indicated the shift of CO₂ emissions from net positive to net negative levels as compared to the conventional A²/O process. This study offers valuable insights into the redistribution and metabolism of various elements involved in the enzymatic anaerobic fermentation, and proposes the potential strategy to recovery C, N, and P from sewage via sludge biorefinery.

Activated sludge (AS) wastewater treatment generates substantial excess sludge which needs to be discarded and thereby increasing operational costs. Extracellular polymeric substances (EPS) within AS present a potential resource for recovery, reducing sludge volume and mass while adding value. Achieving this goal requires a better characterization of EPS, as the relationship between its composition and the microbial communities responsible for its production remains insufficiently understood. Here, we analysed extracted EPS from 16 wastewater treatment plants across 13 countries and 5 continents and found that alkaline extractable EPS yields varied widely (2.81–18.5 wt.% VSS). The microbial community composition of abundant species varied across plants and particularly across continents and did not correlate to the EPS yield. Only sludge retention time had a significant correlation with the EPS yield (p < 0.005). Traditional colorimetric assays failed to detect compositional trends of the EPS, but Fourier Transform Infrared (FTIR) analysis indicated that extracted EPS from biological phosphorus removal systems had higher lipid and polysaccharide content, while chemical phosphorus removal systems had higher relative protein content. Thus, FTIR proved effective for distinguishing extracted EPS composition, demonstrating its potential as a high-throughput characterization tool. These findings highlighted that the wastewater treatment design and operation may shape the functional groups in EPS when using the alkaline method. More investigations are needed to find possible correlations between the composition of extracted EPS and the microbial community structure. Overall, the study presents a baseline for the amount and overall composition of biopolymers that can be extracted from global AS plants for recovery.

Microbial aggregates of different sizes in aerobic granular sludge (AGS) systems have been shown to exhibit distinct microbial community compositions. However, studies comparing the microbial activities of different-sized aggregates in AGS systems remain limited. In this study, genome-resolved metatranscriptomics was used to investigate microbial activity patterns within differently sized aggregates in a full-scale AGS plant. Our analysis revealed a weak correlation between the relative abundance of metagenome-assembled genomes (MAGs) and their transcriptomic activity, indicating that microbial abundance does not directly correspond to metabolic activity within the system. Flocculent sludge (FL; <0.2 mm) predominantly featured active nitrifiers and fermentative polyphosphate-accumulating organisms (PAOs) from Candidatus Phosphoribacter, while small granules (SG; 0.2–1.0 mm) and large granules (LG; >1.0 mm) hosted more metabolically active PAOs affiliated with Ca. Accumulibacter. Differential gene expression analysis further supported these findings, demonstrating significantly higher expression levels of key phosphorus uptake genes associated with Ca. Accumulibacter in granular sludge (SG and LG) compared to flocculent sludge. Conversely, Ca. Phosphoribacter showed higher expression of these genes in the FL fraction. This study highlights distinct functional roles and metabolic activities of crucial microbial communities depending on aggregate size within AGS systems, offering new insights into optimizing wastewater treatment processes.

Identifying structural proteins within the extracellular polymeric substances (EPS) will provide a better understanding of the stability of aerobic granular sludge (AGS) and biofilms in general. In this work, an abundant surface protein was identified and localized in the extracellular matrix of seawater-adapted AGS. Granules with good phosphate removal were cultivated in a sequencing batch bubble column reactor with acetate as a carbon source dissolved in seawater. “Candidatus Accumulibacter” was observed as the most dominant community member through fluorescent in-situ hybridization. A surface protein of 74.5 kDa was identified in the EPS extract of the seawater-adapted AGS by SDS-PAGE and mass spectrometry. The surface protein was produced by an Accumulibacter species and showed homology to S-layer proteins. A type 1 secretion system was found adjacent to the gene encoding for the surface protein, suggesting a possible export system. Antibodies generated from a unique peptide of the surface protein confirmed the extracellular location of the surface protein. Microscopy observations with antibody staining showed the surface protein forms dense structures within the Accumulibacter microcolonies and larger fiber structures around the microcolonies. These observations highlight the importance of the protein for the structural properties of the granule. To detect more structural proteins in the EPS, optimization of the EPS extraction and in situ imaging validation methods are essential.

城市污水集中收集率是城市污水系统运行效率的关键指标，城市污水污染物排放量是计算城市污水集中收集率的必需参数，但城市污水系统较高溢流量和缺乏溢流监测带来计算城市污水排污量的困扰。为此，应用城市污水系统模型和用水、污水处理、常住人口数等常规市政数据，首次提出了根据污水处理厂进水流量和负荷计算城市污水污染物排放量和污水集中收集率的2种方法。以面积约110 km2、常住居民近100×104 人口的城区污水系统为例，展示了应用2种方法的所需数据、具体步骤和计算结果，分析了2种方法各自的适用性。对流动人口、工商业活动和人日排污量等因素对城市污水污染物排污量的影响和估算进行了讨论。2种方法对当下国内和国外一些国家的城市污水排污量、污水集中收集率的计算和城市污水系统运行效率的评估，具有现实意义。

The centralized collection rate of urban sewage is a key indicator of the operational efficiency of urban sewage systems, urban sewage pollutant loading is an essential parameter for calculating the sewage collection ratio. The high overflow of urban sewage systems and lack of monitoring data pose difficulties in calculating sewage collection ratio. By using the urban sewage system model and regular domestic water consumption and sewage treatment information, this study first time proposed two methods, which were based on the influent flow and pollutant loadings of the sewage treatment plant to calculate the urban sewage pollutant loadings and sewage collection ratio. The applicability of two methods was demonstrated including the required data, specific steps, and calculation results by a case of an urban area with almost 110 km2 square meters and nearly 100×104 inhabitants in a city in the Yangtze Delta area. The applicability of each method was discussed. Furthermore, the effects of the commuters, industry and commercial activities, personal discharge load on the estimation of sewage collection ratio in the cities at different levels of economic development are discussed and analyzed. The methods introduced in this study had practical significance for calculating the sewage collection ratio and evaluating the operation efficiency of urban sewage systems in China and part of the other countries.

Polyhydroxyalkanoate (PHA) production is a promising technology fostering the spread of the circular bio-economy approach. However, the environmental implication of the process is usually neglected. This paper shows the results of a membrane-based PHA production pilot plant fed with no-pretreated waste activated sludge (WAS). The system was monitored for effluent water quality, nitrous oxide (N₂O), and PHA production by dynamic accumulation over a long-term period to assess the consistency of the results over several fluctuations. The experimental study was characterized by three C/N ratios of 9, 4.5, and 4 g COD/g N. The system achieved a stable and high removal efficiency for carbon and nitrogen (96.3 ± 2.6 % and 89.9 ± 6.7 %, respectively), despite the only legislation limit respected being the biological oxygen demand concentration discharge limits imposed by 2020/741/EU. Low N₂O gaseous and liquid concentrations were achieved over the 200-day experimental period, never exceeding 0.52 mg N₂O-N/L. Despite the high concentration, the N₂O emission factor accounted for only 0.21 ± 0.14 % of the influent nitrogen. Finally, the system produced an average of 36.3 ± 1.8 % g PHA/g VSS with a storage yield of up to 0.42 g COD_PHA/g COD_VFA. The system revealed a high stability over a long-term experimental period, achieving a considerable amount of PHA while maintaining a low N₂O emission. Promising effluent water quality was achieved, highlighting the potential of applying the water reuse practices.

Microorganisms encoding for the N2O reductase (NosZ) are the only known biological sink of the potent greenhouse gas N2O and are central to global N2O mitigation efforts. Clade II NosZ populations are of particular biotechnological interest as they usually feature high N2O affinities and often lack other denitrification genes. We focus on the yet-unresolved ecological constraints selecting for different N2O-reducers strains and controlling the assembly of N2O-respiring communities. Two planktonic N2O-respiring mixed cultures were enriched at low dilution rates under limiting and excess dissolved N2O availability to assess the impact of substrate affinity and N2O cytotoxicity, respectively. Genome-resolved metaproteomics was used to infer the metabolism of the enriched populations. Under N2O limitation, clade II N2O-reducers fully outcompeted clade I affiliates, a scenario previously only theorized based on pure-cultures. All enriched N2O-reducers encoded and expressed the sole clade II NosZ, while also possessing other denitrification genes. Two Azonexus and Thauera genera affiliates dominated the culture, and we hypothesize their coexistence to be explained by the genome-inferred metabolic exchange of cobalamin intermediates. Under excess N2O, clade I and II populations coexisted; yet, proteomic evidence suggests that clade II affiliates respired most of the N2O, de facto outcompeting clade I affiliates. The single dominant N2O-reducer (genus Azonexus) notably expressed most cobalamin biosynthesis marker genes, likely to contrast the continuous cobalamin inactivation by dissolved cytotoxic N2O concentrations (400 μM). Ultimately, our results strongly suggest the solids dilution rate to play a pivotal role in controlling the selection among NosZ clades, albeit the conditions selecting for genomes possessing the sole nosZ remain elusive. We furthermore highlight the potential significance of N2O-cobalamin interactions in shaping the composition of N2O-respiring microbiomes.

To meet the increasing drinking water demand, membrane technologies are used to treat previously unavailable water sources. A byproduct of membrane technologies is the concentrate stream, containing valuable resources in higher concentrations. We studied the recovery of iron from different groundwater matrices and anaerobic reverse osmosis (RO) concentrates via precipitation of vivianite and the co-removal of other common groundwater divalent cations Mn²⁺, Mg²⁺ and Ca²⁺ during vivianite precipitation. The formed precipitates were characterized using X-Ray Diffraction and Scanning Electron Microscopy. Vivianite precipitation removed a maximum of 89 % of Fe²⁺ in raw groundwater and 52 % Fe²⁺ from RO concentrate. Substantial co-removal of Mn²⁺ (max 91 %) and limited co-removal of Mg²⁺ (max 7 %) were found, without hindering Fe removal efficiencies or altering morphological changes of the vivianite crystal. In contrast, co-removal of Ca²⁺ occurred at the expense of iron removal, forming amorphous calcium phosphate precipitates. This study shows the potential of vivianite precipitation for iron recovery across a wide range of groundwater matrices and highlights the need for further research to optimize this novel method to treat concentrate streams that are challenging to dispose of.

Polyhydroxyalkanoates (PHA) can be produced using fermentation products of an excess sewage sludge fermentation process. An efficient method to enrich a PHA-producing community is an aerobic-feast/anoxic-famine enrichment strategy. The effect of different carbon to nitrogen (C/N) feed ratios of 1, 2 and 3.5 g COD/g N on the process performance was studied. The study was executed on a pilot plant scale using fermented waste activated sludge as the organic carbon source. The system's performance was monitored in terms of removing contaminants, producing PHA, and reducing N2O emissions. The results indicated that a lower C/N ratio results in lower PHA production, with PHA content in the sludge of 20, 24 and 36 % w/w for C/N ratios of 1, 2 and 3.5 g COD/g N, respectively. At the lowest C/N ratio, the highest nitrite accumulation rate (77 %), nitrification efficiency (89 %) and denitrification efficiency (89 %) were observed, but the N2O production was also the highest (0.77 mg N2O-N/L). The long-term comprehensive monitoring carried out in this study revealed high carbon and ammonia removal efficiencies (never below 80 %) despite the C/N shifts and high COD and ammonia concentrations. At the same time, the system showed relatively low PHA production and high environmental impact in terms of high gaseous N2O emission. These findings question the sustainability of the aerobic-feast/anoxic-famine enrichment strategy for PHA production in full-scale plants.

Sensor availability and costs are nowadays no longer limiting data gathering at wastewater treatment plants (WWTPs). However, one should be aware that a higher amount of measured data gathered does not necessarily imply that also more information is obtained. In this light, this contribution assesses the general applicability and the added value of a structured experimental design approach for planning measurement campaigns at WWTPs, in view of mass-balance-based data reconciliation. To this end, the results from full-scale WWTP case studies available in the literature were compared to those obtained with the developed structured experimental design procedure. Planning measurement campaigns comprises the selection of (additional) measurements to meet a pre-set main goal. The need for a structured experimental design procedure replacing past expert judgment approaches became clear from the fact that three out of five case studies available in the literature failed to meet the main goal and/or performed unnecessary additional measurements. Translating the main goal into specific key variables was found essential in this respect. The general applicability of the procedure was proven with three outcomes. First, the procedure, involving well-defined steps, could be applied to different WWTP layouts. Second, it ensured the fulfilment of various main goals. Third, it provided useful outcomes, i.e., optimal measurement campaigns, which reduced the need for additional measurements (40-70% less) compared to expert knowledge approaches, hence more information could be obtained with less analytical data. Overall, the experimental design procedure proved a fast and useful tool ensuring the success of subsequent mass-balance-based data reconciliation.

The role of high-pH conditions in anaerobic digestion (AD) has traditionally been confined to it's use in pre-treatment processes. However, operating AD at elevated pH and alkalinity offers significant advantages, including in-situ upgrading of biogas to biomethane. This study examines the potential and scalability of AD under these conditions (pH ∼ 9.3; alkalinity ∼ 0.5 eq/L). The substrate used was the alkaline waste generated from the extraction of extracellular polymeric substances (EPS) from aerobic granular sludge (AGS), and the inoculum used was a haloalkaliphile microbial community from soda lake sediments. To evaluate the system’s performance, the organic loading rate (OLR) was incrementally increased. The highest methane production obtained was 8.4 ± 0.1 mL/day/gVSadded at a hydraulic retention time (HRT) of 15 days and an OLR of 1 kgVS/day/m3. At this loading rate, methanogenesis became the rate limiting conversion. The maximum volatile solids conversion was 48.1 ± 1.1 %. Throughout the reactor operation, methane purity in the biogas consistently exceeded 90 % peaking at 96.0 ± 0.2 %, showcasing the potential for in-situ biogas purification under these conditions. In addition, no ammonia inhibition was observed, even with free-ammonia (NH3) concentrations reaching up to 14 mM. This study underscores the potential of high-pH anaerobic digestion as a sustainable method for both waste treatment and energy recovery.

The immense microbial diversity on Earth represents a vast genomic resource, yet discovering novel enzymes from complex environments remains challenging. Here, we combine a microbial enrichment with metagenomics and metaproteomics to facilitate the identification of microbial glycoside hydrolases that operate under defined conditions. We enriched microbial communities on the carbohydrate polymer pullulan at elevated temperatures under acidic conditions. Pullulan is a natural polysaccharide composed of maltotriose units linked by α-1,6-glycosidic bonds. Pullulan, along with its hydrolyzing enzymes, has broad applications across various industries. The enrichment inocula were sampled from thermophilic compost and from soil from the bank of a pond. In both cases, Alicyclobacillus was identified as the dominant microorganism. Metaproteomic analysis of the enriched biomass and secretome enabled the identification of several pullulan-degrading enzyme candidates from this organism. These enzymes were absent in the metagenomic analysis of the initial inoculum, which is highly complex with a wide diversity of species. This underscores the effectiveness of combining microbial enrichment with multi-omics for uncovering novel enzymes and sequence variants that operate under defined conditions from complex microbial environments.

As water research and industry shift towards resource recovery plants, comprehensive assessment methods are needed to capture environmental trade-offs. Existing life cycle assessments (LCA) on desalination often neglect key methodological challenges in multi-product zero-liquid-discharge (ZLD) systems, risking misleading conclusions. This study applies LCA to conventional desalination and with three resource recovery scenarios (integrated desalination and brine treatment) in Cyprus: Sc1) maximum water recovery using waste heat (WH), Sc2) integrated desalination plant with brine treatment using WH, Sc3) electricity-based desalination with chemicals recovery, to assess how key methodological decisions influence the results and decisions. Five impact categories were analysed: climate change, human toxicity, marine ecotoxicity, water depletion, and fossil depletion. Without product substitution, multi-product ZLD systems show higher absolute impacts than SWRO due to increased energy and chemical demands. However, when credits for recovered salts and chemicals are considered, Scenarios 2 and 3 achieve large net reductions compared to conventional production, highlighting the sustainability potential of resource recovery. Results proved highly sensitive to methodological choices: functional unit selection (increase up to 59 %), allocation methods (variation from 54 % to 90 %), while excluding WH altered impacts by up to 89 %, emphasizing the need for transparent reporting to support robust decision-making in desalination design. Sensitivity analysis showed that integrating renewable energy could cut climate change and fossil depletion impacts by up to 99 %, though with trade-offs in marine ecotoxicity and water depletion. Rather than proposing new methods, this work provides critical guidance on applying standardized LCA options to complex systems, offering directly relevant insights for practitioners and policy-makers in sustainable desalination design.

A Lactobacillus-rich vaginal microbiome is associated with a reduced risk for sexually transmitted diseases and adverse reproductive health outcomes, with Lactobacillus crispatus identified as particularly beneficial. This study investigated the nutritional requirements of two vaginal isolates, L. crispatus RL09 and RL10, and presents a chemically defined medium (CDM) that supports their growth. This study experimentally validated that L. crispatus relies on exogenous fatty acids, essential B vitamins, including riboflavin (B2), niacin (B3) and pantothenate (B5), and all eighteen amino acids for sustained growth. On one hand, this body of work adds to the physiological understanding of Lactobacilliaceae and lays the groundwork for future quantitative studies on L. crispatus. On the other hand, we have shown that L. crispatus exhibits a high metabolic dependency on its environment. These dependencies underscore the potential sensitivity of L. crispatus to nutrient variations, which may influence its ability to dominate and maintain a healthy vaginal ecosystem.

Optimizing resource use is essential for the survival and fitness of species in microbial communities ubiquitous in natural and engineered ecosystems. These ecosystems are often characterized by the simultaneous presence of multiple substrates such as volatile fatty acids, amino acids and sugars. Yet, the evaluation of metabolic potential for these microbial community members is predominantly based on single substrate utilisation. Metabolic and ecological implications of the interactions of multiple substrates, particularly in environments with changes in redox conditions and substrate availability, remain poorly understood. In this study, we investigate the metabolic interactions resulting from co-substrate utilization in polyphosphate-accumulating organisms within wastewater treatment systems. We combined experimental analysis of highly enriched “Ca. Accumulibacter” mixed cultures with genome-resolved metagenomics and conditional flux balance analysis (cFBA) to quantify the physiological relevance of co-substrate uptake. We observe that anaerobic co-substrate utilisation of acetate and aspartate result in metabolic interactions leading to optimized redox balance, reduced ATP losses and increased biomass yields by up to 8% compared to individual substrate use. Metabolic modelling revealed that these benefits emerge from the network topology, where the interaction of different metabolic routes gives rise to synergistic effects. Extending our analysis to additional substrate pairs, we classify metabolic interactions into three general types: (i) neutral, (ii) one-way synergistic and (iii) reciprocal synergistic. Our findings highlight the importance of metabolic interactions and cellular resource allocation strategies in dynamic microbial ecosystems. This study provides a broader ecological framework for understanding competitive metabolic strategies in environmental organisms. Co-substrate utilization can have direct implications for improving the yield or productivity of bioprocesses.

Aerobic granular sludge (AGS) process is an effective wastewater treatment technology for nutrient and organic matter removal and is being widely applied worldwide. To date, its performance in removing organic micropollutants (OMPs), particularly under wet weather conditions when operation differs, remains poorly understood. This study evaluated the occurrence and removal of OMPs, including 19 pharmaceuticals and 2 industrial compounds, in a full-scale AGS plant during one year under both dry and wet weather conditions. Under dry weather conditions, influent concentrations of 5 pharmaceuticals and 1 industrial compound exceeded 1 μg L−1. Rainfall resulted in diluted OMP influent concentrations, but also caused a significant increase in the influent load of 6 OMPs with positively charged functional groups, likely due to mobilization of sewage sediments that had adsorbed these OMPs. Under dry weather conditions, average removal efficiencies of 14 compounds were greater than 20 %, with 6 of these compounds detected in the sludge phase, and thus likely removed through sorption. Under wet weather conditions, OMP removal efficiencies decreased by 8 % to 38 %. Shortened aeration reaction time significantly reduced (p-value<0.05; R2>0.5) the removal of 8 potentially biodegradable compounds, while the impact on sorption-driven removal was limited for 6 compounds. Effluent OMP load increased under wet weather conditions, mainly due to reduced removal efficiency, rather than the discharge of OMPs adsorbed onto suspended solids. Under dry weather conditions, the AGS plant exhibited comparable or slightly higher OMP removal efficiencyies than activated sludge plants; however, differences in performance under wet weather conditions remain unclear due to limited data on activated sludge systems. Overall, this study is the first to assess OMP removal in a full-scale AGS plant under wet weather, showing the impact of increased flow on the sorption and biotransformation of OMPs.

The hydrolysis of structural extracellular polymeric substances (St-EPS) is considered a major limiting step in the anaerobic fermentation of waste activated sludge (WAS). However, the degradation of heteropolysaccharides, characterized by complex monomers of uronic acids and neutral saccharides in St-EPS, has rarely been reported. In this study, microbial-produced xanthan-like heteropolysaccharides, characterized by a blue filamentary film, were identified. The xanthan-producing bacteria comprised ∼7.2% of total genera present in WAS. An xanthan-degrading consortium (XDC) was enriched in an anaerobic batch reactor. This consortium could degrade Xanthan for over 90% and disrupt the gel structure of xanthan while promoting methane production from WAS by 29%. The xanthan degradation network consisting of extracellular enzymes and bacteria was elucidated by combining high-throughput sequencing, metagenomic, and metaproteomic analyses. Five enzymes were identified as responsible for hydrolyzing xanthan to monomers, including xanthan lyase, β-D-glucosidase, β-D-glucanase, α-D-mannosidase, and unsaturated glucuronyl hydrolase. Seven genera, including Paenibacillus (0.2%) and Clostridium (3.1%), were identified as key bacteria excreting one to five of the aforementioned enzymes. This study thus provides insights into the complex conversions in anaerobic digestion of WAS and gives a foundation for future optimization of this process.

The post-pandemic world still faces ongoing COVID-19 infections, although international travel has returned to pre-pandemic conditions. Wastewater-based epidemiology (WBE) is considered an efficient tool for the population-wide surveillance of COVID-19 infections during the pandemic. However, the performance of WBE in post-pandemic era with travel restrictions lifted remains unknown. Utilizing weekly county-level wastewater surveillance data from June 2021-November 2022 for 222 counties in 49 states (covering 104 million people) in the United States of America, we retrospectively evaluated the correlations between SARS-CoV-2 RNA (CRNA) and reported cases, as well as the impacts of international air travel, demographics, socioeconomic aspects, test accessibility, epidemiological, and environmental factors on reported cases under the corresponding CRNA. The lifting of travel restrictions in June 2022, shifted the correlation between CRNA and COVID-19 incidence in the following 7-day and 14-day from 0.70 (IQR: 0.30–0.88) in June 2021-May 2022 (pandemic) to 0.01 (IQR: -0.31–0.36) in June-November 2022 (post-pandemic), and from 0.74 (IQR: 0.31–0.90) to -0.01 (IQR: -0.38–0.45), respectively. Besides, after lifting the travel restrictions, under the same CRNA, the reported case numbers were impacted by many factors, including the variations of international passengers, test accessibility, Omicron prevalence, ratio of population aged between 18 and 65, minority vulnerability, and healthcare system. This highlights the importance of demographics, infection testing, variants and socioeconomic status on the accuracy and implication of WBE to monitor COVID-19 infection status in post-pandemic era. Our findings facilitate the public health authorities to dynamically adjust their WBE-based tools/strategies to the local contexts to achieve optimal community surveillance.

Protozoa contribute to water purification through predation in wastewater treatment systems. Full-scale aerobic granular sludge (AGS) reactors treating municipal wastewater contain AGS of varying sizes, with those larger than 2 mm dominating. These size fractions exhibit different sludge morphologies and microbial communities. To date, little is known about protozoan communities and their role in the removal of human-associated bacteria (like pathogens) in AGS plants, particularly across different size fractions. This study conducted uptake experiments with fluorescent Escherichia coli, as a model for human-associated bacteria, followed by microscopic observation to investigate protozoan communities and their predatory behavior in six AGS size fractions and activated sludge collected from full-scale municipal wastewater treatment plants. Sessile ciliates, particularly Epistylis and Vorticella, dominated protozoan populations across six AGS size fractions, with Epistylis being more abundant in larger AGS fractions (>1 mm) and Vorticella in smaller fractions (<1 mm). Additionally, microcosm experiments under aerobic (including predation) and anoxic conditions (excluding predation) revealed that predation was likely to be the main E. coli removal pathway, contributing an additional 0.5 to 2.5 log10CFU mL^–1reduction over a combination of non-predatory biological and abiotic processes. Larger AGS fractions showed greater predation capacity, linked to higher Epistylis abundance, while activated sludge, dominated by Vorticella, resembled smaller AGS fractions with lower predation capacity. These findings advance the understanding of the distribution of protozoan communities and their contribution to E. coli removal by predation in AGS wastewater treatment.

Aerobic granular sludge (AGS) is an effective wastewater treatment widely applied worldwide for the removal of nutrients and organic matter. However, limited information is available on the effectiveness of full-scale AGS plants for removing organic micropollutants (OMP), particularly during wet weather. This study investigated the occurrence and removal of OMPs, including 19 pharmaceuticals and 2 industrial compounds, in a full-scale AGS plant during both dry and wet weather over one year. We selected a full-scale AGS plant located in Utrecht, the Netherlands as the targeted AGS plant, and collected monthly 24-hour composite water samples and grab sludge samples f rom May 2023 to April 2024. 21 OMPs were extracted from water and sludge samples and measured by LC/MS. Concentrations of ammonia and organic matter (both particulate and soluble) were measured with HACH Lange GMBH kits.

The results show that Influent concentrations of 5 pharmaceuticals and 1 industrial compound exceeded 1 ?g L-1, which were diluted by rainfall. Influent loadings of 7 OMPs (absolute OMP amount in ?g day -1) were significantly increased during wet weather, likely from sewage sediment resuspen sion and urban runoff. Average removal efficiencies of 11 compounds achieved greater than 20%, with 5 of them exceeding 50%, in the AGS plants. Simple linear regression results between OMP removal efficiencies and flow rates showed that biodegradable OMPs were more strongly affected by increased influent volumes (as indicated by steeper slopes) than OMPs primarily removed through sorption. Additionally, correlation analysis results showed that the removal of soluble organic matter was significantly correlated (p-value < 0.05) with the removal of 14 OMPs, suggesting that organic matter removal may be an indicator for OMP removal in the AGS plant. After AGS treatment, 8 compounds in the effluent remained above their predicted no-effect concentration levels, indicating potential ecological and human health risks in the receiving water. During wet weather, effluent OMP loadings increased mainly driven by reduced removal efficiency. Additionally, compared to activated sludge plants, the AGS plant exhibited comparable or slightly higher OMP removal efficiency during dry weather. Overall, this study is the first to investigate OMP removal during wet weather in a full-scale AGS plant and propose the potential impact of increased flow rates on biodegradable OMPs or OMPs mainly removed through sorption.