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.

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.

Water treatment facilities are bound to incorporate resource recovery in the near future, necessitating novel economic assessments that capture the full economic potential of these systems. This study evaluates three cost calculation methods—Non-allocation, Economic allocation, and Dual allocation— to improve the accuracy of the Levelized Cost for multi-product desalination and brine treatment plants. The methods were tested across three technical scenarios: Sc1) maximum water recovery, Sc2) integrated desalination with brine treatment for resource recovery and Sc3) electricity-based desalination for chemical recovery. Results reveal that the traditional Non-allocation method tends to overestimate production costs by uniformly applying fixed costs across products, leading to inflated levelized costs. The Economic allocation approach reduces the levelized costs of water and other recovered products by up to 81 %, enhancing competitiveness with conventional production methods. The Dual allocation approach is most effective for recovered salts and chemicals, ensuring fair cost distribution and fostering competitiveness with linear systems. Sc2 is the most economically feasible under both novel approaches due to its balanced mix of high-value products and moderate operational costs. These findings suggest that cost calculation methods should align with plant objectives: Economic allocation for scenarios prioritizing water recovery and Dual allocation for maximizing the value of salts and chemicals. This study provides a foundation for tailored economic assessments and guides plant design and investment decisions.

Understanding the relative influence of immigration and species sorting in wastewater treatment systems is essential, as bacteria in influent wastewater can significantly impact treatment system functionality. This study investigated the contribution of immigration to the community assembly of different-sized microbial aggregates in a full-scale aerobic granular sludge (AGS) system using genome-resolved metatranscriptomics. Our novel analysis revealed that negative-net-growth-rate populations, which persist due to immigration, can exhibit substantial activity and potentially contribute to the AGS system’s functionality. The results also highlighted that sulfate-reducing and fermenting bacteria, along with some nitrifiers and glycogen-accumulating organisms (GAOs), were more active in the influent wastewater, serving as a continuous source of both beneficial and competing immigrants to the AGS system. Granular sludge (size >0.2 mm) demonstrated a robust capacity to resist immigration effects from competing immigrants, whereas flocculent sludge (size <0.2 mm) was more susceptible. Importantly, flocculent sludge harbored functional microbial groups such as active nitrifiers and fermentative polyphosphate-accumulating organisms (PAOs) belonging to Ca. Phosphoribacter, while granular sludge enriched for active conventional PAOs such as Ca. Accumulibacter. These findings provide valuable insights for engineers to design and operate AGS systems by optimizing microbial aggregate sizes and emphasizing the importance of influent microbial characterization in the design of wastewater treatment plants to enhance the functionality and activity of AGS systems.

The implementation of mainstream anaerobic ammonium oxidation (anammox) can facilitate the realization of carbon-neutral wastewater treatment. However, this technology remains challenging owing to the inability to stably provide nitrite. In this study, we developed a novel nitrification and elemental sulfur-based partial autotrophic denitrification/anammox (NS⁰DA) process for mainstream nitrogen removal. The NS⁰DA system consists of a nitrification reactor and a combined elemental sulfur-based denitrification and anammox (S⁰DA) reactor. Each reactor was independently initiated and optimized before being integrated. At mainstream nitrogen levels (48.5 ± 1.7 mg NH₄⁺-N/L) and 25 °C, the NS⁰DA system achieved 89.1 ± 5.7 % total nitrogen (TN) removal efficiency, with an effluent TN concentration of 5.4 ± 2.8 mg N/L. The system exhibited a low N₂O emission factor (0.23 %), significantly lower than other anammox-based systems. The S⁰DA reactor achieved a nitrogen removal rate of 0.53 kg N/(m³·d) with a short hydraulic retention time (2 h). Anammox accounted for 87.3 ± 7.0 % of the TN removal in the S⁰DA reactor. Isotope experiments and kinetic analysis revealed the cooperation between anammox and denitrification for nitrogen removal. Polysulfides formed in the S⁰DA reactor enhanced the utilization rate of elemental sulfur. High-throughput sequencing identified Thiobacillus and Candidatus Brocadia as the dominant genera of sulfur oxidation and anammox, respectively. The nitrogen and sulfur metabolic pathways were further verified through metagenomic analysis. Overall, the NS⁰DA process provides a stable and efficient nitrogen removal process, minimizing oxygen demand, eliminating organic carbon requirements, and reducing N₂O emissions compared to conventional nitrification/denitrification. This approach offers a promising solution for mainstream nitrogen removal in wastewater treatment.

>50 % of the organic matter in sewage consist of particulate chemical oxygen demand (pCOD). This study used 250 μm fluorescent microbeads, 130±58 μm microparticles and 100 nm nanobeads to simulate sewage particles, and investigated the fate of these particles under both plug flow feeding and aeration phases in an aerobic granular sludge (AGS) system. Filtration performance was dominantly influenced by the particle size rather than the upflow velocity (V_upflow). The microbeads exhibited 95±3 % filtration efficiency with obvious accumulation around the AGS bed bottom, even as slight fluidization started at the V_upflow of 5.0 m·h^-1. In contrast, the nanobeads filtration efficiency was significantly lower (43±6 %). During the aeration phase, the attachment efficiency increased with the decrease of particle size. The microbeads attachment efficiency variated between 39–49 %, whereas the microparticles and nanobeads achieved better attachment of 89.4–95.2 % and 98.8–99.3 %, respectively. Furthermore, aeration batch tests showed both nanobeads and the irregular microparticles attachment by AGS was strong, and the detach-attach of nanobeads/microparticles between different sized AGS was very limited duration aeration. This work provides insight into the fate of particles in AGS system. The optimal sludge treatment was also evaluated in the scope of this removal of non-biodegradable, and potentially harmful particles.

Nitrous oxide (N2O) is the third most important greenhouse gas and originates primarily from natural and engineered microbiomes. Effective emission mitigations are currently hindered by the largely unresolved ecophysiological controls of coexisting N2O-converting metabolisms in complex communities. To address this, we used biological wastewater treatment as a model ecosystem and combined long-term metagenome-resolved metaproteomics with ex situ kinetic and full-scale operational characterization over nearly 2 years. By leveraging the evidence independently obtained at multiple ecophysiological levels, from individual genetic potential to actual metabolism and emergent community phenotype, the cascade of environmental and operational triggers driving seasonal N2O emissions has ultimately been resolved. We identified nitrifier denitrification as the dominant N2O-producing pathway and dissolved O2 as the prime operational parameter, paving the way to the design and fostering of robust emission control strategies. This work exemplifies the untapped potential of multi-meta-omics in the mechanistic understanding and ecological engineering of microbiomes towards reducing anthropogenic impacts and advancing sustainable biotechnological developments.

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.

The synthesis of chemically derived bituminous-based activated carbons (ACs) was conducted in this research. The study examined the efficacy of the KOH-2 modified AC by conducting batch experiments under different operating settings. Pollutant concentration, adsorbent dosage and contact time were considered as variables. Bovine serum albumin (BSA), dextrose, and methylene blue (MB) were used as model substances to represent protein, carbon, and textile dyes, respectively. Under optimal conditions, the activated carbon modified with a 2:1 impregnation ratio using KOH showed the best performance among two other ACs, therefore, that was introduced as chosen AC. The optimal parameters were determined as pollutant concentration, adsorbent dosage and contact time obtained to be 600 mg/L, 0.22 g/L and 220 min; 200 mg/L, 0.6 g/L and 180 min; 200 mg/L, 1 g/L and 196 min for MB, BSA and dextrose, respectively. This AC obtained the highest adsorption capacity of 2710.6, 283, and 273 mg/g adsorbing MB, BSA and dextrose, respectively. In the following, column studies were conducted using biologically treated wastewater obtained from a new one-stage hybrid internal circulation airlift A2O bioreactor to eliminate soluble microbial products (SMP). It was seen that the adsorption column, which was filled with the AC under optimal experimental conditions (bed height of 4.5 cm and solution flow rate of 5 mL/min), resulted in a decrease in residual organic substances from 35 mg-TOC/L to 6.6 mg-TOC/L. It is worth mentioning that the utilization of an integrated approach involving sophisticated biological treatment and post-treatment technology has demonstrated efficacy in the elimination of residual organic substances.

Anaerobic ammonium-oxidation (anammox) is a typical redox reaction driven by membrane electron transformation. However, the electron transfer mechanism of the core redox reaction and its evolutionary origins are still not thoroughly identified. In this study, a preliminary analysis was conducted for such interaction based on the 64 anammox bacterial genomes representing 12 genera available currently. The results suggested that enzymes involved in anammox reaction share the similar catalytic and electron transfer modes in different lineages, while the electron-carrying proteins shuttled between membrane and soluble enzymes are very different. A comparatively simple electronic shuttle protein system was encoded in the early-branching groundwater lineages Candidatus (Ca.) Avalokitesvara and Ca. Tripitaka, which was replaced by a sophisticated electron carrier scheme in the late-branching marine and terrestrial groups within family Ca. Brocadiaceae. Remarkably, the increasing availability of nitrite after Great Oxidation Event (GOE) potentially drove the adaptive evolution of the core redox systems by successively recruiting the nitrite reductase (NIR) for nitrite balance, a stable complex of two small cytochrome c proteins (NaxL and NaxS homologues) for electron transfer to HZS, as well as optimizing the structure of nitrite oxidoreductase gamma (NxrC) for electron conservation. In particular, a tubule-inducing nitrite oxidoreductase subunit (NxrT homologue) was further formed for electron transformation after the Neoproterozoic Oxygenation Event (NOE). Finally, based on two full-scale anammox-based wastewater treatment systems (WWTPs), we identified core gene transcriptional activities affecting the abundance of the family Ca. Brocadiaceae and their association with environmental factors. Overall, our study not only provides key information for understanding the dynamic patterns and evolutionary mechanisms of the anammox reactions and the associated electron transfers in conjunction with major geological events, but also provides new insights for future enrichment and effective applications.

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.

Methane removal is an essential step in drinking water production from methane-rich groundwaters. Conventional aeration-based stripping results in significant direct methane emissions, contributing up to one-third of a treatment plant's total carbon footprint. To address this, a full-scale trickling filter was operated for biological methane oxidation upstream of a submerged sand filter, and its performance was compared to a conventional aeration–submerged sand filtration set-up. Full-scale data were combined with ex-situ batch assays and metagenome-resolved metaproteomics to quantify the individual contribution of the main (a)biotic processes and characterize the enriched microbial communities. Both treatment setups fully removed methane, iron, ammonium, and manganese, yet the underlying mechanisms differed significantly. Methane was completely removed from the effluent after trickling filtration, with stripping and biological oxidation each accounting for half of the removal, thereby halving overall methane emissions. Methane-oxidizing bacteria not only outcompeted nitrifiers in the trickling filter, but also likely contributed directly to ammonia oxidation. In contrast to the submerged filter preceded by methane stripping, signatures of biological iron oxidation were almost completely absent in the trickling filter, suggesting that the presence of methane directly or indirectly promotes chemical iron oxidation. All systems had similar ex-situ manganese oxidation capacities, yet removal occurred only in the submerged filters but not the trickling filter. Ultimately, our results demonstrate that trickling filtration is effective in promoting biological methane oxidation at comparable produced drinking water quality, highlighting its potential for advancing sustainable drinking water production.

This study investigated the concurrent removal of carbon, nitrogen, and phosphorus (CNP) from milk processing wastewater (MPW). The evaluation was carried out using a newly developed single-stage bioreactor known as the baffled dual internal circulation airlift A2O (B-DCAL-A2O) bioreactor. The bioreactor functionality was monitored to determine the impact of three important factors, i.e., hydraulic retention time (HRT; 7-15 h), air flow rate (AFR; 1-3 L/min), and aerobic volume ratio (AVR, 0.324-0.464). The use of baffles in the anoxic zone identified improved nutrient removal. Specifically, the total chemical oxygen demand (TCOD), total nitrogen (TN), and phosphorus removals of 94.8, 80, and 80%, respectively, as well as a reduction in the effluent turbidity of 8 NTU at optimum circumstances (HRT, AFR, and AVR of 10 h, 3 L/min, and 0.464, respectively) were obtained. From polymerase chain reaction (PCR) analysis, the superior nitrogen removal was attributed to the combined effects of simultaneous nitrification and denitrification (SND), anaerobic ammonium oxidation (anammox), and denitrifying phosphorus-accumulating organisms (DPAOs) and glycogen-accumulating organisms (GAOs). PCR analysis validated the coexistence of diverse functional microbial groups, including ammonia-oxidizing bacteria (AOB: Nitrosospira sp., Nitrosomonas sp., Nitrosococcus), and nitrite-oxidizing bacteria (NOB: Nitrospira [23S rRNA]), denitrifiers (Pseudomonas), anammox bacteria (Candidatus Brocadia and Candidatus Kuenenia), PAOs (Tetrasphaera, Candidatus Microthrix [16S rRNA], and Candidatus Accumulibacter [ppk1]), DPAOs (Pseudomonas), and GAOs (Sphingomonas [16S rRNA]). The phosphorus removal was attributed to the existence of PAOs and DPAOs in a single bioreactor. The results confirmed improved sludge properties, verified with the best results obtained for the turbidity (6 NTU) and SVI (90 mL/g) at an HRT of 15 h, an AFR of 3 L/min, and an AVR of 0.464.

While nowadays a lot of measurements are conducted at wastewater treatment plants, data reliability could further be improved, e.g., through data reconciliation. This study demonstrated the added value of data reconciliation to improve data quality in a full-scale wastewater treatment plant. Also, the effect of the mass balance setting (linear and bilinear mass balances) was quantitatively evaluated, considering data sets with missing measurements and with gross errors. The improvement in the precision of the key variables was higher with bilinear mass balances (40–80 %) compared to the linear setting (0–70 %). Besides, it delivered a higher number of improved key variables, especially when flow measurements were limited (minimum improved variables of 15 and 0, respectively). Bilinear mass balances were also more efficient in gross error detection and played a crucial role in cross-validation based on flow measurements, resulting in lower incorrectly-identified gross errors. Overall, it is recommended to use bilinear mass balances.

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.

The first therapeutic use of antimicrobial agents initiated their endless arms race with antimicrobial resistance (AMR). Although the genes encoding antimicrobial resistance are ancient and ubiquitous in various environmental compartments, including aquatic environments, over eight decades of exposure to selective pressure has changed the way antimicrobial resistance genes (ARGs) emerge and transmit among the three One Health sectors (that is, the intersected sectors of humans, animals and the environment). The dissemination of ARGs has been facilitated by the widespread use of antimicrobials, along with direct and secondary pollution pathways. Current global consensus dictates that AMR should be addressed under a One Health framework. AMR National Action Plans have frequently been formulated. However, the capacity for implementation is not ready in most countries, especially in low- and middle-income regions. This is in part due to the substantial challenges in documenting and controlling cross-sector AMR connectivity. Here we describe the past and current status of AMR, emphasizing the contribution of connectivity to global AMR burden. We discuss connectivity at ecological, microbial and genetic levels; propose an approach based on genomics and metagenomics to assess connectivity; and finally advocate for cross-sector studies to better understand AMR connectivity and mitigate dissemination. We believe that such harmonized connectivity studies will facilitate coordinated actions and investments across sectors and regions to scale up AMR management globally.

The aerobic granular sludge (AGS) is an emerging technology widely spread, since most organic matters in actual domestic sewage were particulate matters, this study aims to determine whether the attachment between micro particles and different sized AGS was influenced by granule surface area. The attachment of micro particles by different sized AGS (2.0–5.0 mm) were investigated. Furthermore, to simulate the attachment by broken fragments of AGS, complete 4.0–5.0 mm AGS were cut into 2,4, and 8 pieces, and the attachment performance between the broken pieces and similar sized complete AGS were compared. Fourier transform infrared (FTIR) and fluorescence staining were applied to analyze the chemical bonds and amyloid-glucan like structure of AGS from outside to inside. The results showed the 3.1–4.0 mm AGS had the best surface area attachment of micro particles, followed by the 2.5–3.1 mm AGS. The attachment performance of micro particles was not determined by specific surface area, but was closely related to the surface roughness caused by the amyloid-glucan like structure. The distribution density of amyloid-glucan like structure decreased from outside to inside, and if an granule was broken into pieces during aeration, micro particles were preferential to be attached by the outer layer of the broken pieces from the initial granule. The micro particles attachment showed little relationship with the hydrophilicity of AGS surface, either the outer layer or the inner layer. This study highlighted the crucial role of AGS outer layer in micro particle attachment, particularly the broken pieces from the original AGS outer layer, which facilitate to attach micro particles and contribute to form new granules.

Aerobic Granular Sludge (AGS) is a relatively new approach to wastewater treatment. As opposed to activated sludge with mainly flocculant biomass of relatively even composition, the gra nules in AGS differ in size and composition. Biomass ranges from flocs similar to activated sludge to granules of >6 mm. Granules also have distinct biomass due to the stratification of redox conditions across the granules. Typically organic micropollutants (OMPs) are removed via sorption and biodegradation during wastewater treatment, however this has yet to be thoroughly invested for AGS systems. We hypothesized that different sized granules have potentially different sorption behaviour due to the differe nces in extracellular polymeric substances (EPS) and have different biodegradation patterns due to the differences in biomass composition among size fractions. Therefore, we investigated the capacity of AGS to remove OMPs via sorption and biodegradation by performing controlled batch experiments in the lab. We separated the sludge into different sizes in order to understand the role of different sludge composition on OMP removal.

We observed notable sorption (>40% removal) for 10 of the 24 OMPs tested in our study (Figure 1). The 10 OMPs include 3 fluoroquinolones (norfloxacin, ofloxacin, and ciprofloxacin), 3 macrolides (clarithromycin, azithromycin, and erythromycin), 2 beta-blockers (propranolol and atenolol), tetracycline, and citalopram. We noted that all of these 10 compounds are ionizable, with 6 positively charged compounds and 4 zwitterionic compounds at pH 7. Considering that sludge biomass typically has a negative charge, this seems to indicate electrostatic interactions between the 10 OMPs and th e sludge. Larger fractions contributed more to sorption than smaller granules and flocs, as expressed per unit of biomass. Specifically, the normalized Kd¬ for large fractions was up to 100%larger than for small fractions, suggesting that larger fractions contribute more to sorption in real AGS systems than smaller fractions and flocs. However, sorption kinetics were most likely retarded by diffusion limitations in large granules. Biodegradation was observed for a number of compounds. Overall, this study elucidated the roles of sorption and biodegradation in the removal of OMPs in AGS WWTPs, showing that these processes are size fraction dependent.

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.

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.