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.

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.

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.

This study compares two membrane bioreactor-based enrichment strategies to produce polyhydroxyalkanoates (PHAs) from domestic waste-activated sludge. The aerobic dynamic feeding was implemented in layout 1 while layout 2 employs an aerobic/anoxic enrichment adopting an additional nitritation reactor. Both systems achieved around 38 % w/w of PHA with storage yields of 0.28–0.42 and 0.35–0.53 gCOD_PHA/gCOD_VFA for layouts 1 and 2, respectively. Layout 2 demonstrated an average N removal efficiency of 88.8 ± 3.9 %, slightly higher than layout 1 (82.7 ± 9.9 %). However, layout 2 showed greater nitrous oxide (N₂O) emissions, averaging 0.7 ± 0.2 mg N₂O-N/L almost doubling layout 1 (0.4 ± 0.1 mg N₂O-N/L). Additionally, layout 2 exhibited a 42 % increase in carbon footprint compared to layout 1, reaching 10.2 kg CO₂/day. This research highlights the high potential and drawbacks of the AE/AN enrichment strategy for integrating PHA production into wastewater treatment plant operations.

The removal of nitrogen from the mature leachate landfills is a major challenge. In this study, a novel aeration control strategy combined with step-wise influent addition was used to successfully implement simultaneous partial nitrification, anaerobic ammonia oxidation and denitrification (SNAD) in a single-stage Sequencing Biofilm Batch Reactor (SBBR) used for nitrogen removal and carbon removal of mature landfill leachate with a low C/N ratio. The SNAD process was rapidly initiated by transitioning from aeration to aeration-anaerobic (OA) phases and stabilized under anaerobic-aeration-anoxic (AOA) conditions. The relative abundance of nitrite-oxidizing bacteria (NOB) in the reactor was consistently suppressed (<0.1 %), while the dominant genus of anaerobic ammonium-oxidizing bacteria (AnAOB), Candidatus Jettenia, reached a relative abundance of 10.9 % in the biofilm. During 160 days of operation with influent NH4+-N up to 600.9 mg/L and COD/TIN of 2.3 ± 0.3, the system achieved 98.3 ± 1.1 % nitrogen removal efficiency and effluent TIN of 10.8 ± 1.3 mg N/L, demonstrating robust synergy among ammonia-oxidizing bacteria (AOB), anammox bacteria, and denitrifying bacteria (DNB). This strategy provides a scalable approach for SNAD system cultivation and sustainable treatment of mature landfill leachate.

Vivianite (Fe₃(PO₄)₂·8 H₂O) has emerged as a promising mineral for phosphorus (P) recovery from digested sludge, and it may also contribute to phosphate management in lake sediments and manure, given the similar anaerobic conditions across these environments. However, organic ligands in these matrices have been proposed to complex with iron (Fe), thereby reducing the efficiency of vivianite formation. This study aims to elucidate the impact of organic ligands on vivianite formation, particularly focusing on their binding strength with iron and the subsequent effects on vivianite formation in pig manure. Organic ligands not only form complexes with iron but also influence the crystal growth process. We investigated how different organic ligands affect the formation and dissolution of vivianite, assuming that ligands with higher iron-binding strength would enhance phosphate solubilization. Our findings revealed that citrate nearly completely inhibited vivianite formation (up to 100 %) and caused a 50 % dissolution of existing vivianite, while humate hindered vivianite formation by 40 % but only led to a 10 % dissolution. Interestingly, pig-derived dissolved organic matter had minimal effects on the precipitation of iron and phosphorus but significantly altered the morphology of the resulting products, which varied depending on the age of the manure filtrate. While the iron binding strength of organic ligands does influence vivianite formation, it does not solely account for the reduced vivianite formation observed in complex matrices like manure. Therefore, a more nuanced assessment of the role of organic matter in vivianite formation is warranted.

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.

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.

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.

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.

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.

Phosphorus runoff from agricultural land is a major driver of eutrophication, with manure serving as a significant source of phosphorus input. In regions such as the Netherlands, high livestock densities and limited land availability pose challenges for manure management, particularly in pig farming. Recovering phosphorus from manure and redistributing it to phosphorus-deficient areas offers a sustainable solution. This study explores phosphate recovery via vivianite (Fe₃(PO₄)₂·8H₂O) precipitation—a method previously demonstrated in municipal wastewater treatment plant sludge—and evaluates its applicability to pig manure. Vivianite formation was investigated in fresh, 4-month-aged, and digested pig manure, as well as in Thermal Hydrolysis Plant (THP) derived digested sewage sludge. A key finding is that high dissolved inorganic carbon (DIC) concentrations inhibit vivianite formation by promoting siderite (FeCO₃) precipitation. In digested manure, a DIC threshold of approximately 3 g/L HCO₃⁻ was identified, below which vivianite formation is favored. THP sludge, characterized by elevated DIC, exhibited similar inhibitory effects. More generally, vivianite was shown to form without significant competition with siderite if the DIC concentration is <2.5 times the iron concentration. Experimental results were compared with thermodynamic predictions using Visual MINTEQ and experiments in ultrapure water, revealing discrepancies which may be attributed to the ionic composition in environmental matrices. Strategies such as combining ammonia and DIC stripping or targeting fresh manure were shown to enhance vivianite formation. These findings can be used to propose the integration of vivianite-based phosphorus recovery into broader resource recovery frameworks, including biomethane production, ammonium recovery, and carbon capture.

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.

The objective of this study was to treat different types of industrial wastewaters (milk processing wastewater, soft drink wastewater, and soybean oil plant wastewater) using modified configurations of a bioreactor called one-stage internal dual circulation airlift A2O (DCAL-A2O) bioreactor. The modification involved the use of physical barriers (baffle and packing media) to enhance the anoxic zone of the bioreactor. The study investigated the performance of bioreactors in simultaneously removing carbon, nitrogen, and phosphorus. The bioreactor was designed in three configurations: ordinary, baffled, and hybrid DCAL-A2O bioreactors, depending on the type of manipulation. Different operating conditions, such as hydraulic retention time (HRT), air flow rate (AFR), and anaerobic volume ratio (AVR), were examined simultaneously. The hybrid DCAL-A2O bioreactor was found to be highly suitable for wastewater treatment due to its superior biological performance, ease of operation, and cost-effectiveness when compared to two other bioreactors. The chosen bioreactor demonstrated high performance in terms of TCOD, TN, phosphorus removal efficiencies, and effluent turbidity. Specifically, it achieved removal efficiencies of 97.0% for TCOD, 92% for TN (with a concentration of 179.4 mg/L), 90% for phosphorus (with a concentration of 50.33 mg/L), and an effluent turbidity of 9 NTU. These results were obtained under optimal conditions, which included a HRT of 10 h, an AFR of 2 L/min, and an AVR of 0.464. The unique setup of the bioreactor demonstrated its undeniable ability to effectively treat wastewater from different feeding points. PCR tests confirmed the co-existence of multiple functional bacterial species, including AOB, NOB, denitrifying bacteria, anammox, PAOs, DPAOs, and GAOs. This provides strong evidence for concurrent removal of organics and nutrients in all three configurations of the integrated unit. In summary, this study emphasizes the need for ongoing research in energy efficiency to safeguard our environmental resources.

Within this research, a one-stage hybrid dual internal circulation airlift A2O (DCAL-A2O) bioreactor was designed and operated to simultaneously remove carbon, nitrogen and phosphorous (CNP) from milk processing wastewater (MPW) in different operational circumstances. The substantial operating variables monitored in this work were including hydraulic retention time (HRT), airflow rate (AFR) and aeration volume ratio (AVR) ranged from 7 to 15 h, 1–3 L/min and 0.324–0.464, respectively. From the view point of economics and process function, the optimum conditions were obtained at the HRT, AFR and AVR of 10 h, 2 L/min and 0.464, respectively. At the optimum conditions TCOD, TN, TP removal efficiencies and effluent turbidity were reported to be 97 %, 90 %, 92 % and 9 NTU, respectively. The impact of wastewater biodegradability (BOD5/COD) was evaluated on the bioreactor performance using two other wastewaters i.e. soft drink (SDW) and soybean oil plant wastewaters (SOW) in comparison with the MPW. Removal efficiencies for TCOD and TN exceeding 80 % were observed. The feeding location revealed a prominent impact on the TN and phosphorous removal efficiencies (both ≥80 %) related to the availability degree of the readily biodegradable organic substrate to denitrifiers and PAOs. The rise in HRT, AFR and AVR resulted in reducing microbial secretions as SMP present in sludge and bioreactor effluent as well as loosely bounded EPS (LB-EPS), reported to be 26, 28, 32.5 and 194.4 mg/L TOC, respectively. Different bacteria species were present at optimum conditions confirming concurrent CNP removal in a single body. Finally, the operating cost evaluation verified the effectiveness of the hybrid airlift A2O treating the MPW.

Aerobic granular sludge (AGS) technology holds great promise of becoming the standard for biological wastewater treatment due to its lower energy consumption, small footprint, and high removal efficiency of nutrients compared to the conventional activated sludge processes. Different-sized aggregates have been shown to harbor a different microbial community composition. The central question is do full-scale AGS wastewater treatment plants (WWTPs) select for core microbial communities across different aggregate sizes and how these selected organisms differ between the different-sized aggregates. This study analyzed samples from nine geographically distributed full-scale AGS WWTPs that consistently perform well in terms of chemical oxygen demand (COD) and nutrient (N and P) removal. The main results showed that site-specific conditions highly influence microbial composition in smaller aggregates (< 1 mm), while larger granules form stable communities independent of WWTP location. Notably, all aggregates contained a small subset of 128–139 core OTUs that were both prevalent and abundant across all sizes. These core OTUs include key functional groups such as fermenters, aerobic heterotrophs, polyphosphate-accumulating organisms (PAOs), glycogen-accumulating organisms (GAOs), and nitrifiers, which play a crucial role in COD and nutrient removal. Additionally, an enrichment pattern was observed, with aerobic heterotrophs dominating in flocs, PAOs in small granules, and GAOs and nitrifiers in large granules. This study offers valuable insights into the core microbiome of different-sized aggregates in full-scale AGS WWTPs and highlights their potential role in overall system performance.

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 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.