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.

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.

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.

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.

Valuable and rare materials in seawater brine are often discarded during desalination. However, there is an increasing focus on recovering these resources, due to the economic and environmental opportunities it can bring. Despite this shift, current Sustainability Assessments (SA) in desalination overlook the brine handling and social dimensions, and brine treatment assessments remain centered on techno-economic dimensions. This work proposes a comprehensive framework for the SA of integrated desalination and resource recovery options, focusing on recovering valuable materials from brine. The framework not only evaluates pre-defined systems but supports the identification of system features of interest, such as products to assess and technologies to include, as well as the transparent selection of indicators, considering specific contexts. To develop this framework, a review of the literature on SA in desalination and brine treatment systems was conducted. Looking at the identified gaps, we synthesized the findings and key messages and proposed the integration of Multi-Criteria Analysis and Value-Sensitive Design in the decision-making process. This allows stakeholders to be involved and incorporates their values at different stages of the assessment, making it distinct from traditional SA methods. This framework offers structured guidance to stakeholders on how to carry out qualitative and quantitative assessments while ensuring transparency in the assessment process.

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 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.

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.

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.

The authors wish to make a rectification of authorship, adding Dr. Peter. J.T. Verheijen as an author. Dr. Peter. J.T. Verheijen (ORCID 0000-0003-2649-1189) introduced the methodological approach regarding statistics and data reconciliation on which this paper was built, was actively involved in the formal analysis, the software and the reviewing and editing of the original drafts. The correct author list reads as follows: Q.H. Le1, P.J.T. Verheijen2, P. Carrera1, M.C.M. van Loosdrecht2, E.I.P. Volcke1* 1 BioCo research group, Department of Green Chemistry and Technology, Ghent University, 9000 Ghent, Belgium 2 Department of Biotechnology, Delft University of Technology, 2600 AA Delft, The Netherlands> *Corresponding author: The authors sincerely apologise for this mistake, arising from a most unfortunate miscommunication.Quan Hong Le: Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing-review & editing Peter J.T. Verheijen (ORCID 0000-0003-2649-1189): Formal analysis, Methodology, Software, Supervision, Writing – original draft, Writing-review & editing Paula Carrera (ORCID ID 0000-0003-0893-5663): Investigation, Validation, Visualization, Writing-review & editing Mark C.M. van Loosdrecht (ORCID ID 0000-0003-0658-4775): Conceptualization, Supervision, Writing-review & editing Eveline I.P.

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.

Municipal wastewater treatment plants are a ubiquitous source of microbial biomass for PHA production. Technological feasibility of directly using municipal activated sludge (WAS) for a PHA accumulation bioprocess is demonstrated in the literature. However, PHA contents and yields may be lower due to a coexistence of PHA-storing and non-PHA-storing microorganisms in WAS. This work focused on metabolic principles for stimulating selective growth of PHA-storing microorganisms during a PHA accumulation bioprocess to enhance PHA productivity. Two model substrates, butyrate and acetate, were used to evaluate conditions and principles that may regulate this selective growth response. Conditions promoting selective growth of the PHA storers were consistently observed in the fed-batch PHA production process fed with butyrate. Productivity was increased to 4 times more PHA produced over 48 h, wherein PHA contents and average yields were improved from 0.39 gPHA/gVSS and 0.25 gCODPHA/gCOD to 0.61 gPHA/gVSS and 0.47 gCODPHA/gCOD, respectively. Respiration monitoring and mass balances, with metabolic modelling, suggest that expected underlying differences in ATP yields between two tested substrates are the main mechanistic drivers of the observed selective growth. This study proposed that if conditions are created such that ATP is produced in sufficient excess of the demands for PHA storage and cell maintenance, then those PHA storers will further grow concurrently. The selective microbial growth allows extra conversion of substrates into PHA. These principles, concerning the metabolic basis of the PHA production pathway, provide a foundation that can be applied to a range of substrates or substrate mixtures for enhanced PHA accumulation.

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.

Extracellular proteins are supposed to play crucial roles in the formation and structure of biofilms and aggregates. However, often little is known about these proteins, in particular for microbial communities. Here, we use two advanced metaproteomic approaches to study the extracellular proteome in a granular Candidatus Accumulibacter enrichment as a proxy for microbial communities that form solid microbial granules, such as those used in biological wastewater treatment. Limited proteolysis of whole granules and metaproteome isolation from the culture's supernatant successfully classified over 50% of the identified protein biomass to be secreted. Moreover, structural and sequence-based classification identified 387 proteins, corresponding to over 50% of the secreted protein biomass, with characteristics that could aid the formation of aggregates, including filamentous, beta-barrel containing, and cell surface proteins. While various of these aggregate-forming proteins originated from Ca. Accumulibacter, some proteins associated with other taxa. This suggests that not only a range of different proteins but also multiple organisms contribute to granular biofilm formation. Therefore, the obtained extracellular metaproteome data from the granular Ca. Accumulibacter enrichment provides a resource for exploring proteins that potentially support the formation and stability of granular biofilms, whereas the demonstrated approaches can be applied to explore biofilms of microbial communities in general.

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.

Extracellular Polymeric Substances (EPS) are ubiquitous in biological wastewater treatment (WWT) technologies like activated sludge systems, biofilm reactors, and granular sludge systems. EPS recovery from sludge potentially offers a high-value material for the industry. It can be utilized as a coating in slow-release fertilizers, as a bio-stimulant, as a binding agent in building materials, for the production of flame retarding materials, and more. P recovered within the extracted EPS is an intrinsic part of the recovered material that potentially influences its properties and industrial applications. P is present in EPS in different speciation (e.g., P esters, poly-P, ortho-P, etc.). Such P species are already intensively used in the chemical industry to enhance thermal stability, viscoelasticity, emulsification, water-holding capacity, and many other properties of some natural and petroleum-derived polymers. The translation of this knowledge to EPS is missing which prevents the full utilization of phosphorus in EPS. This knowledge could allow us to engineer EPS via phosphorus for specific target properties and applications. In this review, we discuss how P could affect EPS properties based on experiences from other industries and reflect on how these P species could be influenced during the EPS extraction process or in the WWTPs.

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 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 emphasis on phosphorus removal and recovery from wastewater treatment plants has intensified in recent years due to the urgent need to reduce dependency on nonrenewable phosphorus reserves. Enhanced biological phosphorus removal (EBPR), driven by a diverse community of polyphosphate-accumulating organisms with distinct metabolic capabilities, offers several advantages over chemical precipitation methods. These benefits include reduced chemical use, lower sludge volumes, decreased reliance on costly chemical precipitants, and improved phosphorus recovery quality. Recent advancements in recovery technologies now enable efficient phosphorus extraction from digester supernatant, dewatered digested sewage sludge, and sewage sludge ash, each yielding different recovery efficiencies. Despite these advances, a comprehensive assessment of the phosphorus recovery potential from these target streams in conjunction with EBPR remains crucial and has yet to be fully explored.

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.