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.

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.

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.

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 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.

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.

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 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.

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.

Division of metabolic labour is a defining trait of natural and engineered microbiomes. Denitrification-the stepwise reduction of nitrate and nitrite to nitrogenous gases-is inherently modular, catalysed either by a single microorganism (termed complete denitrifier) or by consortia of partial denitrifiers. Despite the pivotal role of denitrification in biogeochemical cycles and environmental biotechnologies, the ecological factors selecting for complete versus partial denitrifiers remain poorly understood. In this perspective, we critically review over 1500 published metagenome-assembled genomes of denitrifiers from diverse and globally relevant ecosystems. Our findings highlight the widespread occurrence of labour division and the dominance of partial denitrifiers in complex ecosystems, contrasting with the prevalence of complete denitrifiers only in simple laboratory cultures. We challenge current labour division theories centred around catabolic pathways, and discuss their limits in explaining the observed niche partitioning. Instead, we propose that labour division benefits partial denitrifiers by minimising resource allocation to denitrification, enabling broader metabolic adaptability to oligotrophic and dynamic environments. Conversely, stable, nutrient-rich laboratory cultures seem to favour complete denitrifiers, which maximise energy generation through denitrification. To resolve the ecological significance of metabolic trade-offs in denitrifying microbiomes, we advocate for mechanistic studies that integrate mixed-culture enrichments mimicking natural environments, multi-meta-omics, and targeted physiological characterisations. These undertakings will greatly advance our understanding of global nitrogen turnover and nitrogenous greenhouse gases emissions.

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

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.

Soil amendments and microbial inoculants can affect plant growth, water retention, and crop resilience. This study investigated the effects of two amendments, extracellular polymeric substances (EPS) and biochar, with and without bacterial inoculation, on maize (Zea mays) growth, irrigation needs, and physiological responses. Maize was cultivated in soil with 2.5 % and 5 % (w/w) of wet EPS (Kaumera®) or biochar and inoculated with a bacterial consortium consisting of Arthrobacter nicotinovorans EAPPA and Rhodococcus sp. EC35.

EPS-treated plants exhibited significantly higher shoot biomass, larger stem thickness, while soil plant analysis development (SPAD) values suggest improved nutrient availability and photosynthetic efficiency. In non-inoculated plants, EPS supplementation increased shoot dry biomass by 78 % and stem thickness by 9 % compared to control plants grown without amendments. This enhancement strongly correlated with nutrient uptake, especially in plants supplemented with 5 % of EPS. Particularly, Mg and Ca concentrations increased by 195 % and 73 %, respectively, compared to non-amended controls. Inoculation further amplified these benefits, underscoring its key role in plant development and resilience. In contrast, biochar-treated plants exhibited reduced growth, suggesting stress effects at the tested addition doses. Electrolyte leakage, a key indicator of plant stress, was significantly lower in soils amended with EPS, suggesting that EPS provides a protective effect to the plants. EPS also demonstrated remarkable water retention benefits, reducing irrigation requirements by 30 % with 5 % of EPS application, compared to 9 % reduction with biochar. The use of EPS, combined with microbial inoculants, represents a sustainable agricultural strategy for optimizing maize production in water-limited environments.

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.

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.

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.

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.

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.

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.

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.