The scarcity of electron acceptors in municipal wastewater hampers nitrogen removal in anaerobic ammonium oxidation (anammox) systems, limiting their wider adoption. Electrochemically-assisted anammox (E-AMX) addresses this by using electrodes as alternative electron acceptors to nitrite (NO₂⁻), thereby enhancing electron transfer and reducing NO₂⁻ dependency. However, the underlying nitrogen metabolism and extracellular electron transfer (EET) mechanisms in E-AMX require further exploration. Here, we employed metagenomic sequencing to elucidate nitrogen-metabolic pathways and EET mechanisms in an E-AMX system, focusing on key functional genes and extracellular polymeric substances (EPS) synthesis genes. The genetic potential for pili synthesis in Candidatus (Ca.) Brocadia was, for the first time, revealed through binning analysis. Under a 0 V (vs. Ag/AgCl) electric field, E-AMX achieved stable and efficient operation on day 27 with near 100% ammonia and NO₂⁻ removal. The abundance of Ca. Brocadia increased to 13.9% (1.9× that of the control), accompanied by more abundant core anammox genes (hzs and hdh). These results allow the prediction of the nitrogen-metabolism pathway in E-AMX. Functional gene profiling indicated that direct EET relied chiefly on heme, the Mtr pathway, flagella and pili. Additionally, the electric field increased copies of EPS-associated amino acid metabolism genes (e.g., cysK, thrC), which likely facilitated protein-mediated indirect EET. Binning analysis further revealed selective enrichment of Ca. Brocadia sinica, indicating species-level niche differentiation under the electric field. This study proposes potential mechanisms by which an applied micro-electric-field enhances anammox activity and EET, offering theoretical clues for future optimization of electric-field-driven nitrogen removal processes.

Microplastics (MPs) serve as carriers for microbial community colonization, forming unique ecosystems known as plastispheres in urban aquatic ecosystems. However, interactions among microbes, extracellular polymeric substances (EPS), and MPs remain poorly understood. This study investigates microbial consortia and their EPS secretion behaviors across various plastispheres at two representative coastal urban water sites. Permutational multivariate analysis of variance revealed that MP type significantly influenced microbial community structures in reservoir environments (R² = 0.60, p < 0.001), highlighting the pronounced impact of MP types in high-quality urban waters. Specific microbial phyla and genera were identified as key contributors to EPS compositional variations across different plastispheres. Hierarchical partitioning results identified Acidobacteria, Nitrospirae, and Planctomycetes as influential phyla positively affecting EPS composition. Spearman correlation analysis pinpointed Robiginitialea (positive correlation) and Fimbriiglobus (negative correlation) as critical genera influencing EPS dynamics. Moreover, EPS-related gene abundance corresponded closely with observed EPS compositional differences. Dominant genes associated with protein biosynthesis included xapD in reservoirs and glnA in bays, while glmS and eno were predominant for polysaccharide biosynthesis in bays. This research advances our understanding of microbial-EPS-MP interactions in urban water systems, offering critical insights into ecological remediation and risk assessment of MP pollution.

Granular sludge (GS) is a special self-aggregation biofilm. Extracellular polymeric substances (EPS) are mainly associated with the architectural structure, rheological behaviour and functional stability of fine granules, given that their significance to the physicochemical features of the biomass catalysing the biological purification process. This review targets the EPS excretion from GS and introduces newly identified EPS components, EPS distribution in different granules, how to effectively extract and recover EPS from granules, key parameters affecting EPS production, and the potential applications of EPS-based biomaterials. GS-based EPS components are highly diverse and a series of new contents are highlighted. Due to high diversity, emerging extraction standards are proposed and recovery process is capturing particular attention. The major components of EPS are found to be polysaccharides and proteins, which manifest a larger diversity of relative abundance, structures, physical and chemical characteristics, leading to the possibility to sustainably recover raw materials. EPS-based biomaterials not only act as alternatives to synthetic polymers in several applications but also figure in innovative industrial/environmental applications, including gel-forming materials for paper industry, biosorbents, cement curing materials, and flame retardant materials. In the upcoming years, it is foreseen that productions of EPS-based biomaterials from renewable origins would make a significant contribution to the advancement of the circular economy.

Phosphorus has been successfully eliminated from wastewater by biological techniques of enhanced biological phosphorus removal (EBPR) process, which relies on a specific microbiota of polyphosphate accumulating organisms (PAOs) that accumulate phosphate as polyphosphates (poly-P). Most methods for quantification of poly-P pools suffer from low accuracy and specificity. More powerful and implementable P-analysis tools are required for poly-P quantification, which will help in improved evaluation of processes in laboratory and full-scale EBPR systems. This study developed two methods to quantify poly-P pools by releasing the poly-P from the cell. During experimental optimization, it was observed that two different methods resulted in the highest phosphate release: acetate addition at a pH of 4.8 and exposure to EDTA solution with a concentration of 1% (w/v). Treatment with EDTA resulted in a higher amount of phosphate release from all sludge samples. This was characterized by P-release of 1.5–2.5 times higher than the control tests. In contrast, treatments with acetate addition at a low pH exhibited that P-release depended upon the types of the sludge samples. The highest P-release amount and rate were found in highly-enriched PAO sludge samples, but with fewer influences on the sludge collected from WWTP, which may be attributed to the lower fraction of PAOs in the sludge. Overall, the proposed approaches to quantify the poly-P concentration can be applied in simple, user-friendly, and cost-effective ways.

Selection of appropriate extraction methods of extracellular polymeric substances (EPSs)is crucial, affecting the yield, composition and the properties of EPSs. However, there is still a lack of standard methods for EPS extraction. In this study, seven methods were compared to extract EPS from anaerobic ammonium oxidation (anammox)granules, and the effectiveness and cell lysis of these methods were evaluated. The heating-Na₂CO₃ based method obtained a higher EPS yield with limited cell lysis. Proteins (PN)and polysaccharides (PS)were predominant in extractable EPSs. Particularly, PN-related substances were substantially enriched and high PN/PS ratios resulted in most methods. Different methods resulted in EPSs with a different size distribution. By analysis of Attenuated total reflectance-Fourier transform infrared spectra (ATR-FTIR), amide- and polysaccharide-associated bands were dominant. ATR-FTIR and fluorescence density of excitation-emission matrix spectra indicated EPS compositions and structures varied with the extraction methods. Moreover, the extraction/recovery protocol based on the heating-Na₂CO₃ method was remarkably improved, by varying heating time and using a centrifugal filter device with membrane (CFDM)to replace concentration and purification steps. Eventually, the thermal method with an extraction time of 45 mins was identified to be most effective. The application of CFDM was demonstrated as an alternative approach to concentrate and purify for EPS recovery. To our best knowledge, this is the first original study to systematically evaluate the different EPS extraction methods from anammox granules.