Metagenomic Analysis Revealed Community-level Metabolic Differences Between Full-scale EBPR and S2EBPR Systems

Side-Stream Enhanced Biological Phosphorus Removal (S2EBPR) has emerged as a promising technology addressing certain challenges of conventional Enhanced Biological Phosphorus Removal (EBPR), notably stability in phosphorus removal, yet the underlying mechanisms are not fully understood. Metagenomic analysis presents a powerful approach to elucidate community-level metabolic differences between EBPR and S2EBPR configurations. In this study, we compared three EBPR and three S2EBPR activated sludge communities using metagenomic analysis at taxonomy, key functional pathways/genes, and polyphosphate-metabolism marker genes. Our analysis revealed larger genus-level diversity variance in S2EBPR communities, indicating distinct microbial community compositions influenced by different operational configurations. A higher diversity index in the S2EBPR than the EBPR was observed, and a higher Ca. Accumulibacter abundance was detected in EBPRs, whereas the fermentative candidate PAOs genera, including Ca. Phosphoribacter and Ca. Promineifilum, were more abundant in S2EBPR systems. EBPR and S2EBPR groups displayed similar gene and pathway abundance patterns related to core metabolisms essential for carbon and nitrogen metabolism. PolyP-metabolism marker gene phylogeny analysis suggested that exopolyphosphatase gene (ppx) showed better distinctions between EBPR and S2EBPR communities than polyphosphate kinase gene (ppk). This also highlighted the needs in fine-cale microdiversity analysis and finding novel Ca. Accumulibacter clades and species as resolved using the ppk gene. These findings provide valuable insights into AS community dynamics and metabolic functionalities, paving the way for further research into optimizing phosphorus removal processes in wastewater treatment systems.