Unveiling Metabolic Insights and Niche Differentiation of Microbial Communities in EBPR-Anammox Reactor through Integration of Long-Read Metagenomics and Metatranscriptomics

In this manuscript, we report niche differentiation among important groups of organisms including polyphosphate accumulating organisms, anaerobic ammonium oxidizers, aerobic ammonium oxidizers, and the interplay between relevant metabolic pathways. Microbes are a vital part of biological wastewater treatment systems in engineered reactors. Although, engineering data incorporating typical water quality parameters could tell the role and engagement of specific microbial groups in bioreactor performance, this data does not inform about the potential interplay between different groups. We employed nanopore-based long-read and Illumina-based short-read sequencing techniques for metagenomics and metatranscriptomics, respectively on samples collected from an integrated fixed film activated sludge (IFAS) bioreactor run the conventional A2O mode. We recovered 130 metagenome-assembled genomes (MAGs) from the floc community and 123 MAGs from the biofilm community. Among them, 89 MAGs from each community were high-quality, including 37 and 26 MAGs were close-circularized, from the floc and biofilm communities, respectively. With these high-quality MAGs and metatranscriptomic data, we revealed niche differentiation among the Candidatus Accumulibacter strains, some encoded for either full or partial denitrification. We found that the most enriched Ca. Accumulibacter was very efficient in aerobic inorganic phosphorus (Pi) uptake, while the second highly enriched Ca. Accumulibacter was as competitive for denitrification metabolism. This study also provided valuable insights into the contributions of biofilm communities to denitrification processes, even during the aerobic phase. Additionally, our findings provided evidence of potential reductions in nitrous oxide (N2O) emissions in both the floc and biofilm communities. This study also sheds further light on the core metabolic pathways of anammox bacteria and the mechanisms underlying N2O emissions in ammonium-oxidizing bacteria.