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 involved in phosphorus and nitrogen cycling, and the interplay between relevant metabolic pathways carried out by these key organisms. 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 in the conventional A2O mode under low dissolved oxygen (DO) conditions. Among the recovered metagenome-assembled genomes (MAGs), >90 MAGs from each community were high-quality, including 39 and 30 MAGs that were close-circularized, from the floc and biofilm communities, respectively, with no or minimal contamination. Some Candidatus Accumulibacter strains encoded for either full or partial denitrification. Ca. Accumulibacter were very efficient in aerobic and anoxic inorganic phosphorus (Pi) uptake, while the second highly enriched Ca. Accumulibacter was as competitive for denitrification metabolism. The potential of nitrous oxide (N2O) emissions in both the floc and biofilm communities was nearly 20 times higher in the aerobic zone than in the anoxic zone. As opposed to our initial hypothesis that slow growers will mostly reside in biofilms, the expression of ammonium monooxygenase (amoABC) was higher in flocs than in biofilm communities.