Phylogenetic and metabolic diversity of microbial communities performing anaerobic ammonium and methane oxidations under different nitrogen loadings.

The microbial guild coupling anammox and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) is an innovative process to achieve energy-efficient nitrogen removal with the beneficial use of methane in biogas or in anaerobically treated wastewater. Here, metagenomics and metatranscriptomics were used to reveal the microbial ecology of two biofilm systems, which incorporate anammox and n-DAMO for high-level nitrogen removal in low-strength domestic sewage and high-strength sidestream wastewater, respectively. We find that different nitrogen loadings (i.e., 0.1 vs. 1.0 kg N/m3/d) lead to different combinations of anammox bacteria and anaerobic methanotrophs ("Candidatus Methanoperedens" and "Candidatus Methylomirabilis"), which play primary roles for carbon and nitrogen transformations therein. Despite methane being the only exogenous organic carbon supplied, heterotrophic populations (e.g., Verrucomicrobiota and Bacteroidota) co-exist and actively perform partial denitrification or dissimilatory nitrate reduction to ammonium (DNRA), likely using organic intermediates from the breakdown of methane and biomass as carbon sources. More importantly, two novel genomes belonging to "Ca. Methylomirabilis" are recovered, while one surprisingly expresses nitrate reductases, which we designate as "Ca. Methylomirabilis nitratireducens" representing its inferred capability in performing nitrate-dependent anaerobic methane oxidation. This finding not only suggests a previously neglected possibility of "Ca. Methylomirabilis" bacteria in performing methane-dependent nitrate reduction, and also challenges the previous understanding that the methane-dependent complete denitrification from nitrate to dinitrogen gas is carried out by the consortium of bacteria and archaea.