Quest for Nitrous Oxide-reducing Bacteria Present in an Anammox Biofilm Fed with Nitrous Oxide.

N₂O-reducing bacteria have been examined and harnessed to develop technologies that reduce the emission of N₂O, a greenhouse gas produced by biological nitrogen removal. Recent investigations using omics and physiological activity approaches have revealed the ecophysiologies of these bacteria during nitrogen removal. Nevertheless, their involvement in‍ ‍anammox processes remain unclear. Therefore, the present study investigated the identity, genetic potential, and activity‍ ‍of N₂O reducers in an anammox reactor. We hypothesized that N₂O is limiting for N₂O-reducing bacteria‍ ‍and an‍ ‍exogeneous N₂O supply enriches as-yet-uncultured N₂O-reducing bacteria. We conducted a 1200-day incubation of N₂O-reducing bacteria in an anammox consortium using gas-permeable membrane biofilm reactors (MBfRs), which efficiently supply N₂O in a bubbleless form directly to a biofilm grown on a gas-permeable membrane. A ¹⁵N tracer test indicated that the supply of N₂O resulted in an enriched biomass with a higher N₂O sink potential. Quantitative PCR and 16S rRNA amplicon sequencing revealed Clade II nosZ type-carrying N₂O-reducing bacteria as protagonists of N₂O sinks. Shotgun metagenomics showed the genetic potentials of the predominant Clade II nosZ-carrying bacteria, Anaerolineae and Ignavibacteria in MBfRs. Gemmatimonadota and non-anammox Planctomycetota increased their abundance in MBfRs despite their overall lower abundance. The implication of N₂O as an inhibitory compound scavenging vitamin B12, which is essential for the synthesis of methionine, suggested its limited suppressive effect on the growth of B12-dependent bacteria, including N₂O reducers. We identified Dehalococcoidia and Clostridia as predominant N₂O sinks in an anammox consortium fed exogenous N₂O because of the higher metabolic potential of vitamin B12-dependent biosynthesis.