Abstract
N(2)O-reducing bacteria have been examined and harnessed to develop technologies that reduce the emission of N(2)O, 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 N(2)O reducers in an anammox reactor. We hypothesized that N(2)O is limiting for N(2)O-reducing bacteria and an exogeneous N(2)O supply enriches as-yet-uncultured N(2)O-reducing bacteria. We conducted a 1200-day incubation of N(2)O-reducing bacteria in an anammox consortium using gas-permeable membrane biofilm reactors (MBfRs), which efficiently supply N(2)O in a bubbleless form directly to a biofilm grown on a gas-permeable membrane. A (15)N tracer test indicated that the supply of N(2)O resulted in an enriched biomass with a higher N(2)O sink potential. Quantitative PCR and 16S rRNA amplicon sequencing revealed Clade II nosZ type-carrying N(2)O-reducing bacteria as protagonists of N(2)O 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 N(2)O 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 N(2)O reducers. We identified Dehalococcoidia and Clostridia as predominant N(2)O sinks in an anammox consortium fed exogenous N(2)O because of the higher metabolic potential of vitamin B12-dependent biosynthesis.