Although nitrogen removal by partial nitritation and anammox is more cost-effective than conventional nitrification and denitrification, one downside is the production and accumulation of nitrous oxide (N₂O). The potential exploitation of N₂O-reducing bacteria, which are resident members of anammox microbial communities, for N₂O mitigation would require more knowledge of their ecophysiology. This study investigated the phylogeny of resident N₂O-reducing bacteria in an anammox microbial community and quantified individually the processes of N₂O production and N₂O consumption. An up-flow column-bed anammox reactor, fed with NH₄⁺ and NO₂^- and devoid of oxygen, emitted N₂O at an average conversion ratio (produced N₂O: influent nitrogen) of 0.284%. Transcriptionally active and highly abundant nosZ genes in the reactor biomass belonged to the Burkholderiaceae (clade I type) and Chloroflexus genera (clade II type). Meanwhile, less abundant but actively transcribing nosZ strains were detected in the genera Rhodoferax, Azospirillum, Lautropia, and Bdellovibrio and likely act as an N₂O sink. A novel ¹⁵N tracer method was adapted to individually quantify N₂O production and N₂O consumption rates. The estimated true N₂O production rate and true N₂O consumption rate were 3.98 ± 0.15 and 3.03 ± 0.18 mg_N·g_VSS^-1·day^-1, respectively. The N₂O consumption rate could be increased by 51% (4.57 ± 0.51 mg_N·g_VSS^-1·day^-1) with elevated N₂O concentrations but kept comparable irrespective of the presence or absence of NO₂^-. Collectively, the approach allowed the quantification of N₂O-reducing activity and the identification of transcriptionally active N₂O reducers that may constitute as an N₂O sink in anammox-based processes.

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