Reduction of the greenhouse gas N₂O to N₂ is a trait among denitrifying and non-denitrifying microorganisms having an N₂O reductase, encoded by nosZ. The nosZ phylogeny has two major clades, I and II, and physiological differences among organisms within the clades may affect N₂O emissions from ecosystems. To increase our understanding of the ecophysiology of N₂O reducers, we determined the thermodynamic growth efficiency of N₂O reduction and the selection of N₂O reducers under N₂O- or acetate-limiting conditions in a continuous culture enriched from a natural community with N₂O as electron acceptor and acetate as electron donor. The biomass yields were higher during N₂O limitation, irrespective of dilution rate and community composition. The former was corroborated in a continuous culture of Pseudomonas stutzeri and was potentially due to cytotoxic effects of surplus N₂O. Denitrifiers were favored over non-denitrifying N₂O reducers under all conditions and Proteobacteria harboring clade I nosZ dominated. The abundance of nosZ clade II increased when allowing for lower growth rates, but bacteria with nosZ clade I had a higher affinity for N₂O, as defined by μ_max/K_s. Thus, the specific growth rate is likely a key factor determining the composition of communities living on N₂O respiration under growth-limited conditions.

Nitrous oxide (N₂O) reducing microorganisms may be key in the mitigation of N₂O emissions from managed ecosystems. However, there is still no clear understanding of the physiological and bioenergetic implications of microorganisms possessing either of the two N₂O reductase genes (nosZ), clade I and the more recently described clade II type nosZ. It has been suggested that organisms with nosZ clade II have higher growth yields and a lower affinity constant (K_s) for N₂O. We compared N₂O reducing communities with different nosZI/nosZII ratios selected in chemostat enrichment cultures, inoculated with activated sludge, fed with N₂O as a sole electron acceptor and growth limiting factor and acetate as electron donor. From the sequencing of the 16S rRNA gene, FISH and quantitative PCR of nosZ and nir genes, we concluded that betaproteobacterial denitrifying organisms dominated the enrichments with members within the family Rhodocyclaceae being highly abundant. When comparing cultures with different nosZI/nosZII ratios, we did not find support for (i) a more energy conserving N₂O respiration pathway in nosZ clade II systems, as reflected in the growth yield per mole of substrate, or (ii) a higher affinity for N₂O, defined by μ_max/K_s, in organisms with nosZ clade II.