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 (Ks) 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 μmax/Ks, in organisms with nosZ clade II.