N₂O is a potent greenhouse gas, but also a potent electron acceptor. In search of thermodynamically favourable – yet undescribed – metabolic pathways involving N₂O reduction, we set up a continuous microbial enrichment, inoculated with activated sludge, fed with N₂O as the sole electron acceptor and acetate as an electron donor. A nitrogen-free mineral medium was used with the intention of creating a selective pressure towards organisms that would use N₂O directly as source of nitrogen for cell synthesis. Instead, we obtained a culture dominated by microorganisms of the Rhodocyclaceae family growing by N₂O reduction to N₂ coupled to N₂ fixation. Biomass yields of this culture were 40% lower than those of a previously reported culture grown under comparable conditions but with an NH⁺ ₄ -amended medium, as expected from the extra energy expense of N₂ fixation. Interestingly, we found no significant difference in yields whether N₂O or acetate was the growth-limiting substrate in the chemostat in contrast to the study with NH⁺ ₄-amended medium, in which biomass yields were roughly 30% lower during acetate limiting conditions.