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 μmax/Ks. Thus, the specific growth rate is likely a key factor determining the composition of communities living on N2O respiration under growth-limited conditions.

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Nitrous oxide (N O) reducing microorganisms may be key in the mitigation of N O emissions from managed ecosystems. However, there is still no clear understanding of the physiological and bioenergetic implications of microorganisms possessing either of the two N O 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 ) for N O. We compared N O reducing communities with different nosZI/nosZII ratios selected in chemostat enrichment cultures, inoculated with activated sludge, fed with N O 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 N O respiration pathway in nosZ clade II systems, as reflected in the growth yield per mole of substrate, or (ii) a higher affinity for N O, defined by μ /K , in organisms with nosZ clade II.

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N O is a potent greenhouse gas, but also a potent electron acceptor. In search of thermodynamically favourable - yet undescribed - metabolic pathways involving N O reduction, we set up a continuous microbial enrichment, inoculated with activated sludge, fed with N O 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 N O directly as source of nitrogen for cell synthesis. Instead, we obtained a culture dominated by microorganisms of the Rhodocyclaceae family growing by N O reduction to N coupled to N fixation. Biomass yields of this culture were 40% lower than those of a previously reported culture grown under comparable conditions but with an NH4+-amended medium, as expected from the extra energy expense of N fixation. Interestingly, we found no significant difference in yields whether N O or acetate was the growth-limiting substrate in the chemostat in contrast to the study with NH4+-amended medium, in which biomass yields were roughly 30% lower during acetate limiting conditions.