Selective enrichment of high-affinity clade II N2O-reducers in a mixed culture

Microorganisms encoding for the N2O reductase (NosZ) are the only known biological sink of the potent greenhouse gas N2O, and are central to global N2O mitigation efforts. Yet, the ecological constraints selecting for different N2O-reducers strains and controlling the assembly of N2O-respiring communities remain largely unknown. Of particular biotechnological interest are clade II NosZ populations, which usually feature high N2O affinities and often lack other denitrification genes. Two planktonic N2O-respiring mixed cultures were enriched under limiting and excess dissolved N2O availability to assess the impact of substrate affinity and N2O cytotoxicity, respectively. Genome-resolved metaproteomics was used to infer the metabolism of the enriched populations. We show that clade II N2O-reducers outcompete clade I affiliates for N2O at sufficiently low sludge dilution rates (0.006 h-1), a scenario previously only theorized based on pure-cultures. Under N2O limitation, all enriched N2O reducers encoded and expressed only clade II NosZ, while also possessing other denitrification genes. Two Azonexus and Thauera genera affiliates dominated the culture. We explain their coexistence with the genome-inferred metabolic exchange of cobalamin intermediates. Conversely, under excess N2O, clade I and II populations coexisted. Notably, the single dominant N2O reducer (genus Azonexus ) expressed most cobalamin biosynthesis marker genes, likely to contrast the continuous cobalamin inactivation by dissolved cytotoxic N2O concentrations (400 μM). Ultimately, we demonstrate that the solids dilution rate controls the selection among NosZ clades, albeit the conditions selecting for genomes possessing the sole nosZ remain elusive. Additionally, we suggest the significance of N2O-cobalamin interactions in shaping the composition of N2O-respiring microbiomes. ### Competing Interest Statement The authors have declared no competing interest.