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. Clade II NosZ populations are of particular biotechnological interest as they usually feature high N₂O affinities and often lack other denitrification genes. We focus on the yet-unresolved ecological constraints selecting for different N₂O-reducers strains and controlling the assembly of N₂O-respiring communities. Two planktonic N₂O-respiring mixed cultures were enriched at low dilution rates 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. Under N₂O limitation, clade II N₂O-reducers fully outcompeted clade I affiliates, a scenario previously only theorized based on pure-cultures. All enriched N₂O-reducers encoded and expressed the sole clade II NosZ, while also possessing other denitrification genes. Two Azonexus and Thauera genera affiliates dominated the culture, and we hypothesize their coexistence to be explained by the genome-inferred metabolic exchange of cobalamin intermediates. Under excess N₂O, clade I and II populations coexisted; yet, proteomic evidence suggests that clade II affiliates respired most of the N₂O, de facto outcompeting clade I affiliates. The single dominant N₂O-reducer (genus Azonexus) notably expressed most cobalamin biosynthesis marker genes, likely to contrast the continuous cobalamin inactivation by dissolved cytotoxic N₂O concentrations (400 μM). Ultimately, our results strongly suggest the solids dilution rate to play a pivotal role in controlling the selection among NosZ clades, albeit the conditions selecting for genomes possessing the sole nosZ remain elusive. We furthermore highlight the potential significance of N₂O-cobalamin interactions in shaping the composition of N₂O-respiring microbiomes.