Unveiling the overlooked nitrous oxide reduction enzyme in wastewater treatment systems

Nitrous oxide (N₂O) is a potent greenhouse gas with steadily rising atmospheric concentrations. N₂O reductase (N₂OR), encoded by the nosZ gene, is the key enzyme capable of reducing N₂O to dinitrogen. While the canonical nosZ clades (I and II) have been extensively investigated, the recently proposed clade III (L-nosZ) remains largely uncharacterized in engineered ecosystems. Here, by analyzing 226 metagenomes from globe 114 wastewater treatment plants (WWTPs), we demonstrate that l-nosZ is both widespread and phylogenetically diverse within activated sludge communities. Although its abundance is generally lower than that of clade II nosZ, integrating l-nosZ into emission estimates reduces the predicted microbial N₂O emission potential by up to 12% in some systems. Environmental association analyses show that the distribution and functional contribution of l-nosZ are shaped primarily by sludge retention time and temperature (Rs > 0.2; p < 0.001), and that its response to dissolved oxygen differs from that of canonical nosZ. Comparisons across representative habitats further reveal that WWTPs act as important reservoirs for nosZ genes, whereas l-nosZ exhibits broad environmental occurrence and marked taxonomic conservatism. Collectively, our findings uncover a previously overlooked component of microbial N₂O reduction in WWTPs and highlight the need to incorporate l-nosZ into current emission assessments to improve predictions and inform mitigation strategies in engineered nitrogen-cycling systems.