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.