N2O production and novel nitric oxide reductases influenced by electron donors during denitrification in rivers

N 2 O is a potent greenhouse gas with strong impacts on global warming and ozone layer depletion. The well characterized nitric oxide reductases, cNor and qNor, have known roles in denitrification, while several additional clades—eNor, gNor, nNor, and sNor—have recently been discovered, but their environmental drivers are unknown. In this study, we investigated N 2 O production in eutrophic rivers under different electron donor amendments and in rivers receiving varying nutrient inputs. We also examined the enrichment of nitric oxide reductases across different incubation setups. Iron addition resulted in elevated N 2 O production, due to the combined effects of chemodenitrification and microbial activity involving cNor and qNor. In contrast, thiosulfate addition led to reduced N 2 O levels and enriched eNor, nNor, and sNor along with cNor and qNor. The reduction in N 2 O release was also observed during thiosulfate phases in incubations with alternating additions of acetate and thiosulfate. Under electron donor-limiting conditions, N 2 O accumulated, and eNor and sNor were co-expressed with cNor and qNor. Many organisms frequently associated with these newly identified nitric oxide reductases were from the Burkholderiales order. Overall, their presence and transcriptional activity suggested that previously unrecognized enzymes could contribute to substantial production of N 2 O and shape denitrification outcomes in riverine systems. • Thiosulfate addition resulted in lower N 2 O production with cNor, eNor, and sNor. •Iron induced higher N 2 O, due to chemodentrification and the function of cNor and qNor. •Different NO reductases were enriched and expressed in response to electron donors. •Diverse Burkholderiales populations encoded eNor, nNor, and sNor.