Assimilatory N2O reduction by Nostoc sp. strain MS1 isolated from a river: insights from genome and 15N tracer analysis

Direct evidence for the assimilation of nitrous oxide (N₂O), a potent greenhouse gas, by freshwater cyanobacteria has been lacking. Here, we report a cyanobacterium, isolated from a nitrogen-polluted river, that fixes N 2 O via dinitrogen (N 2 ) gas by nitrogenase activity. N 2 O-reducing bacteria were enriched from river samples, under alternating light/dark conditions in the presence of atmospheric N 2 and the absence of oxygen (O 2 ), followed by isolation using fluorescence-activated cell sorting. The isolated strain, Nostoc sp. strain MS1 (NIES-4466), consists of moniliform coccoid cells and is phylogenetically affiliated with the genus Nostoc . A high-quality draft genome of strain MS1 revealed the presence of nitrogenase genes encoding the MoFe protein but the absence of N 2 O reductase genes, i.e., clades I, II, and III nosZ . When incubated in a He (95%)/CO 2 (5%) atmosphere with 0.01% 15 N-labeled N 2 O, the cells exhibited elevated 15 N content relative to natural abundance (0.36%). The degree of 15 N incorporation positively correlated with ethylene production from acetylene, implicating nitrogenase in N 2 O assimilation by strain MS1. While replacing He with N 2 reduced N 2 O uptake, likely due to substrate competition, N 2 O consumption activity persisted, suggesting that freshwater cyanobacteria can function as an N 2 O sink. These findings, supported by genomic and 15 N tracer analyses, highlight the previously unrecognized role of cyanobacteria in mitigating N₂O emissions in freshwater environments.