Abstract
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.