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 N₂O via dinitrogen (N₂) gas by nitrogenase activity. N₂O-reducing bacteria were enriched from river samples, under alternating light/dark conditions in the presence of atmospheric N₂ and the absence of oxygen (O₂), followed by isolation using fluorescence-activated cell sorting. The isolated strain, <i>Nostoc</i> sp. strain MS1 (NIES-4466), consists of moniliform coccoid cells and is phylogenetically affiliated with the genus <i>Nostoc</i>. A high-quality draft genome of strain MS1 revealed the presence of nitrogenase genes encoding the MoFe protein but the absence of N₂O reductase genes, i.e., clades I, II, and III <i>nosZ</i>. When incubated in a He (95%)/CO₂ (5%) atmosphere with 0.01% ¹⁵N-labeled N₂O, the cells exhibited elevated ¹⁵N content relative to natural abundance (0.36%). The degree of ¹⁵N incorporation positively correlated with ethylene production from acetylene, implicating nitrogenase in N₂O assimilation by strain MS1. While replacing He with N₂ reduced N₂O uptake, likely due to substrate competition, N₂O consumption activity persisted, suggesting that freshwater cyanobacteria can function as an N₂O sink. These findings, supported by genomic and ¹⁵N tracer analyses, highlight the previously unrecognized role of cyanobacteria in mitigating N₂O emissions in freshwater environments.