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

Kazumi Suda¹Toshikazu Suenaga^2*Soichiro Matsuzaki¹Shohei Riya¹Kento Ishii³Manami Nomachi³Hirotsugu Fujitani¹Satoshi Tsuneda³Kartik Chandran⁴Akihiko Terada^5*

• ¹Tokyo Noko Daigaku Kogakubu Daigakuin Kogakufu, Koganei, Japan
• ²Hiroshima Daigaku, Higashihiroshima, Japan
• ³Waseda Daigaku, Shinjuku, Japan
• ⁴Columbia University, New York, United States
• ⁵Tokyo University of Agriculture and Technology, Fuchu, Japan

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, 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 N2O reductase genes, i.e., clades I, II, and III nosZ. 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.