Nitrous oxide (N₂O) is a potent greenhouse gas, which has long been thought to be only consumed by the last step of denitrification in anoxic environments. Since the last decade, N₂O assimilation into diazotrophic biomass in oxic environments has been reported in the marine and a freshwater system, representing a novel N₂O sink. However, there’s a lack of understanding of the regulating factors for this new biological N₂O consumption pathway, despite the fact that assimilation of N₂O is thermodynamically more favorable over dinitrogen (N₂) fixation. Here, we used isotopic tracing experiments to detect the N₂O assimilation in cultured diazotrophs, Crocosphaera (WH8501) and Trichodesmium (IMS101), and water samples from a eutrophic estuary. Results showed that only under elevated [N₂O]/[N₂] conditions (0.0005 to 0.01) can N₂O assimilation be detected on cultured diazotrophs, with specific rates from 1.27 × 10^-4 to 2.00 × 10^-4 h^-1. The eutrophic estuarine water samples lacking active N₂ fixation were not detected for N₂O assimilation. A competitive substrate kinetic model calibrated by experimental data was used to quantify the rate ratios of N₂O assimilation and N₂ fixation in varying [N₂O]/[N₂] conditions. It also well characterizes the N₂O assimilation and N₂ fixation rate ratios reported in former studies. The model explains the absence of N₂O assimilation in natural [N₂O]/[N₂] ratios and enhances insights among present and previous studies on the mechanism of this aerobic biological N₂O sink, which will assist in improving the N₂O flux characterization for aquatic ecosystems.

nitrous oxide, nitrogen fixation, diazotroph