Low salinity and weak hypoxia interactively amplify N₂O production in nitrogen-enriched estuarine ecosystems

Human activities and climate change are intensifying nitrogen loading, hypoxia, and salinity intrusion in estuarine systems, with important implications for nitrous oxide (N₂O) emissions. However, the interactive effects of these co-occurring stressors on N₂O production remain poorly understood. Here, we combined ¹⁵N-¹⁸O tracing and molecular analyses to investigate N₂O production pathways and their microbial regulation in estuarine sediment based on a series of nitrogen-oxygen-salinity incubation treatments and field observations. Results showed that low salinity under weak hypoxia significantly increased the abundance of nirS and norB genes, indicating an enhanced potential for nitrite reduction to N₂O, which in turn promoted N₂O production, whereas high salinity mitigated this effect by suppressing Pseudomonas abundance. Nitrate input further stimulated denitrification, amplifying N₂O production under weak hypoxia treatments. Heterotrophic denitrification was the major N₂O production pathway overall, while under severe hypoxia, the contribution of nitrifier denitrification increased and could reach 65.57%. Elevated oxygen promoted complete nitrification, thereby reducing the contribution of the nitrifier nitrification and nitrifier denitrification pathways. In contrast, elevated salinity enhanced nitrifier denitrification and nitrification-coupled denitrification, associated with increased abundance of Nitrosomonas and Nitrospina. Notably, the low salinity and weak hypoxia zone emerged as a hotspot of N₂O production, with a doubling of nitrogen input increasing production rates by approximately 78%. Overall, these findings demonstrate that nitrogen pollution, oxygen depletion, and salinity shifts interactively regulate N₂O production in estuarine sediments, highlighting high-risk conditions for N₂O emissions and providing insights for mitigating greenhouse gas emissions in human-impacted estuaries.