Temperature differentially regulates estuarine microbial N₂O production along a salinity gradient

Nitrous oxide (N₂O) is atmospheric trace gas that contributes to climate change and affects stratospheric and ground-level ozone concentrations. Ammonia oxidizers and denitrifiers contribute to N₂O emissions in estuarine waters. However, as an important climate factor, how temperature regulates microbial N₂O production in estuarine water remains unclear. Here, we have employed stable isotope labeling techniques to demonstrate that the N₂O production in estuarine waters exhibited differential thermal response patterns between nearshore and offshore regions. The optimal temperatures (T_opt) for N₂O production rates (N₂OR) were higher at nearshore than offshore sites. ¹⁵N-labeled nitrite (¹⁵NO₂^-) experiments revealed that at the nearshore sites dominated by ammonia-oxidizing bacteria (AOB), the thermal tolerance of ¹⁵N-N₂OR increases with increasing salinity, suggesting that N₂O production by AOB-driven nitrifier denitrification may be co-regulated by temperature and salinity. Metatranscriptomic and metagenomic analyses of enriched water samples revealed that the denitrification pathway of AOB is the primary source of N₂O, while clade II N₂O-reducers dominated N₂O consumption. Temperature regulated the expression patterns of nitrite reductase (nirK) and nitrous oxide reductase (nosZ) genes from different sources, thereby influencing N₂O emissions in the system. Our findings contribute to understanding the sources of N₂O in estuarine waters and their response to global warming.