Temperature differentially regulates estuarine microbial N2O production along a salinity gradient

•Divergent thermal response patterns for N2O production were found between nearshore/offshore regions.•Temperature and salinity co-regulate N2O production by AOB via nitrifier denitrification pathway.•Temperature regulate sources of N2O production in estuarine water. 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 (Topt) 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. [Display omitted]