Depth-temperature coupling shapes denitrifier community assembly and metabolic adaptation in drinking water reservoir...

Sediments in freshwater reservoirs serve as dynamic biogeochemical interfaces, regulating nitrogen transformations and greenhouse gas emissions. However, the microbial mechanisms driving these processes under concurrent carbon limitation and climate warming are not fully understood. In this study, sediments from fourteen source drinking water reservoirs across three distinct climatic zones in China were analyzed to investigate the impact of environmental factors on denitrifying microbial communities and their metabolic potential. Temperature and depth together explained 33.4% of the variance in denitrification gene abundance. Proteobacteria dominated the denitrifying assemblages (> 82%), with Burkholderiales, Hyphomicrobiales, and Nitrosomonadales identified as the primary carriers of denitrification genes. Despite having 22.9% lower total organic carbon, sediments from deep-water reservoirs (> 15 m), characterized by thermal stratification, exhibited 1.33-fold higher carbon metabolic activity, reflecting an oligotrophic adaptation to carbon scarcity due to persistent anoxia and limited organic carbon inputs. Depth-driven carbon limitation and anoxia favored oligotrophic and chemolithoautotrophic taxa, which adopted hybrid chemosynthesis-fermentation metabolisms, while shallow sediments supported resource-generalist heterotrophs adapted to oxygen fluctuations. Cold conditions enhanced the abundance of enzyme-encoding genes as a form of thermal compensation, while warming stimulated aerobic diversification and truncated denitrification, thereby elevating the risk of N₂O emissions. Warming also reduced nosZ-type diversity and niche breadth, highlighting the vulnerability of deep sediments to impaired N₂O reduction. These findings underscore that deterministic environmental selection, primarily driven by physical gradients, governs denitrifier community assembly and metabolic plasticity. The results provide mechanistic insights into nitrogen transformation processes and offer a mechanistic framework for mitigating N₂O emissions and improving microbial management in stratified freshwater reservoirs.