Thermal stratification-mediated DOM transformation shapes the vertical coupling-to-decoupling of carbon and nitrogen in an oxic alpine reservoir.

Owing to depth and distinctive geography, alpine reservoirs develop persistent seasonal thermal stratification that reshapes habitats and shapes carbon (C) and nitrogen (N) cycling, yet the fine-scale vertical mechanisms of C-N coupling remain unresolved. Combining FT-ICR MS, dual N-O isotopes, and multi-omic microbial analyses, we resolved biogeochemical dynamics across a full stratification cycle, targeting its stable phase to decipher C-N coupling and decoupling. In the epilimnion, intense radiation and full oxygenation sustained active dissolved organic matter (DOM) transformation; its high bioavailability, rather than hypoxia, appeared to relieve heterotrophic carbon limitation and accelerate mineralization, supporting ammonia-oxidizing archaea (AOA) nitrification. Incomplete denitrification potential and oxygen suppression of nosZ favored N2O accumulation via nitrification by-products, reflecting labile-carbon-associated C-N coupling. In the metalimnion, density barriers intercepted settling particulates and promoted reductive DOM transformation. Resulting substrate heterogeneity fostered niche differentiation between autotrophic (amoA) and heterotrophic (nirS) groups, co-enriching nitrification and denitrification potential and defining this layer as the biogeochemical hotspot. In the hypolimnion, prolonged isolation forged an oligotrophic, oxic habitat where carbon deprivation and oxygenation impeded denitrification while favoring chemolithoautotrophic AOA. Carbon-fixation potential co-occurred with autochthonous reduced lipids, implicating endogenous organic carbon via the microbial carbon pump (MCP). Largely independent of labile carbon, ammonia oxidation alongside stagnant denitrification coincided with nitrate accumulation, manifesting C-N decoupling. This study establishes a vertical framework of epilimnetic coupling, metalimnetic transition, and hypolimnetic decoupling, offering new insights into element cycling and greenhouse-gas management under global warming.