Dissolved organic matter molecular composition links water chemistry, microbial functional potential, and nitrous oxide variability in a temperate estuary.

Estuaries receive substantial carbon and nitrogen inputs and are sources of nitrous oxide (N₂O), but salinity-driven conservative mixing can obscure biogeochemical links between DOM molecular composition and estuarine N₂O accumulation. We integrated ultrahigh-resolution DOM molecular characterization, high-throughput sequencing, and functional gene quantification to examine how water chemistry, DOM molecular composition, and microbial functional potential were associated with N₂O dynamics after accounting for conservative mixing in the Luanhe River Estuary, China. Estuarine waters were consistently supersaturated in N₂O and showed positive deviations from conservative mixing (ΔN₂O > 0), indicating non-conservative N₂O accumulation, especially in summer. Estuarine ΔN₂O variability was associated with a coupled physicochemical-DOM-microbial framework rather than with any single factor: the shared effects of water chemistry, DOM molecular composition, and microbial structure and function explained 75.3% of ΔN₂O variance. Specifically, nitrate availability and low-oxygen conditions provided the dominant physicochemical context for elevated ΔN₂O; DOM composition provided molecular information on substrate quality. Net removal of labile, aliphatic, and nitrogen-containing fractions was positively associated with ΔN₂O, whereas recalcitrant fraction accumulation was negatively associated with ΔN₂O, consistent with declining electron-donor favorability along the salinity gradient. Microbial evidence supported denitrification-related functional potential, including nirK/nirS predominance over AOA/AOB amoA, network-identified keystone genera associated with nitrate and dissolved organic carbon, and DOM-microbe associations linking substrate quality shifts to denitrification-associated taxa. Overall, these results highlight DOM molecular composition as an interface linking estuarine nutrient and redox conditions to microbial functional potential and N₂O variability, supporting the incorporation of DOM quality indices into coastal biogeochemical models.