Hydrological Seasonality Drives DOM–Bacteria Interactions in the Rushan River Basin

To unravel hydrological controls on dissolved organic matter (DOM)–microbe interactions in river ecosystems, this study integrated 3D excitation–emission matrix spectroscopy (3D-EEMs), parallel factor analysis (PARAFAC), and 16S rRNA sequencing to characterize seasonal DOM dynamics and microbial assembly in China’s Rushan River Basin. PARAFAC resolved contrasting DOM signatures between dry (four protein-like, two humic-like components) and wet seasons (three protein-like, three humic-like components). Dry-season DOM was dominated by tyrosine-like substances (58.03%), reflecting microbial degradation and phytoplankton activity, while wet-season DOM showed elevated tryptophan-like components (34.38%) and terrestrial fulvic acids (17.14%), which may be related to rain-driven external inputs. The α -diversity of the microbiota is relatively high in the wet season, mainly consisting of Proteobacteria (34.06–68.10%) and Actinobacteriota (9.15–20.76%). In the dry season community, there are Bacteroidota (14.71–38.45%) and Verrucomicrobiota (6.13–14.32%). The structural equation model (SEM) semi-quantified the comprehensive pathways by which microorganisms inhibit unstable proteins and enhance humification. These results reveal the synergistic regulatory role of hydrological seasonality on DOM and microorganisms, and provide a basis for adaptive water quality management.