Decoding dissolved organic matter reorganization and multitrophic regulation of biogeochemical cycling in effluent-dominated hyporheic sediments

Continuous wastewater effluent discharge sustains pronounced redox heterogeneity in the hyporheic zone (HZ), with consequences for dissolved organic matter (DOM) transformation and sediment biogeochemistry. However, the ecological pathways linking redox-dependent DOM transformation to multitrophic organization and coupled nitrogen (N) and sulfur (S) cycling remain poorly understood. We integrated fluorescence and molecular DOM characterization, bacterial and eukaryotic community profiling, metagenomics, ecological network analysis, and structural equation modeling across oxic and anoxic zones. The summed relative contribution of protein-like fluorescence did not differ significantly between zones, while tyrosine-like fluorescence was 31% higher in the oxic zone and the median tryptophan/tyrosine ratio was 52% higher under anoxia. FT-ICR MS analysis showed that formulas positively associated with HIX had higher O/C ratios and a median NOSC of 0.35, whereas negatively associated formulas had a median NOSC of -0.90, linking optical humification to molecular carbon oxidation characteristics. Bacterial genera exhibited broader estimated niche breadths than eukaryotic genera, with median Levins' B values of 7.19 and 5.86, respectively. Under oxic conditions, DOM was associated with basal communities within a connected sequence of bacterial, protozoan, and metazoan associations. Bacterial and algal communities were primarily associated with N and S cycling potentials, respectively. Under anoxia, significant DOM associations with basal groups and the bacteria-to-protozoa association were not detected, while both cycling potentials shifted toward bacteria-centered regulation. N and S cycling potentials remained strongly coordinated, while modeled network robustness was 20.5% lower under anoxia. These findings reveal that redox-associated DOM reorganization shifts multitrophic regulation of coupled N and S cycling, thus providing a framework for integrating DOM composition and multitrophic organization when assessing aquatic interfaces exposed to sustained organic matter loading and redox heterogeneity.