The Response Mechanism of Microbial Community Characteristics and Nitrogen Cycle Function in River Hyporheic Zones to Nitrate Pollution

Excessive nitrate pollution poses a significant threat to aquatic ecosystems, particularly in river hyporheic zones where microbially mediated nitrogen cycling critically governs water quality. In this study, we integrated metagenomic analysis and indoor soil column simulations to investigate microbial responses and nitrogen-cycling regulatory mechanisms across four nitrate concentrations and three river hyporheic zone depths. The results demonstrated that exogenous nitrate input reduced microbial diversity and altered the abundance of nitrogen-cycling functional genes, with changes primarily occurring in the shallow and middle layers. Under low-nitrate concentrations, the community possesses multiple nitrogen transformation potentials, such as nitrogen fixation, nitrification, and assimilatory nitrate reduction to ammonium (ANRA), which results in the accumulation of ammonium nitrogen (NH 4 + –N) in the deep layer. As nitrate levels increased, denitrifying taxa such as Thauera and Dechloromonas became enriched, accompanied by elevated abundances of denitrification genes ( norB and norC ) and dissimilatory nitrate reduction to ammonium (DNRA) genes ( nirB and nirD ). The nitrate concentration, dissolved oxygen (DO), and redox potential (Eh) emerged as the key environmental drivers shaping microbial community composition and functional gene distribution. These findings provide critical insights for nitrogen pollution mitigation and river water quality management.