Understanding the biogeochemical processes governing carbon (C) and nitrogen (N) cycling in karst aquifers is critical, yet the specific pathways through which dissolved organic matter (DOM) influences nitrogen cycling under varying degrees of aquifer confinement remain poorly understood. This study addresses this knowledge gap by analyzing geochemical characteristics, DOM composition, and microbial communities in three types of aquifer media, fissure groundwater (KFW), conduit groundwater (KCW), and surface water (KSW), at the Zengpiyan karst site in southern China, using carbon isotopes, FT-ICR MS, and amplicon sequencing. Results indicate that hydraulic residence time is the primary factor driving differences in DOM molecular characteristics and biodegradation, with KFW exhibiting conditions favoring denitrification and KCW/KSW supporting nitrification. DOM in karst aquifers is predominantly composed of terrestrial, low NOSC lignin compounds (-0.3 < NOSCwa < -0.1), which degrade in relation to hydraulic retention. Longer residence times enhance degradation of lignin and nitrogenous molecules by key microbial taxa. Potential Molecular Transformation analysis and Spearman correlation network analysis highlight the specific contributions of ASV9 (Comamonas sp.), ASV79 (Nitrospira sp.), and ASV95 (Comamonadaceae sp.) to CHON compound transformation, linking microbial-driven DOM degradation to nitrogen cycling. These findings underscore the interconnected C-N biogeochemical processes in karst aquifers and offer insights into the roles of specific microbial communities in mediating nitrogen transformations, with implications for groundwater remediation in polluted karst systems.

Groundwater (GW)-surface water (SW) interactions in karst areas may have a strong impact on the quantity and quality of the groundwater system. Although knowledge of karst hydrology has improved in recent decades, the interaction patterns of GW-SW, and understanding of the hyporheic zone (HZ) on improving or deteriorating groundwater remains very limited. Here, we document HZ in a karst basin through study of hydrological, hydrochemical, and biological processes. The depths of some sinkholes or karst windows in the karst plain are more than 100 m, which are dozens of meters lower than the river elevations. The HZ is not limited to the riverbed, but extends into aquifer along the karst conduits. And their interaction patterns are not limited to the mixing GW and SW, but also include mixing of groundwater with other water bodies. Due to the existence of karst conduits, the types of HZ and the dynamic process of hyporheic flow are unique within karst aquifers. We defined these generally in karst groundwater systems dominated by conduits as karst cave hyporheic zones (KCHZ), with the meaning of the place or area where conduit flow interacts with other types of water bodies. The KCHZ was further classified into four types. Research on the five springs in the study area showed the formation of KCHZ is related to the karst development and the hydrogeochemical gradient of water environment. Once the quality of one type of water deteriorates, or the amount of water decreases, the function of hyporheic zone will degenerate.