Linking Microbial Communities and Molecular Transformations of Dissolved Organic Matter to the Fate of Nitrogen in Karst Aquifers

Abstract Understanding the biogeochemical processes governing carbon and nitrogen cycling in karst aquifers is critical. However, 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 compositions, and microbial communities in three types of aquifer media, karst fissure media (KFM), karst conduit media (KCM), and karst window groundwater (KWG), at the Zengpiyan karst site in southern China, using carbon isotopes, Fourier transform ion cyclotron resonance mass spectrometry (FT‐ICR MS), and amplicon sequencing. Results indicated that longer hydraulic residence time in KFM was associated with enhanced denitrification, whereas shorter residence in KCM/KWG favored nitrification. DOM was dominated by terrestrial lignin‐like molecular formulas (55.92%), but residence time shaped molecular composition and biodegradation: KFM showed lower nominal oxidation state of carbon (NOSC wa , −0.60 ± 0.37) yet a higher end‐member index (I bio , 0.37 ± 0.05), reflecting microbial preferential consumption of labile, high NOSC fractions, leaving recalcitrant lignin‐like molecular formulas. Core taxa Comamonas and an unclassified Comamonadaceae genus were associated with lignin‐like molecular formulas degradation and denitrification, while Nitrospira contributed to nitrification via lignin‐like molecular formula‐derived intermediates. Together they formed a “lignin‐like molecular formulas degradation, ammonification, nitrification” cascade, with 53.82% of DOM degradation reactions involving nitrogen loss through CHON transformations. Overall, extended residence time in karst aquifers enables sequential degradation of recalcitrant lignin‐like molecular formulas under low NOSC conditions via synergistic interactions between microbes, refining conceptual models of C‐N coupling in hydrologically heterogeneous karst systems.