Humic-like dissolved organic matter as a potential driver of microbial denitrification in carbon-limited groundwater

Groundwater nitrate pollution is increasingly severe, creating a significant stoichiometric imbalance between carbon and nitrogen. Yet, how dissolved organic carbon-to-nitrate ratios (DOC:NO₃⁻) regulate dissolved organic matter (DOM) composition, microbial community structure, and nitrogen transformation remains unclear, especially in oligotrophic aquifers with scarce carbon sources. By integrating Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and high-throughput quantitative PCR (HT-qPCR), we elucidated the effects of DOC:NO₃⁻ on DOM composition and nitrogen cycling. Our results revealed that extreme carbon limitation in the LDN group (low dissolved organic carbon-to-nitrate ratio, DOC:NO₃⁻ < 0.1) promoted the selective preservation and accumulation of humic-like DOM. The LDN group contained more unique molecular formulas than the HDN group (0.1 ≤ DOC:NO₃⁻ < 1.0) (1194 vs 848), predominantly distributed in the highly unsaturated structures with high oxygen region, suggesting greater humification and structural complexity under persistent carbon limitation. Moreover, the microbial co-occurrence network in the LDN group showed higher connectivity with more positive associations than that in the HDN group, implying greater reliance on metabolic cross-feeding to overcome energy constraints. Notably, denitrification genes were 1.98 times more abundant in the LDN group than in the HDN group (P < 0.05), suggesting a robust potential for nitrogen removal despite the low DOC availability. Partial least squares structural equation modeling (PLS-SEM) further identified DOM composition and microbial community structure as key drivers of nitrogen reduction functions. These findings challenge the conventional view that only labile carbon supports denitrification, demonstrating instead that humic-like DOM may serve as a persistent carbon reservoir and potential redox mediator to sustain nitrogen cycling in carbon-limited groundwater. This study provides a new mechanistic perspective for nitrate pollution control and the management of deep subsurface ecosystems.