Deciphering Sulfur-Based Denitrification in Confined Alluvial-Lacustrine Aquifers through Multi-isotope ( ¹⁵ N, ³⁴ S, ¹³ C, and ¹⁸ O) and Metagenomic Analyses

Sulfur-based denitrification offers unique advantages in nitrogen removal in low-carbon environments. Despite its importance, the prevalence, controlling factors, and microbial mediators of sulfur-based denitrification in aquifers remain largely unknown. By integrating multiple stable isotopes and genome-resolved metagenomics, we investigated the coupling of nitrogen and sulfur cycling along the groundwater flowpath from unconfined to confined alluvial-lacustrine aquifers in the Poyang Lake Plain, China. Isotope (i.e., δ 15 N–NO 3 –, δ 18 O–NO 3 –, δ 34 S–SO 4 2–, δ 18 O–SO 4 2–, δ 18 O–H 2 O, δ 13 C-DIC, and δ 13 C-DOC) fractionation and hydrogeochemical trends indicated a transition from nitrification-dominated processes in the unconfined aquifer to denitrification, sulfide oxidation, and sulfate reduction in the confined aquifer. A significant negative correlation between δ 34 S–SO 4 2– and δ 15 N–NO 3 –, along with a significant positive correlation between genes involved in sulfide oxidation and nitrate reduction, suggested cryptic coupling between sulfide oxidation and nitrate reduction. Sulfur-autotrophic denitrifying metagenome assembly genomes encoding Calvin–Benson–Bassham carbon fixation and rTCA pathways, such as Burkholderiales, Rhodocyclaceae, and Sulfurimonas, were enriched in the confined aquifer and co-occurred with sulfate reducers ( Pseudomonadota and Desulfobacterota ). These results highlighted sulfur cycling consortia when sulfate was reduced to facilitate removal of nitrate via sulfur-based denitrification. These findings reveal the role of cryptic sulfur cycling in sustaining denitrification in aquifers, offering new insights into nitrogen–sulfur interactions in subsurface ecosystems.