loading . . . Deciphering Sulfur-Based Denitrification in Confined Alluvial-Lacustrine Aquifers through Multi-isotope (15N, 34S, 13C, and 18O) 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., Ī“15NāNO3ā, Ī“18OāNO3ā, Ī“34SāSO42ā, Ī“18OāSO42ā, Ī“18OāH2O, Ī“13C-DIC, and Ī“13C-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 Ī“34SāSO42ā and Ī“15NāNO3ā, 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 CalvināBensonāBassham 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 nitrogenāsulfur interactions in subsurface ecosystems. https://pubs.acs.org/doi/10.1021/acs.est.5c09851?utm_source=SendGrid_ealert&utm_medium=ealert&utm_campaign=TOC_esthag_v60_i4
