Groundwater microbial community structure under contrasting hydrological conditions in a sulfate-rich karst aquifer

Karst aquifers represent dynamic subsurface environments where hydrological fluctuations and redox variability can strongly influence groundwater microbiomes and associated biogeochemical processes. However, microbial sulfur cycling in sulfate-rich karst aquifers remains poorly understood, particularly from a genome-resolved perspective that links hydrochemical gradients to microbial metabolic potential. Here, we investigated how hydrochemical heterogeneity and contrasting hydrological conditions relate to microbial community composition and sulfur-cycling potential in the Irecê Basin karst aquifer (Salitre Formation, northeastern Brazil), a sulfur-rich non-hydrothermal karst system. Hydrochemical analyses, 16S rRNA gene sequencing, and genome-resolved metagenomics were integrated to characterize groundwater microbial communities and evaluate relationships between hydrochemical variability and sulfur-related metabolic potential. Samples displayed significant hydrochemical heterogeneity, including remarkable variation in oxidation-reduction potential, dissolved oxygen, sulfate concentrations, and dissolved sulfide accumulation. Amplicon sequencing of 10 samples generated more than 10,000 high-quality reads each and revealed substantial variation in community composition among groundwater samples, with substantial differences among environments and contrasting hydrological conditions, highlighting pronounced spatial and habitat-level heterogeneity. A total of 185 dereplicated medium- and high-quality metagenome-assembled genomes (MAGs) were recovered from three samples, many representing potentially novel microbial lineages. M00596-associated sulfur-transformation modules were detected in MAGs from all three metagenomic samples, whereas complete SOX modules were restricted to the comparatively more oxidizing groundwaters. Several MAGs contained both M00596 and SOX modules, indicating the potential for multiple sulfur-transformation pathways within the same genomes. Overall, these findings indicate that sulfur cycling represents a central component of groundwater biogeochemistry in this sulfate-rich karst aquifer and that hydrochemical heterogeneity is associated with variation in microbial community structure and sulfur-cycling metabolic potential. Together, the combination of sulfur-rich carbonate lithologies, heterogeneous redox conditions, and diverse sulfur-cycling microbial populations highlights the potential importance of sulfur transformations in the ecological and biogeochemical functioning of the Irecê Basin karst aquifer, with possible implications for local geochemical processes.