Expanded global groundwater microbial diversity reveals bioprospecting potential.

Although the terrestrial subsurface harbors a substantial fraction of Earth's microbial biomass, the genomic diversity of groundwater microbiomes and their potential for bioprospecting remain poorly characterized. Here, we recovered 44,320 bacterial and archaeal genomes from in-house and publicly available metagenomic datasets, establishing a large-scale groundwater microbiota catalog (GWMC) spanning 167 phyla, including four candidate phyla and over 12,000 previously uncharacterized species. This unprecedented phylogenetic diversity was accompanied by a bimodal genome size distribution (0.3-12.8 Mbp), revealing divergent strategies of genomic allocation. By mining extensive genomic resources, we found that small genomes prioritized molecular defense and redox regulation, whereas large genomes frequently harbored greater biosynthetic potential. Notably, we establish the largest selenoprotein catalog to date and highlight groundwater as an overlooked hotspot of microbial selenium metabolism. Overall, this work advances our understanding of microbial diversity in aquifers and uncovers underexplored genomic resources with potential for biotechnology and biomedicine.