The deep terrestrial subsurface, hundreds of meters to kilometers below the surface, is characterized by oligotrophic conditions, dark and often anoxic settings, with fluctuating pH, salinity, and water availability. Despite this, microbial populations are detected and active, contributing to biogeochemical cycles over geological time. Because it is extremely difficult to access the deep biosphere, little is known about the identity and metabolisms of these communities, although they likely possess unknown pathways and might interfere with deep waste deposits. Therefore, we analyzed rock and groundwater microbial communities from deep, isolated brine aquifers in two regions dating back to the Ordovician and Devonian, using amplicon and whole genome sequencing. We observed significant differences in diversity and community structure between both regions, suggesting an impact of site age and composition. The deep hypersaline groundwater did not contain typical halophilic bacteria, and genomes suggested pathways involved in protein and hydrocarbon degradation, and carbon fixation. We identified mainly one strategy to cope with osmotic stress: compatible solute uptake and biosynthesis. Finally, we detected many bacteriophage families, potentially indicating that bacteria are infected. However, we also found auxiliary metabolic genes in the viral genomes, probably conferring an advantage to the infected hosts.
Keywords: brine; deep terrestrial subsurface; endolithic bacteria; groundwater bacteria; metagenomics.