Dissolved inorganic carbon supports robust anabolism and methanogenesis in actively serpentinizing rocks.

Serpentinites, hydrated ultramafic rocks that produce [hyper]alkaline, reducing, H2-rich groundwaters, host subsurface microbial ecosystems. Though in the presence of enormous reducing power, life in serpentinizing systems is limited by oxidant and carbon availability. The forms of carbon that support the serpentinite-hosted microbiome, and their rates of biological assimilation, remain poorly understood. In this work, we quantify the habitability of subsurface environments shaped by serpentinization and examine the forms of carbon that support their microbial constituents, focusing specifically on dissolved inorganic carbon, acetate, and formate. We access reacted groundwater from Earth's largest terrestrial serpentinizing body and measure carbon assimilation at the single-cell level. Across all conditions, we consistently observe robust assimilation of dissolved inorganic carbon into microbial biomass. Notably, we find that dissolved inorganic carbon supports the majority of methanogenic activity in the system, even at hyperalkaline conditions (pH > 11). Inferred bioenergetic fluxes suggest that rates of biological hydrogen-consumption and methanogenesis are relevant at the landscape scale. We identify a strong potential for the microbiome to be stimulated by increases in H and CO, a finding with implications for the search for life on other planetary bodies and for the growing deployment of fluid injection technologies in ultramafic rocks, such as geological hydrogen production or carbon mineralization.