Old but not ancient: Rock-leached organic carbon drives groundwater microbiomes.

More than 90% of earth's microbial biomass resides in the continental subsurface, where sedimentary rocks provide the largest source of organic carbon (C). While many studies indicate microbial utilization of fossil C sources, the extent to which rock-organic C is driving microbial activities in aquifers remains largely unknown. Here we incubated oxic and anoxic groundwater with crushed carbonate rocks from the host aquifer and an outcrop rock of the unsaturated zone characterized by higher organic C content, and compared the natural abundance of radiocarbon (C) of available C pools and microbial biomarkers. The ancient rocks surprisingly released organic substances with up to 72.6 ± 0.3% modern C into the groundwater, suggesting leachable fresh organic material from surface transport was preserved within rock fractures. Over half of the rock-leached compounds were also found in the original groundwater dissolved organic carbon (DOC), indicating in situ release of material stored in rock fractures through weathering processes. In addition to aliphatic and aromatic hydrocarbons, rock-leachates were rich in lipids, peptides, and carbohydrates. Radiocarbon analysis of phospholipid-derived fatty acids showed a rapid microbial response to this 'younger' organic material, comprising up to 31% (anoxic) and 51% (oxic) of their biomass C from the rock-leachate after 18 days of incubation. Predictive functional profiling of rock-enriched taxa, including species of Desulfosporosinus, Ferribacterium and Rhodoferax, also suggested metabolic potential for aliphatic and aromatic hydrocarbon degradation. PLFAs of the original groundwater were highly C-depleted, indicating utilization of a mixture of fossil and 'younger' C sources. Our findings suggest that carbonate rocks act as temporal sink for 'younger' organic matter, that leaches with fossil hydrocarbons from sedimentary rocks, driving microbial metabolism in subsurface ecosystems.