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Deeply fractured rocks of meteorite impact craters are suggested as prime niches for subsurface microbial colonization. Methane can be a product of such microbial communities and seeps of methane from impact craters on Earth are of strong interest as they act as analogs for Mars. Previous studies report signs of ancient microbial methanogenesis in the Devonian Siljan meteorite impact structure in Sweden, but the proportion of microbial methane, metabolic pathways, and potential modern activity remain elusive. In this study, gas composition, hydrochemistry, oil organic geochemistry, and microbial community analyses are reported in 400 m deep fractures of the Siljan impact structure. The results showed a dominantly microbial origin for methane, which was supported by highly negative δ¹³C_CH4 and positive δ¹³C_CO2 values along with multiply substituted isotopologues (Δ¹³CH₃D) that indicated disequilibrium fractionation due to microbial kinetic isotope effects. The presence of C₂ to C₅ hydrocarbons suggested a minor thermogenic input in the gas mix. Characterization of the microbial community via 16S rRNA gene amplicon sequencing and real-time PCR indicated a low abundance of several methanogenic archaeal populations, which is common for settings with active methanogenesis. Evidence of oil biodegradation suggested that secondary microbial hydrocarbon utilization was involved in the methanogenesis. Low sulfate and high alkalinity in the groundwaters also suggested a dominantly microbial methane formation driven by infiltration of freshwater that was coupled to sulfate reduction and secondary utilization of early mature thermogenic hydrocarbons.

The continental bedrock contains groundwater-bearing fractures that are home to microbial populations that are vital in mediating the Earth’s biogeochemical cycles. However, their diversity is poorly understood due to the difficulty of obtaining samples from this environment. Here, a groundwater-bearing fracture at 975 m depth was isolated by employing packers in order to characterize the microbial community via metagenomes combined with prokaryotic and eukaryotic marker genes (16S and 18S ribosomal RNA gene). Genome-resolved analyses revealed a community dominated by sulfate-reducing Bacillota, predominantly represented by Candidatus Desulforudis audaxviator and with Wood-Ljungdahl as the most prevalent pathway for inorganic carbon fixation. Moreover, the eukaryotic community had a considerable diversity and was comprised of mainly flatworms, chlorophytes, crustaceans, ochrophytes, and fungi. These findings support the important role of the Bacillota, with the sulfate reducer Candidatus Desulforudis audaxviator as its main representative, as primary producers in the often energy-limited groundwaters of the continental subsurface.