Abstract. Deep subsurface microorganisms constitute over 80 % of Earth's prokaryotic biomass and play an important role in global biogeochemical cycles. Geochemical processes driven by geothermal heating are key factors influencing their biomass and activities, yet their full breadth remains uncaptured. Here, we investigated the microbial community composition and metabolism in microbial-natural-gas-bearing aquifers at temperatures ranging from 38 to 81 °C, situated above nonmicrobial-gas- and oil-bearing sediments at temperatures exceeding 90 °C. Cultivation-based and molecular gene analyses, including radiotracer measurements, of formation water indicated variations in predominant methanogenic pathways across different temperature regimes of upper aquifers: high potential for hydrogenotrophic–methylotrophic, hydrogenotrophic, and acetoclastic methanogenesis at temperatures of 38, 51–65, and 73–81 °C, respectively. The potential for acetoclastic methanogenesis correlated with elevated acetate concentrations with increasing depth, possibly due to the decomposition of sedimentary organic matter. In addition to acetoclastic methanogenesis, in aquifers at temperatures as high as or higher than 65 °C, acetate is potentially utilized by microorganisms responsible for the dissimilatory reduction of sulfur compounds other than sulfate because of their high relative abundance at greater depths. The stable sulfur isotopic analysis of sulfur compounds in water and oil samples suggested that hydrogen sulfide, generated through the thermal decomposition of sulfur compounds in oil, migrates upward and is subsequently oxidized with iron oxides present in sediments, yielding elemental sulfur and thiosulfate. These compounds are consumed by sulfur-reducing microorganisms, possibly reflecting elevated microbial populations in aquifers at temperatures as high as or higher than 73 °C. These findings reveal previously overlooked geothermal-heat-driven geochemical and microbiological processes involved in carbon and sulfur cycling in the deep sedimentary biosphere.
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