Hydrogen and carbon isotope fractionation by thermophilic hydrogenotrophic methanogens from a deep aquifer under coculture with fermenters

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To elucidate the isotope geochemistry of CH₄ production in deep subsurface environments, we investigated the relation between H₂ concentration and hydrogen and carbon isotope fractionation by CO₂ reduction using microbial communities obtained from groundwater in a deep aquifer associated with an accretionary prism. Incubation experiments were conducted under anaerobic culture conditions of two types. In one experiment, a coculture of H₂-producing fermenters and hydrogenotrophic methanogens was established in groundwater treated with organic substrates. The other experiment used groundwater under H₂ + CO₂ (80:20, v/v) to produce CH₄ under high H₂ concentrations. In the cocultures, H₂ concentrations increased in the initial phases, then decreased gradually and remained low during CH₄ production, indicating H₂ consumption by hydrogenotrophic methanogens to produce CH₄. This study revealed for the first time that cocultures with fermenters and hydrogenotrophic methanogens producing CH₄ in low H₂ concentration cause smaller hydrogen isotope fractionations (0.663 < α_H < 0.725) than in monocultures under high H₂ concentrations (0.629 < α_H < 0.656). Carbon isotope fractionation in cocultures was greater (1.052 < α(CO₂-CH₄) < 1.074) than in monocultures under high H₂ concentrations (1.021 < α(CO₂-CH₄) < 1.023). The large carbon fractionation was thought to result from low levels of H₂, supporting the hypothesis of differential reversibility of multiple enzymatic processes in CH₄ production. Although lack of agreement remains between results of incubation experiments and field observations especially in hydrogen isotope fractionations, both hydrogen and carbon isotope fractionation in cocultures were close to the fractionations of field observation in which CO₂ reduction is a dominant pathway in CH₄ production compared with those in monoculture.