A comparison between the C-14 content of the methane and dissolved inorganic carbon (DIC) in deep, terrestrial subsurface systems was used to assess the timing of microbial methanogenesis contributing to gases in fracture water samples from three mines in the Witwatersrand Basin, South Africa. The results demonstrated that the majority of methane was produced over geologic timescales. In four of the samples, the methane contained no significant radiocarbon, indicating that the estimated 90% microbial methane in these samples was produced in the geologic past by indigenous microbial communities. In two samples from different mines, methane Delta C-14 levels indicated a primarily ancient origin for the microbial methane with the potential for more recent contributions from ongoing indigenous microbial activities constrained to between 0 and 40%, and 0 and 24%, respectively. Microbiological evidence for methanogenic archaea was observed in both of these samples. One sample had a Delta C-14 CH4 that was higher than the corresponding DIC, indicating an extreme decoupling between these species and raising concerns over the representative quality of this sample. The variations in the Delta C-14 of DIC and CH4 between and within mines demonstrate the need for a thorough assessment of each sample to obtain an accurate understanding of the role and timing of microbiological gas production in these complex, heterogeneous, terrestrial subsurface systems. The approach detailed here introduces timing as a new and widely applicable signature for the recognition of a major geochemical marker of indigenous life in the deep subsurface.
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