Continental subsurface waters support unique but diverse C-acquisition strategies

Terrestrial subsurface environments generally support two orders of magnitude fewer microorganisms than submarine environments where energy and C sources are more abundant. However, our research on the geochemistry and stable isotopes has suggested that the microbial communities residing in the continental subsurface waters, aged more than thousands of years, do not live by a monotypic metabolic network across sites. We evaluated the taxonomic and functional diversity of microbial communities from three localities in South Africa and their relationship to the environmental parameters associated with each fracture water. The borehole at Tau Tona Au mine (TT107; 3,100 mbls), Masimong Au mine (MM5; 1,900 mbls) and Zondereinde Pt mine (NO14; 2,100 mbls) contain saline fracture water of paleometeroic origin but the anaerobic ecosystems were driven by distinctive C-assimilation strategies. Archaea and Bacteria are present in all samples with the latter being dominant (>75%). The similarity between the Δ14C and δ13C-PLFA with those of the DIC indicates that the majority of cellular C in the TT107 sample was derived from the DIC (0.6 mM), even though dissolved CH4 (8.8 mM) is more available. The DIC may have supported a wide variety of chemoautotrophs including the predominant firmicutes, e.g. Thermincola sp. and Ca. Desulforudis audaxviator. Interestingly, a considerable percentage of sequences related to oligotrophic α-proteobacteria Caulobacter sp. was detected, which warrants further investigation as the aerobic heterotrophic microorganism has a unique dimorphic life cycle. For the MM5 sample, the δ13C and δ2H of the CH4 indicate it was produced via CO2 reduction from DIC, which is consistent with the relatively high abundance of hydrogenotrophic methanogen Methanothermobacter sp. that scavenged the abiogenic H2 and utilized the DIC (0.43 mM) leading to its enriched δ13C signature. In contrast to the TT107 sample, the much-depleted δ13C-CH4 indicates that the biogenic CH4 (8.9 mM) was consumed but the responsible methanotrophs are not apparent in the metagenome data. Nonetheless, functional genes most closely related to aerobic CH4 oxidizers Methylacidiphiales (~70% of similarity) within Verrucomicrobia were found in the MM5 sample but not other two. NO14 is the most high temperature (65°C) and energy-rich sample (3.9 μM H2, 0.6 mM DOC and 0.2 mM S), which supported a heterotrophic community populated with S-dependent euryarcheaote Thermococci and γ-proteobacteria Pseudoalteromonas sp. that has thiosulfate-oxidizing strains.