Fractured rock aquifers within the continental crust can harbor oligotrophic fluid environments at depth that are largely isolated from surface photosynthate. Depending on the thermal history of the system, dissolved organic carbon (DOC) in these aquifers can be biotic in origin from in situ microbial production or thermal degradation, and/or abiotically contributed from the mantle or water-rock reactions (e.g. Fischer-Tropsch Type Synthesis). Fracture systems with low water/rock ratios at great depth (>2 km) can accumulate abiogenic methane and C₂₊ hydrocarbons that can serve as precursors in production pathways for various other abiogenic organics, leading to large quantities of fluid DOC. As these systems cool (<150°C), they can also support organics contribution from low biomass (≤10⁴ cells/mL) microbial communities. Low biomass living in systems with high quantities of organic substrates, suggests microbial utilization of high DOC pools is constrained by environmental stressors.

Hypersalinity is one large constraint often found in subsurface fluids in regions of low porosity and permeability, where water radiolysis and clay hydration reactions can establish elevated ion concentrations over geologic time. Additionally, differences in microbial organics utilization have been found in subsurface fluid settings of various salinity. Previous microbial characterization of <2 km deep, mesophilic brackish fracture fluids in the Witwatersrand Basin of South Africa found a microbial community reliant on primary production by a small group of hydrogenotrophic methanogens. Alternatively, 1.7 Ga hypersaline brines from a 2.4 km deep and mesophilic fracture system at Kidd Creek mine in the Canadian Shield, supported sulfate reducing microorganisms utilizing abiogenic alkanes.These systems suggest abiogenic carbon may sustain mesophilic communities in hypersaline subsurface environments vs. biotically sustained communities in younger, brackish environments with reduced salinity stress. For deep subsurface fluids with multiple abiotic stressors (e.g. high salinity and temperature), it is essential to characterize the source (abiotic vs. biotic contribution) and identity of DOC components to understand the role of organics in microbial habitability on Earth and in planetary subsurface fluid settings.

In 2018, a brine system extending to 3.2 km depth in the Moab Khotsong gold and uranium mine was discovered in South Africa’s Witwatersrand Basin. This brine system combines hypersalinity (215-246 g/L) with high temperatures (45-55℃) and long noble gas residence times in the subsurface (1.2 Ga). Preliminary analyses revealed a similar trend to previous deep terrestrial subsurface fluids, of high DOC alongside low biomass (10²-10³ cells/mL). Additionally, the Moab Khotsong system was found to contribute redox species, including H₂ and O₂, via water radiolysis acting on the system over its long isolation. In our paper now out in Nature Communications, we investigate the extent of old biotic, current biotic, and abiotic contribution to DOC, along with consideration for dissolved inorganic carbon (DIC), to this hypersaline and high temperature brine system. 21-Tesla Negative Electrospray Ionization (- ESI) Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) analysis revealed the majority of DOC in the brine is ‘old biotic’ input from proximal kerogen-enriched shale or reef zones, with which they share a light δ¹³C signature (~30‰). Evidence for extended radiolytic oxidation of the DOC pool was observed in high overall O/C content for FT-ICR MS identified organic molecular classes. Tyrosine and tryptophan-like protein alongside some autochthonous microbial DOC peaks (revealed through excitation-emission matrices), and aspartic acid D/L enantiomeric ratios <1, suggest minor contribution from microorganisms currently inhabiting the brine system. Characterization of the volatile organic carbon pool revealed C_1-6 alkanes with abiogenic  δ²H and  δ¹³C signatures, a low CH₄/C₂₊ ratio, and potentially abiotic akylsulfides. These results suggest that abiotic processes dominate the current DOC contribution and alteration processes in the Moab Khotsong brine system, helping to support a meager ‘slow’ biosphere along with radiolytic contribution of redox species.

            This characterization of the Moab Khotsong brines presents a long-isolated organic environment distinct from the formate and acetate dominated abiotic Kidd Creek brines in the Canadian Shield, as well as from previously characterized Witwatersrand Basin fluids with primary contribution from low biomass microbial communities. These results may also suggest that under conditions of high salinity and temperature stress, a combination of biotic and abiotic contribution is necessary to maintain habitable conditions. Additionally, it may also signal that long-isolated planetary brines with higher organics content and radiolytic input are good targets in the search for microbial life. Some prospective planetary systems may include near-surface regions on Mars exposed to galactic radiation, or in brine regions underneath the ice crusts of Europa, Enceladus, and Ceres where there may be redox species contribution from local radionuclide decay and/or serpentinization processes. Future investigations will require an energetic evaluation of microbial metabolism amongst the physicochemical and inorganic/organic conditions of Moab Khotsong to determine supported catabolic strategies, and to enhance our understanding of microbial habitability in the deep subsurface on Earth and beyond.

Funding for organics characterization in the presented article was provided by the NSF, NASA, Princeton University’s High Meadows Environmental Institute, NSERC, and CIFAR. Drilling of the boreholes was provided by ICDP and shipment of samples by SABDI.

Photo credit: Dr. Devan Nisson, Princeton University, 2019