Potential energy sources for the deep continental biosphere in isolated anoxic brines

In isolated fracture networks in the Precambrian Shield, long-term water and rock interactions produce saline anoxic fluids that host extant microbial communities deep within the continental subsurface. Light and oxygen (O₂) are absent in these environments. Thus, chemotrophic organisms inhabiting these systems rely on anaerobic reactions for energy. Viable electron donors include short-chain alkanes, such as methane (CH₄) and C₂₊ alkanes, while alternative electron acceptors include sulfate (SO2−4), nitrate (NO−3), and ferric iron (Fe³⁺). Here, we constrain the potential sources of energy for microorganisms in Neoarchean bedrock on the 27th level west drift of the Soudan Underground Mine State Park, MN, USA (713.5 meters below the surface). The Gibbs Free Energy (ΔG) of 11 reactions are modeled and expressed as available chemical potential energy per mass fluid (J/kg_fluid). Metabolic reactions involving CH₄ oxidation by SO2−4 would yield the highest potential energy of reactions modeled in this study (−111 J/kg_fluid). The free energy for methanogenesis via the breakdown of dimethylamine (DMA; ∑(CH₃)₂NH_(aq)) is exergonic but with near-zero available energy per mass fluid, suggesting that DMA may be cycled quickly to produce biological CH₄ at Soudan. We examine all the possible pathways by which CH₄ and other short-chain alkanes may be formed. Conventional δ¹³C_CH4 values and C₁/C₂₊ abundance ratios support a mixed biological and non-biological origin of CH₄. Doubly substituted ‘clumped’ CH₄ isotope ¹³CH₃D values are consistent with formation temperatures of 84-89 °C that exceed current environmental conditions of 11.5-12.1 °C. These estimated formation temperatures are too low for CH₄ to be formed solely through thermogenic degradation of organic matter. Further, low or undetectable H₂ rules out active abiogenesis of CH₄ from CO₂ reduction. It is more likely that the bulk CH₄ pool reflects a mixture of microbial CH₄ with Δ¹³CH₃D values equilibrated at 11.5-12.1 °C and thermogenic CH₄ formed at temperatures >100 °C. Understanding the origin and cycling of these electron donors contributes to a fundamental understanding of how microbial activity may promote, maintain, or suppress the habitability of these isolated systems over long timescales.