Hydrogenotrophic Methanogenesis at 7 to 12 mbar by Methanosarcina barkeri under Simulated Martian Atmospheric Conditions

It is widely accepted that ancient Mars hosted long-lived habitable environments, and that habitable refugia may persist in the subsurface today. The presence and behavior of methane gas in the Martian atmosphere makes a compelling case to study methanogenesis as a potential biosignature under simulated Mars surface or shallow subsurface conditions. Methanogenic archaea have demonstrated extremotolerance to prolonged exposure to desiccation, high salinity, strong oxidants, UVC irradiation, and extremes in temperature, pH, and pressure. We investigated hydrogenotrophic methanogenesis and accompanying gene expression patterns in Methanosarcina barkeri in response to sustained hypobaria (7 to 12 mbar), low temperature (0C), and a CO2-dominated gas mixture simulating Martian atmospheric conditions. We observed statistically significant CH4 production under all assayed conditions, including at 7-12 mbar. Comparative transcriptomics to control conditions (i.e., 30C, 1500 mbar, 80:20 H2:CO2) revealed that atmospheric gas composition to be the most important factor in regulating methanogenesis. Constrained only by the distribution of liquid water and sufficient H2 availability, we conclude an extant methanogenic biosphere could be supported throughout the Martian lithosphere.