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

Abstract Mars, with its ancient history of long-lived habitable environments, continues to captivate researchers exploring the potential for extant life. This study investigates the viability of hydrogenotrophic methanogenesis, a potential biosignature, in Methanosarcina barkeri MS under simulated Martian surface conditions. We expose M. barkeri to sustained hypobaria (7–12 mbar), low temperature (0˚C), and a CO 2 -dominated gas mixture mimicking the Martian atmosphere. The results demonstrate statistically quantifiable CH 4 production under all tested conditions, including at 7-12 mbar. Transcriptomics reveal that hypobaria alone did not significantly impact gene expression, highlighting the resilience of M. barkeri . However, atmospheric gas composition, specifically Mars gas with 2.9% pH 2 , led to significant down-regulation of methanogenesis genes, hindering growth over a 14-day experiment. Notably, CH 4 production scaled with the partial pressure of H 2 , indicating its critical role in methanogenesis. We propose that Mars subsurface could harbor habitable refugia, sustaining microbial life at low metabolic steady states. The findings challenge assumptions about the habitability of present-day Mars and have implications for Planetary Protection and in situ resource generation for future human exploration. Overall, this work contributes to understanding the habitability of Mars and informs future astrobiological and exploration endeavors.