Growth of Methanogens on Different Mars Regolith Analogues and Stable Carbon Isotope Fractionation during Methanogenesis

Introduction: The detection of methane in the Martian atmosphere (1-4) and the probability of the existence of liquid water during the early history of Mars (5-10) prompted enthusiasm about plausible life forms on Mars. The half-life of methane in a planetary atmosphere is about 300 years (11), so in order to be detected on Mars, methane would need to be replenished continuously. Several potential sources of methane in the martian atmosphere have been suggested, such as volcanic, meteoritic, cometary, hydrogeochemical, and biogenic (12). On Earth, however, about 90 to 95% of atmospheric methane has a biological origin, either from living organisms or decay of organic matter (12). Hence, one explanation for the finding and non-uniform distribution of methane on Mars could be localized microbial sources, either extinct or extant, such as methanogens. Methanogens have been considered models for possible martian life-forms even before the discovery of methane in Mars’ atmosphere (13-16). Methanogens are anaerobes, and certain strains can tolerate low pressure, desiccation (17), and very cold temperature (18), like conditions present on Mars. Methanogenic archaea are anaerobic chemoautotrophs that mostly consume CO2 as a carbon source and H2 as an energy source and produce methane as an end product of metabolism. Stable carbon isotope fractionation is one of the important techniques that can distinguish various potential sources of methane (19). Here, we present the carbon isotope fractionation pattern of methane produced by three different strains of methanogens, Methanothermobacter wolfeii, Methanosarcina barkeri, and Methanobacterium formicicum, growing on four different Mars regolith analogues. Methanogens were provided CO2, in the form of bicarbonate buffer, and molecular hydrogen in the gaseous form. Methods: The anaerobic stock cultures in growthsupporting media such as MM, MS, and MSF for M. wolfeii, M. barkeri, and M. formicicum, respectively, were prepared using protocols described previously (20). They were transferred into their respective new growth media every two weeks. Four different Mars regolith analogues utilized in this experiment were JSC Mars-1 (21); JSC Mars-2 (22), which is a mixture of 45% smectite, 45% basalt, and 10% hematite; montmorillonite (WA: 46 E 0438, size <63μm); and Mojave Mars Simulant (MMS) (23). Montmorillonite, a clay mineral, is abundant on Mars (24). A total of thirtysix 150 mL serum bottles were used for three different strains of methanogens and four different Mars simulants. Samples were in triplicate. Each bottle contained 3g of the regolith analog. They were left overnight in the anaerobic chamber to deoxygenate. On the following day, 60 mL of bicarbonate buffer were added to each bottle. Bottles were sealed with butyl rubber stoppers, secured with aluminum crimps and autoclaved. For positive controls, three bottles of each medium containing 60 mL of MM, MS, and MSF were also prepared. About an hour prior to inoculation, 1.5 mL of sterile Na2S were added to each sample bottle to remove any remaining molecular oxygen. Actively growing microbial cells were centrifuged at 6000 rpm for 10 min and washed two times with reduced sterile bicarbonate buffer (25). Washing of cells with buffer ensures that they do not carry over any residual growth media. The washed cells of each species were then suspended in 15 mL of sterile bicarbonate buffer. Each bottle containing Mars regolith analogues received a 1 mL aliquot of their respective cell suspension. All bottles were pressurized with 200 kPa of H2 and incubated at their respective optimum growth temperatures. Headspace gas was analyzed periodically for methane concentration and stable carbon isotopic fractionation using a Varian CP-4900 Micro-GC, and a Cavity Ringdown Spectrometer G2201-1 isotopic CO2/CH4 (University of Arkansas Stable Isotope Laboratory). Results: The carbon isotope fractionation, δC, was calculated using the following equation: