Simulating Martian Conditions: Methanogen Survivability during Freeze-thaw Cycles

Introduction: Methanogens are obligate anaerobes that use molecular hydrogen as an energy source and carbon dioxide as a carbon source to produce methane. They are classified as Archaea and are found in many extreme environments, including hydrothermal vents, volcanoes, and also the human microflora. The current Martian atmosphere is low in pressure, very dry (hyper-arid), and high in radiation, and thus the surface is not suitable for life. However, the subsurface contains permafrost, liquid water [1], and trace amounts of methane [2, 3]. Thus, it is proposed that these Archaea are able to persist in Martian conditions. According to data obtained from NASA’s Mars Science Laboratory between August 2012 and the present time, the maximum and minimum temperatures on Mars have ranged from +20°C to 100°C (Fig. 1) [4]. These conditions might be suitable for methanogenic growth. The goals of this experiment are to use freeze-thaw cycles and measure methanogen growth using gas chromatography in order to further understand temperature constraints on growth. Methods: Four types of methanogen growth media were prepared according to Kendrick and Kral [5]. The strains used were Methanothermobacter wolfeii, Methanosarcina barkeri, Methanobacterium formicicum, and Methanococcus maripaludis. The media were autoclaved for sterilization, inoculated with 0.5 mL of each respective methanogen, and grown in their respective media and optimal temperature at ambient pressure: M. wolfeii at 55°C, M. barkeri/M. formicicum at 37°C, and M. maripaludis at room temperature (22°C). Growth was monitored in the form of methane production throughout the duration of the experiments via gas chromatography. Experiment 1. Following seven days at their respective incubation temperatures, all four of the methanogen inoculums were exposed to 4°C for seven days and incubated for six days, respectively (Fig. 2). Experiment 2. Each inoculum was placed in either five grams of sand or ten grams of sand. Following fifteen days at their respective incubation temperatures, all four of the methanogen inoculums were exposed to 4°C for eight days, -20°C for eleven days, -80°C for eight days, and then incubated for seven days, respectively (Fig. 3). Figure 1. Ground temperature data from the Rover Environmental Monitoring System from earlyAugust 2012 to the present time at Mars’ Gale Crater [4]. Results: Only methane concentrations above 1% were considered. Experiment 1. Following seven days of incubation at their respective temperatures, M. wolfeii, M. maripaludis, and M. formicicum showed appreciable growth after exposure to 4°C and incubation. M. barkeri did not show appreciable growth during the experiment and these data were not included (Fig. 2).