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Polygonal ground and other geomorphological features reminiscent of recent freeze/thaw cycling are evident on Mars, despite the widespread belief that the planet is currently inhospitably cold and dry. On Earth, permafrost microbial communities are subjected to wide ranges in temperature and are often active at subfreezing temperatures. The existence of active microbial communities within permafrost on Earth suggests that permafrost on Mars may constitute a habitable environment. Terrestrial microbial permafrost communities typically contain methane-producing Archaea, which is cause for concern as global temperatures rise, resulting in permafrost thaw and the release of the potent greenhouse gas. Similarly, on Mars, the overlap between patterned ground and detections of localized methane plumes suggest that the compound may have been released from thawing permafrost. Analyses of permafrost ice cores and soil samples on Earth note that (1) archaeal communities often contain both mesophiles and psychrophiles at different depths and (2) active methane is being produced at subfreezing temperatures over geological timescales. Thus, the purpose of the experiments described here was to determine the effect of extreme temperature changes (reminiscent of the martian diurnal temperature cycle) on the growth and survival of four non-psychrophilic methanogens previously used as models for potential life on Mars. The results indicate that non-psychrophilic methanogens are capable of survival during extreme diurnal and 48-h temperature changes, similar to those on Mars.

Low pressure is often overlooked in terms of possible biocidal effects when considering a habitable environment on Mars. Few experiments have investigated the ability for microorganisms to actively grow under low pressure conditions, despite the atmosphere being a location on Earth where organisms could be exposed to these pressures. Three species of methanogens (Methanobacterium formicicum, Methanosarcina barkeri, Methanococcus maripaludis) were tested for their ability to actively grow (demonstrate an increase in methane production and optical density) within low-pressure microenvironments at 50 mbar or 100 mbar. M. formicicum was the only species to demonstrate both an increase in methane and an increase in optical density during the low-pressure exposure period for experiments conducted at 50 mbar and 100 mbar. In certain experiments, M. barkeri showed an increase in optical density during the low-pressure exposure period, likely due to the formation of multicellular aggregates, but minimal methane production (

Life as we know it requires liquid water and sufficient liquid water is highly unlikely on the surface of present-day Mars. However, according to thermal models there is a possibility of liquid water in the deep subsurface of Mars. Thus, the martian subsurface, where the pressure and temperature is higher, could potentially provide a hospitable environment for a biosphere. Also, methane has been detected in the Mars’ atmosphere. Analogous to Earth’s atmospheric methane, martian methane could also be biological in origin. The carbon and energy sources for methanogenesis in the subsurface of Mars could be available by downwelling of atmospheric CO2 into the regolith and water-rock reactions such as serpentinization, respectively. Corresponding analogs of the martian subsurface on Earth might be the active sites of serpentinization at depths where methanogenic thermophilic archaea are the dominant species. Methanogens residing in Earth’s hydrothermal environments are usually exposed to a variety of physiological stresses including a wide range of pressures, temperatures, and pHs. Martian geochemical models imply that the pH of probable groundwater varies from 4.96 to 9.13. In this work, we used the thermophilic methanogen, Methanothermobacter wolfeii, which grows optimally at 55oC. Therefore, a temperature of 55oC was chosen for these experiments, possibly simulating Mars’ subsurface temperature. A martian geophysical model suggests depth and pressure corresponding to a temperature of 55 °C would be between 1–30 km and 100-3,000 atm respectively. Here, we have simulated Mars deep subsurface pH, pressure, and temperature conditions and have investigated the survivability, growth rate, and morphology of M. wolfeii after exposure to a wide range of pH 5–9) and pressure (1−1200 atm) at a temperature of 55 °C. Interestingly, in this study we have found that M. wolfeii was able to survive at all the pressures and pHs tested at 55 °C. In order to understand the effect of different pHs and pressures on the metabolic activities of M. wolfeii, we also calculated their growth rate by measuring methane concentration in the headspace gas samples at regular intervals. In acidic conditions, the growth rate (γ) of M. wolfeii increased with the increase in pressure. In neutral and alkaline conditions, the growth rate (γ) of M. wolfeii initially increased with pressure, but decreased upon further increase of pressure. To investigate the effect of combined pH, pressure, and temperature on the morphology of M. wolfeii, we took phase contrast images of the cells. We did not find any obvious significant alteration in the morphology of M. wolfeii cells. Methanogens, chemolithoautotrophic anaerobic microorganisms, are considered as ideal model microorganisms for Mars. In light of research presented here, we suggest that at least one methanogen, M. wolfeii, could survive in the deep subsurface environment of Mars.

In order to examine the potential survivability of life in the Martian deep subsurface, we have investigated the effects of temperature (45°C, 55°C, and 65°C) and pressure (1 atm, 400 atm, 800 atm, and 1200 atm) on the growth, carbon isotopic data, and morphology of chemolithoautotrophic anaerobic methanogenic archaea,Methanothermobacter wolfeii. The growth and survivability of this methanogen were determined by measuring the methane concentration in headspace gas samples after the cells were returned to their conventional growth conditions. Interestingly, this methanogen survived at all the temperatures and pressures tested.M. wolfeiidemonstrated the highest methane concentration following exposure to pressure of 800 atm and a temperature of 65°C. We found that the stable carbon isotopic fractionation of methane, δ13C(CH4), was slightly more enriched in12C at 1 atm and 55°C than the carbon isotopic data obtained in other temperature and pressure conditions. A comparison of the images of the cells before and after the exposure to different temperatures and pressures did not show any obvious alteration in the morphology ofM. wolfeii. The research reported here suggests that at least one methanogen,M. wolfeii, may be able to survive under hypothetical Martian subsurface conditions with respect to temperature and pressure.

In 2008, the Mars Phoenix Lander discovered perchlorate at its landing site, and in 2012, the Curiosity rover confirmed the presence of perchlorate on Mars. The research reported here was designed to determine if certain methanogens could grow in the presence of three different perchlorate salt solutions. The methanogens tested were Methanothermobacter wolfeii, Methanosarcina barkeri, Methanobacterium formicicum and Methanococcus maripaludis. Media were prepared containing 0%, 0.5%, 1.0%, 2%, 5% and 10% wt/vol magnesium perchlorate, sodium perchlorate, or calcium perchlorate. Organisms were inoculated into their respective media followed by incubation at each organism׳s growth temperature. Methane production, commonly used to measure methanogen growth, was measured by gas chromatography of headspace gas samples. Methane concentrations varied with species and perchlorate salt tested. However, all four methanogens produced substantial levels of methane in the presence of up to 1.0% perchlorate, but not higher. The standard procedure for growing methanogens typically includes sodium sulfide, a reducing agent, to reduce residual molecular oxygen. However, the sodium sulfide may have been reducing the perchlorate, thus allowing for growth of the methanogens. To investigate this possibility, experiments were conducted where stainless steel nails were used instead of sodium sulfide as the reducing agent. Prior to the addition of perchlorate and inoculation, the nails were removed from the liquid medium. Just as in the prior experiments, the methanogens produced methane at comparable levels to those seen with sodium sulfide as the reductant, indicating that sodium sulfide did not reduce the perchlorate to any significant extent. Additionally, cells metabolizing in 1% perchlorate were transferred to 2%, cells metabolizing in 2% were transferred to 5%, and finally cells metabolizing in 5% were transferred to 10%. All four species produced methane at 2% and 5%, but not 10% indicating some success in adapting cells to concentrations higher than 1%. The results reported here indicate that the presence of perchlorate on Mars does not rule out the possible existence of methanogens.

In order to characterize stable carbon (13C/12C) isotope fractionation of metabolically produced methane by methanogens in martian settings, Methanothermobacter wolfeii, Methanosarcina barkeri, and Methanobacterium formicicum were cultured on four different Mars regolith analogs – JSC Mars-1, Artificial Mars Simulant, montmorillonite, and Mojave Mars Simulant – and also in their growth supporting media. These chemoautotrophic methanogens utilize CO2 for their carbon source and H2 for their energy source. When compared to the carbon isotope signature of methane when grown on their respective growth media, M. wolfeii and M. barkeri demonstrated variability in carbon isotope fractionation values during methanogenesis on the Mars analogs, while M. formicicum showed subtle or negligible difference in carbon isotope fractionation values. Interestingly, M. wolfeii and M. barkeri have shown relatively consistent enriched values of 12C on montmorillonite, a kind of clay found on Mars, compared to other Mars regolith analogs. In general, M. barkeri showed large carbon isotope fractionation compared to M. wolfeii and M. formicicum during methanognesis on various kinds of analogs. Stable carbon isotope fractionation is one of the techniques used to infer different origins, environments, and pathways of methanogensis. The results obtained in this novel research can provide clues to determine ambiguous sources of methane on Mars.

Methanogens, microorganisms in the domain Archaea, have been studied as life forms that might inhabit the subsurface of Mars. These organisms can use carbon dioxide as a carbon source, a compound that is abundant in the martian atmosphere. But if they exist in the deep subsurface where the carbon dioxide may not penetrate, they would have to rely on another source of carbon. Magnesium carbonate and calcium carbonate have been detected at the martian surface, and there is no reason to believe that they would not be in the subsurface as well. In the research reported here, we asked if these carbonates could possibly serve as carbon sources for four species of methanogens. Methanothermobacter wolfeii, Methanobacterium formicicum and Methanococcus maripaludis were able to produce a small amount of methane (approximately 0.4–0.8% headspace gas) when either carbonate was the carbon source available while Methanosarcina barkeri only produced significant methane (also 0.4–0.8%) when calcium carbonate was the carbon source. The amounts produced were dependent on methanogenic species, carbonate used and pH. At equilibrium, a small amount of carbon dioxide (approximately 0.05–0.15% headspace gas as well as in liquid media) was generated by these carbonates, and this carbon dioxide was most likely the carbon compound that was being metabolized. Background carbon dioxide from the atmosphere was not sufficient for measureable methane production.

Any life existing in the martian environment must be able to deal with relatively extreme factors including desiccation, low pressure, and the presence of different martian regoliths. We have been studying methanogens, microorganisms in the domain Archaea, as models for life on Mars. Previously, we have examined methanogens in the presence of these three factors individually. Here, four methanogen species were tested for survival under the three conditions combined. Two of the methanogens survived desiccation at low pressure. One survived desiccation at low pressure on different martian regolith analogs.

The low pressure at the surface of Mars (average: 6 mbar) is one potentially biocidal factor that any extant life on the planet would need to endure. Near subsurface life, while shielded from ultraviolet radiation, would also be exposed to this low pressure environment, as the atmospheric gas-phase pressure increases very gradually with depth. Few studies have focused on low pressure as inhibitory to the growth or survival of organisms. However, recent work has uncovered a potential constraint to bacterial growth below 25 mbar. The study reported here tested the survivability of four methanogen species (Methanothermobacter wolfeii, Methanosarcina barkeri, Methanobacterium formicicum, Methanococcus maripaludis) under low pressure conditions approaching average martian surface pressure (6 mbar – 143 mbar) in an aqueous environment. Each of the four species survived exposure of varying length (3 days – 21 days) at pressures down to 6 mbar. This research is an important stepping-stone to determining if methanogens can actively metabolize/grow under these low pressures. Additionally, the recently discovered recurring slope lineae suggest that liquid water columns may connect the surface to deeper levels in the subsurface. If that is the case, any organism being transported in the water column would encounter the changing pressures during the transport.

We consider six possible sources of CH 4 and other lowmass (C 2 –C 5 ) organics in the plume of Enceladus: three of these sources represent initial endowments of organics: cometary organics, Titan-like tholin, and the Fisher–Tropsch type reactions in the gases from which Enceladus formed. The other three sources represent processes inside Enceladus: water–rock reactions, microbiology, and thermogenesis. We report on new laboratory results for C 2 hydrocarbons released by thermogenesis of laboratory tholin and the Fisher–Tropsch type synthesis. Thermal processing of Titan-like tholin produced ratios of CH 4 /C 2 H 4 and CH 4 /C 2 H 6 of about two for temperatures up to 450 °C and about six for a temperature of 650 °C. The low pressure (∼1 atm) Fisher–Tropsch type experiments produced CH 4 /C 2 H 4 of ∼1.5, similar to previous results. C 2 H 2 was not produced by either process. Tests of gas production by four strains of methanogens confirmed the absence of any detectable production of non-methane hydrocarbons. Cometary endowment, the Fisher–Tropsch type synthesis, and Titan-like tholin incorporation could be primary inputs of organics and subsequent thermal processing of any of these all are possible sources of low mass organics in the plume. Biological production and water–rock reactions are an alternative source of CH 4 . Aqueous reactions with CO and H 2 can produce C 2 –C 5 organics even at the low pressures of the interior of Enceladus. If there is a confirmed detection of CO and C 2 H 2 in the plume of Enceladus, this provides an important constraint on sources, as we have identified no process, other than the initial volatile component of cometary organics, which can supply these gases. Precise determination of the relative concentrations of C 1 –C 5 hydrocarbons may provide additional constraints on sources, but a detailed isotopic analysis of C and H in these organics and a search for amino acids constitute the next important steps in resolving the sources of the organics in Enceladus' plume.

Zero-valent iron, montmorillonite-like smectites, and CO 2 gas are known to exist on Mars, and work was performed to investigate the ability of methanogens to subsist on these materials. After 71 days of incubation at 55 °C, mean methane concentration as percent of headspace volume was 19.80 ± 1.76% (mean ± SE) for replicates containing elemental iron and 0.50 ± 0.15% for those lacking elemental iron.

Methanogens have been considered models for life on Mars for many years. In order to survive any exposure at the surface of Mars, methanogens would have to endure Martian UVC radiation. In this research, we irradiated hydrated and desiccated cultures of slightly halophilic Methanococcus maripaludis and non-halophilic Methanobacterium formicicum for various time intervals with UVC (254 nm) radiation. The survivability of the methanogens was determined by measuring methane concentrations in the headspace gas samples of culture tubes after re-inoculation of the methanogens into their growth-supporting media following exposure to UVC radiation. Hydrated M. maripaludis survived 24 h of UVC exposure, while in a desiccated condition they endured for 16 h. M. formicicum also survived UVC radiation for 24 h in a liquid state; however, in a desiccated condition, the survivability of M. formicicum was only 12 h. Some of the components of the growth media could have served as shielding agents that protected cells from damage caused by exposure to ultraviolet radiation. Overall, these results suggest that limited exposure (12–24 h) to UVC radiation on the surface of Mars would not necessarily be a limiting factor for the survivability of M. maripaludis and M. formicicum.

The jets of icy particles and water vapor issuing from the south pole of Enceladus are evidence for activity driven by some geophysical energy source. The vapor has also been shown to contain simple organic compounds, and the south polar terrain is bathed in excess heat coming from below. The source of the ice and vapor, and the mechanisms that accelerate the material into space, remain obscure. However, it is possible that a liquid water environment exists beneath the south polar cap, which may be conducive to life. Several theories for the origin of life on Earth would apply to Enceladus. These are (1) origin in an organic-rich mixture, (2) origin in the redox gradient of a submarine vent, and (3) panspermia. There are three microbial ecosystems on Earth that do not rely on sunlight, oxygen, or organics produced at the surface and, thus, provide analogues for possible ecologies on Enceladus. Two of these ecosystems are found deep in volcanic rock, and the primary productivity is based on the consumption by methanogens of hydrogen produced by rock reactions with water. The third ecosystem is found deep below the surface in South Africa and is based on sulfur-reducing bacteria consuming hydrogen and sulfate, both of which are ultimately produced by radioactive decay. Methane has been detected in the plume of Enceladus and may be biological in origin. An indicator of biological origin may be the ratio of non-methane hydrocarbons to methane, which is very low (0.001) for biological sources but is higher (0.1-0.01) for nonbiological sources. Thus, Cassini's instruments may detect plausible evidence for life by analysis of hydrocarbons in the plume during close encounters.

The recent discovery of methane in the martian atmosphere is arguably one of the most important discoveries in the field of astrobiology. One possible source of this methane could be a microorganism analogous to those on Earth in the domain Archaea known as methanogens. Methanogens are described as obligately anaerobic, and methods developed to work with methanogens typically include anaerobic media and buffers, gassing manifolds, and possibly anaerobic chambers. To determine if the time, effort, and supplies required to maintain anaerobic conditions are necessary to maintain viability, we compared anaerobically washed cells with cells that were washed in the presence of atmospheric oxygen. Anaerobic tubes were opened, and cultures were poured into plastic centrifuge tubes, centrifuged, and suspended in fresh buffer, all in the presence of atmospheric oxygen. Washed cells from both aerobic and anaerobic procedures were inoculated into methanogenic growth media under anaerobic conditions and incubated at temperatures conducive to growth for each methanogenic strain tested. Methane production was measured at time intervals using a gas chromatograph. In three strains, significant differences were not seen between aerobically and anaerobically washed cells. In one strain, there was significantly less methane production observed following aerobic washing at some time points; however, substantial methane production occurred following both procedures. Thus, it appears that aerobic manipulations for relatively short periods of time with at least a few species of methanogens may not lead to loss of viability. With the discovery of methane in the martian atmosphere, it is likely that there will be an increase in astrobiology-related methanogen research. The research reported here should simplify the methodology.

The relatively recent discoveries that liquid water likely existed on the surface of past Mars and that methane currently exists in the martian atmosphere have fueled the possibility of extant or extinct life on Mars. One possible explanation for the existence of the methane would be the presence of methanogens in the subsurface. Methanogens are microorganisms in the domain Archaea that can metabolize molecular hydrogen as an energy source and carbon dioxide as a carbon source and produce methane. One factor of importance is the arid nature of Mars, at least at the surface. If one is to assume that life exists below the surface, then based on the only example of life that we know, liquid water must be present. Realistically, however, that liquid water may be seasonal just as it is at some locations on our home planet. Here we report on research designed to determine how long certain species of methanogens can survive desiccation on a Mars soil simulant, JSC Mars-1. Methanogenic cells were grown on JSC Mars-1, transferred to a desiccator within a Coy anaerobic environmental chamber, and maintained there for varying time periods. Following removal from the desiccator and rehydration, gas chromatographic measurements of methane indicated survival for varying time periods. Methanosarcina barkeri survived desiccation for 10 days, while Methanobacterium formicicum and Methanothermobacter wolfeii were able to survive for 25 days.

Due to the hostile conditions at the surface, any life forms existing on Mars today would most likely inhabit a subsurface environment where conditions are potentially wetter and warmer, but organic compounds may be lacking and light energy for photosynthesis would be absent. Methanogens, members of the domain Archaea, are microorganisms from planet Earth that can grow under these relatively extreme conditions. We have demonstrated that certain methanogenic species can indeed grow on a Mars soil simulant, JSC Mars-1, with limited amounts of water, under conditions approaching a possible subsurface environment on Mars.

The recently established Arkansas–Oklahoma Center for Space and Planetary Science has been given a large planetary simulation chamber by the Jet Propulsion Laboratory, Pasadena, California. When completely refurbished, the chamber will be dubbed Andromeda and it will enable conditions in space, on asteroids, on comet nuclei, and on Mars, to be reproduced on the meter-scale and surface and subsurface processes monitored using a range of analytical instruments. The following projects are currently planned for the facility. (1) Examination of the role of surface and subsurface processes on small bodies in the formation of meteorites. (2) Development of in situ sediment dating instrumentation for Mars. (3) Studies of the survivability of methanogenic microorganisms under conditions resembling the subsurface of Mars to test the feasibility of such species surviving on Mars and identify the characteristics of the species most likely to be present on Mars. (4) The nature of the biochemical “fingerprints” likely to have been left by live organisms on Mars from a study of degradation products of biologically related molecules. (5) Testing local resource utilization in spacecraft design. (6) Characterization of surface effects on reflectivity spectra for comparison with the data from spacecraft-borne instruments on Mars orbiters.

One of the five lines of evidence used by McKay et al. (1996) for relic life in the Martian meteorite Allan Hills (ALH) 84001 was the presence of objects thought to be microfossils. These ovoid and elongated forms are similar to structures found in terrestrial rocks and described as "nanobacteria" (Folk, 1993; McBride et al., 1994). Using the same procedures and apparatus as McKay et al. (1996), we have found structures on internal fracture surfaces of lunar meteorites that cannot be distinguished from the objects described on similar surfaces in ALH 84001. The lunar surface is currently a sterile environment and probably always has been. However, the lunar and Martian meteorites share a common terrestrial history, which includes many thousands of years of exposure to Antarctic weathering. Although we do not know the origin of these ovoid and elongated forms, we suggest that their presence on lunar meteorites indicates that the objects described by McKay et al. (1996) are not of Martian biological origin.

It is possible that the first autotroph used chemical energy rather than light. This could have been the main source of primary production after the initial inventory of abiotic organic material had been depleted. The electron acceptor most readily available for use by this first chemoautotroph would have been CO2. The most abundant electron donor may have been H2 that would have been outgassing from volcanoes at a rate estimated to be as large as 10(12) moles yr-1, as well as from photo-oxidation of Fe+2. We report here that certain methanogens will consume H2 down to partial pressures as low as 4 Pa (4 x 10(-5) atm) with CO2 as the sole carbon source at a rate of 0.7 ng H2 min-1 microgram-1 cell protein. The lower limit of pH2 for growth of methanogens can be understood on the basis that the pH2 needs to be high enough for one ATP to be synthesized per CO2 reduced. The pH2 values needed for growth measured here are consistent with those measured by Stevens and McKinley for growth of methanogens in deep basalt aquifers. H2-consuming autotrophs are likely to have had a profound effect on the chemistry of the early atmosphere and to have been a dominant sink for H2 on the early Earth after life began rather than escape from the Earth's atmosphere to space.

Certain methanogens have been shown to grow on a Mars soil simulant following a washing procedure using a carbonate buffer. In experiments where liquid fractions from the soil simulant and water mixtures were used in place of the buffer, two out of three of the species demonstrated significantly greater methane production compared to the buffer.