Arkansas-Oklahoma
Center for Space and Planetary Sciences
 
 

Introduction

      Current ideas on the origin of life start with the synthesis of the biogenic elements in stars, and proceed through the formation of organic molecules in the interstellar medium and their incorporation onto planetesimals and planets in the solar nebula and end with the formation of self-replicating molecules that ultimately formed the microbial communities (Allamendola et al., 1988; Cronin et al., 1988; Miller and Orgil, 1974; Schopf, 1983; McKay et al., 1992).  Theories of nucleosynthesis, interstellar chemistry, meteorite organic analysis and laboratory simulation experiments are broadly consistent with this outline.  It is thus reasonable to assume that life will evolve on any planet with conditions even remotely similar to those on Earth.
 The Viking experiments have shown that although the surface dust on Mars has unusual chemical properties, it is unlikely that life currently exists on the surface of the planet (Klein et al., 1992).  Of course, it is impossible to argue that this is true of the entire surface, and that rare oases of life may exist near the poles or elsewhere, but this is thought unlikely.
 The Viking Landers only examined the surface of Mars where conditions are probably too dry, too cold, and too oxidizing for known life forms to exist (Klein, 1978; Klein, 1979). It may be possible that liquid water can exist and persist below the surface. There is evidence that liquid water has been present in subsurface aquifers on Mars throughout its history (McKay and Stoker, 1989; McKay, 1997). Lifeforms existing below the surface could not obtain their energy from photosynthesis, but rather they would have to utilize chemical energy. Because the Viking Landers found no measurable quantities of organic matter, lifeforms might be limited to oxidation of inorganic matter for energy. Organisms that fall into this category are referred to as chemoautotrophs. Methanogens, microorganisms in the domain Archaea, are chemoautotrophs that consume molecular hydrogen and carbon dioxide and produce methane as a waste product. In addition to requiring liquid water, many terrestrial microbes require oxygen, but this is not true of the methanogens, some of which are found around deep sea hydrothermal vents in the Pacific Ocean (Jones et al., 1983). The methane-producing reaction is highly energetic and could proceed in the dark at depth in the martian soil.  If this process is occurring on Mars, it is not occurring on a widespread global scale because methane has not been detected above a 0.02 ppm detection limit (Maguire, 1977). The half-life of methane in the atmosphere is only 300 years (Hunten, 1979b), so "hot-spots" would quickly disappear.
A potential habitat for existence of methanogens on Mars might be based on a geothermal source of hydrogen, possibly due to volcanic or hydrothermal activity, or the reaction of basalt and anaerobic water (Boston et al., 1992; Stevens and McKinley, 1995; Anderson et al., 1998), carbon dioxide, which is abundant in the martian atmosphere, and subsurface liquid water. Knowing that certain methanogens can grow on Mars soil simulant with hydrogen, carbon dioxide, and very little water, we want to ultimately subject the methanogens to a simulated martian environment.

Objectives and Significance

 Unpublished preliminary studies in our laboratory indicate that certain methanogens grow well on Mars soil simulant with carbon dioxide, molecular hydrogen, and very limited amounts of water. If molecular hydrogen is present below the surface of Mars, with even small amounts of liquid water, then all requirements for methanogenic growth are present. Even if hydrogen is not present, carbon monoxide is known to be present, and some methanogens can use carbon monoxide instead of molecular hydrogen. Studies to determine the minimal amount of water that will allow for growth on Mars soil simulant under various environmental conditions (temperature, pH, pressure, inoculum size) are currently underway and will certainly have relevance to the possibility of life surviving on Mars, currently or in the past.
 The present work seeks to explore the range of martian conditions over which methanogenic microorganisms can grow and/or survive in order to determine the likelihood of such organisms currently existing on Mars.  Even if it is found that it is unlikely that methanogens could exist today on Mars, the present experiments should better enable us to determine how likely it was that such organisms could have existed on Mars 3.5 Ga or more ago when conditions may have been wetter (Fanale et al., 1992).

Methods and Discussion

 With respect to growth on Mars soil simulant, preliminary results show that of the four species of methanogens tested, M. wolfei  (Kral and Bekkum, 1999a) and M. barkeri are able to grow on Mars soil simulant with reduced amounts of water quite well while M. formicicum grows more slowly (Kral and Bekkum, 1999a; 1999b). So far, M. maripaludis has shown no growth on Mars soil simulant. M. wolfei shows methane production when 2 mL or more of cell suspension are added to 5 g of Mars soil simulant. As the water content increases up to standing liquid, the methane increases. (Volumes greater than 3.5 mL result in standing liquid.) M. barkeri grows fairly well when 1 mL is added to the 5 g of soil simulant. A small amount of methane is even evident at 0.5 mL.

Table 1.  Methane production by three methanogens on Mars
soil simulant with variable amounts of water.
______________________________________________
Organism        Liquid     Volume      Adjusted     Methane (%)
                      Added   (% Watera) 7 days         14 days
                      (mL)
______________________________________________
M. wolfei         0.5             21         NDb           ND
M. wolfei         1.0             28         0.003          0.009
M. wolfei         2.0             38         0.131          5.862
M. wolfei         3.0             46         5.928          7.400
M. wolfei         4.0             52         3.513          8.437
M. barkeri        0.5            21         0.020          0.056
M. barkeri        1.0            28         0.609          1.335
M. barkeri        2.0            38         2.789          6.270
M. barkeri        3.0            46         1.523          6.331
M. barkeri        4.0            52         1.002          2.591
M. formicicum  0.5            21          0.005          0.015
M. formicicum  1.0            28          0.003          0.003
M. formicicum  2.0            38          0.003          0.064
M. formicicum  3.0            46          0.075          0.566
M. formicicum  4.0            52          0.104          0.275
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a    The Mars soil simulant already contains considerable water
      (Allen et al., 1998). Direct determination gives a water
      content   of 13 wt% (Dreibus-Kapp, personal communication).
      Thus, the adjusted % water (wt/wt) is the sum of the 13%
      water content plus the added water.
b    ND indicates that methane was not detected by the gas
      chromatograph.

     Of significance to this proposal is the fact that M. barkeri and M. wolfei grow well when the soil is not totally saturated with water. Rather, a fraction of the soil is saturated (defined as the least concentration of water where the micropores and macropores of the soil are filled with water [Brady, 1990]) while the surrounding soil is dry.
     The overall plan is to add approximately one cubic meter of Mars soil simulant to the Andromeda chamber. We will then add small volumes of liquid methanogenic cultures to Mars soil simulant at various depths. Since these organisms are anaerobic and will be added while the chamber is open and exposed to oxygen, the cultures will be added in the frozen state. Cultures in tubes or bottles (depending on the volume to be added) will be frozen at –80C. Immediately prior to adding to the soil simulant, the aluminum crimps and stoppers will be removed. The tubes or bottles will be placed inverted into the soil simulant at predetermined depths. (Once they melt, gravity will cause the liquid cultures to flow into the soil simulant and saturate only a small volume of simulant at their locations.) Previous experiments in our laboratory have shown that these methanogens have remained viable in the frozen state for at least18 months. Immediately following placement of the tubes/bottles, the chamber will be closed and evacuated to remove oxygen (before the cultures melt). The chamber will be equipped with detectors at various depths and locations for temperature, methane, hydrogen, carbon dioxide, carbon monoxide, and oxygen (there is a small fraction of molecular oxygen in the martian atmosphere, therefore we will add that same fraction to some of our experiments). Thus, we will know the temperature at depths below the surface and we will be able to measure any methane production by specific cultures. We will also be able to measure hydrogen, carbon dioxide, carbon monoxide, and oxygen concentrations at various locations in the soil simulant.
     In initial experiments, we will attempt to simulate the ideal conditions for growth of these methanogens as determined by our previous research. The atmosphere will be 75:25 hydrogen:carbon dioxide at ambient pressure. The atmospheric temperature will coincide with the ideal temperature for each organism being tested (55oC for M. wolfei, 37oC for M. formicium and M. barkeri, 25oC for M. maripaludis, and 15oC for Methanogenium frigidum (a methanogen not described above which shows growth down to 0oC). Cultures will be placed on the surface of the soil simulant.
     Once it is determined that any or all of these organisms can grow in the vacuum chamber, we will begin to systematically change individual factors (temperature, pressure, atmospheric composition, depth of culture, radiation) in follow-up experiments such that the new conditions more closely resemble those on Mars. (It should be noted all of these factors will differ in the soil simulant from what they are in the atmosphere depending on the depth.) Temperature in follow-up experiments will be lowered, but only to the extent that water below the surface does not freeze. (Temperatures on the surface of Mars do range up to greater than 20C [Barth et al., 1992].) Pressure will be lowered to 6mb (Barth et al., 1992). The atmospheric composition will be shifted to 95% carbon dioxide with smaller amounts of nitrogen, argon, carbon monoxide and even traces of oxygen to mimic the martian atmosphere. The objective is to determine if any of the methanogens tested can grow (or just survive) at any depth in the soil simulant while conditions at the surface of the simulant mimic or approach those on the surface of Mars. If we reach conditions where grow ceases (as measured by methane production), methanogens that have been subjected to those conditions for various lengths of time can be “shifted up” to conditions that allow growth to test for viability. This may be an important consideration knowing that surface conditions on Mars change regularly with seasonal changes.

     The major equipment requirements are a "bucket" that would hold the Mars soil simulant and be lowered into the chamber, and a detector that could measure methane, carbon dioxide, carbon monoxide, hydrogen, and oxygen at various depths and locations in the soil simulant.

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