Methane Production on Rock and Soil Substrates by Methanogens: Implications for Life on Mars

Bioastronomy 2007: Molecules, Microbes, and Extraterrestrial Life ASP Conference Series, Vol. 420, 2009 K. J. Meech, J. V. Keane, M. J. Mumma, J. L. Siefert, and D. J. Werthimer, eds.

H. A. Kozup and T. A. Kral 1West Virginia University, WV 26505 2Arkansas Center for Space and Planetary Sciences, University of Arkansas, Fayetteville 72701

Abstract. In order to understand the methanogens as models for possible life on Mars, and some of the factors likely to be important in determining their abundance and distribution, we have measured their ability to produce methane on a few types of inorganic rock and soil substrates. Since organic materials have not been detected in measurable quantities at the surface of Mars, there is no reason to believe that they would exist in the subsurface. Samples of three methanogens (Methanosarcina barkeri, Methanobacterium formicicum, and Methanothermobacter wolfeii) were placed on four substrates (sand, gravel, basalt, and a Mars soil simulant, JSC Mars-1) and methane production measured. Glass beads were used as a control substrate. As in earlier experiments with JSC Mars-1 soil simulant, a crushed volcanic tephra, methane was produced by all three methanogens when placed on the substrates, sand and gravel. None produced methane on basalt in these experiments, a mineral common in Martian soil. While these substrates do not represent the full range of materials likely to be present on the surface of Mars, the present results suggest that while some surface materials on Mars may not support this type of organism, others might.

1. Introduction

The possibility of life on Mars has been a subject of intense interest for many decades, but only recently have we learned enough about Martian surface conditions to seriously address the issue. The Viking missions of the mid-1970s, though a spectacular engineering accomplishment, shed little light on the issue, yielding results that initially appeared to be positive, but were subsequently interpreted by most researchers as reflecting an unexpected inorganic chemistry. However, the Viking missions did confirm that conditions at the surface are rather harsh, conceivably too harsh to support any known life forms (Klein, 1978; Klein, 1979; Klein et al., 1992). The possibility of life below the surface is another matter. Recent evidence for global distributions of subsurface ice (Boynton et al., 2002; Feldman et al., 2002; Mitrofanov et al., 2002) and indica- tions that liquids flowed on the surface in recent times (Christensen et al., 2004; Herkenhoff et al., 2004; Klingelhofer et al., 2004; Rieder et al., 2004; Squyres et al., 2004a; Squyres et al., 2004b) mean that the possibility of extremophiles existing below the surface cannot be entirely excluded. We have been exploring the possibility that methanogens, microorganisms in the domain Archaea, could survive on Mars in subsurface environments. In general, methanogens metabo- 137 138 Kozup and Kral lize H2, using CO2 as carbon source, without a need for organic carbon (even though they are ubiquitous in environments associated with the decomposition of organic matter [Sowers, 1995]). The recent discovery of methane in the atmosphere of Mars is highly significant in connection with the possible presence of these organisms on Mars (Formisano et al., 2004; Malik, 2004; Krasnopolsky et al., 2004; Krasnopolsky, 2005). One factor that must be considered is whether there are reasonable soil/rock substrates – reasonable in terms of what we know about the surface of Mars – that would support methanogenesis on the planet. We have shown that three species of methanogens produce methane when incubated on the Mars soil simulant, JSC Mars-1 (Kral et al, 2004), a crushed volcanic tephra from Hawaii. However, as in all simulation experiments, there is an issue as to whether this substrate is relevant to Mars, and if so, how widespread such material might be. We have therefore proposed to examine the suitability of a few other inorganic substrates, in order to explore whether the identity of the substrate is important and, if so, gain some insight into the type of materials likely to support methanogens on Mars. Since organic materials have not been detected in measurable quantities at the surface of Mars (Bie- mann et al., 1977; Biemann, 1979), there is no reason to believe that they would exist in the subsurface. Here we report attempts to measure methanogenesis on a number of substrates, sand and gravel which are typical streambed deposits, basalt which might be expected in the vicinity of the huge volcanic regions on Mars, and a Mars soil simulant, JSC Mars-1.

2. Materials and Methods

2.1. Cultures and Growth Media The methanogens were obtained from David Boone, Portland State Univer- sity, OR. Each methanogenic strain was grown in a medium that supported growth (MS medium [Boone et al., 1989] for Methanosarcina barkeri [OCM 38] and Methanobacterium formicicum [OCM 55]; MM medium [Xun et al., 1988] for Methanothermobacter wolfeii [OCM 36; formerly Methanobacterium wolfei]. Growth media were prepared under 90% carbon dioxide and 10% hydrogen in a Coy anaerobic environmental chamber. Media made in the described atmosphere were saturated with carbon dioxide. This resulted in a pH of 6.6, which allows for good growth of the methanogens being studied. The 10% hydrogen is only required for the oxygen-removing palladium catalysts to function prop- erly. The catalysts facilitate the reaction of the residual molecular oxygen with the molecular hydrogen to form water. (Methanogens are strict anaerobes and will not grow or produce methane in the presence of molecular oxygen [Zin- der, 1993].) The anaerobically-prepared media were added to anaerobic culture tubes and sealed as described by Boone et al. (1989). At least one hour prior to inoculation, a sterile 2.5% sodium sulfide solution was added to each tube (0.15 mL per 10 mL medium) to eliminate any residual molecular oxygen (Boone et al., 1989). 2.2. Preparation of the Soil and Rock Samples Four different soil and rock samples were used in these experiments. They in- cluded a Mars soil simulant [JSC Mars-1], sand, gravel, and basalt. A fifth Methane Production 139 substrate, glass beads (Pyrex brand), served as a negative control. JSC Mars-1 is a fraction from altered volcanic ash from a Hawaiian cinder cone that approx- imates the reflectance spectrum, composition, grain size, density, and magnetic properties for the oxidized soil of Mars (Allen et al., 1998). The sand (silicate [SiO2] mineral [Leet, 1982]) and the gravel (natural mixture of various rock frag- ments resulting from erosion [Leet, 1982]) were from Quickrete Companies, Inc., Atlanta GA. The basalt (pulverized) was obtained from Todd Stevens, Portland State University. It is composed of plagioclase feldspars and ferromagnesium silicates (Leet, 1982). The sand and gravel were washed in deionized water to remove any loose de- bris and fine-grained dust. Because of the small grain size of the JSC Mars-1 and the basalt, washing was not feasible. Following the washing procedure, the sand, gravel and basalt were analyzed for total organic compounds at the Arkansas Wa- ter Resource Center, Water Quality Laboratory, University of Arkansas, Fayet- teville, using an Inductively Coupled Argon Plasma Spectrophotometer. (JSC Mars-1 had previously been tested for total organic compounds [Kral et al., 2004]). Five grams of each type of soil or rock were placed into anaerobic culture tubes. All of the tubes containing soil and rock samples were placed into a Coy anaerobic chamber, allowed to acclimate to the hydrogen:carbon dioxide atmosphere for 24 hours, then stoppered. The sealed tubes were removed from the chamber, crimped, and autoclaved at 121C for 30 minutes at 100 kPa above ambient.

2.3. Methane Production on Different Soil and Rock Substrates Actively growing cells (approximately 0.1 O.D. at 675nm) of all three methanogens were centrifuged at 4200 rpm for 45 min, and then washed with sterile carbonate buffer (the same buffer used to make methanogenic growth medium). At least an hour earlier, sterile sodium sulfide solution was added to the buffer to eliminate residual oxygen. This washing procedure was repeated three times. Following the final washing, the cell pellets were suspended in the same buffer. Four milliliters of each organism were added to individual tubes of each of the soil and rock substrates. (We have shown previously [Kral et al., 2004] that 4 mL is ideal for growth on 5 g of JSC Mars-1.) All tubes were pressurized with 180 kPa (above ambient) of 75:25 H2:CO2. The tubes were incubated at temperatures within the growth range for the respective methanogens (37 oC for M. barkeri and M. formicicum; 55 oC for M. wolfeii). Methane was measured as a percentage of the total headspace gas. Headspace gas samples (1 mL) were removed with a plastic syringe at time intervals of 0, 72, 144, 312 and 480 hours after inoculation and manually injected into the port of a Hewlett Packard model 5890 gas chromatograph with a thermal conductivity detector at an oven temperature of 40 oC using argon as the carrier gas.

2.4. Transfer Experiments Washed cell cultures mixed with soil and rock samples were prepared as described in the previous section. Gas chromatographic measurements were recorded at 0, 72, and 144 hours. At 168 hours after inoculation, 4 mL of sterile methanogenic medium buffer (with sodium sulfide) were added to each culture tube in the anaerobic chamber using a sterile syringe. The tubes were inverted 140 Kozup and Kral a few times and then allowed to settle for five minutes. The tubes were then carefully inverted, one at a time, allowing the liquid fraction to collect at the stopper end of each tube. Using a clean syringe, 4 mL were removed and trans- ferred to a fresh, sterile tube of the exact same soil or rock substrate. All tubes were pressurized with 75:25 H2:CO2 at 180 kPa and incubated at their respective temperatures. Gas chromatographic measurements were again recorded at 0, 72 and 144 hours. This transfer procedure was repeated two more times. All transfer experiments were performed in triplicate.

3. 2.5. pH Measurements

One gram of each type of substrate was added to an anaerobic tube. Each tube was placed into the Coy anaerobic chamber and allowed to acclimate for three hours. They were then sealed with rubber stoppers, crimped and autoclaved. They were placed back into the anaerobic chamber, crimps and rubber stoppers removed, and then 1 mL of sterile buﬀer solution (with sodium sulﬁde) was added to each. The contents of each tube were agitated at 5-minute interval for 25 minutes. The tubes were then sealed with rubber stoppers, removed from the chamber, and the contents of each tube were immediately subjected to pH measurement.

4. Results

In the first set of experiments, where medium-grown washed cells were added directly to soil and rock substrates, increasing methane concentrations were observed in all tubes including the glass beads (data not shown). Methane production in the negative control led to the transfer experiments where, theoretically, any residual nutrients from the original growth medium would eventually be diluted out and endogenous energy reserves from the medium-grown cultures would be exhausted. All three organisms showed no methane production following two transfers on glass beads (M. barkeri showed no methane production after the first transfer). Figures 1, 2 and 3 show methane production for M. barkeri, M. formicicum and M. wolfeii, respectively, following the third transfer on various soil and rock substrates (the third transfer increases our confidence that residual nutrient carryover and endogenous energy stores are insignificance). All three methanogens produced methane on sand, gravel, and JSC Mars-1. M. barkeri (Figure 1) and M. wolfeii (Figure 3) showed no significant differences on the three substrates as indicated by overlapping error bars. M. formicicum (Fig- ure 2) produced significantly greater methane on sand than on the other two substrates. All three organisms failed to produce methane on basalt in these experiments. Analysis of the sand, gravel and basalt revealed no measurable organic compounds, thus the methanogenesis observed on sand and gravel was not due to metabolism of organic material. Even though JSC Mars-1 contains traces of organic matter (79 mg/kg), we have demonstrated (Kral et al., 2004) that these organic compounds cannot support methane production in the absence of hydrogen, carbon dioxide or both. Results from the pH studies showed a rather Methane Production 141

Figure 1. Methane production by Methanosarcina barkeri on diﬀerent soil and rock substrates following a third transfer on the same substrates. Each point represents the average of three measurements. Error bars represent +/- one standard deviation. narrow range of pH values from a low of 6.6 for basalt to a high of 6.8 for sand and gravel.

5. Discussion

Results from the first set of experiments, where washed cells were added directly to soil and rock substrates, showed methane production on all substrates, including the glass beads. Possible explanations for methane production on the negative control include endogenous energy reserves and residual nutrients following the washing procedure. With respect to endogenous energy reserves, most organisms store reserve material when in excess for times when there are limited or exhausted energy sources. Reserve polymers of glycogen and polyphosphate have been detected in methanogens (Zinder, 1993). Research on Methanosarci- naceae has shown that they contain storage granules, as described by Sprott and Beveridge (1993) that are polyphosphate-like electron dense particles. Using X- ray microanalysis, these particles were shown to contain stored macronutrients of P, Ca, Fe and sometimes Mg, S and Cl. In one species, 14% of the cell’s weight was made up of the stored nutrients in these granules. With respect to the washing procedure, it may have been incorrectly assumed in the first set of experiments that washing the cells three times with a buffer would have been sufficient to dilute the nutrient pool to insignificant levels. Nonetheless, the follow-up transfer experiments were very successful in demonstrating which substrates supported methane production for each of the three methanogens. 142 Kozup and Kral

Figure 2. Methane production by Methanobacterium formicicum on diﬀer- ent soil and rock substrates following a third transfer on the same substrates. Each point represents the average of three measurements. Error bars represent +/- one standard deviation.

In all cases, the second transfer resulted in lack of methane production on glass beads. We chose to report the results for the third transfer, two removed from the negative controls that were positive for methane. Since we were only interested in knowing which substrates supported methane production, we only have three measurements for each organism on each substrate for 144 hours of incubation. However, with three replicates for each experiment, we are confident in concluding which substrates supported methane production. Results from pH measurements would seem to eliminate pH as a factor contributing to the observed differences. The narrow range of pH values (6.6 – 6.8) falls within the ranges for optimal growth of these organisms (6.5-7.5 for M. barkeri, 6.6-7.8 for M. formicicum [Sowers, 1995], 5.8-7.7 for M. wolfeii [data from our laboratory]). It should be noted that methane production is what was measured and reported here. Measurement of cell growth by gas chromatographic analysis of methane production is unique to the methanogens and is the most rapid and commonly used technique to measure growth (Sowers, 1995). It is certainly conceivable that methane production could be uncoupled from actual growth (increase in cell mass) under certain conditions. However, we have demonstrated (Kral et al., 2004) that increasing methane production on JSC Mars-1 is correlated with increase in total organic carbon. In light of the insignificant differences in methane production by the three methanogens on most of the substrates (the only case of significantly greater methane production was by M. formicicum on sand [Figure 2]) reported here, we have no reason to believe that Methane Production 143

Figure 3. Methane production by Methanothermobacter wolfeii on different soil and rock substrates following a third transfer on the same substrates. Each point represents the average of three measurements. Error bars represent +/- one standard deviation. the methanogens are not growing in these experiments. The observation that all three methanogens produced methane on sand, gravel and JSC Mars-1 means that they must be obtaining their nutrient requirements (other than H2 and CO2) from these substrates. (There is typically about 1% atmospheric N2 in the headspace gas [from the anaerobic chamber where the buffer is stored]. Some methanogens including M. barkeri [Murray and Zinder, 1984] and M. formicicum [Magingo and Stumm, 1991] have been shown to fix nitrogen.) In addition to nitrogen, methanogens growing on H2 and CO2 typically require phosphate, sulfur, magnesium, iron, cobalt, nickel and molybdenum (Scherer et al., 1982; Schonheit et al., 1979) and/or tungsten (Winter et al., 1984; van Bruggen et al., 1986; Widdel, 1986). The sodium sulfide added to scavenge residual oxygen would supply sulfur. Sodium hydroxide pellets (J.T. Baker, 98% min) are used to make the buffer, and like any chemical reagent, contain trace impuri- ties. Analyses show the presence of nitrogen (<3 ppm), phosphate (<1 ppm), sulfate (<5 ppm), iron (<2 ppm), and nickel (<5 ppm). Other possible sources of trace contaminants are the sodium sulfide solution (sodium sulfide, nonahy- dride, Sigma, trace analysis not listed), the carbon dioxide gas that is bubbled through the buffer, the deionized water, and even the stainless steel needle on the syringe used for inoculum transfer. The bottom line is that there was no measurable methane production by any of the three methanogens on glass beads. Additionally, there was no methane production on basalt which contains iron and magnesium as part of its chemical makeup (Leet, 1982). The combina- 144 Kozup and Kral tion of contaminant nutrients along with the H2, CO2 and N2 are apparently not sufficient to support methanogenesis alone. We can also rule out some in- hibitory factor in the basalt that might be preventing methanogenesis because in the first set of experiments, where washed cells were added directly to the substrates, there was methane production by all three methanogens, compara- ble to that seen on the other substrates (data not shown). The “additional” required nutrients must be present in sand, gravel and JSC Mars-1, but not in basalt. Methanogens are commonly associated with environments with high carbon loads (sewage digestors, peat bogs, cattle pastures, bovine rumens), and are ubiquitous in environments associated with decomposition of organic matter (Sowers, 1995). In these rich organic environments, methanogens are the terminal members of a three-member consortium of microorganisms. The first two members, using interspecies hydrogen transfer, ultimately produce hydrogen and acetate as the major intermediates, which the methanogens then consume (Sowers, 1995; Bryant et al., 1967; Bryant, 1979). Thus, the observation that all three methanogens did produce methane on inorganic substrates is not neces- sarily unexpected. Today, one of the most widely favored hypotheses concerning the origin of life on Earth is that the first microorganisms were chemoautotrophs (Bennett et al, 2003), and that methanogens may have been one of the first of these to evolve (Ehrlich, 1990). Hydrogen and iron compounds were abundant on the early Earth, so energy sources were available (Bennett et al., 2003). They probably would have existed at a time when our planet was more Mars-like, before atmospheric oxygen concentrations began to rise (between 2 and 3 billion years ago) due to oxygenic photosynthesis, and before the deposition of the tremendous quantities of organic matter that we observe today (Bennett et al., 2003).

6. Summary

In summary, these three methanogenic species metabolize (methanogenesis) on sand, gravel and JSC Mars-1, substrates that may have similar counterparts on Mars. The observation that the methanogens tested here did not metabolize on basalt is interesting, especially since Mars is known to contain a considerable quantity of this mineral (McSween, 1994). However, this may be a moot point since methanogens are ubiquitous on Earth (Sowers, 1995), another basalt-rich planet (Leet, 1982). While these substrates do not represent the full range of materials likely to be present on the surface of Mars, the present results suggest that while some surface materials on Mars may not support this type of organism, others might.

Acknowledgments. This work was supported by a grant from the NASA- Ames University Consortium Oﬃce, and funding from the Arkansas Center for Space and Planetary Sciences, University of Arkansas, Fayetteville. Special thanks go to Derek Sears for reviewing this manuscript. Methane Production 145

References

Allen, C.C., Jager, K.M., Morris, R.V., Lindstrom, D.J., Lindstrom, M.M., Lockwood, J.P. 1998, EOS, 79, 405. Bennett, J., Shostak, S., Jakosky, B. 2003, in Life in the Universe. Addison Wesley, San Francisco, p. 127. Biemann, K. 1979, J. Mol. Evol. 14, 65. Biemann, K., Oro, J., Toulmin, P., III, Orgel, L.E., Nier, A.O., Anderson, D.M., Sim- monds, P.G., Flory, D., Diaz, A.V., Rushneck, D.R., Biller, J.E., LaFleur, A.L. 1977, J. Geophys. Res. 82, 4641. Boone, D.R., Johnson, R.L., Liu, Y. 1989, Appl. Environ. Microbio. 55, 1735. Boynton, W.V., Feldman, W.C., Squyres, S.W., Prettyman, T., Bruckner, J., Evans, L.G., Reedy, R.C., Starr, R., Arnold, J.R., Drake, D.M., Englert, P.A.J., Met- zger, A.E., Mitrofanov, I., Trombka, J.I., d’Uston, C., Wanke, H., Gasnault, O., Hamara, D.K., Janes, D.M., Mancialis, R.L., Maurice, S., Mikheeva, I., Taylor, G.J., Tokar, R., Shinohara, C. 2002, Science Online, http://www.sciencemag.org/cgi/content/abstract/1073722v1 http://www.sciencemag.org/cgi/content/abstract/1073722v1. Bryant, M.P. 1979, J. Animal Sci. 48, 193. Bryant, M.P., Wolin, E.A., Wolin, M.J., Wolfe, R.S. 1967, Arch. Microbiol. 59, 20. Christensen, P.R, Wyatt, M.B., Glotch, T.D., Rogers, A.D., Anwar, S., Arvidson, R.E., Bandfield, J.L., Blaney, D.L., Budney, C., Calvin, W.M., Fallacaro, A., Ferga- son, R.L., Gorelick, N., Graft, T.G., Hamilton, V.E., Hayes, A.G., Johnson, J.R., Knudson, A.T., McSween, H.Y., Mehall, G.L., Mehall, L.K., Moersch, J.E., Mor- ris, R.V., Smith, M.D., Squyres, S.W., Ruff, S.W., Wolff, M.J. 2004, Science 306, 1733. Ehrlich, H.L. 1990, Geomicrobiology (2nd ed.) Marcel Dekker, Inc. New York. Feldman, W.C., Boynton, W.V., Tokar, R.L., Prettyman, T.H., Gasnault, O., Squyres, S.W., Elphic, R.C., Lawrence, D.J., Lawson, S.L., Maurice, S., McKinney, G.W., Moore, K.R., Reedy, R.C. 2002, Science Online, http://www.sciencemag.org/cgi/content/abstract/1073541v1. Formisano, V., Atreya, S., Encrenaz, T., Ignatiev, N., and Giuranna, M. 2004, Science 306, 1758. Herkenhoff, K.E., Squyres, S.W., Arvidson, R., Bass, D.S., Bell, J.F., Bertelsen, P., Ehlmann, B.L., Farrand, W., Gaddis, L., Greeley, R., Grotzinger, J., Hayes, A.G., Hviid, S.F., Johnson, J.R., Jolliff, B., Kinch, K.M., Knoll, A.H., Madsen, M.B., Maki, J.N., McLennan, S.M., McSween, H.Y., Ming, D.W., Rice, J.W., Richter, L., Sims, M., Smith, P.H., Soderblom, L.A., Spanovich, N., Sullivan, R., Thompson, S., Wdowiak, T., Weitz, C., Whelley, P. 2004, Science 306, 1727. Klein, H.P. 1978, Icarus 34, 666. Klein, H.P. 1979, Rev. Geophys. Space Phys. 17, 1655. Klein, H.P., Horowitz, N.H. and Biemann, K. 1992, in Mars, ed. H.H. Kieffer, B.M. Jakosky, C.W. Snyder, M.S. Matthews, University of Arizona Press, Tucson, 1221. Klingelhofer, G., Morris, R.V., Bernhardt, B., Schroder, C., Rodonov, D.S., de Souza, P.A., Yen, A., Gellert, R., Evlanov, E.N., Zubkov, B., Foh, J., Bonnes, U., Kankeleit, E., Gutlich, P., Ming, D.W., Renz, F., Wdowiak, T., Squyres, S.W., Arvidson, R.E. 2004, Science 306, 1740. Kral, T.A., Bekkum, C.R., McKay, C.P. 2004, Origins Life Evol. Biosphere. 34, 615. Krasnopolsky, V.A., Maillard, J.P., Owen, T.C. 2004, Icarus. 172, 537. Krasnopolsky, V.A. 2005, Icarus. 180, 359. 146 Kozup and Kral

Leet, L.D. 1982, Physical Geology, 6th Ed. Prentice Hall, Englewood Cliffs, N.J. Magingo, F.S.S., Stumm, C.K. 1991, FEMS Microbiol. Lett. 81, 273. Malik, T. 2004, CNN.com. http://www.cnn.com/2004/TECH/space/03/30/mars.methane/index.html http://www.cnn.com/2004/TECH/space/03/30/mars.methane/index.html McSween H. Y., Jr. 1994, Meteoritics 29 , 757. Mitrofanov, I., Anfimov, D., Kozyrev, A., Litvak, M., Sanin, A., Tret’yakov, V., Krylov, A., Shvetsov, V., Boynton, W., Shinohara, C., Hamara, D., Saunders, R.S. 2002, Mars Odyssey. Science Online, http://www.sciencemag.org/cgi/content/abstract/107361v1. Murray, P.A., Zinder, S.H. 1984, Nature 312, 284. Rieder, R., Geller, R., Anderson, R.C., Bruckner, J., Clark, B.C., Dreibus, G., Economou, T., Klingelhofer, G., Lugmair, G.W., Ming, D.W., Squyres, S.W., d’Uston, C., Wanke, H., Yen, A., Zipfel, J. 2004, Science 306, 1746. Scherer, P., Lippert, H., Wolff, G. 1983, Biol. Trace Elem. Res. 5, 149. Sowers, K.R. 1995, Methanogenic Archaea: An Overview. in Archaea: A Laboratory Manual, Methanogens, ed. K.R. Sowers and H.J. Schreier, Cold Spring Harbor Laboratory Press, Plainview, NY, 3, 93. Sprott, G.D., Beveridge, T.J. 1993, in Methanogens, ed J.G. Ferry, Chapman and Hall, NY. 81. Schonheit, P., Moll, J., Thauer, R.K. 1979, Arch. Microbiol. 123, 105. Squyres, S.W., Arvidson, R.E., Bell, J.F., Bruckner, J., Cabrol, N.A., Calvin, W., Carr, M.H., Christensen, P.R., Clark, B.C., Crumpler, L., Des Marais, D.J., d’Uston, C., Economou, T., Farmer, J., Farrand, W., Folkner, W., Golombek, M., Corevan, S., Grant, J.A., Greeley, R., Grotzinger, J., Haskin, L., Herkenhoff, K.E., Hviid, S., Johnson, J., Klingelhofer, G., Knoll, A.H., Landis, G., Lemmon, M., Li, R., Madsen, M.B., Malin, M.B., McLennan, S.M., McSween, H.Y., Ming, D.W., Moersch, J., Morris, R.V., Parker, T., Rice, J.W., Richter, L., Rieder, R., Sims. M., Smith, M., Smith, P., Soderblom, L.A., Sullivan, R., Wanke, H., Wdowiak, T., Wolff, M., Yen, A. 2004a, Science 306, 1698. Squyres, S.W., Grotzinger, J.P., Arvidson, R.E., Bell, J.F., Calvin, W., Christensen, P.R., Clark, B.C., Crisp, J.A., Farrand, W.H., Herkenhoff, K.E., Johnson, J.R., Klingelhofer, G., Knoll, A.H., McLennan, S.M., McSween, H.Y., Morris, R.V., Rice, J.W., Rieder, R., Soderblom, L.A. 2004b, Science 306, 1709. Van Bruggen, J.J.A., Zwart, K.B., Hermans, J.G.F., van Hove, E.M., Stumm, C.K., Vogels, G.D. 1986, Arch. Microbiol. 144, 367. Widdel, F. 1986, Appl. Environ. Microbiol. 51, 1056. Winter, J., Lerp, C., Zabel, H-P., Wildenauer, F.X., Konig, H., Schindler, F. 1984, System. Appl. Microbiol. 5, 457. Xun, L., Boone, D.R., Mah, R.A. 1988, Appl. Environ. Microbio. 54, 2064. Zinder, S.H. 1993, in Methanogens, ed J.G. Ferry, Chapman and Hall, NY. 128.