Desert methane: Implications for life detection on Mars

Icarus 178 (2005) 277–280 www.elsevier.com/locate/icarus

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Desert methane: Implications for life detection on Mars Mark Moran a,∗ , Joseph D. Miller b , Tim Kral c , Dave Scott d a 606 South Alder Ave., Sterling, VA 20164, USA b Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, 1333 San Pablo St., BMT401,

Los Angeles, CA 90089-9112, USA c Department of Biological Sciences, 632 Science and Engineering Building, University of Arkansas, Fayetteville, AR 72701, USA d 420 Euclid Ave, Apt 3, Toronto, ON, M6G 2S9, Canada

Received 8 March 2005; revised 2 June 2005 Available online 11 August 2005

Abstract Methane, a potential biosignature, has recently been detected in the martian atmosphere. This Note focuses on field investigations/operational simulations and laboratory studies which resulted in successful detection of methane within arid terrestrial soils, as distinct from the usual methanogen environment, but in at least partial analogy to martian conditions.  2005 Elsevier Inc. All rights reserved. Keywords: Mars; Earth; Regolith; Exobiology

1. Introduction The Viking landers (1976) were designed to detect life on Mars through a variety of experimental approaches. The Viking I and II Labeled Release (LR) experiments (Levin and Straat, 1977, 1979) detected gas production with a scintillation detector following incubation of soil samples with a 14 C-labeled nutrient medium. The scintillation detector may have measured either CO2 or CH4 or some combination of these gases. The kinetics of the response and its abolition by heat sterilization suggested microbial generation of gas metabolites. Some investigators attributed the results to superoxide reactions in martian soil (Warmflash et al., 2002), but more recent re-analysis (Miller et al., 2002) of the same data found gas accumulation to be oscillatory, with a period of 24.66 h, one martian sol, indicative of a circadian rhythm (an excellent biosignature). A biological interpretation of the Viking results gains further support from recent observations of large amounts of water ice on Mars as well as geological evidence for flowing water in the “recent” past. Furthermore, certain terrestrial microbes can live in environments at least as extreme (e.g., low temperature, intense ultraviolet exposure) as Mars. Kral et al. (2004) have demonstrated that certain terrestrial methanogens survive in a simulated martian atmosphere and soil. The Mars Express Planetary Fourier Spectrometer group (Formisano et al., 2004) and two other independent groups (Krasnopolsky et al., 2004; Mumma et al., paper presented at American Astronomical Society Division

* Corresponding author.

E-mail address: [email protected] (M. Moran). 0019-1035/$ – see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.icarus.2005.06.008

of Planetary Science Meeting, Monterey, CA, 2003) have recently observed methane in the martian atmosphere at a concentration of 10–11 ppb. Assuming a half-life of 300 years and generation by methanogens in the upper 1 km of the regolith, atmospheric methane could be completely replenished if the generating capacity of the soil is on average about 1 attmol/cc/wk or about 0.05 ppb of the Viking LR labeled gas. A similar calculation by Krasnopolsky et al. (2004), assuming a 100-m layer, suggested this would correspond to an average density of 1 microbe/10 cc, although it is more likely that microbes would be concentrated in patches, probably juxtaposed to sources of water. In fact, water vapor and methane seem to colocalize in distinct areas (Formisano et al., 2004). While non-biological explanations (cryptic volcanism, methane clathrates) might explain such data, a biological interpretation is plausible. Terrestrial methanogens are typically found in anaerobic environments (e.g., ruminant intestines, stagnant water, methane clathrates). Presence of methanogens or their methane signature in an arid terrestrial soil would suggest that such organisms could be present in somewhat analogous arid environments on Mars. Thus, the sensitive and accurate detection of methaneproducing microbes in Earth soil is a proof of principle for similar biodetection experiments in future Mars missions.

2. Methods The prospects for detecting methanogens in arid terrestrial sites were thought comparable to the prospects for detecting methanogens in future studies on Mars, should it exist there. In light of this possibility, investiga-

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Table 1 Drilling method for gas vapor sampling from Utah Desert and Idaho High Desert 1. 2. 3. 4.

Locate site near or in the vicinity of a dry wash. Drill at least 0.5 m down; if not feasible, then reject site. Insert gas vapor probe into drilled hole without Tedlar sample bag. Clear pre-existing air from gas vapor probe by doing five purges on the hand pump. 5. Attach the Tedlar sample bag. 6. Deliver 20 squeezes of the hand pump to inflate the Tedlar sample bag. 7. Seal Tedlar valve and label the sample.

Five of 40 Utah soil samples produced methane when the samples were inoculated with a growth medium (MS). Table 3 describes the results of the soil sample analyses. Methane production was variable (extending over three orders of magnitude) in comparison with levels in Table 2. Methane production generally increased over time, strongly implying the presence of methanogenic microorganisms. However, in samples 1 and 2, methane percentages decreased in MS medium from week 3 to week 4. If methane production slows down or ceases, removal of headspace gas will result in CO2 coming out of solution, thus lowering the relative percentage of methane.The Arctic Circle, Death Valley, and Atacama soil samples were all negative for methane.

Note. All Utah samples were taken in or near dry washes. 4. Conclusions tors extracted gas vapors from drillings in the upper regolith at the Mars Desert Research Station in Utah and in the Idaho High Desert. During each drilling, the field procedure was as shown in Table 1. In Utah, four sites were scouted, 18 holes drilled, and 5 vapor samples were drawn into Tedlar plastic sample bags. Five vapor samples were also collected from the Idaho High Desert in similar fashion. Sample bags were shipped overnight to the Kral lab. Upon receipt 1-ml gas samples were drawn for gas chromatographic analysis (Varian micro-GC). Soil samples were also gathered from the Utah site (n = 40) as well as from sites in Death Valley (n = 1), the Arctic Circle (Devon Island; n = 19), and the Atacama desert in Chile (n = 1). For the Utah site a 1-in diameter drill was employed (AMS Gas Vapor Probe) over a drilling period of approximately 20 min to yield a soil sample from a depth of approximately 100 cm (Table 1). Samples from other sites were obtained in similar fashion, but at a more shallow depth (15 cm). In analogy with potential future Mars missions, terrestrial working conditions and drilling sites at the Utah site were arranged to approximate those expected on Mars—including the use of Mars analog space suits, NASA EVA operational requirements, local topography and regolith, and all-terrain vehicles. Simulation guidelines included Hoffman (2001). Investigator-crew entered the full simulation mode on April 29, 2003. Soil samples arrived at the Kral laboratory in sealed plastic ziplock bags. Upon arrival, the samples were placed into a Coy anaerobic chamber containing between 5 and 10% hydrogen gas with the remainder volume being carbon dioxide. Approximately 3–4-g samples were removed from each bag with sterile spatulas and placed into sterile anaerobic culture tubes containing 10 ml MS medium (Boone et al., 1989) or a pH 6.7 carbonate buffer that is used to make the MS medium. Thus each soil sample was inoculated into a rich organic medium or a control buffer. Each tube was sealed with a sterile rubber stopper. The tubes were removed from the chamber followed by the addition of aluminum crimps. Sterile sodium sulfide solution (125 ml of a 2.5% stock) was added to each tube using a sterile 3 ml disposable syringe to remove any residual oxygen. All tubes were pressurized with 200 kpa (above ambient) of hydrogen gas (the combination of hydrogen gas with the carbon dioxide-saturated medium supplies both an energy source and carbon source for methanogens). All culture tubes were incubated at room temperature. At 3 and 4 weeks of incubation, 1-ml headspace gas samples were injected into a Hewlett Packard 5890 gas chromatograph with a thermal conductivity detector at an oven temperature of 40 ◦ C using argon as the carrier gas. The gas chromatograph was calibrated to detect and measure hydrogen, carbon dioxide, and methane down to 10 ppm.

3. Observations Gas chromatography of the five vapor samples from four drilling sites indicated the presence of methane in three of the Utah gas samples, but in none of the Idaho gas samples. The Varian micro-GC detected nitrogen and oxygen as expected, carbon dioxide at the normal levels in the atmosphere (0.04%), and trace amounts of methane from three of the samples as seen in Table 2. For the three positive gas samples the mean and standard error was 0.059 ± 0.006% methane.

Anaerobic decomposition of organics is associated with the presence of methanogens. This condition requires a combination of muted aeration of detritus, deposition of more organic matter than can be decomposed aerobically, and/or relative cold to inhibit decomposition. The best terrestrial sites for methanogens are peat bogs where stagnant water acts as a barrier to air and the shear bulk of detritus requires far too much oxygen for aerobic respiration. The best locations for such bogs are in wet, or at least temperate, environments. Biological oxygen demand (BOD) in such sites is far too high for all the matter to be decomposed aerobically. Utah appears at first glance to be a poor terrestrial source for methane production. It is dry, with aerated soils, although its topography and soil porosity are similar to those of at least some sites in which anaerobic metabolism can occur. The Utah research site is dominated by erosional rather than depositional processes, so accumulation of organics is limited or non-existent. Also, deposition of organics deep enough to avoid aeration is probably unlikely. Good aeration of soils is evidenced by yellow/orange/red coloration in some soils, indicating oxidation of soil minerals. Given such constraints, we determined that the best places to look for methane production at the Utah site were poorly aerated channel deposits in river and creek valleys. Thus, the field research crew of Mars Desert Research Station (MDRS) examined dry washes. This region of the Mars Desert Research Station (MDRS) borders the Colorado plateau; hence hydrothermal activity is improbable as a local source of methane, as is volcanic activity. Likewise, methane production by ruminants seemed unlikely, since the dry washes were remote from grazing areas, little or no vegetation was present, and cow dung deposits were absent. We cannot exclude the possibility that cow dung at some point in the past allowed the establishment of a founder colony of methanogens. However, the main conclusion is that methanogens can survive in desert regolith, as indicated by the detection of methane in the gas samples and the production of methane in desert soil samples inoculated with an appropriate growth medium. In fact, the positive cultures continue to produce methane and are undergoing further characterization in the Kral laboratory. This is the strongest evidence for biological production of methane in these soil samples. Future studies will aim to examine the 12 C/13 C ratio as a biosignature and determine whether cold sterilization eliminates methane production. Atmospheric methane on earth is about 1800 ppb. The vapor samples exhibit about 300 times that concentration. Gas samples positive for methane were usually obtained from relatively low depths in the soil, suggesting some shielding from oxygen may be necessary for methanogen viability, consistent with the observation that oxygen is toxic to methanogens. In contrast, only 12.5% of the relatively shallow Utah soil samples (and none of the Arctic samples) were methane-positive. The depth effect and the variable presence of methane in the soil samples suggest that methanogens may be present in patches or “hot spots” in some desert soils, much as Formisano et al. (2004) has suggested may be the case for putative methanogens on Mars. But variability in methanogen density may occur even in samples taken at the same depth, explaining the considerable heterogeneity in the culture results. We conclude that the detection of methane, apparently of biological origin, in terrestrial desert regolith bodes well for

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Table 2 Description of Utah gas vapor probed sites Sample 1 May 3, 2003

Sample 2 May 3, 2003

Sample 3 May 3, 2003

Sample 4 May 3, 2003

Sample 5 May 8, 2003

GPS coordinates of drilled hole: 38◦ 22 48 N by 110◦ 46 8 W

GPS coordinates of drilled hole: 38◦ 22 48 N by 110◦ 46 8 W

GPS coordinates of drilled hole: 38◦ 23 49 N by 110◦ 45 57 W

GPS coordinates of drilled hole: 38◦ 23 49 N by 110◦ 45 54 W

GPS coordinates of drilled hole: 38◦ 26 2 N by 110◦ 47 42 W

Elevation: 1350 m

Elevation: 1350 m

Elevation 1350 m

Elevation: 1352 m

Elevation: 1365 m

Depth of sample: 75 cm

Depth of sample: 70 cm

Depth of sample: 77 cm

Depth of sample: 95 cm

Depth of sample: 155 cm

Channel deposit on river bank of White Rock Canyon. Substrate: sand, silt. Geologist reports some minor organics and some clay; very dry and aerated. Geologist infers poor chance for anaerobic respiration/decomposition.

Channel deposit on river bank of White Rock Canyon. Substrate: sand, silt. Geologist reports some minor organics and some clay; very dry and aerated. Geologist infers little chance for anaerobic respiration/decomposition.

Anthill in old reservoir bed. Reservoir not in use (nor collecting water) during previous 5+ years. To the north of White Rock Reservoir. Substrate: sand, silt, clay. Geologist reports very little organics; well aerated, dry, some orange coloration.

Old reservoir bed, same reservoir as in Sample 3 through the artificial berm that used to be the reservoir wall. Substrate: sand, silt, clay, some organics. Geologist reports very dry and “not as orange as Sample 3.”

Channel deposit in Muddy Creek tributary. Substrate: channel deposit with silt, sand, organics, and clay. Geologist reports a dark gray, rather than orange/brown color seen in the other samples; soil very dry and likely to be aerated.

Chromatography findings: 0.0480% methane, or 480 ppm

Chromatography findings: no methane detected, 0 ppm Note: This Tedlar bag shipped to the lab totally deflated. Methane undetectable even by the more sensitive Varian CP-4900 Micro-GC.

Chromatography findings: no methane detected, 0 ppm

Chromatography findings: 0.0591% methane, or 591 ppm

Chromatography findings: 0.0693% methane, or 693 ppm

Table 3 Soil samples: Percent methane concentration following incubation at room temperature in buffer or methanogen medium (MS) Sample 1 July 17, 2003

Sample 2 July 17, 2003

Sample 3 July 17, 2003

Sample 25 July 17, 2003

Sample 29 July 17, 2003

GPS coordinates: 38◦ 25 00 N, 110◦ 52 51 W

GPS coordinates: 38◦ 24 59 N, 110◦ 52 55 W

GPS coordinates: 38◦ 24 59 N, 110◦ 52 55 W

GPS coordinates: 38◦ 27 45 N, 110◦ 47 15 W

GPS coordinates: 38◦ 26 29 N, 110◦ 52 48 W

Elevation: 1438 m

Elevation: 1430 m

Elevation: 1432 m

Elevation: 1323 m

Elevation: 1463 m

Sample depth: 15 cm

Sample depth: 15 cm

Sample depth: 15 cm

Sample depth: 15 cm

Sample depth: 15 cm

Perennial spring and pond nearby

Perennial spring and pond nearby

Perennial spring and pond nearby

Confluence of Lith Canyon and Muddy Creek

Level area near the pond

Substrate: sand, clay, silt, and organics. Moderate moisture

Substrate: sand, silt, and organics. Moderate moisture

Substrate: sand, silt, and organics. Moderate moisture

Substrate: sand, clay, silt, and organics. High moisture

Substrate: sand and silt. Low moisture

3 weeks 9.246 (buffer) 22.436 (MS)

3 weeks 0 (buffer) 0.310 (MS)

3 weeks 0.553 (buffer) 0.033 (MS)

3 weeks 0.099 (buffer) 1.872 (MS)

3 weeks 0 (buffer) 0.021 (MS)

4 weeks 32.020 (buffer) 15.358 (MS)

4 weeks 0 (buffer) 0.189 (MS)

4 weeks 0.922 (buffer) 0.480 (MS)

4 weeks 7.824 (buffer) 30.839 (MS)

4 weeks 0 (buffer) 0.188 (MS)

future biodetection experiments in at least partially analogous martian environments. It is clear that the AMS Gas Vapor Probe works well for vapors extracted from terrestrial drilled holes, but the hardware and procedure would have to be adapted to Mars conditions. For example, the extraction

of vapors under Mars atmospheric pressure might provide a better measure of vapor pressure in the hole if an inert gas not present in the atmosphere were pumped behind the extracted vapors and collected along with the vapors.

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Acknowledgments We thank AMS Inc. of Idaho and General Manager Ms. Artha Chipps who waived rental fees for an AMS Soil Vapor Probe with Retract-a-Tip and Associated Soil Drilling Hardware; Carol Stoker of NASA helped in the crew selection; Brent Bos of NASA helped with the adherence to NASA procedures throughout the operational simulation aspects of the Utah experiment. We also thank Robert Zubrin and the Mars Society for help in procuring soil samples from Devon Island and Dr. Chris McKay for the samples from Death Valley and Atacama. Special thanks to John Barainca who helped with collection and geological characterization of the Utah samples. References Boone, D.R., Johnson, R.L., Liu, Y., 1989. Diffusion of the interspecies electron carriers H2 and formate in methanogenic ecosystems and its implications in the measurement of Km for H2 or formate uptake. Appl. Environ. Microbiol. 55, 1735–1741.

Formisano, V., Atreya, S., Encrenaz, T., Ignatiev, N., Giuranna, M., 2004. Detection of methane in the atmosphere of Mars. Science 306, 1758– 1761. Hoffman, S. (Ed.), 2001. The Mars Surface Reference Mission: A Description of Human and Robotic Surface Activities, NASA Document. Johnson Space Center, Houston. Kral, T.A., Bekkum, C.R., McKay, C.P., 2004. Growth of methanogens on a Mars soil simulant. Orig. Life Evol. Biosph. 34, 615–626. Krasnopolsky, V.A., Maillard, J.P., Owen, T.C., 2004. Detection of methane in the martian atmosphere: Evidence for life? Icarus 172, 537–547. Levin, G.V., Straat, P.A., 1977. Recent results from the Viking labeled release experiment on Mars. J. Geophys. Res. 82, 4663–4667. Levin, G.V., Straat, P.A., 1979. Completion of the Viking labeled release experiment on Mars. J. Mol. Evol. 14, 167–183. Miller, J.D., Straat, P.A., Levin, G.V., 2002. Periodic analysis of the Viking Lander labeled release experiment. Proc. SPIE 4495, 96–107. Warmflash, D.M., Clemett, S.J., McKay, D.S., 2002. Progress in the search for organic matter on Mars: Implications for the interpretation of the Viking labeled release data. Proc. SPIE 4495, 89–95.