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Low pressure microenvironments: Methane production at 50 mbar and 100 mbar by methanogens
Planetary and Space Science xxx (2017) 1–10
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Low pressure microenvironments: Methane production at 50 mbar and 100 mbar by methanogens Rebecca L. Mickol a, Timothy A. Kral a, b, * a b
Arkansas Center for Space and Planetary Sciences, University of Arkansas, Fayetteville, AR, USA Department of Biological Sciences, University of Arkansas, Fayetteville, AR, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Methane Methanogens Low pressure Astrobiology 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 (<1%). During incubation following exposure to low pressure, cultures of all species resumed methane production and increased in optical density. Thus, low pressure may not be a biocidal factor for certain methanogen species, with growth possible under low-pressure conditions. Results indicate that low pressure exposure may just be inhibitory during the exposure itself, and metabolism may resume following incubation under more ideal conditions. Further work is needed to address growth/survival under Mars surface pressures.
1. Introduction Few experiments have considered the biocidal nature of the lowpressure atmosphere of Mars or the effect that low pressure (<100 mbar) might have on microorganism growth or metabolism (Fajardo-Cavazos et al., 2012; Kral et al., 2011; Mickol and Kral, 2016; Nicholson et al., 2010, 2013; Schuerger and Nicholson, 2006; Schuerger and Nicholson, 2016; Schuerger et al., 2013; Waters et al., 2014, 2015). Schuerger and Nicholson (2016) and Schuerger et al. (2013) have previously proposed a potential “25 mbar limit” to growth at low pressure based on the analysis of over 150 bacterial strains. However, recent experiments suggest that survival and growth at low pressure may be more common amongst certain genera such as Serratia or Carnobacterium (Nicholson et al., 2013; Schuerger and Nicholson, 2016). The effect on growth and metabolism of Archaea, however, remains relatively unknown as most studies have focused on bacterial isolates from diverse soils or spacecraft cleanrooms and do not include Archaea (Fajardo-Cavazos et al., 2012; Nicholson et al., 2010, 2013; Schuerger and Nicholson, 2006; Schuerger and Nicholson, 2016;
Schuerger et al., 2013; Waters et al., 2014, 2015). Only four studies have analyzed archaeal growth or survival under low-pressure conditions, all of which have focused on methanogens and three of which have come from this lab (Kral and Altheide, 2013; Kral et al., 2011; Mickol and Kral, 2016; Schirmack et al., 2014). Interest in methanogen growth at low pressure relates to the discovery of methane in the martian atmosphere (Fonti and Marzo, 2010; Formisano et al., 2004; Geminale et al., 2008, 2011; Krasnopolsky et al., 1997; Krasnopolsky et al., 2004; Maguire, 1977; Mumma et al., 2009; Webster et al., 2015) tied to the fact that methanogenic activity is responsible for the majority of methane in Earth's atmosphere (Conrad, 2009; IPCC, 2013). The surface pressure on Mars averages between 1 and 10 mbar (Hess et al., 1979, 1980; Spiga et al., 2007) and increases very slowly with depth (Schuerger et al., 2013). Methanogens are anaerobic and non-photosynthetic chemolithotrophs, many of which utilize hydrogen (H2) and carbon dioxide (CO2) to produce methane (CH4). The harsh ionizing radiation at the surface of Mars dictates that any extant life would likely be subsurface. Despite the overlying regolith, pore space
* Corresponding author. University of Arkansas, Department of Biological Sciences, SCEN 601, Fayetteville, AR 72701, USA. E-mail address: [email protected] (T.A. Kral). https://doi.org/10.1016/j.pss.2017.12.014 Received 7 September 2017; Received in revised form 14 November 2017; Accepted 20 December 2017 Available online xxxx 0032-0633/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Mickol, R.L., Kral, T.A., Low pressure microenvironments: Methane production at 50 mbar and 100 mbar by methanogens, Planetary and Space Science (2017), https://doi.org/10.1016/j.pss.2017.12.014
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Planetary and Space Science xxx (2017) 1–10
Simulation Chamber, housed within the Arkansas Center for Space and Planetary Sciences at the University of Arkansas, previously described (Kral et al., 2011). Experiments described here were initially modeled after the microenvironment experiment in Mickol and Kral (2016). Pressure setpoints (Table 1) were maintained using a MKS Type 651C pressure controller and MKS Type 253B throttling valve (MKS Instruments Inc., Andover, MA). All experiments were conducted at room temperature and the chamber was vented with room air following the period of exposure to low pressure. Pressures and exposure times are seen in Table 1.
between soil grains would still resemble the low-pressure conditions at the planet's surface, only reaching 25 mbar at 13.8 km below the martian surface (Schuerger et al., 2013). Higher pressures can be achieved in the near-subsurface of Mars, but this would require complete seclusion from the atmosphere so that gas-exchange and equilibration is not possible (Schuerger et al., 2013). Methane production at 400 mbar and 50 mbar by two of the three methanogens (Methanobacterium formicicum, Methanosarcina barkeri) tested here has been shown previously by experiments in this lab, although experiments only lasted 12 days (50 mbar) or 18 days [400 mbar] (Kral et al., 2011). Additionally, the chamber environment was heated to 35 C to promote growth and cultures were subject to desiccation over the course of the experiment. Schirmack et al. (2014) have also assessed methanogen growth under low-pressure conditions. These experiments utilized the permafrost isolate, Methanosarcina soligelidi, and assessed methane production at low temperatures (-5 to 20 C) at 500 mbar. In two experiments, cultures were initially incubated for one day at either 20 C or 10 C, during which most methane was produced. The temperature was then stepped down to 10 C or 0 C for 12 h, before the temperature was lowered to 0 C or -5 C for the remainder of the experiment (5 days). Cultures of M. soligelidi produced about 100 ppm methane at 0 C for five days and about 350 ppm methane during five days at -5 C, both at 500 mbar (Schirmack et al., 2014). Three species of methanogens (M. formicicum, M. barkeri, Methanococcus maripaludis) were tested for their ability to actively grow (increase in both methane abundance and optical density) within lowpressure microenvironments at 50 mbar or 100 mbar. The experiments conducted here were modeled after the microenvironment experiment in Mickol and Kral (2016). Previous experiments conducted at low pressure with methanogens in their typical, liquid, anaerobic growth media were subject to rapid evaporation due to the instability of water, when temperature is not maintained at low enough values (~0 C). To resolve this issue, experiments discussed here only incorporated pressures down to 50 mbar (above the vapor pressure of water at ~30 C) and test tubes were equilibrated to low pressure, then re-sealed, effectively creating individual microenvironments within each test tube.
2.3. Experiment 1: exposure of M. barkeri, M. formicicum, and M. maripaludis to 100 mbar for 28 days Methanogens were initially grown in their respective anaerobic growth media, as described above (2.1 Microbial procedures). The tubes were incubated at the methanogens’ respective growth temperatures for 7 days. After the initial incubation period, each tube was tested for both methane production and optical density to determine initial growth. There were five replicates for each of the three methanogen species, with two separate sets (inoculated from separate sources) for M. maripaludis (e.g., there were five replicates for Set A for M. maripaludis and five replicates for Set B for M. maripaludis). Next, tubes were placed inside the Pegasus Planetary Simulation Chamber with a palladium catalyst box to remove residual oxygen (Fig. 1A). Within the chamber, twenty tubes (five replicates each for M. barkeri and M. formicicum, ten replicates for M. maripaludis [see above]) were situated inside a test tube rack, sorted randomly. A second test tube rack was placed over the top of the tubes to secure their position for use with a specialized puncture device (see Mickol and Kral (2016) for a description of the device; also see Fig. 1B). As a control, an additional seventeen tubes (five replicates each for M. barkeri, M. formicicum, and M. maripaludis Set A, and two replicates for the M. maripaludis Set B cultures) were situated inside an adjacent test tube rack. These tubes would not be punctured (i.e., exposed to low pressure), but would be exposed to the same temperature as the punctured (low pressure) test tubes. Within both sets, one test tube for each species was not initially inoculated to serve as a blank for optical density measurements. The chamber door was closed and duct seal putty (Rainbow Technology, Pelham, AL) was applied around the seal as a further safeguard against oxygen contamination. The seal between the puncture device and the chamber was also covered with duct seal putty. The chamber was evacuated to 100 mbar, 80/20 H2/CO2 gas was bled into the chamber, and the system was left to equilibrate for 30 min. Next, the experimental tubes were punctured with the specialized puncture device containing one-inch, 22-gauge syringe needles for 60 min to allow equilibration between the chamber pressure and the pressure inside the tubes (see Mickol and Kral, 2016). After 60 min, the needles were removed from the tubes, effectively creating “low-pressure microenvironments” within each test tube, and the H2/CO2 gas was turned off. The chamber was actively maintained at 100 mbar for the duration of the experiment. After 28 days, the chamber was slowly vented with room air to atmospheric pressure. Test tubes were removed from the chamber and immediately tested for methane concentration and optical density. All tubes were re-pressurized with 2 bar H2 and put at the organisms’ respective incubation temperatures. Tubes were then monitored over time for methane production and optical density.
2. Materials and methods 2.1. Microbial procedures Methanogens were initially obtained from the American Type Culture Collection (ATCC; Manassas, VA), cultured in their respective anaerobic growth media, and kept at a temperature within the organisms' ideal growth ranges: Methanococcus maripaludis (ATCC 43000), MSH medium (Ni and Boone, 1991), 22 C; Methanosarcina barkeri (ATCC 43569), MS medium (Boone et al., 1989), 37 C; Methanobacterium formicicum (ATCC 33274), MSF medium (MS medium supplemented with formate), 37 C. Media were prepared as described in Kendrick and Kral (2006) under anaerobic conditions in a 90/10 CO2/H2 gas Coy Anaerobic Chamber (Coy Laboratory Products Inc., Grass Lake Charter Township, MI) and dispensed into anaerobic culture tubes at a rate of 10 mL per tube. The tubes were fitted with rubber stoppers and sealed with aluminum crimps as described in Boone et al. (1989), and autoclaved for sterilization. Prior to inoculation with the respective organisms, a solution of 2.5% w/v sodium sulfide (~125 μL per 10 mL medium) was added to each culture tube to remove residual oxygen (Boone et al., 1989). The tubes were then pressurized with 2 bar H2 and incubated at the organisms’ respective growth temperatures. For each experiment, growth was monitored by methane production via gas chromatography (Shimadzu Scientific Instruments Inc., model GC-2014, Columbia, MD) and via optical density (OD600, Spectronic 20Dþ, Spectronic Instruments, USA).
2.4. Experiment 2: exposure of M. barkeri, M. formicicum, and M. maripaludis to 50 mbar for 35 days Media were prepared as described in 2.1 Microbial procedures, however, the oxygen indicator, resazurin, was not included. Although this removed the ability to determine the extent of oxygen contamination within each test tube, it allowed for continued optical density measurements over time as the color of the media remained unchanged.
2.2. Pegasus planetary simulation chamber Low pressure experiments were conducted in the Pegasus Planetary 2
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Table 1 Experimental conditions for each of four experiments, including pressures and time exposed to low pressure. Each experiment consisted of five replicates for each of the methanogen species tested in 10 mL of their respective anaerobic growth medium. Pre- and post-exposure incubations were conducted at the methanogens’ respective growth temperatures (Methanosarcina barkeri, 37 C, MS medium; Methanobacterium formicicum, 37 C, MSF medium; Methanococcus maripaludis, 22 C, MSH medium). Expt.
Species Used
Pre-exposure Incubation Period (days)
Evacuation/ Experimental Pressure (mbar)
# of Evacuation Cycles
Time for Equilibration of Chamber at Low Pressure (min)
Time for Equilibration Between Punctured Tubes and Chamber (min)
Length of Exposure to Low Pressure (days)
Average Temperature Inside Pegasus Chamber ( C)
1
M. barkeri, M. formicicum, M. maripaludis M. barkeri, M. formicicum, M. maripaludis M. formicicum
7
100.4 0.1
0
30
60
28
26.6 0.5
7
49.8 0.1
2
30
60
35
28.9 2.0
7
50.1 0.1
1, CO2 2, H2/CO2 1, CO2 2, H2/CO2
30
60
49
27.7 1.7
30
60
49
27.5 1.7
2
3 4
M. formicicum
7
49.9 0.2
a
a The pressure control valve malfunctioned on Day 30 (of 49), dropping the chamber pressure to ~0.8 mbar. The tubes remained sealed and no significant evaporation was witnessed in any tubes. Thus, it is believed the ~50 mbar microenvironments remained intact for the duration of the experiment.
experiment. After 35 days, the chamber was vented to the atmosphere and the test tubes were removed. All test tubes were immediately measured for methane concentration and optical density. After measurement, tubes were re-pressurized with 2 bar H2 and stored at their incubation temperatures. Additional methane and optical density measurements were taken over time.
2.5. Experiment 3: exposure of M. formicicum to 50 mbar for 49 days This experiment utilized only cultures of M. formicicum and media did not contain resazurin, as in Expt. 2. Additionally, cells were washed before inoculation in order to remove residual resazurin. McAllister and Kral (2006) demonstrated that methanogenic cells could be exposed to atmospheric oxygen for at least 1.5 h during a washing procedure without significant loss of viability. Two, one-month-old cultures consisting of 10 mL MSF medium were combined, then poured into two large centrifuge tubes. Tubes were centrifuged at 5000 rpm for 45 min. Next, the supernatant was poured off, and 5 mL CO2 buffer containing 2.5% sodium sulfide (Na2S) were added to each centrifuge tube. The tubes were shaken to redistribute cells and centrifuged again at 5000 rpm for 45 min. The supernatant was poured off and 10 mL CO2 buffer þ Na2S were added to each centrifuge tube. The centrifuge tubes were combined to form a single inoculum source. Thirty-three test tubes containing sterile MSF medium without resazurin were then inoculated with 0.5 mL of washed M. formicicum cells (seven tubes were not inoculated to serve as blanks). Each test tube was pressurized with 2 bar H2 and incubated at 37 C for 7 days. After incubation, tubes were measured for methane concentration and optical density to determine initial growth. Chamber procedures were similar to those for Expts. 1 and 2. Tubes were placed into the Pegasus Planetary Simulation Chamber and the chamber was evacuated to 50 mbar while CO2 was bled into the system. The chamber was filled to atmospheric pressure with CO2, then the CO2 was turned off and the H2/CO2 gas was turned on. The chamber was evacuated again to 50 mbar, then filled to atmospheric pressure with H2/CO2. This cycle was repeated, the chamber pressure was lowered to 50 mbar, and the system was left to equilibrate at 50 mbar for 30 min. Next, the test tubes were punctured and allowed to equilibrate with the low pressure of the chamber for 60 min, after which the needles were removed from the test tubes and the H2/CO2 gas turned off. The chamber was actively maintained at 50 mbar for the duration of the experiment. After 49 days, the chamber was vented to the atmosphere and the tubes were removed. The test tubes were immediately measured for methane concentration and optical density. Following measurement, tubes were re-pressurized with 2 bar H2 and incubated at 37 C. Methane concentration and optical density continued to be monitored over time.
Fig. 1. Photos depicting arrangement of test tubes within the Pegasus Planetary Simulation Chamber for each experiment. A. Experimental setup for each experiment: pd ¼ puncture device, pc ¼ palladium catalyst. B. Close-up view of the puncture device. The puncture device contains 22-gauge, 1-inch syringe needles to puncture tubes and allow equilibration with the chamber atmosphere.
Experimental procedures were similar to those for Expt. 1, except for additional cycling between low pressure and atmospheric pressure before the tubes were punctured in order to ensure removal of the ambient atmosphere. Test tubes were inoculated with 0.5 mL culture, pressurized with 2 bar H2 and incubated for 7 days. Tubes were then measured for methane concentration and optical density to determine initial growth. Tubes were placed inside the chamber as described above for Expt. 1. The chamber was closed and evacuated to 50 mbar while CO2 gas was bled into the system. The chamber was then filled with CO2 to atmospheric pressure and evacuated again. This cycle was repeated, the chamber was again evacuated to 50 mbar, and the system was left to equilibrate for 30 min. Next, the CO2 gas was turned off and the H2/CO2 (80/20) gas was turned on while the chamber was maintained at 50 mbar. The test tubes were punctured as in Expt. 1. After 60 min, the needles were removed from the test tubes and the H2/CO2 gas was turned off. The chamber was actively maintained at 50 mbar for the duration of the 3
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Fig. 2. Methane production (left) and optical density (right) for Methanobacterium formicicum, Methanosarcina barkeri, Methanococcus maripaludis (Set A), and M. maripaludis (Set B) [top to bottom] measured immediately before and immediately after exposure to low pressure (100 mbar). An initial incubation period took place at 37 C (M. formicicum, M. barkeri) or 22 C (M. maripaludis) for 7 days. Low Pressure tubes were exposed to 100 mbar for 28 days. Control tubes were subjected to the same environmental conditions as the low-pressure tubes, except 4
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Simulation Chamber, the chamber was evacuated to ~50 mbar, and the tubes were punctured to directly expose them to the chamber atmosphere. After 1 h, the needles were removed from the test tubes, resealing the test tubes. The tubes were then removed from the chamber and immediately tested for methane concentration.
2.6. Experiment 4: exposure of M. formicicum to 50 mbar for 49 days with and without formate-supplemented media This experiment was intended to replicate Expt. 3, with slight modification. Two sets of media were prepared: Fourteen test tubes contained 10 mL MSF medium (MS medium supplemented with formate), while fourteen test tubes contained 10 mL MS medium (no formate). Thus, one set of tubes contained two energy sources, H2 and formate, while the second set contained only H2. Similarly to Expts. 2 and 3, the media did not contain the oxygen indicator, resazurin. Cells were also washed prior to inoculation, as in Expt. 3. Three, twomonth-old 10-mL stock cultures were poured into large centrifuge tubes. The tubes were centrifuged for 45 min at 5000 rpm, after which the supernatant was poured off. Ten milliliters of sterile CO2 buffer þ Na2S were added to each centrifuge tube and the tubes were shaken to redistribute pelleted cells. The tubes were centrifuged again at 5000 rpm for 45 min. Next, the supernatant was poured off and 10 mL CO2 buffer þ Na2S were added to each centrifuge tube. The tubes were then combined to form one inoculum source. An aliquot of 0.5 mL of washed cells was added to each of twenty test tubes (ten containing media with formate, ten containing media without formate). Eight test tubes remained un-inoculated to serve as blanks. The test tubes were pressurized with 2 bar H2 and incubated at 37 C for 7 days. After incubation, tubes were tested for methane concentration and optical density to determine initial growth. Chamber procedures were similar to those for Expt. 3. Tubes were placed within test tubes racks, assorted randomly. The chamber door was closed, sealed with duct seal putty, and the palladium catalyst box was turned on. The procedures for the initial evacuation of the chamber and the equilibration of the low-pressure tubes to the low pressure atmosphere of the chamber were identical to those in Expt. 3. After 49 days, the chamber was vented to the atmosphere and the tubes were removed. The low-pressure test tubes were immediately filled to just above atmospheric pressure (~0.07 bar) with CO2 gas using a gassing manifold. This was done for two reasons: 1. To bring cultures to atmospheric pressure for headspace analysis and 2. To minimize oxygen contamination when headspace samples were removed from the low-pressure test tubes. All test tubes were then equilibrated for 2 h before methane concentration was measured via gas chromatography. Following methane measurements, all tubes were re-pressurized with 2 bar H2 and incubated at 37 C. Methane concentration and optical density continued to be monitored over time. It is important to note that on Day 30 (of 49) for Expt. 4 (49 days at 50 mbar), the pressure control valve malfunctioned and dropped the internal chamber pressure to ~0.8 mbar. The individual test tubes remained sealed at this point, and there was no evidence of extensive leakage (evaporation) of the media. Thus, it is believed that the microenvironments remained intact at ~50 mbar for the following 19 days of the experiment, despite the lower pressure of the chamber. As the cultures were not subjected to this lower pressure, the data were analyzed without alteration.
3. Results The number of replicates for each species within each experiment are given as n ¼ #, below. In cases where optical density measurements were below zero, likely due to inaccuracies in light transmission through the test tubes, or where tubes containing resazurin showed signs of oxygen contamination, data were not included in the averages given. 3.1. Measurement of methane remaining within test tubes following puncture under low pressure The results indicate that the majority of the initial methane within each test tube escaped during the period of direct exposure to low pressure, as expected: M. formicicum, 0.18 0.10% CH4; M. barkeri, 0.22 0.01% CH4; M. maripaludis, 0.25 0.09% CH4. Thus, methane abundances measured after the initial 7-day incubation period, but prior to puncture under low pressure, only serve to indicate that cultures were active and growing at the time they were placed in the chamber. Those initial methane measurements can be found in Figs. 2–5. Comparing methane concentrations immediately following low-pressure exposure to methane concentrations of cultures prior to puncture would be meaningless. All methane concentrations following low-pressure exposure in Experiments 1-4 have been compared to the average methane concentration following puncture and 1 h direct exposure to 50 mbar (average: 0.22 0.07%; Figs. 2–5). This average methane concentration is derived from methane measurements of 12 cultures including three species. Measurements for all three species were very consistent. 3.2. Experiment 1: exposure of M. barkeri, M. formicicum, and M. maripaludis to 100 mbar for 28 days In this experiment, cultures of M. barkeri, M. formicicum, and M. maripaludis were maintained at 100 mbar for 28 days. Methane concentration for M. formicicum cultures initially measured 2.93 0.73% (n ¼ 3) on the day the tubes were placed inside the chamber (Day 7). Again, the starting concentration of methane following puncture was determined to be approximately 0.22% based on the results from the control experiment. Following exposure to 100 mbar for 35 days, methane abundance measured 4.82 0.41% within the low-pressure tubes (Fig. 2). Methane in control tubes increased from 3.69 0.13% to 33.71 12.36% (n ¼ 4) over the course of the experiment (Fig. 2). One low-pressure replicate was not adequately punctured during the experiment, and thus, not exposed to the low-pressure environment. It is not included in the data shown here. The optical density for M. formicicum low-pressure replicates also increased during exposure to low pressure (0.051 0.004 to 0.100 0.013; Fig. 2). This increase in optical density is similar to the increase in optical density for the control tubes. After exposure to low pressure, methane abundance in M. barkeri cultures (n ¼ 4) measured 0.78 0.17% while methane abundance within control tubes (n ¼ 4) measured ~6.5% (Fig. 2). Optical density within low-pressure tubes (n ¼ 4) increased from 0.003 0.003 to 0.012 0.006 over the period of exposure (Fig. 2). Optical density ranged widely for the control tubes with one replicate measuring 0.044
2.7. Measurement of methane remaining within test tubes following puncture under low pressure A separate control study was conducted in order to determine the amount of methane remaining in tubes following puncture under low pressure. Active cultures of the three species (M. formicicum, n ¼ 3; M. barkeri, n ¼ 4; M. maripaludis, n ¼ 5) were measured for methane abundance. The tubes were then placed into the Pegasus Planetary
for pressure. The specific number of replicates (typically, n ¼ 4) for both low pressure and control tubes for each methanogen are given in the text. In a separate experiment, active cultures (M. formicicum, n ¼ 3; M. barkeri, n ¼ 4; M. maripaludis, n ¼ 5) were directly exposed to low pressure (~50 mbar) for 1 h, then removed from the chamber and immediately measured for methane abundance (middle bars in graphs on left). These averages indicate that methane abundance within the low-pressure tubes was negligible (0.22 0.07%) at the start of the experiment. Error bars indicate one standard deviation. Note: there are no error bars for the optical density data for the M. maripaludis (Set B) Low Pressure tubes “Following Initial 7-day Growth Period” and “Following Length of Experiment (28 days)” nor for the Control tubes “Following Initial 7-day Growth Period” because these data reflect n ¼ 1 replicate. 5
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to 0.050 0.017) and control replicates (0.019 0.016 to 0.085 0.033, Fig. 4). Methane abundance and optical density also continued to increase in all tubes (low pressure, control) following removal from the chamber, repressurization with 2 bar H2 and incubation at 37 C (data not shown).
on Day 35 and the other three replicates measuring ~0.02 (Fig. 2). With respect to M. maripaludis (Sets A and B), there were minor increases in methane concentration for cultures exposed to 100 mbar for 28 days (Set A: 0.78 0.34%, n ¼ 4; Set B: 0.65 0.20%, n ¼ 3). M. maripaludis control cultures (Set A: n ¼ 4, Set B: n ¼ 2) showed increases in both methane concentration (~43%) and optical density (0.12–0.15) (Fig. 2). Optical density measurements decreased for the low-pressure replicates for M. maripaludis Set A over the course of exposure to low pressure, but increased greatly for the control replicates (n ¼ 2; Fig. 2).
3.5. Experiment 4: exposure of M. formicicum to 50 mbar for 49 days with and without formate-supplemented media Experiment 4 was intended to duplicate Experiment 3, but contained two major differences: 1. Media with or without formate and 2. Lowpressure replicates were brought to just above atmospheric pressure (~0.07 bar) with CO2 immediately following removal from the Pegasus Planetary Simulation Chamber. Both low-pressure groups (þformate, -formate) measured greater methane abundance following 49 days’ exposure to 50 mbar compared to 0.220.07% (Fig. 5). Low-pressure tubes supplemented with formate (n ¼ 5) measured 2.12 0.58% methane after the course of the experiment (49 days). The low-pressure tubes without formate (n ¼ 5) measured 1.08 0.54% methane following exposure to low pressure (Fig. 5). This is not surprising since formate acted as an additional energy source. Both control groups (þformate, -formate; n ¼ 5) increased by over ~40% methane over 49 days (Fig. 5). Optical density increased from 0.028 0.002 to 0.040 0.013 within low-pressure replicates supplemented with formate, while replicates lacking formate decreased slightly in optical density (0.023 0.003 to 0.019 0.011) over the period of exposure to low pressure (Fig. 5). Optical density increased in control tubes, from 0.013 0.002 to 0.139 0.019 in replicates supplemented with formate and from 0.022 0.005 to 0.112 0.025 in replicates lacking formate (Fig. 5). Methane abundance and optical density also continued to increase in all tubes (low pressure, control) following removal from the chamber, repressurization with 2 bar H2 and 28 days’ incubation at 37 C (data not shown).
3.3. Experiment 2: exposure of M. barkeri, M. formicicum, and M. maripaludis to 50 mbar for 35 days This experiment exposed cultures of M. barkeri, M. formicicum, and M. maripaludis to 50 mbar for 35 days. The media for this experiment did not include the oxygen indicator, resazurin. Therefore, possible oxygen contamination did not affect optical density values. However, the extent to which tubes may have been contaminated with oxygen is unknown. Both methane abundance and optical density increased for cultures of M. formicicum exposed to 50 mbar for 35 days (Fig. 3). Methane within low-pressure replicates (n ¼ 4) increased from 0.22 0.07% to 2.36 1.08% following exposure to 50 mbar. Methane production within control replicates (n ¼ 4) was nearly identical to production within low-pressure replicates (Fig. 3). Optical density of low-pressure tubes (n ¼ 4) increased from 0.023 0.015 to 0.046 0.023 during exposure to low pressure. All four low-pressure replicates demonstrated increased methane abundance and optical density within individual tubes, but the variation amongst the four tubes accounts for the large error bars (Fig. 3). Surprisingly, optical density within control replicates (n ¼ 4) decreased slightly during the length of the experiment (Fig. 3). With respect to M. barkeri, there was no difference in methane concentration following low-pressure exposure (n ¼ 4; Fig. 3). Optical density increased from 0.009 0.001 to 0.022 0.014 within low-pressure tubes (n ¼ 4) during the 35-day exposure to 50 mbar. Optical density only increased in three of the four low-pressure replicates, resulting in the large error bar seen in Fig. 3. Both methane abundance and optical density increased in control tubes (n ¼ 4) over the course of the experiment (Fig. 3). Methane concentration measured 0.82 0.05% in cultures of M. maripaludis (n ¼ 4) following exposure to 50 mbar for 35 days whereas optical density decreased (Fig. 3). Optical density increased from 0.023 0.002 to 0.106 0.020 within control tubes over the course of the experiment while methane abundance increased from 1.66 0.41% to 41.28 10.00% (Fig. 3). Methane production and optical density continued to be monitored for all species in both Experiment 1 (Section 3.2) and Experiment 2 (Section 3.3) following removal from the chamber. Increases in both methane and optical density were seen for all three species up to 49 days following the length of the experiment (data not shown).
4. Discussion When considering the habitability of Mars, low pressure is often overlooked as a potentially biocidal factor in favor of more potentially lethal conditions such as exposure to UV radiation and desiccation. However, there are currently no surface environments on Earth where microorganisms would be subjected to the low pressures present on Mars. For example, the lowest surface pressure on Earth reaches only 330 mbar at the top of Mount Everest (Fajardo-Cavazos et al., 2012), whereas the surface pressure on Mars averages ~7 mbar (Schuerger and Nicholson, 2016). Several studies have noted decreased growth rates for certain bacteria under 100 mbar and complete lethality below 25 mbar (Schuerger and Nicholson, 2016). Of over 150 bacterial strains studied, only a few species such as Carnobacterium spp. (Nicholson et al., 2013), Pseudomonas fluorescens (Schuerger and Nicholson, 2016) and Serratia liquefaciens (Schuerger et al., 2013) have exhibited growth below 25 mbar. These results suggest that low pressure may have more of a lethal or inhibitory effect on certain bacteria, warranting further research into growth under low pressure. Additionally, few studies have included Archaea in low pressure experiments (Kral and Altheide, 2013; Kral et al., 2011; Mickol and Kral, 2016; Schirmack et al., 2014), illustrating a need for further research in this area. The use of methanogens in studies of martian habitability provides a possible biological explanation for the methane detected in the martian atmosphere. However, demonstrating active methane production within methanogenic cultures at low pressure is difficult considering methane is typically monitored via gas chromatography and removing a gas sample from a low-pressure environment (e.g., vacuum chamber) for analysis is problematic. In the experiments conducted here, low-pressure “microenvironments” were created by equilibrating the headspace within anaerobic test tubes with the low-pressure atmosphere inside a planetary simulation chamber. Tubes were equilibrated to low pressure (100 mbar,
3.4. Experiment 3: exposure of M. formicicum to 50 mbar for 49 days This experiment solely utilized cultures containing M. formicicum and cells were washed prior to inoculation. There were initially 17 replicates that were exposed to low pressure, however, one tube was not sufficiently punctured to allow equilibrium between the low-pressure environment of the chamber and the test tube and so this replicate is not included in the data shown here (n ¼ 16). There were also 16 tubes that were not exposed to low pressure that served as control replicates (n ¼ 16). The average methane produced among the low-pressure replicates increased during the exposure period to 3.37 1.26% (Fig. 4). Methane increased by greater amounts in control tubes (average: 25.48 22.23%, Fig. 4), but with considerable variation among replicates, ranging from 5% to 57%. Optical density increased in both low-pressure replicates (0.019 0.011 6
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Fig. 3. Methane production (left) and optical density (right) for Methanobacterium formicicum, Methanosarcina barkeri, and Methanococcus maripaludis (top to bottom) measured immediately before and immediately after exposure to low pressure (50 mbar). An initial incubation period took place at 37 C (M. formicicum, M. barkeri) or 22 C (M. maripaludis) for 7 days. Low Pressure tubes (n ¼ 4) were exposed to 50 mbar for 35 days. Control tubes (n ¼ 4) were subjected to the same environmental conditions as the low-pressure tubes, except for pressure. In a separate experiment, active cultures (M. formicicum, n ¼ 3; M. barkeri, n ¼ 4; M. maripaludis, n ¼ 5) were directly exposed to low pressure (~50 mbar) for 1 h, then removed from the chamber and immediately measured for methane abundance (middle bars in graphs on left). These averages indicate that methane abundance within the low-pressure tubes was negligible (0.22 0.07%) at the start of the experiment. For M. barkeri, optical density measurements reflect n ¼ 3 replicates for the “Following Initial 7-day Growth Period” control tubes only. Error bars indicate one standard deviation. 7
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Fig. 4. Methane production (left) and optical density (right) for Methanobacterium formicicum measured immediately before and immediately after exposure to low pressure (50 mbar). Cells were washed before inoculation. An initial incubation period took place at 37 C for 7 days. Low Pressure tubes (n ¼ 16) were exposed to 50 mbar for 49 days. Control tubes (n ¼ 16) were subjected to the same environmental conditions as the low-pressure tubes, except for pressure. In a separate experiment, active cultures of M. formicicum (n ¼ 3), Methanosarcina barkeri (n ¼ 4), and Methanococcus maripaludis (n ¼ 5) were directly exposed to low pressure (~50 mbar) for 1 h, then removed from the chamber and immediately measured for methane abundance (middle bars in graphs on left). These averages indicate that methane abundance within the low-pressure tubes was negligible (0.22 0.07%) at the start of the experiment. Error bars indicate one standard deviation.
Fig. 5. Methane production (left) and optical density (right) for Methanobacterium formicicum measured immediately before and immediately after exposure to low pressure (50 mbar). Cells were washed prior to inoculation. An initial incubation period took place at 37 C for 7 days. Low Pressure tubes (n ¼ 10) were exposed to 50 mbar for 49 days. Control tubes (n ¼ 10) were subjected to the same environmental conditions as the low-pressure tubes, except for pressure. Both Low Pressure and Control tubes were separated into two groups: þF replicates (n ¼ 5) contain formate in the medium, -F replicates (n ¼ 5) lack formate. In a separate experiment, active cultures of M. formicicum (n ¼ 3), Methanosarcina barkeri (n ¼ 4), and Methanococcus maripaludis (n ¼ 5) were directly exposed to low pressure (~50 mbar) for 1 h, then removed from the chamber and immediately measured for methane abundance (middle bars in graphs on left). These averages indicate that methane abundance within the low-pressure tubes was negligible (0.22 0.07%) at the start of the experiment. Error bars indicate one standard deviation.
height (volume) of the liquid medium was detected from the individual cultures. Additionally, in Expts. 1-3, negative pressure within each replicate (as headspace samples were removed for methane detection) further demonstrated that each individual test tube was under low pressure. Low pressure tubes in Expt. 4 were also verified as being under low pressure by the ability to add ~0.07 bar CO2 to cultures following removal from the chamber. Methane abundance within anaerobic test tubes was measured by
Expt. 1 or 50 mbar, Expts. 2-4) for 60 min using syringe needles. After the period of equilibration, the needles were removed, resealing the anaerobic test tubes and effectively creating low-pressure microenvironments within each replicate. The chamber atmosphere was actively maintained at 100 mbar (Expt. 1) or 50 mbar (Expts. 2-4) for the duration of the experiment so that there was no change in pressure in the event of test tube leakage. Further, visual inspection during and after the exposure period ensured that microenvironments were intact as no change in 8
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against one another, for differences in growth rates or cell clumping may exacerbate differences. That being said, the apparent differences in active growth at low pressure between the three species tested here may be the result of physical attributes of the cells such as cell wall composition and affinity for clumping. M. maripaludis was the only species that did not demonstrate appreciable methane production or increased optical density at either pressure tested, which may be due to a relatively weaker cell wall. The cell walls of Methanococcus species consist of a single electron dense outer layer that is both osmotically fragile and susceptible to detergents (Jones et al., 1983). In comparison, Methanosarcinales is the only methanogenic order that forms multicellular aggregates due to the formation of a heteropolysaccharide matrix called methanochondroitin (Milkevych et al., 2015). This extracellular polysaccharide matrix is believed to offer additional protection against environmental stresses. In a study comparing individual cells and multicellular aggregates of M. barkeri, Anderson et al. (2012) discovered that cells that produced methanochondroitin (and thus formed multicellular aggregates) had significantly higher survival rates than individual cells in response to desiccation, high temperatures, and oxygen exposure. Additionally, because variations in growth proxies exist across replicates within the same experiment, comparison across experiments may also be difficult when precise cell numbers are not used. This is apparent for M. formicicum in Expt. 2 where methane abundance only reaches ~2% within both control and low pressure tubes after 35 days (Fig. 3). Expected methane production within control tubes would be about 30%. Optical density for these cultures was also significantly lower in Expt. 2 than in the other experiments described here. The explanation for the lower methane and optical density measurements are currently unknown, but are not uncommon with respect to variation in methanogenic pure cultures. This variation is also evident in the methane production for M. formicicum in Expt. 3 which encompasses very large error bars (Fig. 4). Ultimately, these experiments aimed to qualitatively demonstrate the ability of one species, M. formicicum, to both produce methane and increase in optical density at low pressure (50 mbar, 100 mbar) despite variation both within and between experiments. The pressures utilized in the experiments described here (50 mbar, 100 mbar) may be achievable in the martian subsurface. Schuerger et al. (2013) describe the atmospheric gas-phase pressure increasing very slowly in the martian subsurface, reaching 25 mbar at 13.8 km. However, should there be complete seclusion from the atmosphere either by inclusion in rock or ice, the lithographic pressure increases rapidly, reaching 25 mbar at a depth of 19.5 cm (Schuerger et al., 2013). In a subsurface environment, microorganisms would be shielded from the harsh ionizing radiation at the surface of the planet and may experience more stable temperature regimes (Schorghofer and Aharonson, 2005). Additionally, observations of recurring slope lineae at the surface of Mars suggest that there is a mobile liquid brine water column within the subsurface that operates on seasonal timescales (McEwen et al., 2014; Ojha et al., 2015). This water column could provide liquid water to any extant microorganisms (dependent on salt tolerances), as well as transport microorganisms to various depths (e.g., pressures) in the martian subsurface. Future experiments would benefit from the inclusion of additional martian parameters such as temperature and salt concentration, as well as lower pressures, in order to better mimic the current martian environment. Due to the variation amongst replicates under identical conditions, future experiments may need to utilize additional growth proxies besides methane concentration and optical density. The anaerobic nature of methanogens, the high number of replicates within most experiments, and the slow doubling time of the organisms makes cell counting or MPN procedures a daunting task. However, accurate cell counts would provide a necessary comparison to methane abundances and optical densities.
removing a 0.5-mL sample of gas from the headspace and injecting this into a gas chromatograph. In Expts. 1-3, following exposure to low pressure, headspace samples were removed from the test tubes while still under low pressure. Since the test tubes were themselves below atmospheric pressure, it is possible that air began to enter each test tube as soon as the external pressure rose above 50 mbar (or 100 mbar, Expt. 1). The test tubes remained sealed with aluminum crimps and rubber stoppers, although it is possible that air began to seep into each tube through pores within the stoppers or if the seal between the stopper and the test tube was not adequately robust. Whether or not air is able to seep into the test tubes themselves following removal from the chamber, it is known that air will rush into the syringe when a headspace sample is removed from the test tube and transferred to the gas chromatograph. Airtight syringes were not used in this research. If airtight syringes had been used, a similar effect would have occurred. Once the low-pressure sample had been injected into the gas chromatograph, air would have diluted the sample in the injection port. Thus, the samples removed from the lowpressure tubes were diluted, including the control tubes designed to measure remaining methane following puncture. In the case of Expt. 1, the dilution would occur at a factor as high as ~10 between the experimental pressure (100 mbar) and atmospheric pressure (~1013 mbar). For Expts. 2-4, the dilution factor may have reached ~20 (1013 mbar/50 mbar). As a gas chromatograph measures methane concentration as a percentage of the headspace within the test tube, the recorded values for methane abundance seen here are lower estimates for the true abundances contained within the test tubes. M. formicicum was the only methanogen out of the three species to demonstrate both substantial increases in methane production (>1%) and optical density during exposure to 100 mbar and 50 mbar. In a separate control study, growing cultures were placed inside the Pegasus Planetary Simulation Chamber, punctured, and exposed to low pressure (~50 mbar) for 1 h. The needles were removed from the test tubes and the cultures were immediately tested for methane abundance. The methane initially present in each test tube had escaped, as expected. Average methane abundance was 0.22% for each of the three methanogens (see Results). Thus, measured methane abundances above 1% within low-pressure replicates following the length of the experiment indicate active methane production at low pressure. M. barkeri demonstrated a small increase in both methane concentration and optical density at 100 mbar, but not at 50 mbar (Figs. 2 and 3). Cells of Methanosarcina spp. typically form irregular multicellular aggregates, especially under stressed conditions (Maestrojuan and Boone, 1991), which may account for the increases in optical density seen here. Although M. barkeri and M. maripaludis did not demonstrate active growth (>1% CH4) under low-pressure conditions, methane production resumed during the post-exposure incubation period and cultures also increased in optical density (data not shown). However, survival was likely not difficult as cells remained within a 10-mL water column for the duration of the experiment. Mickol and Kral (2016) also note that as the tubes were upright and the cells likely congregated at the bottom of the test tubes, the pressure at the bottom of the water column was, in actuality, about 6 mbar higher than the experimental pressure (50 mbar or 100 mbar) due to the weight of the water. On the other hand, the ideal gas law (PV ¼ nRT; Quagliano and Vallarino, 1969) would dictate that combining five molecules of gas to create three molecules would slightly decrease the headspace pressure within the test tubes: 4H2 þ CO2 → CH4 þ 2H2O (1). Working with methanogens, we witness this decrease in pressure regularly. Methane abundance and optical density measurements were not tightly coupled in the experiments described here, with increases in methane abundance not correlating to increases in optical density, and vice versa (for example, Expt. 2: M. barkeri, 35 days at 50 mbar). This is not uncommon with respect to methanogenic pure cultures where identical replicates are often seen to vary in both methane production and optical density (Gunnigle et al., 2013; Maestrojuan and Boone, 1991). Thus, these experiments are not intended to compare species
5. Conclusions Low pressures between 50 mbar and 100 mbar may be achievable in 9
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the martian subsurface and could potentially house extant methanogens, given the availability of liquid water. M. formicicum is capable of active growth at 50 mbar as verified by concurrent methane production and increased optical density. Although M. barkeri did not demonstrate growth at 50 mbar and M. maripaludis did not demonstrate growth during either low pressure tested, all cultures produced methane and increased in optical density during the post-exposure incubation period, indicating that all species survived exposure to low pressure. These experiments suggest that low pressure environments on Mars may not be lethal to certain species of methanogens and increase the possibility of a habitable subsurface environment on the planet.
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Low pressure microenvironments: Methane production at 50 mbar and 100 mbar by methanogens Rebecca L. Mickol a, Timothy A. Kral a, b, * a b
Arkansas Center for Space and Planetary Sciences, University of Arkansas, Fayetteville, AR, USA Department of Biological Sciences, University of Arkansas, Fayetteville, AR, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Methane Methanogens Low pressure Astrobiology 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 (<1%). During incubation following exposure to low pressure, cultures of all species resumed methane production and increased in optical density. Thus, low pressure may not be a biocidal factor for certain methanogen species, with growth possible under low-pressure conditions. Results indicate that low pressure exposure may just be inhibitory during the exposure itself, and metabolism may resume following incubation under more ideal conditions. Further work is needed to address growth/survival under Mars surface pressures.
1. Introduction Few experiments have considered the biocidal nature of the lowpressure atmosphere of Mars or the effect that low pressure (<100 mbar) might have on microorganism growth or metabolism (Fajardo-Cavazos et al., 2012; Kral et al., 2011; Mickol and Kral, 2016; Nicholson et al., 2010, 2013; Schuerger and Nicholson, 2006; Schuerger and Nicholson, 2016; Schuerger et al., 2013; Waters et al., 2014, 2015). Schuerger and Nicholson (2016) and Schuerger et al. (2013) have previously proposed a potential “25 mbar limit” to growth at low pressure based on the analysis of over 150 bacterial strains. However, recent experiments suggest that survival and growth at low pressure may be more common amongst certain genera such as Serratia or Carnobacterium (Nicholson et al., 2013; Schuerger and Nicholson, 2016). The effect on growth and metabolism of Archaea, however, remains relatively unknown as most studies have focused on bacterial isolates from diverse soils or spacecraft cleanrooms and do not include Archaea (Fajardo-Cavazos et al., 2012; Nicholson et al., 2010, 2013; Schuerger and Nicholson, 2006; Schuerger and Nicholson, 2016;
Schuerger et al., 2013; Waters et al., 2014, 2015). Only four studies have analyzed archaeal growth or survival under low-pressure conditions, all of which have focused on methanogens and three of which have come from this lab (Kral and Altheide, 2013; Kral et al., 2011; Mickol and Kral, 2016; Schirmack et al., 2014). Interest in methanogen growth at low pressure relates to the discovery of methane in the martian atmosphere (Fonti and Marzo, 2010; Formisano et al., 2004; Geminale et al., 2008, 2011; Krasnopolsky et al., 1997; Krasnopolsky et al., 2004; Maguire, 1977; Mumma et al., 2009; Webster et al., 2015) tied to the fact that methanogenic activity is responsible for the majority of methane in Earth's atmosphere (Conrad, 2009; IPCC, 2013). The surface pressure on Mars averages between 1 and 10 mbar (Hess et al., 1979, 1980; Spiga et al., 2007) and increases very slowly with depth (Schuerger et al., 2013). Methanogens are anaerobic and non-photosynthetic chemolithotrophs, many of which utilize hydrogen (H2) and carbon dioxide (CO2) to produce methane (CH4). The harsh ionizing radiation at the surface of Mars dictates that any extant life would likely be subsurface. Despite the overlying regolith, pore space
* Corresponding author. University of Arkansas, Department of Biological Sciences, SCEN 601, Fayetteville, AR 72701, USA. E-mail address: [email protected] (T.A. Kral). https://doi.org/10.1016/j.pss.2017.12.014 Received 7 September 2017; Received in revised form 14 November 2017; Accepted 20 December 2017 Available online xxxx 0032-0633/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Mickol, R.L., Kral, T.A., Low pressure microenvironments: Methane production at 50 mbar and 100 mbar by methanogens, Planetary and Space Science (2017), https://doi.org/10.1016/j.pss.2017.12.014
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Simulation Chamber, housed within the Arkansas Center for Space and Planetary Sciences at the University of Arkansas, previously described (Kral et al., 2011). Experiments described here were initially modeled after the microenvironment experiment in Mickol and Kral (2016). Pressure setpoints (Table 1) were maintained using a MKS Type 651C pressure controller and MKS Type 253B throttling valve (MKS Instruments Inc., Andover, MA). All experiments were conducted at room temperature and the chamber was vented with room air following the period of exposure to low pressure. Pressures and exposure times are seen in Table 1.
between soil grains would still resemble the low-pressure conditions at the planet's surface, only reaching 25 mbar at 13.8 km below the martian surface (Schuerger et al., 2013). Higher pressures can be achieved in the near-subsurface of Mars, but this would require complete seclusion from the atmosphere so that gas-exchange and equilibration is not possible (Schuerger et al., 2013). Methane production at 400 mbar and 50 mbar by two of the three methanogens (Methanobacterium formicicum, Methanosarcina barkeri) tested here has been shown previously by experiments in this lab, although experiments only lasted 12 days (50 mbar) or 18 days [400 mbar] (Kral et al., 2011). Additionally, the chamber environment was heated to 35 C to promote growth and cultures were subject to desiccation over the course of the experiment. Schirmack et al. (2014) have also assessed methanogen growth under low-pressure conditions. These experiments utilized the permafrost isolate, Methanosarcina soligelidi, and assessed methane production at low temperatures (-5 to 20 C) at 500 mbar. In two experiments, cultures were initially incubated for one day at either 20 C or 10 C, during which most methane was produced. The temperature was then stepped down to 10 C or 0 C for 12 h, before the temperature was lowered to 0 C or -5 C for the remainder of the experiment (5 days). Cultures of M. soligelidi produced about 100 ppm methane at 0 C for five days and about 350 ppm methane during five days at -5 C, both at 500 mbar (Schirmack et al., 2014). Three species of methanogens (M. formicicum, M. barkeri, Methanococcus maripaludis) were tested for their ability to actively grow (increase in both methane abundance and optical density) within lowpressure microenvironments at 50 mbar or 100 mbar. The experiments conducted here were modeled after the microenvironment experiment in Mickol and Kral (2016). Previous experiments conducted at low pressure with methanogens in their typical, liquid, anaerobic growth media were subject to rapid evaporation due to the instability of water, when temperature is not maintained at low enough values (~0 C). To resolve this issue, experiments discussed here only incorporated pressures down to 50 mbar (above the vapor pressure of water at ~30 C) and test tubes were equilibrated to low pressure, then re-sealed, effectively creating individual microenvironments within each test tube.
2.3. Experiment 1: exposure of M. barkeri, M. formicicum, and M. maripaludis to 100 mbar for 28 days Methanogens were initially grown in their respective anaerobic growth media, as described above (2.1 Microbial procedures). The tubes were incubated at the methanogens’ respective growth temperatures for 7 days. After the initial incubation period, each tube was tested for both methane production and optical density to determine initial growth. There were five replicates for each of the three methanogen species, with two separate sets (inoculated from separate sources) for M. maripaludis (e.g., there were five replicates for Set A for M. maripaludis and five replicates for Set B for M. maripaludis). Next, tubes were placed inside the Pegasus Planetary Simulation Chamber with a palladium catalyst box to remove residual oxygen (Fig. 1A). Within the chamber, twenty tubes (five replicates each for M. barkeri and M. formicicum, ten replicates for M. maripaludis [see above]) were situated inside a test tube rack, sorted randomly. A second test tube rack was placed over the top of the tubes to secure their position for use with a specialized puncture device (see Mickol and Kral (2016) for a description of the device; also see Fig. 1B). As a control, an additional seventeen tubes (five replicates each for M. barkeri, M. formicicum, and M. maripaludis Set A, and two replicates for the M. maripaludis Set B cultures) were situated inside an adjacent test tube rack. These tubes would not be punctured (i.e., exposed to low pressure), but would be exposed to the same temperature as the punctured (low pressure) test tubes. Within both sets, one test tube for each species was not initially inoculated to serve as a blank for optical density measurements. The chamber door was closed and duct seal putty (Rainbow Technology, Pelham, AL) was applied around the seal as a further safeguard against oxygen contamination. The seal between the puncture device and the chamber was also covered with duct seal putty. The chamber was evacuated to 100 mbar, 80/20 H2/CO2 gas was bled into the chamber, and the system was left to equilibrate for 30 min. Next, the experimental tubes were punctured with the specialized puncture device containing one-inch, 22-gauge syringe needles for 60 min to allow equilibration between the chamber pressure and the pressure inside the tubes (see Mickol and Kral, 2016). After 60 min, the needles were removed from the tubes, effectively creating “low-pressure microenvironments” within each test tube, and the H2/CO2 gas was turned off. The chamber was actively maintained at 100 mbar for the duration of the experiment. After 28 days, the chamber was slowly vented with room air to atmospheric pressure. Test tubes were removed from the chamber and immediately tested for methane concentration and optical density. All tubes were re-pressurized with 2 bar H2 and put at the organisms’ respective incubation temperatures. Tubes were then monitored over time for methane production and optical density.
2. Materials and methods 2.1. Microbial procedures Methanogens were initially obtained from the American Type Culture Collection (ATCC; Manassas, VA), cultured in their respective anaerobic growth media, and kept at a temperature within the organisms' ideal growth ranges: Methanococcus maripaludis (ATCC 43000), MSH medium (Ni and Boone, 1991), 22 C; Methanosarcina barkeri (ATCC 43569), MS medium (Boone et al., 1989), 37 C; Methanobacterium formicicum (ATCC 33274), MSF medium (MS medium supplemented with formate), 37 C. Media were prepared as described in Kendrick and Kral (2006) under anaerobic conditions in a 90/10 CO2/H2 gas Coy Anaerobic Chamber (Coy Laboratory Products Inc., Grass Lake Charter Township, MI) and dispensed into anaerobic culture tubes at a rate of 10 mL per tube. The tubes were fitted with rubber stoppers and sealed with aluminum crimps as described in Boone et al. (1989), and autoclaved for sterilization. Prior to inoculation with the respective organisms, a solution of 2.5% w/v sodium sulfide (~125 μL per 10 mL medium) was added to each culture tube to remove residual oxygen (Boone et al., 1989). The tubes were then pressurized with 2 bar H2 and incubated at the organisms’ respective growth temperatures. For each experiment, growth was monitored by methane production via gas chromatography (Shimadzu Scientific Instruments Inc., model GC-2014, Columbia, MD) and via optical density (OD600, Spectronic 20Dþ, Spectronic Instruments, USA).
2.4. Experiment 2: exposure of M. barkeri, M. formicicum, and M. maripaludis to 50 mbar for 35 days Media were prepared as described in 2.1 Microbial procedures, however, the oxygen indicator, resazurin, was not included. Although this removed the ability to determine the extent of oxygen contamination within each test tube, it allowed for continued optical density measurements over time as the color of the media remained unchanged.
2.2. Pegasus planetary simulation chamber Low pressure experiments were conducted in the Pegasus Planetary 2
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Table 1 Experimental conditions for each of four experiments, including pressures and time exposed to low pressure. Each experiment consisted of five replicates for each of the methanogen species tested in 10 mL of their respective anaerobic growth medium. Pre- and post-exposure incubations were conducted at the methanogens’ respective growth temperatures (Methanosarcina barkeri, 37 C, MS medium; Methanobacterium formicicum, 37 C, MSF medium; Methanococcus maripaludis, 22 C, MSH medium). Expt.
Species Used
Pre-exposure Incubation Period (days)
Evacuation/ Experimental Pressure (mbar)
# of Evacuation Cycles
Time for Equilibration of Chamber at Low Pressure (min)
Time for Equilibration Between Punctured Tubes and Chamber (min)
Length of Exposure to Low Pressure (days)
Average Temperature Inside Pegasus Chamber ( C)
1
M. barkeri, M. formicicum, M. maripaludis M. barkeri, M. formicicum, M. maripaludis M. formicicum
7
100.4 0.1
0
30
60
28
26.6 0.5
7
49.8 0.1
2
30
60
35
28.9 2.0
7
50.1 0.1
1, CO2 2, H2/CO2 1, CO2 2, H2/CO2
30
60
49
27.7 1.7
30
60
49
27.5 1.7
2
3 4
M. formicicum
7
49.9 0.2
a
a The pressure control valve malfunctioned on Day 30 (of 49), dropping the chamber pressure to ~0.8 mbar. The tubes remained sealed and no significant evaporation was witnessed in any tubes. Thus, it is believed the ~50 mbar microenvironments remained intact for the duration of the experiment.
experiment. After 35 days, the chamber was vented to the atmosphere and the test tubes were removed. All test tubes were immediately measured for methane concentration and optical density. After measurement, tubes were re-pressurized with 2 bar H2 and stored at their incubation temperatures. Additional methane and optical density measurements were taken over time.
2.5. Experiment 3: exposure of M. formicicum to 50 mbar for 49 days This experiment utilized only cultures of M. formicicum and media did not contain resazurin, as in Expt. 2. Additionally, cells were washed before inoculation in order to remove residual resazurin. McAllister and Kral (2006) demonstrated that methanogenic cells could be exposed to atmospheric oxygen for at least 1.5 h during a washing procedure without significant loss of viability. Two, one-month-old cultures consisting of 10 mL MSF medium were combined, then poured into two large centrifuge tubes. Tubes were centrifuged at 5000 rpm for 45 min. Next, the supernatant was poured off, and 5 mL CO2 buffer containing 2.5% sodium sulfide (Na2S) were added to each centrifuge tube. The tubes were shaken to redistribute cells and centrifuged again at 5000 rpm for 45 min. The supernatant was poured off and 10 mL CO2 buffer þ Na2S were added to each centrifuge tube. The centrifuge tubes were combined to form a single inoculum source. Thirty-three test tubes containing sterile MSF medium without resazurin were then inoculated with 0.5 mL of washed M. formicicum cells (seven tubes were not inoculated to serve as blanks). Each test tube was pressurized with 2 bar H2 and incubated at 37 C for 7 days. After incubation, tubes were measured for methane concentration and optical density to determine initial growth. Chamber procedures were similar to those for Expts. 1 and 2. Tubes were placed into the Pegasus Planetary Simulation Chamber and the chamber was evacuated to 50 mbar while CO2 was bled into the system. The chamber was filled to atmospheric pressure with CO2, then the CO2 was turned off and the H2/CO2 gas was turned on. The chamber was evacuated again to 50 mbar, then filled to atmospheric pressure with H2/CO2. This cycle was repeated, the chamber pressure was lowered to 50 mbar, and the system was left to equilibrate at 50 mbar for 30 min. Next, the test tubes were punctured and allowed to equilibrate with the low pressure of the chamber for 60 min, after which the needles were removed from the test tubes and the H2/CO2 gas turned off. The chamber was actively maintained at 50 mbar for the duration of the experiment. After 49 days, the chamber was vented to the atmosphere and the tubes were removed. The test tubes were immediately measured for methane concentration and optical density. Following measurement, tubes were re-pressurized with 2 bar H2 and incubated at 37 C. Methane concentration and optical density continued to be monitored over time.
Fig. 1. Photos depicting arrangement of test tubes within the Pegasus Planetary Simulation Chamber for each experiment. A. Experimental setup for each experiment: pd ¼ puncture device, pc ¼ palladium catalyst. B. Close-up view of the puncture device. The puncture device contains 22-gauge, 1-inch syringe needles to puncture tubes and allow equilibration with the chamber atmosphere.
Experimental procedures were similar to those for Expt. 1, except for additional cycling between low pressure and atmospheric pressure before the tubes were punctured in order to ensure removal of the ambient atmosphere. Test tubes were inoculated with 0.5 mL culture, pressurized with 2 bar H2 and incubated for 7 days. Tubes were then measured for methane concentration and optical density to determine initial growth. Tubes were placed inside the chamber as described above for Expt. 1. The chamber was closed and evacuated to 50 mbar while CO2 gas was bled into the system. The chamber was then filled with CO2 to atmospheric pressure and evacuated again. This cycle was repeated, the chamber was again evacuated to 50 mbar, and the system was left to equilibrate for 30 min. Next, the CO2 gas was turned off and the H2/CO2 (80/20) gas was turned on while the chamber was maintained at 50 mbar. The test tubes were punctured as in Expt. 1. After 60 min, the needles were removed from the test tubes and the H2/CO2 gas was turned off. The chamber was actively maintained at 50 mbar for the duration of the 3
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Fig. 2. Methane production (left) and optical density (right) for Methanobacterium formicicum, Methanosarcina barkeri, Methanococcus maripaludis (Set A), and M. maripaludis (Set B) [top to bottom] measured immediately before and immediately after exposure to low pressure (100 mbar). An initial incubation period took place at 37 C (M. formicicum, M. barkeri) or 22 C (M. maripaludis) for 7 days. Low Pressure tubes were exposed to 100 mbar for 28 days. Control tubes were subjected to the same environmental conditions as the low-pressure tubes, except 4
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Simulation Chamber, the chamber was evacuated to ~50 mbar, and the tubes were punctured to directly expose them to the chamber atmosphere. After 1 h, the needles were removed from the test tubes, resealing the test tubes. The tubes were then removed from the chamber and immediately tested for methane concentration.
2.6. Experiment 4: exposure of M. formicicum to 50 mbar for 49 days with and without formate-supplemented media This experiment was intended to replicate Expt. 3, with slight modification. Two sets of media were prepared: Fourteen test tubes contained 10 mL MSF medium (MS medium supplemented with formate), while fourteen test tubes contained 10 mL MS medium (no formate). Thus, one set of tubes contained two energy sources, H2 and formate, while the second set contained only H2. Similarly to Expts. 2 and 3, the media did not contain the oxygen indicator, resazurin. Cells were also washed prior to inoculation, as in Expt. 3. Three, twomonth-old 10-mL stock cultures were poured into large centrifuge tubes. The tubes were centrifuged for 45 min at 5000 rpm, after which the supernatant was poured off. Ten milliliters of sterile CO2 buffer þ Na2S were added to each centrifuge tube and the tubes were shaken to redistribute pelleted cells. The tubes were centrifuged again at 5000 rpm for 45 min. Next, the supernatant was poured off and 10 mL CO2 buffer þ Na2S were added to each centrifuge tube. The tubes were then combined to form one inoculum source. An aliquot of 0.5 mL of washed cells was added to each of twenty test tubes (ten containing media with formate, ten containing media without formate). Eight test tubes remained un-inoculated to serve as blanks. The test tubes were pressurized with 2 bar H2 and incubated at 37 C for 7 days. After incubation, tubes were tested for methane concentration and optical density to determine initial growth. Chamber procedures were similar to those for Expt. 3. Tubes were placed within test tubes racks, assorted randomly. The chamber door was closed, sealed with duct seal putty, and the palladium catalyst box was turned on. The procedures for the initial evacuation of the chamber and the equilibration of the low-pressure tubes to the low pressure atmosphere of the chamber were identical to those in Expt. 3. After 49 days, the chamber was vented to the atmosphere and the tubes were removed. The low-pressure test tubes were immediately filled to just above atmospheric pressure (~0.07 bar) with CO2 gas using a gassing manifold. This was done for two reasons: 1. To bring cultures to atmospheric pressure for headspace analysis and 2. To minimize oxygen contamination when headspace samples were removed from the low-pressure test tubes. All test tubes were then equilibrated for 2 h before methane concentration was measured via gas chromatography. Following methane measurements, all tubes were re-pressurized with 2 bar H2 and incubated at 37 C. Methane concentration and optical density continued to be monitored over time. It is important to note that on Day 30 (of 49) for Expt. 4 (49 days at 50 mbar), the pressure control valve malfunctioned and dropped the internal chamber pressure to ~0.8 mbar. The individual test tubes remained sealed at this point, and there was no evidence of extensive leakage (evaporation) of the media. Thus, it is believed that the microenvironments remained intact at ~50 mbar for the following 19 days of the experiment, despite the lower pressure of the chamber. As the cultures were not subjected to this lower pressure, the data were analyzed without alteration.
3. Results The number of replicates for each species within each experiment are given as n ¼ #, below. In cases where optical density measurements were below zero, likely due to inaccuracies in light transmission through the test tubes, or where tubes containing resazurin showed signs of oxygen contamination, data were not included in the averages given. 3.1. Measurement of methane remaining within test tubes following puncture under low pressure The results indicate that the majority of the initial methane within each test tube escaped during the period of direct exposure to low pressure, as expected: M. formicicum, 0.18 0.10% CH4; M. barkeri, 0.22 0.01% CH4; M. maripaludis, 0.25 0.09% CH4. Thus, methane abundances measured after the initial 7-day incubation period, but prior to puncture under low pressure, only serve to indicate that cultures were active and growing at the time they were placed in the chamber. Those initial methane measurements can be found in Figs. 2–5. Comparing methane concentrations immediately following low-pressure exposure to methane concentrations of cultures prior to puncture would be meaningless. All methane concentrations following low-pressure exposure in Experiments 1-4 have been compared to the average methane concentration following puncture and 1 h direct exposure to 50 mbar (average: 0.22 0.07%; Figs. 2–5). This average methane concentration is derived from methane measurements of 12 cultures including three species. Measurements for all three species were very consistent. 3.2. Experiment 1: exposure of M. barkeri, M. formicicum, and M. maripaludis to 100 mbar for 28 days In this experiment, cultures of M. barkeri, M. formicicum, and M. maripaludis were maintained at 100 mbar for 28 days. Methane concentration for M. formicicum cultures initially measured 2.93 0.73% (n ¼ 3) on the day the tubes were placed inside the chamber (Day 7). Again, the starting concentration of methane following puncture was determined to be approximately 0.22% based on the results from the control experiment. Following exposure to 100 mbar for 35 days, methane abundance measured 4.82 0.41% within the low-pressure tubes (Fig. 2). Methane in control tubes increased from 3.69 0.13% to 33.71 12.36% (n ¼ 4) over the course of the experiment (Fig. 2). One low-pressure replicate was not adequately punctured during the experiment, and thus, not exposed to the low-pressure environment. It is not included in the data shown here. The optical density for M. formicicum low-pressure replicates also increased during exposure to low pressure (0.051 0.004 to 0.100 0.013; Fig. 2). This increase in optical density is similar to the increase in optical density for the control tubes. After exposure to low pressure, methane abundance in M. barkeri cultures (n ¼ 4) measured 0.78 0.17% while methane abundance within control tubes (n ¼ 4) measured ~6.5% (Fig. 2). Optical density within low-pressure tubes (n ¼ 4) increased from 0.003 0.003 to 0.012 0.006 over the period of exposure (Fig. 2). Optical density ranged widely for the control tubes with one replicate measuring 0.044
2.7. Measurement of methane remaining within test tubes following puncture under low pressure A separate control study was conducted in order to determine the amount of methane remaining in tubes following puncture under low pressure. Active cultures of the three species (M. formicicum, n ¼ 3; M. barkeri, n ¼ 4; M. maripaludis, n ¼ 5) were measured for methane abundance. The tubes were then placed into the Pegasus Planetary
for pressure. The specific number of replicates (typically, n ¼ 4) for both low pressure and control tubes for each methanogen are given in the text. In a separate experiment, active cultures (M. formicicum, n ¼ 3; M. barkeri, n ¼ 4; M. maripaludis, n ¼ 5) were directly exposed to low pressure (~50 mbar) for 1 h, then removed from the chamber and immediately measured for methane abundance (middle bars in graphs on left). These averages indicate that methane abundance within the low-pressure tubes was negligible (0.22 0.07%) at the start of the experiment. Error bars indicate one standard deviation. Note: there are no error bars for the optical density data for the M. maripaludis (Set B) Low Pressure tubes “Following Initial 7-day Growth Period” and “Following Length of Experiment (28 days)” nor for the Control tubes “Following Initial 7-day Growth Period” because these data reflect n ¼ 1 replicate. 5
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to 0.050 0.017) and control replicates (0.019 0.016 to 0.085 0.033, Fig. 4). Methane abundance and optical density also continued to increase in all tubes (low pressure, control) following removal from the chamber, repressurization with 2 bar H2 and incubation at 37 C (data not shown).
on Day 35 and the other three replicates measuring ~0.02 (Fig. 2). With respect to M. maripaludis (Sets A and B), there were minor increases in methane concentration for cultures exposed to 100 mbar for 28 days (Set A: 0.78 0.34%, n ¼ 4; Set B: 0.65 0.20%, n ¼ 3). M. maripaludis control cultures (Set A: n ¼ 4, Set B: n ¼ 2) showed increases in both methane concentration (~43%) and optical density (0.12–0.15) (Fig. 2). Optical density measurements decreased for the low-pressure replicates for M. maripaludis Set A over the course of exposure to low pressure, but increased greatly for the control replicates (n ¼ 2; Fig. 2).
3.5. Experiment 4: exposure of M. formicicum to 50 mbar for 49 days with and without formate-supplemented media Experiment 4 was intended to duplicate Experiment 3, but contained two major differences: 1. Media with or without formate and 2. Lowpressure replicates were brought to just above atmospheric pressure (~0.07 bar) with CO2 immediately following removal from the Pegasus Planetary Simulation Chamber. Both low-pressure groups (þformate, -formate) measured greater methane abundance following 49 days’ exposure to 50 mbar compared to 0.220.07% (Fig. 5). Low-pressure tubes supplemented with formate (n ¼ 5) measured 2.12 0.58% methane after the course of the experiment (49 days). The low-pressure tubes without formate (n ¼ 5) measured 1.08 0.54% methane following exposure to low pressure (Fig. 5). This is not surprising since formate acted as an additional energy source. Both control groups (þformate, -formate; n ¼ 5) increased by over ~40% methane over 49 days (Fig. 5). Optical density increased from 0.028 0.002 to 0.040 0.013 within low-pressure replicates supplemented with formate, while replicates lacking formate decreased slightly in optical density (0.023 0.003 to 0.019 0.011) over the period of exposure to low pressure (Fig. 5). Optical density increased in control tubes, from 0.013 0.002 to 0.139 0.019 in replicates supplemented with formate and from 0.022 0.005 to 0.112 0.025 in replicates lacking formate (Fig. 5). Methane abundance and optical density also continued to increase in all tubes (low pressure, control) following removal from the chamber, repressurization with 2 bar H2 and 28 days’ incubation at 37 C (data not shown).
3.3. Experiment 2: exposure of M. barkeri, M. formicicum, and M. maripaludis to 50 mbar for 35 days This experiment exposed cultures of M. barkeri, M. formicicum, and M. maripaludis to 50 mbar for 35 days. The media for this experiment did not include the oxygen indicator, resazurin. Therefore, possible oxygen contamination did not affect optical density values. However, the extent to which tubes may have been contaminated with oxygen is unknown. Both methane abundance and optical density increased for cultures of M. formicicum exposed to 50 mbar for 35 days (Fig. 3). Methane within low-pressure replicates (n ¼ 4) increased from 0.22 0.07% to 2.36 1.08% following exposure to 50 mbar. Methane production within control replicates (n ¼ 4) was nearly identical to production within low-pressure replicates (Fig. 3). Optical density of low-pressure tubes (n ¼ 4) increased from 0.023 0.015 to 0.046 0.023 during exposure to low pressure. All four low-pressure replicates demonstrated increased methane abundance and optical density within individual tubes, but the variation amongst the four tubes accounts for the large error bars (Fig. 3). Surprisingly, optical density within control replicates (n ¼ 4) decreased slightly during the length of the experiment (Fig. 3). With respect to M. barkeri, there was no difference in methane concentration following low-pressure exposure (n ¼ 4; Fig. 3). Optical density increased from 0.009 0.001 to 0.022 0.014 within low-pressure tubes (n ¼ 4) during the 35-day exposure to 50 mbar. Optical density only increased in three of the four low-pressure replicates, resulting in the large error bar seen in Fig. 3. Both methane abundance and optical density increased in control tubes (n ¼ 4) over the course of the experiment (Fig. 3). Methane concentration measured 0.82 0.05% in cultures of M. maripaludis (n ¼ 4) following exposure to 50 mbar for 35 days whereas optical density decreased (Fig. 3). Optical density increased from 0.023 0.002 to 0.106 0.020 within control tubes over the course of the experiment while methane abundance increased from 1.66 0.41% to 41.28 10.00% (Fig. 3). Methane production and optical density continued to be monitored for all species in both Experiment 1 (Section 3.2) and Experiment 2 (Section 3.3) following removal from the chamber. Increases in both methane and optical density were seen for all three species up to 49 days following the length of the experiment (data not shown).
4. Discussion When considering the habitability of Mars, low pressure is often overlooked as a potentially biocidal factor in favor of more potentially lethal conditions such as exposure to UV radiation and desiccation. However, there are currently no surface environments on Earth where microorganisms would be subjected to the low pressures present on Mars. For example, the lowest surface pressure on Earth reaches only 330 mbar at the top of Mount Everest (Fajardo-Cavazos et al., 2012), whereas the surface pressure on Mars averages ~7 mbar (Schuerger and Nicholson, 2016). Several studies have noted decreased growth rates for certain bacteria under 100 mbar and complete lethality below 25 mbar (Schuerger and Nicholson, 2016). Of over 150 bacterial strains studied, only a few species such as Carnobacterium spp. (Nicholson et al., 2013), Pseudomonas fluorescens (Schuerger and Nicholson, 2016) and Serratia liquefaciens (Schuerger et al., 2013) have exhibited growth below 25 mbar. These results suggest that low pressure may have more of a lethal or inhibitory effect on certain bacteria, warranting further research into growth under low pressure. Additionally, few studies have included Archaea in low pressure experiments (Kral and Altheide, 2013; Kral et al., 2011; Mickol and Kral, 2016; Schirmack et al., 2014), illustrating a need for further research in this area. The use of methanogens in studies of martian habitability provides a possible biological explanation for the methane detected in the martian atmosphere. However, demonstrating active methane production within methanogenic cultures at low pressure is difficult considering methane is typically monitored via gas chromatography and removing a gas sample from a low-pressure environment (e.g., vacuum chamber) for analysis is problematic. In the experiments conducted here, low-pressure “microenvironments” were created by equilibrating the headspace within anaerobic test tubes with the low-pressure atmosphere inside a planetary simulation chamber. Tubes were equilibrated to low pressure (100 mbar,
3.4. Experiment 3: exposure of M. formicicum to 50 mbar for 49 days This experiment solely utilized cultures containing M. formicicum and cells were washed prior to inoculation. There were initially 17 replicates that were exposed to low pressure, however, one tube was not sufficiently punctured to allow equilibrium between the low-pressure environment of the chamber and the test tube and so this replicate is not included in the data shown here (n ¼ 16). There were also 16 tubes that were not exposed to low pressure that served as control replicates (n ¼ 16). The average methane produced among the low-pressure replicates increased during the exposure period to 3.37 1.26% (Fig. 4). Methane increased by greater amounts in control tubes (average: 25.48 22.23%, Fig. 4), but with considerable variation among replicates, ranging from 5% to 57%. Optical density increased in both low-pressure replicates (0.019 0.011 6
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Fig. 3. Methane production (left) and optical density (right) for Methanobacterium formicicum, Methanosarcina barkeri, and Methanococcus maripaludis (top to bottom) measured immediately before and immediately after exposure to low pressure (50 mbar). An initial incubation period took place at 37 C (M. formicicum, M. barkeri) or 22 C (M. maripaludis) for 7 days. Low Pressure tubes (n ¼ 4) were exposed to 50 mbar for 35 days. Control tubes (n ¼ 4) were subjected to the same environmental conditions as the low-pressure tubes, except for pressure. In a separate experiment, active cultures (M. formicicum, n ¼ 3; M. barkeri, n ¼ 4; M. maripaludis, n ¼ 5) were directly exposed to low pressure (~50 mbar) for 1 h, then removed from the chamber and immediately measured for methane abundance (middle bars in graphs on left). These averages indicate that methane abundance within the low-pressure tubes was negligible (0.22 0.07%) at the start of the experiment. For M. barkeri, optical density measurements reflect n ¼ 3 replicates for the “Following Initial 7-day Growth Period” control tubes only. Error bars indicate one standard deviation. 7
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Fig. 4. Methane production (left) and optical density (right) for Methanobacterium formicicum measured immediately before and immediately after exposure to low pressure (50 mbar). Cells were washed before inoculation. An initial incubation period took place at 37 C for 7 days. Low Pressure tubes (n ¼ 16) were exposed to 50 mbar for 49 days. Control tubes (n ¼ 16) were subjected to the same environmental conditions as the low-pressure tubes, except for pressure. In a separate experiment, active cultures of M. formicicum (n ¼ 3), Methanosarcina barkeri (n ¼ 4), and Methanococcus maripaludis (n ¼ 5) were directly exposed to low pressure (~50 mbar) for 1 h, then removed from the chamber and immediately measured for methane abundance (middle bars in graphs on left). These averages indicate that methane abundance within the low-pressure tubes was negligible (0.22 0.07%) at the start of the experiment. Error bars indicate one standard deviation.
Fig. 5. Methane production (left) and optical density (right) for Methanobacterium formicicum measured immediately before and immediately after exposure to low pressure (50 mbar). Cells were washed prior to inoculation. An initial incubation period took place at 37 C for 7 days. Low Pressure tubes (n ¼ 10) were exposed to 50 mbar for 49 days. Control tubes (n ¼ 10) were subjected to the same environmental conditions as the low-pressure tubes, except for pressure. Both Low Pressure and Control tubes were separated into two groups: þF replicates (n ¼ 5) contain formate in the medium, -F replicates (n ¼ 5) lack formate. In a separate experiment, active cultures of M. formicicum (n ¼ 3), Methanosarcina barkeri (n ¼ 4), and Methanococcus maripaludis (n ¼ 5) were directly exposed to low pressure (~50 mbar) for 1 h, then removed from the chamber and immediately measured for methane abundance (middle bars in graphs on left). These averages indicate that methane abundance within the low-pressure tubes was negligible (0.22 0.07%) at the start of the experiment. Error bars indicate one standard deviation.
height (volume) of the liquid medium was detected from the individual cultures. Additionally, in Expts. 1-3, negative pressure within each replicate (as headspace samples were removed for methane detection) further demonstrated that each individual test tube was under low pressure. Low pressure tubes in Expt. 4 were also verified as being under low pressure by the ability to add ~0.07 bar CO2 to cultures following removal from the chamber. Methane abundance within anaerobic test tubes was measured by
Expt. 1 or 50 mbar, Expts. 2-4) for 60 min using syringe needles. After the period of equilibration, the needles were removed, resealing the anaerobic test tubes and effectively creating low-pressure microenvironments within each replicate. The chamber atmosphere was actively maintained at 100 mbar (Expt. 1) or 50 mbar (Expts. 2-4) for the duration of the experiment so that there was no change in pressure in the event of test tube leakage. Further, visual inspection during and after the exposure period ensured that microenvironments were intact as no change in 8
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against one another, for differences in growth rates or cell clumping may exacerbate differences. That being said, the apparent differences in active growth at low pressure between the three species tested here may be the result of physical attributes of the cells such as cell wall composition and affinity for clumping. M. maripaludis was the only species that did not demonstrate appreciable methane production or increased optical density at either pressure tested, which may be due to a relatively weaker cell wall. The cell walls of Methanococcus species consist of a single electron dense outer layer that is both osmotically fragile and susceptible to detergents (Jones et al., 1983). In comparison, Methanosarcinales is the only methanogenic order that forms multicellular aggregates due to the formation of a heteropolysaccharide matrix called methanochondroitin (Milkevych et al., 2015). This extracellular polysaccharide matrix is believed to offer additional protection against environmental stresses. In a study comparing individual cells and multicellular aggregates of M. barkeri, Anderson et al. (2012) discovered that cells that produced methanochondroitin (and thus formed multicellular aggregates) had significantly higher survival rates than individual cells in response to desiccation, high temperatures, and oxygen exposure. Additionally, because variations in growth proxies exist across replicates within the same experiment, comparison across experiments may also be difficult when precise cell numbers are not used. This is apparent for M. formicicum in Expt. 2 where methane abundance only reaches ~2% within both control and low pressure tubes after 35 days (Fig. 3). Expected methane production within control tubes would be about 30%. Optical density for these cultures was also significantly lower in Expt. 2 than in the other experiments described here. The explanation for the lower methane and optical density measurements are currently unknown, but are not uncommon with respect to variation in methanogenic pure cultures. This variation is also evident in the methane production for M. formicicum in Expt. 3 which encompasses very large error bars (Fig. 4). Ultimately, these experiments aimed to qualitatively demonstrate the ability of one species, M. formicicum, to both produce methane and increase in optical density at low pressure (50 mbar, 100 mbar) despite variation both within and between experiments. The pressures utilized in the experiments described here (50 mbar, 100 mbar) may be achievable in the martian subsurface. Schuerger et al. (2013) describe the atmospheric gas-phase pressure increasing very slowly in the martian subsurface, reaching 25 mbar at 13.8 km. However, should there be complete seclusion from the atmosphere either by inclusion in rock or ice, the lithographic pressure increases rapidly, reaching 25 mbar at a depth of 19.5 cm (Schuerger et al., 2013). In a subsurface environment, microorganisms would be shielded from the harsh ionizing radiation at the surface of the planet and may experience more stable temperature regimes (Schorghofer and Aharonson, 2005). Additionally, observations of recurring slope lineae at the surface of Mars suggest that there is a mobile liquid brine water column within the subsurface that operates on seasonal timescales (McEwen et al., 2014; Ojha et al., 2015). This water column could provide liquid water to any extant microorganisms (dependent on salt tolerances), as well as transport microorganisms to various depths (e.g., pressures) in the martian subsurface. Future experiments would benefit from the inclusion of additional martian parameters such as temperature and salt concentration, as well as lower pressures, in order to better mimic the current martian environment. Due to the variation amongst replicates under identical conditions, future experiments may need to utilize additional growth proxies besides methane concentration and optical density. The anaerobic nature of methanogens, the high number of replicates within most experiments, and the slow doubling time of the organisms makes cell counting or MPN procedures a daunting task. However, accurate cell counts would provide a necessary comparison to methane abundances and optical densities.
removing a 0.5-mL sample of gas from the headspace and injecting this into a gas chromatograph. In Expts. 1-3, following exposure to low pressure, headspace samples were removed from the test tubes while still under low pressure. Since the test tubes were themselves below atmospheric pressure, it is possible that air began to enter each test tube as soon as the external pressure rose above 50 mbar (or 100 mbar, Expt. 1). The test tubes remained sealed with aluminum crimps and rubber stoppers, although it is possible that air began to seep into each tube through pores within the stoppers or if the seal between the stopper and the test tube was not adequately robust. Whether or not air is able to seep into the test tubes themselves following removal from the chamber, it is known that air will rush into the syringe when a headspace sample is removed from the test tube and transferred to the gas chromatograph. Airtight syringes were not used in this research. If airtight syringes had been used, a similar effect would have occurred. Once the low-pressure sample had been injected into the gas chromatograph, air would have diluted the sample in the injection port. Thus, the samples removed from the lowpressure tubes were diluted, including the control tubes designed to measure remaining methane following puncture. In the case of Expt. 1, the dilution would occur at a factor as high as ~10 between the experimental pressure (100 mbar) and atmospheric pressure (~1013 mbar). For Expts. 2-4, the dilution factor may have reached ~20 (1013 mbar/50 mbar). As a gas chromatograph measures methane concentration as a percentage of the headspace within the test tube, the recorded values for methane abundance seen here are lower estimates for the true abundances contained within the test tubes. M. formicicum was the only methanogen out of the three species to demonstrate both substantial increases in methane production (>1%) and optical density during exposure to 100 mbar and 50 mbar. In a separate control study, growing cultures were placed inside the Pegasus Planetary Simulation Chamber, punctured, and exposed to low pressure (~50 mbar) for 1 h. The needles were removed from the test tubes and the cultures were immediately tested for methane abundance. The methane initially present in each test tube had escaped, as expected. Average methane abundance was 0.22% for each of the three methanogens (see Results). Thus, measured methane abundances above 1% within low-pressure replicates following the length of the experiment indicate active methane production at low pressure. M. barkeri demonstrated a small increase in both methane concentration and optical density at 100 mbar, but not at 50 mbar (Figs. 2 and 3). Cells of Methanosarcina spp. typically form irregular multicellular aggregates, especially under stressed conditions (Maestrojuan and Boone, 1991), which may account for the increases in optical density seen here. Although M. barkeri and M. maripaludis did not demonstrate active growth (>1% CH4) under low-pressure conditions, methane production resumed during the post-exposure incubation period and cultures also increased in optical density (data not shown). However, survival was likely not difficult as cells remained within a 10-mL water column for the duration of the experiment. Mickol and Kral (2016) also note that as the tubes were upright and the cells likely congregated at the bottom of the test tubes, the pressure at the bottom of the water column was, in actuality, about 6 mbar higher than the experimental pressure (50 mbar or 100 mbar) due to the weight of the water. On the other hand, the ideal gas law (PV ¼ nRT; Quagliano and Vallarino, 1969) would dictate that combining five molecules of gas to create three molecules would slightly decrease the headspace pressure within the test tubes: 4H2 þ CO2 → CH4 þ 2H2O (1). Working with methanogens, we witness this decrease in pressure regularly. Methane abundance and optical density measurements were not tightly coupled in the experiments described here, with increases in methane abundance not correlating to increases in optical density, and vice versa (for example, Expt. 2: M. barkeri, 35 days at 50 mbar). This is not uncommon with respect to methanogenic pure cultures where identical replicates are often seen to vary in both methane production and optical density (Gunnigle et al., 2013; Maestrojuan and Boone, 1991). Thus, these experiments are not intended to compare species
5. Conclusions Low pressures between 50 mbar and 100 mbar may be achievable in 9
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the martian subsurface and could potentially house extant methanogens, given the availability of liquid water. M. formicicum is capable of active growth at 50 mbar as verified by concurrent methane production and increased optical density. Although M. barkeri did not demonstrate growth at 50 mbar and M. maripaludis did not demonstrate growth during either low pressure tested, all cultures produced methane and increased in optical density during the post-exposure incubation period, indicating that all species survived exposure to low pressure. These experiments suggest that low pressure environments on Mars may not be lethal to certain species of methanogens and increase the possibility of a habitable subsurface environment on the planet.
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