Non-Psychrophilic Methanogens Capable of Growth Following Long-Term Extreme Temperature Changes, with Application to Mars

Abstract

Although the martian environment is currently cold and dry, geomorphological features on the surface of the planet indicate relatively recent (<4 My) freeze/thaw episodes. Additionally, the recent detections of near-subsurface ice as well as hydrated salts within recurring slope lineae suggest potentially habitable micro-environments within the martian subsurface. On Earth, microbial communities are often active at sub-freezing temperatures within permafrost, especially within the active layer, which experiences large ranges in temperature. With warming global temperatures, the effect of thawing permafrost communities on the release of greenhouse gases such as carbon dioxide and methane becomes increasingly important. Studies examining the community structure and activity of microbial permafrost communities on Earth can also be related to martian permafrost environments, should life have developed on the planet. Here, two non-psychrophilic methanogens, Methanobacterium formicicum and Methanothermobacter wolfeii, were tested for their ability to survive long-term (~4 year) exposure to freeze/thaw cycles varying in both temperature and duration, with implications both for climate change on Earth and possible life on Mars.

Keywords: Mars; freeze/thaw; methane; methanogens; permafrost.

Figure 1

Methane production (% headspace) over time for two species of methanogens (Methanobacterium formicicum, Methanothermobacter wolfeii) at 22 °C. Error bars indicate ± one standard deviation (n = 4).

Figure 2

Methane production by Methanobacterium formicicum following an initial incubation period for each of three sets. Test tubes for the Original Set and Transfer Sets 1 and 2 contain 5 g sand and 10 mL MSF medium. Transfer Set 3 tubes contain 10 mL MSF medium only. Transfer Set 1 tubes (n = 4) were inoculated from one tube in the Original Set (n = 4) following 105 days of freeze/thaw cycles. Transfer Set 2 tubes (n = 4) were inoculated from three separate test tubes from Transfer Set 1 following 74 days of freeze/thaw cycles. Transfer Set 3 tubes (n = 4) were inoculated on Day 1494 from the corresponding tube in Transfer Set 2. Specific freeze/thaw cycles (time, temperature) are given in Table 2. Specific inoculation schemes between sets are given in the supplementary material. The asterisk indicates that no methane was detected. Error bars indicate ± one standard deviation.

Figure 3

Methane production by Methanothermobacter wolfeii following an initial incubation period for each of three sets. Test tubes for the Original Set and for Transfer Sets 1 and 2 contain 5 g sand and 10 mL MM medium. Transfer Set 3 tubes contain 10 mL MM medium only. Transfer Set 1 tubes (n = 5) were inoculated from one tube in the Original Set (n = 4) following 105 days of freeze/thaw cycles. Transfer Set 2 tubes (n = 5) were inoculated from the corresponding tube within Transfer Set 1 following 74 days of freeze/thaw cycles. Transfer Set 3 tubes (n = 5 *) were inoculated from the corresponding tube within Transfer Set 2 on Day 1494. Specific freeze/thaw cycles (time, temperature) are given in Table 2. Specific inoculation schemes between sets are given in the supplementary material. Error bars indicate ± one standard deviation. * One replicate did not produce any methane and is not included in the data shown here.

Figure 4

Methane production by Methanobacterium formicicum following an initial incubation period for each of three sets. Test tubes for the Original Set and for Transfer Sets 1 and 2 contain 10 g sand and 5 mL MSF medium. Transfer Set 3 tubes contain 10 mL MSF medium only. Transfer Set 1 tubes (n = 4) were inoculated from one tube in the Original Set (n = 4) following 91 days of freeze/thaw cycles. Transfer Set 2 tubes (n = 4) were inoculated from three separate test tubes from Transfer Set 1 following 99 days of freeze/thaw cycles. Transfer Set 3 tubes (n = 4) were inoculated from the corresponding tube in Transfer Set 2 after 1474 days of freeze/thaw cycles. Specific freeze/thaw cycles (time, temperature) are given in Table 3. Specific inoculation schemes between sets are given in the supplementary material. The asterisk indicates that no methane was detected. Error bars indicate ± one standard deviation.

Figure 5

Methane production by Methanothermobacter wolfeii following an initial incubation period for each of three sets. Test tubes for the Original Set and for Transfer Sets 1 and 2 contain 10 g sand and 5 mL MM medium. Transfer Set 3 tubes contain 10 mL MM medium only. Transfer Set 1 tubes (n = 3) were inoculated from two separate test tubes in the Original Set (n = 3) following 91 days of freeze/thaw cycles. Transfer Set 2 tubes (n = 5) were inoculated from two separate test tubes from Transfer Set 1 following 99 days of freeze/thaw cycles. Transfer Set 3 tubes (n = 5) were inoculated from the corresponding tube in Transfer Set 2 after 1474 days of freeze/thaw cycles. Specific freeze/thaw cycles (time, temperature) are given in Table 4. Specific inoculation schemes between sets are given in the supplementary material. The asterisk indicates that no methane was detected. Error bars indicate ± one standard deviation.

Figure 6

Methane production by Methanothermobacter wolfeii following an initial incubation period for each of two sets. Original Set test tubes contained 5 mL MM medium. Transfer Set 1 test tubes contained 10 mL MM medium. Transfer Set 1 tubes (n = 5 *) were inoculated from the corresponding replicate in the Original Set (n = 5) following 1284 days of freeze/thaw cycles (Table 5). Error bars indicate ± one standard deviation. * Two of five replicates within Transfer Set 1 failed to produce methane after 28 days’ incubation and are not included in the data shown here.

Figure 7

Methane production by Methanobacterium formicicum following an initial incubation period for each of two sets. Original Set test tubes contained 5 mL MSF medium. Transfer Set 1 test tubes contained 10 mL MSF medium. Transfer Set 1 tubes (n = 5) were inoculated from the corresponding replicate in the Original Set (n = 5) following 1284 days of freeze/thaw cycles (Table 5). The asterisk indicates that no methane was detected. Error bars indicate ± one standard deviation.