Next Article in Journal
Effect of Lemongrass Essential Oil Vapors on Microbial Dynamics and Listeria monocytogenes Survival on Rocket and Melon Stored under Different Packaging Conditions and Temperatures
Next Article in Special Issue
Membrane Association and Catabolite Repression of the Sulfolobus solfataricus α-Amylase
Previous Article in Journal / Special Issue
A Three-Component Microbial Consortium from Deep-Sea Salt-Saturated Anoxic Lake Thetis Links Anaerobic Glycine Betaine Degradation with Methanogenesis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microorganisms
Volume 3
Issue 3
10.3390/microorganisms3030518
microorganisms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

The Effects of Perchlorates on the Permafrost Methanogens: Implication for Autotrophic Life on Mars

by
Viktoria Shcherbakova
1,2,*,
Viktoria Oshurkova
1 and
Yoshitaka Yoshimura
2,3
1
Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki 5, Pushchino, Moscow, 142290, Russia
2
Japan Aerospace Exploration Agency, Institute of Space and Astronautical Science (ISAS), 3-1-1 Yoshinodai, Chuo-ku, Sagamihara, Kanagawa, 252-5210, Japan
3
College of Agriculture, Tamagawa University, 6-1-1 Tamagawagakuen, Machida, Tokyo, 194-8610, Japan
*
Author to whom correspondence should be addressed.
Microorganisms 2015, 3(3), 518-534; https://doi.org/10.3390/microorganisms3030518
Submission received: 30 June 2015 / Revised: 30 July 2015 / Accepted: 1 September 2015 / Published: 9 September 2015
(This article belongs to the Special Issue Extremophiles)
Browse Figures
Versions Notes

Abstract

:
The terrestrial permafrost represents a range of possible cryogenic extraterrestrial ecosystems on Earth-like planets without obvious surface ice, such as Mars. The autotrophic and chemolithotrophic psychrotolerant methanogens are more likely than aerobes to function as a model for life forms that may exist in frozen subsurface environments on Mars, which has no free oxygen, inaccessible organic matter, and extremely low amounts of unfrozen water. Our research on the genesis of methane, its content and distribution in permafrost horizons of different ages and origin demonstrated the presence of methane in permanently frozen fine-grained sediments. Earlier, we isolated and described four strains of methanogenic archaea of Methanobacterium and Methanosarcina genera from samples of Pliocene and Holocene permafrost from Eastern Siberia. In this paper we study the effect of sodium and magnesium perchlorates on growth of permafrost and nonpermafrost methanogens, and present evidence that permafrost hydogenotrophic methanogens are more resistant to the chaotropic agent found in Martian soil. In this paper we study the effect of sodium and magnesium perchlorates on the growth of permafrost and nonpermafrost methanogens, and present evidence that permafrost hydogenotrophic methanogens are more resistant to the chaotropic agent found in Martian soil. Furthermore, as shown in the studies strain M2T M. arcticum, probably can use perchlorate anion as an electron acceptor in anaerobic methane oxidation. Earth’s subzero subsurface environments are the best approximation of environments on Mars, which is most likely to harbor methanogens; thus, a biochemical understanding of these pathways is expected to provide a basis for designing experiments to detect autotrophic methane-producing life forms on Mars.

1. Introduction

In 1974 Cameron and Morelli were the first to advance the idea for solving the exobiology problems by using the terrestrial permafrost model [1]. Permafrost is a stable and balanced environment, which maintains life incomparably longer than any other known habitats. It does not depend on the composition of the ground and acts as a physical and biogeochemical barrier that limits infiltration of both surface water and external environmental factors [2,3].Viable microorganisms have been isolated from permanently frozen sediments at lowest temperatures in the Arctic (−17 °C) and Antarctica (−27 °C) [4,5,6], down to 400 m depth in the Mackenzie Delta [4], and up to 4700 m elevation in the Tibetan Plateau [7]. The age of the isolates indicates the longevity of the permanently frozen state of the sediments, and dates back from a few thousand to 2–3 million years in eastern Arctic, and to 5–8 million years in Antarctica. This is why permafrost on Earth has been considered a terrestrial analogue for possible extraterrestrial ecosystems within frozen subsurfaces, such as is assumed for Mars.
Mars presents hostile environments for mesophilic terrestrial life [8,9,10], but it seems to be capable of supporting liquid water, which is generally regarded as a prerequisite for life. Results from the Neutron Spectrometer on Mars Odyssey indicate the presence of extensive deposits of water ice [11,12,13,14]. As it is known by now, the top 20–50 cm of Martian surface is friable dry frozen ground, and the subsequent 1 m that was investigated is permafrost [10]. Methane was discovered in the Martian atmosphere in 2003, and, to date, several separate groups have reported its detection [15,16,17,18]. Because of the photochemical dissociation of tropospheric methane, methane has a lifetime of only several hundred years in the troposphere. Hitchcock and Lovelace [19] were the first to point out that, because of its short lifetime, the presence of atmospheric methane indicates its constant replenishment. This replenishment could originate either from biological metabolism or from non-biological processes (volcanoes, etc.).
Thus, the anaerobic chemolithotrophic psychrotolerant–methanogenic microorganisms with their mechanisms to assimilate CO2 and other inorganic compounds are more likely than aerobes to function as a model for life forms that may exist in frozen subsurface environments on Mars, which has no free oxygen, inaccessible organic matter, and extremely low amounts of unfrozen water. Earlier, we isolated and described four strains of psychrotolerant methanogens from the samples of Pliocene and Holocene permafrost from Eastern Siberia [20,21,22]. Methane-producing Archaea obtain energy in converting a limited number of substrates [23,24]. The main substrates are CO2, methyl-containing compounds and acetate. All of these substrates are stoichiometrically converted to methane. The ability of methanogens to use H2 as an electron donor for the restoration of CO2 is widespread in natural ecosystems.
A methanogen that metabolizes carbon dioxide is classified as hydrogenotrophic, while those that metabolize the methyl group of acetate are called acetotrophic or aceticlastic. For the past two decades, methanogens isolated from terrestrial habitats have been studied as a model for extraterrestrial life [25,26]. As shown in Mars-modeling experiments, methanogens could exist on a low concentration of molecular hydrogen [27], on a Mars soils stimulant [28,29,30,31,32], survive following desiccation at various pressure values [33,34,35,36], and be sensitive to ultraviolet radiation [37,38].
In May 2008, the Wet Chemistry Laboratory on the board of the 2007 Phoenix Mars Lander performed the first wet chemical analysis of Martian soil. The analysis of three samples, two from the surface and one from a depth of 5 cm, revealed slightly alkaline soil and low levels of salts typically found on Earth [39]. The remaining anions included small concentrations of chloride, bicarbonate, and sulfate. Cations were dominated by Mg2+ and Na+, with small contributions from K+ and Ca2+. Unexpected though was the presence of ~0.6% by weight of perchlorate (ClO4−), most likely as a Ca(ClO4)2 or Mg(ClO4)2 phase.
Perchloric acid salts are chaotropic compounds causing the weakening of the hydrogen bonds between water and macromolecules [40,41,42] unlike kosmotropic (stabilizing) salts or substances which may have a comparable effect [42,43]. All chaotropic substances thus far tested have been shown to act on macromolecular systems in vivo in studies of diverse microorganisms [44,45,46,47,48]. On the other hand, it has been known for more than 50 years that microorganisms can reduce oxyanions of chlorine such as chlorate (ClO3−) and perchlorate under anaerobic conditions [49]. The high reduction potential of perchlorate (ClO4−/Cl−Eo = 1.287 V) makes it an ideal electron acceptor for the microbial metabolism. The metabolic capability of perchlorate reduction is widespread throughout the Proteobacteria [50], which has some interesting evolutionary implications in the light of the assumed limited geographical distribution of natural sources of chlorate and perchlorate. However, so far, we know about several representatives of nonmethanogenic Archaea that reduces perchlorate [51,52,53], and it is not clear whether these compounds are stressors to methanogens.
In this paper we study the effect of sodium and magnesium perchlorates on growth of permafrost and nonpermafrost methanogens, and present evidence that permafrost hydogenotrophic methanogens are more resistant to the chaotropic agent found in Martian soil.

2. Experimental Section

2.1. Archaeal Strains

Three strains of methanogenic archaea Methanobacterium articum M2T VKM B-2372T, Methanobacterium veterum MK4T VKM B-2440T, and Methanosarcina sp. JL01 VKM B-2370 isolated in our laboratory from permafrost of different ages [20,21,22] were tested in the study. Methanobacterium bryantii strain M.o.H.T VKM B-1629T and Methanosarcina mazei S6T VKM B-1636T from All-Russian collection of microorganisms (VKM) were used for comparative experiments (Table 1).
Table
Table 1. Characteristics of methanogenic archaea used in the study.
Table 1. Characteristics of methanogenic archaea used in the study.
MethanogensSubstrates for MethanogenesisSource of IsolationReferences
Methanobacterium bryantii M.o.H.TH2 + CO2Syntrophic association[54]
Methanosarcina mazei S-6TAcetate, methanol, methylamine, trimethylamine, H2 + CO2Anaerobic sewage digester[55]
Methanobacterium arcticum M2TH2+CO2, formateHolocene permafrost[22]
Methanobacterium veterum MK4TH2 + CO2, H2 + methanol, H2 + methylaminePlieocene permafrost[21]
Methanosarcina sp. JL01Acetate, methanol, methylamine, trimethylamineHolocene permafrost[20]

2.2. Media and Cultivation

Media for archaeal strains were prepared according to the Hungate anaerobic technique [56]. Strains M.arcticum M2T and M. bryantii M.o.H.T were grown in MB medium containing (g·L−1) 0.05 g sodium acetate trihydrate, 0.45 g (NH4)2SO4, 0.29 g K2HPO4, 0.18 g KH2PO4, 0.12 g MgSO4.7H2O, 0.06 g CaCl2. 2H2O, 5.0 g NaCl, 10 mL vitamin solution (medium 141; DSMZ), 10 mL trace element solution (medium 141; DSMZ), 0.001 g resazurin, 0.25 g cysteine hydrochloride hydrate, 0.25 g Na2S·9H2O; H2/CO2 (80:20) at 200 kPa; [54] and M. veterum strain MK4T was grown in DSMZ medium 506 with the same modification [21]. A basal medium of the following composition was used for cultivation of M. mazei strain S6T and Methanosarcina sp. JL01 (g·L−1): 4.0 g sodium acetate trihydrate, 0.29 K2HPO4, 0.29 KH2PO4, 1.0 NaCl, 0.2 MgCl2 × 6H2O, 1.0 NH4Cl, 0.1 CaCl2 × 2H2O, 0.5 cysteine hydrochloride, plus 5 mL vitamin solution and 10 mL trace elements solution [54].
Methanogens were grown in Hungate tubes or flasks at temperatures that are optimal for the growth of each strain: 37 °C (M. bryantii M.o.H.T, M. arcticum M2T and M. mazei S6T), 30 °C (M. veterum MK4T) and 25–28 °C (Methanosarcina sp. JL01).Growth was determined by the methane formation in the gas phase, or spectrophotometrically at 600 nm.

2.3. Determination of Inhibitory Concentrations

Inhibitory effect of substances on methanogens was assessed by measuring their influence on the rate of methanogenesis from CO2 + H2 and acetate (Figure 1). Methanogenic strains were grown in 150 mL flasks with 100 mL medium volume under optimal conditions, medium and the substrate for each strain until the optical density (OD600) was 0.2 for Methanobacterium spp. and 0.3 for Methanosarcina spp. It should be noted that both methanosarcina strains grow with a milky white turbidity formation, and do not form large clamps in the used medium.
The scheme of the experiment for determining the inhibitory concentrations of perchlorates can be seen in Figure 1. Grown cultures of methanogens were added to 30-mL glass flasks containing 10 mL of cultivation medium for the respective strain supplemented with 30 mM acetate or with H2 + CO2 gas phase (80:20, at 200 kPa). After purging methane from the medium with nitrogen, the flasks were incubated anaerobically at temperatures corresponding to each strain.
Microorganisms 03 00518 g001 1024
Figure 1. Scheme of the experiment for determining the inhibitory concentrations of perchlorates.
Figure 1. Scheme of the experiment for determining the inhibitory concentrations of perchlorates.
Microorganisms 03 00518 g001
On the following day, the incubation medium was analyzed for acetate and H2 and their concentrations in medium and gas phase was replenished. The perchlorates were added to assay flasks at desired concentrations, except for control flasks free of tested substances. After flushing the flasks with nitrogen, the incubation was resumed. Following two days of incubation, the measurement and replenishment of acetate and hydrogen were repeated as described above. After a 1 h reincubation, the headspaces of all the assay flasks were analyzed for the methane produced during this time period. The methane production assay was continued for two days at 12- to 24-h intervals. The maximal specific rate of acetoclastic and hydrogenotrophic methanogenesis was determined from the slope of the time curve of methane production. Inhibited methanogenic activity was expressed as a percentage of the methane production rate in the control flasks, %ACT. The percentage of inhibition (%I) was defined as %I = 100 − %ACT. The concentrations of compounds that caused 20%, 50%, and 80% inhibition of methanogenesis were referred to as, IC20, IC50, and IC80, respectively.

2.4. Microscopic Examination

Light microscopy. Cell morphology was examined under light microscope Opton ICM 405 (Carl Zeiss, Oberkochen, Germany) using the phase-contrast method at a magnification of 100 × 3.5.
Electron microscopy. Bacterial cells were pre-fixed with 1.5% (v/v) glutaraldehyde in 50 mM cacodylate buffer (pH 7.2) at 4°C for 1 h, washed thrice in the same buffer, and refixed in a 1% (w/v) solution of OsO4in the buffer at 20 °C for 4 h. The sections were dehydrated in a series of alcohol solutions of increasing concentration, embedded in Epon 812 epoxy resin, mounted on a grid, and contrasted in a 3% (w/v) solution of uranyl acetate in 70% (v/v) ethanol and then with lead citrate [57] at 20 °C for 4–5 min.

2.5. Analytical Procedures

Acetic acid and methane were analyzed using a Pye-Unicam gas chromatograph (United Kingdom). The glass column (1 m × 2 mm ID) for methane assay was packed with Porapak Q, 80–100 mesh (Fluka, Buchs, Switzerland). The column, injector, and flame-ionization detector were kept at 90, 150, and 180 °C, respectively. The carrier gas was nitrogen at a flow rate of 20 mL/min. Acetic acid was assayed on a glass column (2 m × 2 mm ID) packed with a 20 wt% neopentylglycol succinate on Chromosorb W/AW-DMCS, 100–200 mesh (Fluka, Buchs, Switzerland). The pH of samples was reduced to 4.0 by adding ortophosphoric acid. The column temperature was raised from 80 to 175 °C at a rate of 6 °C/min. The injector and detector were kept at 150 and 180 °C, respectively.
Hydrogen content was determined by a gas chromatograph Shimadzu 8A equipped with a thermal conductivity detector (TCD) and a stainless steel column packed with a molecular sieve 5Å, 60–80 mesh. The temperatures of the detector and column were maintained at 100 and 60 °C, respectively.
Perchlorate concentrations were determined with an IC-2010 ion chromatograph system equipped with a TSKgel SuperIC-Anion HS (Tosoh, Tokyo, Japan). The eluent contained 10 mM NaHCO3, 8 mM Na2CO3, and 30% (v/v) CH3CN. The analytical conditions were as follows: flow rate, 1.2 mL/min; column temperature, 40 °C; and injection volume, 30 μL. The limit of quantification was 0.001%. In order to determine perchlorates, 1 mL of methanogenic culture was collected and centrifuged (12,000 rpm) to separate the biomass.

3. Results and Discussion

Methane-producing archaea under study were isolated from different habitats (Table 1). Reference strains of hydrogenotrophic archaeon M. bryantii M.o.H.T and acetate-consuming archaeon M. mazei S-6T are the most abundant methanogenic species widespread in garden soil, sewage sludge, black mud, and feces of herbivorous animals and also isolated from urban solid waste, various sewage and animal waste digesters, swine waste lagoons, and kelp enrichments. Three other methanogenic strains were isolated from Arctic permafrost of different ages. M. articum M2T and Methanosarcina sp. JL01, hydrogen-and acetate-consuming strain, respectively, were isolated from Holocene permafrost. In contrast, hydrogenotrophic archaeon M. veterum MK4T was isolated from ancient permafrost which never thawed during 3 million years. All isolates were psychrotolerant mesophiles most probably responsible for the production of methane in permafrost samples from which they were isolated [20].

3.1. Determination of Perchlorates Inhibitory Concentrations

To determine the inhibitory effect of sodium and magnesium perchlorates and a mixture thereof, we used concentrations of up to 10 mM, since the aqueous extract Mars soil found precisely this total amount salt [39] and it is the maximum amount of perchlorates that could be there. Table 2 provides calculations of concentrations of NaClO4, Mg (ClO4)2 and their mixture (1:1), which reduce the methane formation by methanogens by 20, 50, and 80%, respectively. The results shows that, if 2.1 to 9.0 mM Mg(ClO4)2 was added into the culture medium we observe 20% reduction in the amount of methane by all methanogenic strains. It should be noted that the M. arcticum M2T was the most resistant to the perchlorates action. Adding 9.0 and 9.8 mM magnesium and sodium salt, respectively, reduced the methanogenesis M. bryantii MoHT by 80%. The joint addition of two perchlorates in all cases increased the inhibition effect. The concentration at which these salts inhibit the cell growth (Table 2) is consistent with their activity as chaotropic stressors [42,48] disordering cellular macromolecules.
Table
Table 2. Inhibitory effect of Mg(ClO4)2 and NaClO4 on the growth of permafrost and nonpermafrost methanogens.
Table 2. Inhibitory effect of Mg(ClO4)2 and NaClO4 on the growth of permafrost and nonpermafrost methanogens.
StrainsMg(ClO4)2, mMNaClO4, mMMg(ClO4)2 + NaClO4, mM
IC20IC50IC80IC20IC50IC80IC20IC50IC80
M. bryantii M.o.HT3.56.29.02.86.09.82.24.08.1
M. arcticum M2T9.0>10.0>10.0>10.0>10.0>10.0>10.0>10.0>10.0
M. veterum MK4T2.66.6>10.04.18.4>10.02.55.69.0
M. mazei S-6T5.09.2>10.07.8>10.0>10.04.8>10.0>10.0
Methanosarcina sp. JL012.15.2>10.03.99.7>10.01.84.8>10.0

3.2. Methane Generation in Perchlorate-Supplemented Medium

All studied microorganisms were used to test the possible growth and methanogenesis in media supplemented with two perchlorates salts which were added in final concentration of 5 mM. Growth of methanogens is characterized by an extremely low biomass accumulation and usually it is judged by the formation of methane in the gas phase and the amount of methane formed by microorganisms being proportional to the biomass produced [58]. Investigation of the influence of sodium perchlorate and magnesium on methanogenesis by hydrogen-consuming methanogens confirmed the stability of methanogens isolated from permafrost in the perchlorate-supplemented media (Figure 2A–C). The growth of M. bryantii is inhibited to a much greater extent than the growth of M. veterum MK4T and M. arcticum M2T. The growth of strain M. arcticum M2T was characterized by a greater rate in the presence of perchlorates (Figure 2C); however, there was 20% less methane formed by the strain than in the control that does not contain perchlorates.
Study of the NaClO4 and Mg(ClO4)2 effect on methanogenesis from acetate by Methanosarcina spp. showed, that in this case, perchlorates slow down the process more heavily, and additionally also result in the elongation of the lag phase to nine days for strain JL01 (Figure 3). The exception was M. mazei S-6T growth in the presence of sodium perchlorate, when the growth rate exceeded the growth rate of methanosarcina in the medium with no perchlorates.
It is known that a chaotrope-induced stress can then trigger oxidative stress [42,44], in particular with oxygen, which in this case may be formed from perchlorate anion [50]. Salt tolerance is expected to be a significant requirement for survival on Mars, and hypersaline environments on Earth, especially in permanently cold regions, are primarily discussed as analogs for Martian brines [10]. These include cryopegs within permafrost [59], brine veins within glacial ice [60], or evaporation ponds in the dry regions of Antarctica [61] and deep-sea hypersaline brine lakes [45]. A recent study of microbiology within the seawater: brine interface at deep-sea hypersaline brine Lake Kryos reports recovery of mRNA at higher levels of MgCl2 (2.27–3.03 M) [62]. It is halophilic species that are most likely to be able to grow under these conditions, and indeed at subzero temperatures, because ion concentrations are increased [63,64].
Earlier the study of microorganisms from Arctic cryopeg showed that the majority of their inhabitants are not halophilic but halotolerant representatives of Eukarya, Bacteria, and Archaea [59]. We have also obtained data that halotolerance of nonhalophilic psychrophilic and psychrotrophic bacteria isolated from cryopeg increases when the cultivation temperature decreases [65]. In the meantime, there is evidence that chaotropic substances (including salts/ions) can actually increase metabolic activity and multiplication of microbes at very low temperatures because they enable the cell to retain biologically permissive membrane fluidity [42,64].
Microorganisms 03 00518 g002 1024
Figure 2. Methane formation by hydrogenotrophic strains M. bryantii M.o.H.T (A), M. veterum MK4T (B) and M. arcticum M2T (C) with NaClO4 (square), Mg(ClO4)2 (triangle) and without perchlorates (rhombus).
Figure 2. Methane formation by hydrogenotrophic strains M. bryantii M.o.H.T (A), M. veterum MK4T (B) and M. arcticum M2T (C) with NaClO4 (square), Mg(ClO4)2 (triangle) and without perchlorates (rhombus).
Microorganisms 03 00518 g002
Microorganisms 03 00518 g003 1024
Figure 3. Methane formation by acetoclastic strains M. mazei S-6T (A) and Methanosarcina sp. JL01 (B) with NaClO4 (square), Mg(ClO4)2 (triangle) and without perchlorates (rhombus).
Figure 3. Methane formation by acetoclastic strains M. mazei S-6T (A) and Methanosarcina sp. JL01 (B) with NaClO4 (square), Mg(ClO4)2 (triangle) and without perchlorates (rhombus).
Microorganisms 03 00518 g003
The methanogens used in the study are not halophiles, but all the strains are tolerant of the NaCl concentration up to 0.3 M in the medium [22]. But, unlike M. bryantii M.o.H.T and M. veterum MK4T, which grow best in fresh medium, M. arcticum M2T showed the highest growth rate at a NaCl concentration of 0.1 M. Perhaps this difference in physiology of the studied methanogens explains why the response to stressors such as perchlorates differs. Further studies will clarify whether perchlorate- and other chaotropic salts are stressors or they may actually promote or enable growth at some temperatures [10].
Our study results of the methanogens growth in the presence of strong oxidants have put a new question forward: can methanogens use the anion ClO4− as an electron acceptor in the oxidation of methane which they were produced? It was in as early as 1979 that methanogenic archaea of Methanobacterium, Methanospirillum, and Methanosarcina genera were found to form and oxidize methane at the same time [66]. As compared to the quantity of methane formed, the amount of methane simultaneously oxidized varied between 0.3% and 0.001%, depending on the strain used.
It is now known that the anaerobic oxidation of methane (AOM) is a significant process in the global carbon cycle and an important sink of methane on Earth. As shown by recent studies, this process is carried out by anaerobic methanotrophic archaea (ANME). Initially, ANME were defined as a separate branch within Euryarchaeota, capable of receiving energy from the anaerobic methane oxidation while using sulfate as a terminal electron acceptor [67]. However, recently it was reported that as a terminal electron acceptor, these microorganisms could utilize nitrate [68] and compounds of iron and manganese [69], not only in marine, but also in freshwater habitats. Using molecular detection tools it was found that methane-producing archaea of Methanosarcina genus are the main group of methanogens found in permafrost grounds [70,71], and many of the detected ANMEs are genetically close to the Methanosarcinales order. Thus, the assumption about the use perchlorates as acceptors instead of sulfates in the anaerobic oxidation of methane is not unreasonable, given the recently appeared first publication describing the use of magnesium perchlorate by Archaeglobus sp. as an electron acceptor instead of sulfate [51]. Due to these speculations, we measured how perchlorates concentration changed after nine days of the Methanobacterium spp. cultivation and 16 days of Methanosarcina spp. cultivation. The medium without the addition of methanogen inoculum incubated the same time was served as an abiotic control. It should be noted that in the case of Methanosarcina spp. the decrease of the perchlorates content was less than 10% of the amount initially added in all cases, including control.
Another pattern (Figure 4) was observed in determining the content perchlorates before and after cultivation of Methanobacterium spp. When strains M. bryantii M.o.H.T and M. veterum MK4T were cultured, the change in NaClO4 (5.7%–16.1%, respectively) and Mg(ClO4)2 (16.0%–7.2%, respectively) content did not exceed the reduction of the perchlorates concentration in the control (19.0% and 17.6%, respectively). In contrast, the content of perchlorates in the culture medium M. arcticum M2T decreased by 31.8% and 45.6%, respectively. Therefore, the decrease of the perchlorates concentration during the strain growth suggests the possible use of the perchlorate anion as an electron acceptor for the oxidation of methane. This remarkable result, of course, requires experimental confirmation, but also offers opportunities to explore new unusual ways of obtaining energy for the methanogens, including in extraterrestrial environments.
Microorganisms 03 00518 g004 1024
Figure 4. The perchlorates content during the cultivation of hydrogen-consuming methanogens. Black bars—perchlorate concentration in the medium at the initial point; gray bars—the perchlorate concentration after nine days of growth. The control was MB medium supplemented with perchlorate salts.
Figure 4. The perchlorates content during the cultivation of hydrogen-consuming methanogens. Black bars—perchlorate concentration in the medium at the initial point; gray bars—the perchlorate concentration after nine days of growth. The control was MB medium supplemented with perchlorate salts.
Microorganisms 03 00518 g004

3.3. Effect of Perchlorate on Morphology of M. arcticum M2T Cells

In order to understand the reasons for the high resistance of the strain M. arcticum M2T to perchlorates, we carried out a microscopic study and examined the morphology of cells grown in the medium supplemented with Mg(ClO4)2 as a stronger oxidizer. Typically, cells of the strain were non-motile, slightly curved rods (Figure 5A), 0.45–0.50 µm wide and 3.0–6.0 µmlong, often forming chains and filaments more than 30 µm long. Previously we have shown that during long-term storage, strain M. arcticum M2T formed cyst-like coccoid cells [22]. However, in the presence of perchlorate the formation of cyst-like cells began during the logarithmic growth phase. The cytoplasm of cyst-like cells was denser, and differentiated surface layers were observed (Figure 5C). The ability to form such morphotypes differentiates M. arcticum M2T from other methanogens used in the study and are probably makes this strain resistant to perchlorates.
Microorganisms 03 00518 g005 1024
Figure 5. Micrographs of strain M. arcticum M2T cells without perchlorate (A) and with Mg(ClO4)2 in the medium (B,C): A,B—phase contrast, bar 10 µm; C—ultrathin section. Abbreviations: CLC—cyst-likes cells.
Figure 5. Micrographs of strain M. arcticum M2T cells without perchlorate (A) and with Mg(ClO4)2 in the medium (B,C): A,B—phase contrast, bar 10 µm; C—ultrathin section. Abbreviations: CLC—cyst-likes cells.
Microorganisms 03 00518 g005
If life existed during the early stages of Martian development, the remnants of primitive forms may be found within frozen material that protects them against unfavorable conditions [10]. Here we consider terrestrial permafrost analogues as a bridge to possible Martian life forms and shallow subsurface habitats where the probability of finding life is the highest. Since there is a place for water, the requisite condition for life, even in solid phase, these analog models are more or less realistic. Permafrost on Earth and Mars vary in age, from a few million years found on the Earth to a few billion years on Mars; such a difference in time scale would have a significant impact on the possibility of preserving life on Mars because the number and biodiversity of microorganisms decrease with increasing permafrost age. This is why the known longevity of life forms preserved within the permafrost can only work as an approximate analogue for Mars.
However, because at subzero temperatures the rate of biochemical reactions and biological process becomes extremely low, microbiological methods are not sufficiently sensitive to detect cell activity. Using 14C-labeled substrates, the possibility of methane formation within permafrost was experimentally demonstrated at subzero temperatures as low as −16.5 °C [20,72]. The experiments with radiolabeled substrates showed that methanogenic archaea not only preserve their viability in the permafrost, but also are able to realize metabolic reactions at subzero temperatures, perhaps it involves chaotropic agents. At these temperatures, autotrophic microorganisms from both the relatively young (2920 years) Holocene permafrost (M. arcticum M2T and Methanosarcina sp. JL01) and the old (~3 million years) Plieocene deposits (M.veterum MK4T) a metabolically active [73].

4. Conclusions

Furthermore, as shown in the studies presented here, methane-producing microorganisms isolated from a permafrost were resistant to oxidizing agents such as a perchlorates, and strain M2T M. arcticum, probably can use perchlorate as an electron acceptor in the anaerobic methane oxidation.
Methane-producing microorganisms from the Archaea domain of life have evolved features distinct from all other life forms, particularly cofactors and several enzymes essential for the pathways to produce CH4 from reduction of the methyl group of acetate or CO2. Although understanding of these pathways has derived primarily with methanogens isolated from organic rich halophilic and nonhalophilic surface environments, features of the pathways also apply to organic-poor environments [24]. Earth’s subzero subsurface environments are the best approximation of environments on Mars, which is most likely to harbor methanogens; thus, a biochemical understanding of these pathways are expected to provide a basis for designing experiments to detect autotrophic methane-producing life forms on Mars.

Acknowledgments

The work was supported by JAXA professorship grant for V. Shcherbakova and the grant of Russian Foundation of Basic Research #15-04-08612. We like to thank Nataliya Suzina for help with the microscopic examinations.

Author Contributions

Victoria Oshurkova is the PhD student who was in charge of the experimental design and wrote the initial draft. Viktoria Shcherbakova is the PhD supervisor of Viktoria Oshurkova and helped with the setup of the experimental design as well as the formulation of the final version of the submitted paper. Yoshitaka Yoshimura provided useful discussion during the practical work and wrote the supporting grant under which the work was performed.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Cameron, R.E.; Morelli, F.A. Viable microorganisms from ancient Ross Island and Taylor Valley drill core. Antarct. J. U. S. 1974, 9, 113–116. [Google Scholar]
  2. Gilichinsky, D. Permafrost as a microbial habitat. In Encyclopedia of Environmental Microbiology; Willey-Interscience: New York, NY, USA, 2002; pp. 932–956. [Google Scholar]
  3. Janssen, J.K.; Tas, N. The microbial ecology of permafrost. Nat. Rev. Microbiol. 2014, 12, 414–425. [Google Scholar] [CrossRef] [PubMed]
  4. Gilichinsky, D.A.; Wilson, G.S.; Friedmann, E.I.; McKay, C.P.; Sletten, R.S.; Rivkina, E.M.; Vishnivetskaya, T.A.; Erokhina, L.G.; Ivanushkina, N.E.; Kochkina, G.A.; et al. Microbial populations in Antarctic permafrost: Biodiversity, state, age, and implication for astrobiology. Astrobiology 2007, 7, 275–311. [Google Scholar] [CrossRef] [PubMed]
  5. Steven, B.; Leveille, R.; Pollard, W.H.; Whyte, L.G. Microbial ecology and biodiversity in permafrost. Extremophiles 2006, 10, 259–267. [Google Scholar] [CrossRef] [PubMed]
  6. Gilichinsky, D.A. Permafrost model of extraterrestrial habitat. In Astrobiology, 2nd ed.; Horneck, G., Baumstark-Khan, C., Eds.; Springer: Berlin, Germany, 2002; pp. 125–142. [Google Scholar]
  7. Zhang, G.; Ma, X.; Niu, F.; Dong, M.; Feng, H.; An, L.; Cheng, G. Diversity and distribution of alkaliphilic psychrotolerant bacteria in the Qinghai-Tibet Plateau permafrost region. Extremophiles 2007, 11, 415–424. [Google Scholar] [CrossRef] [PubMed]
  8. Reid, I.; Sparks, W.; Lubow, S.; McGrath, M.; Livio, M.; Valenti, J.; Sowers, K.; Shukla, H.; MacAuley, S.; Miller, T. Terrestrial models for extraterrestrial life: Methanogens and halophiles at Martian temperatures. Int. J. Astrobiol. 2006, 5, 89–97. [Google Scholar] [CrossRef]
  9. Demidov, N.E.; Gilichinsky, D.A. Terrestrial permafrost models and analogues of Martian habitats and inhabitans. In Permafrost Soil, 6th ed.; Margesin, R., Ed.; Springer Berlin Heidelberg: Berlin, Germany, 2009. [Google Scholar]
  10. Rummel, J.D.; Beaty, D.W.; Jones, M.A.; Bakermans, C.; Barlow, N.G.; Boston, P.J.; Chevrier, V.F.; Clark, B.C.; de Vera, J.P.; Gough, R.V.; et al. A new analysis of Mars “Special Regions”: Findings of the second MEPAG Special Regions Science Analysis Group (SR-SAG2). Astrobiology 2014, 14, 887–968. [Google Scholar] [CrossRef] [PubMed]
  11. Boynton, W.V.; Feldman, W.C.; Mitrofanov, I.; Evans, L.G.; Reedy, R.C.; Squyres, S.W.; Starr, R.; Trombka, J.I.; D’Uston, C.; Arnold, J.R.; et al. Distribution of hydrogen in the near surface of Mars: Evidence for subsurface ice deposits. Science 2002, 297, 81–85. [Google Scholar] [CrossRef] [PubMed]
  12. Demidov, N.; Boynton, W.; Gilichinsky, D.; Zuber, M.; Kozyrev, A.; Litvak, M.; Mitrofanov, I.; Sanin, A.; Saunders, R.; Smith, D. Water distribution in Martian permafrost regions from joint analysis of HEND (Mars Odyssey) and MOLA (Mars Global Surveyor) data. Astron. Lett. 2008, 34, 713–723. [Google Scholar] [CrossRef]
  13. Cull, S.; Arvidson, R.E.; Catalano, J.G.; Ming, D.W.; Morris, R.V.; Mellon, M.T.; Lemmon, M. Concentrated perchlorate at the Mars Phoenix landing site: Evidence for thin film liquid water on Mars. Geophys. Res. Lett. 2010, 37, L22203. [Google Scholar] [CrossRef]
  14. Fischer, E.; Martínez, G.M.; Elliott, H.M.; Rennó, N.O. Experimental evidence for the formation of liquid saline water on Mars. Geophys. Res. Lett. 2014, 41, 4456–4462. [Google Scholar] [CrossRef] [PubMed]
  15. Mumma, M.; Novak, R.; DiSanti, M.; Bonev, B. A sensitive search for methane on Mars. Bull. Am. Astron. Soc. 2003, 35, 937. [Google Scholar]
  16. Formisano, V.; Atreya, S.; Encrenaz, T.R.; Ignatiev, N.; Giuranna, M. Detection of methane in the atmosphere of Mars. Science 2004, 306, 1758–1761. [Google Scholar] [CrossRef] [PubMed]
  17. Krasnopolsky, V.A.; Maillard, J.P.; Owen, T.C. Detection of methane in the Martian atmosphere: Evidence for life? Icarus 2004, 172, 537–547. [Google Scholar] [CrossRef]
  18. Mumma, M.J.; Villanueva, G.L.; Novak, R.E.; Hewagama, T.; Bonev, B.P.; Disanti, M.A.; Mandell, A.M.; Smith, M.D. Strong release of methane on Mars in northern summer 2003. Science 2009, 323, 1041–1045. [Google Scholar] [CrossRef] [PubMed]
  19. Hitchcock, D.R.; Lovelace, J.E. Life Detection by Atmospheric Analysis. Icarus 1967, 7, 149–159. [Google Scholar] [CrossRef]
  20. Rivkina, E.; Shcherbakova, V.; Laurinavichius, K.; Petrovskaya, L.; Krivushin, K.; Kraev, G.; Pecheritsina, S.; Gilichinsky, D. Biogeochemistry of methane and methanogenic archaea in permafrost. FEMS Microbiol. Ecol. 2007, 61, 1–15. [Google Scholar] [CrossRef] [PubMed]
  21. Krivushin, K.V.; Shcherbakova, V.A.; Petrovskaya, L.E.; Rivkina, E.M. Methanobacterium veterum sp. nov., from ancient Siberian permafrost. IJSEM 2010, 60, 455–459. [Google Scholar] [CrossRef] [PubMed]
  22. Shcherbakova, V.; Rivkina, E.; Pecheritsyna, S.; Laurinavichius, K.; Suzina, N.; Gilichinsky, D. Methanobacterium arcticum sp. nov., a methanogenic archaeon from Holocene Arctic permafrost. IJSEM 2011, 61, 144–147. [Google Scholar] [CrossRef] [PubMed]
  23. Costa, K.C.; Leigh, J.A. Metabolic versatility in methanogens. Curr. Opin. Biotechnol. 2014, 29, 70–75. [Google Scholar] [CrossRef] [PubMed]
  24. Ferry, J.G. The chemical biology of methanogenesis. Planet. Space Sci. 2010, 58, 1775–1783. [Google Scholar] [CrossRef]
  25. Wagner, D.; Spieck, E.; Bock, E.; Pfeiffer, E.-M. Microbial Life in Terrestrial Permafrost: Methanogenesis and Nitrification in Gelisols as Potentials for Exobiological Process. In Astrobiology, 2nd ed.; Horneck, G., Baumstark-Khan, C., Eds.; Springer Berlin Heidelberg: Berlin, Germany, 2002; pp. 143–159. [Google Scholar]
  26. Levin, G.V.; Straat, P.A. Methane and life on Mars. In Proceedings of the SPIE Optical Engineering + Applications, International Society for Optics and Photonics, San Diego, CA, USA, 2 August 2009.
  27. Kral, T.A.; Brink, K.M.; Miller, S.L.; McKay, C.P. Hydrogen consumption by methanogens on the early Earth. Orig. Life Evol. B 1998, 28, 311–319. [Google Scholar] [CrossRef]
  28. Kral, T.A.; Bekkum, C.R.; McKay, C.P. Growth of methanogens on a Mars soil simulant. Orig. Life Evol. B 2004, 34, 615–626. [Google Scholar] [CrossRef]
  29. Ormond, D.R.; Kral, T.A. Washing methanogenic cells with the liquid fraction from a Mars soil simulant and water mixture. J. Microbiol. Methods 2006, 67, 603–605. [Google Scholar] [CrossRef] [PubMed]
  30. Howe, K.; Gavin, P.; Kral, T.; Goodhart, T.; Chevrier, V. Methanogen Growth in Perchlorate-Supplemented Media and Implications for Life in Phoenix-Type Soils. LPI Contrib. 2009, 1502, 19. [Google Scholar]
  31. Chastain, B.K.; Kral, T.A. Zero-valent iron on Mars: An alternative energy source for methanogens. Icarus 2010, 208, 198–201. [Google Scholar] [CrossRef]
  32. Sinha, N.; Kral, T. Growth of Methanogens on Different Mars Regolith Analogues and Stable Carbon Isotope Fractionation during Methanogenesis. In Proceedings of the Lunar and Planetary Science Conference, Woodlands, TX, USA, 16–20 March 2015.
  33. Kendrick, M.G.; Kral, T.A. Survival of methanogens during desiccation: Implications for life on Mars. Astrobiology 2006, 6, 546–551. [Google Scholar] [CrossRef] [PubMed]
  34. Altheide, T.; Kral, T. Low-pressure desiccation effects on methane production by methanogens. In Proceedings of the Lunar and Planetary Science Conference, League City, TX, USA, 10–14 March 2008.
  35. Kral, T.A.; Altheide, T.S.; Lueders, A.E.; Schuerger, A.C. Low pressure and desiccation effects on methanogens: Implications for life on Mars. Planet. Space Sci. 2011, 59, 264–270. [Google Scholar] [CrossRef]
  36. Schirmack, J.; Alawi, M.; Wagner, D. Influence of Martian regolith analogs on the activity and growth of methanogenic archaea, with special regard to long-term desiccation. Front. Microbiol. 2015, 6. [Google Scholar] [CrossRef] [PubMed]
  37. Morozova, D.; Möhlmann, D.; Wagner, D. Survival of methanogenic archaea from Siberian permafrost under simulated Martian thermal conditions. Orig. Life Evol. Biosph. 2007, 37, 189–200. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  38. Sinha, N.; Kral, T. Methanogen sensitivity to ultraviolet radiation: Implications for life on Mars. In Proceedings of the 76th Annual Meeting of the Meteoritical Society, Edmonton, AB, Canada, 29 July–7 August 2013.
  39. Hecht, M.; Kounaves, S.; Quinn, R.; West, S.; Young, S.; Ming, D.; Catling, D.; Clark, B.; Boynton, W.; Hoffman, J. Detection of perchlorate and the soluble chemistry of Martian soil at the Phoenix lander site. Science 2009, 325, 64–67. [Google Scholar] [PubMed]
  40. Zhang, Y.; Cremer, P.S. Interactions between macromolecules and ions: The Hofmeister series. Curr. Opin. Chem. Biol. 2006, 10, 658–663. [Google Scholar] [CrossRef] [PubMed]
  41. Cray, J.A.; Russell, J.T.; Timson, D.J.; Singhal, R.S.; Hallsworth, J.E. A universal measure of chaotropicity and kosmotropicity. Environ. Microbiol. 2013, 15, 287–296. [Google Scholar] [CrossRef] [PubMed]
  42. Cray, J.A.; Stevenson, A.; Ball, P.; Bankar, S.B.; Eleutherio, E.C.; Ezeji, T.C.; Singhal, R.S.; Thevelein, J.M.; Timson, D.J. Hallsworth, J.E. Chaotropicity: A key factor in product tolerance of biofuel-producing microorganisms. Curr. Opin. Biotechnol. 2015, 33, 228–259. [Google Scholar] [CrossRef] [PubMed]
  43. Rangel, D.E.; Alder-Rangel, A.; Dadachova, E.; Finlay, R.D.; Kupiec, M.; Dijksterhuis, J.; Gilberto, U.L.; Braga, G.U.L.; Luis, M.; Corrochano, L.M.; et al. Fungal stress biology: A preface to the Fungal Stress Responses special edition. Curr. Genet. 2015, 61, 231–238. [Google Scholar] [CrossRef] [PubMed]
  44. Hallsworth, J.E.; Heim, S.; Timmis, K.N. Chaotropic solutes cause water stress in Pseudomonas putida. Environ. Microbiol. 2003, 5, 1270–1280. [Google Scholar] [CrossRef] [PubMed]
  45. Duda, V.I.; Danilevich, V.N.; Suzina, N.E.; Shorokhova, A.P.; Dmitriev, V.V.; Mokhova, O.N. Akimov, V.N. Changes in the fine structure of microbial cells induced by chaotropic salts. Mikrobiologiya 2004, 73, 341–349. [Google Scholar]
  46. Hallsworth, J.E.; Yakimov, M.M.; Golyshin, P.N.; Gillion, J.L.; D’Auria, G.; de Lima Alves, F.; la Cono, V.; McKew, B.A.; Hayes, S.L.; Harris, G.; et al. Limits of life in MgCl2-containing environments: Chaotropicity defines the window. Environ. Microbiol. 2007, 9, 801–813. [Google Scholar] [CrossRef] [PubMed]
  47. Williams, J.P.; Hallsworth, J.E. Limits of life in hostile environments: No barriers to biosphere function. Environ. Microbiol. 2009, 11, 3292–3308. [Google Scholar] [CrossRef] [PubMed]
  48. Bhaganna, P.; Volkers, R.J.; Bell, A.N.; Kluge, K.; Timson, D.J.; McGrath, J.W.; Ruijssenaars, H.J.; Hallsworth, J.E. Hydrophobic substances induce water stress in microbial cells. Microbiol. Biotechnol. 2010, 3, 701–716. [Google Scholar] [CrossRef] [PubMed]
  49. Coates, J.D.; Michaelidou, U.; Bruce, R.A.; Connor, S.M.; Crespi, J.N.; Achenbach, L.A. Ubiquity and diversity of dissimilatory (per) chlorate-reducing bacteria. Appl. Environ. Microbiol. 1999, 65, 5234–5241. [Google Scholar] [PubMed]
  50. Coates, J.D.; Achenbach, L.A. Microbial perchlorate reduction: Rocket-fuelled metabolism. Nat. Rev. Microbiol. 2004, 2, 569–580. [Google Scholar] [CrossRef] [PubMed]
  51. Liebensteiner, M.G.; Pinkse, M.W.; Schaap, P.J.; Stams, A.J.; Lomans, B.P. Archaeal (per) chlorate reduction at high temperature: An interplay of biotic and abiotic reactions. Science 2013, 340, 85–87. [Google Scholar] [CrossRef] [PubMed]
  52. Oren, A.; Bardavid, R.E.; Mana, L. Perchlorate and halophilic prokaryotes: Implications for possible halophilic life on Mars. Extremophiles 2014, 18, 75–80. [Google Scholar] [CrossRef] [PubMed]
  53. Martínez-Espinosa, R.M.; Richardson, D.J.; Bonete, M.J. Characterization of chlorate reduction in the haloarchaeon Haloferax mediterranei. BBA-Gen. Subj. 2015, 1850, 587–594. [Google Scholar] [CrossRef] [PubMed]
  54. Balch, W.E.; Fox, G.; Magrum, L.; Woese, C.; Wolfe, R. Methanogens: Reevaluation of a unique biological group. Microbiol. Rev. 1979, 43, 260. [Google Scholar] [PubMed]
  55. Mah, R.A.; Kuhn, D.A. Transfer of the type species of the genus Methanococcus to the genus Methanosarcina, naming it Methanosarcina mazei (Barker 1936) comb. nov. et emend and conservation of the genus Methanococcus (approved lists 1980) with Methanococcus vannielii (approved lists 1980) as the type species request for an opinion. IJSB 1984, 34, 263–265. [Google Scholar]
  56. Hungate, R. A roll tube method for cultivation of strict anaerobes. Methods Microbiol. 1969, 3B, 117–132. [Google Scholar]
  57. Reynolds, E.S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 1963, 17, 208–212. [Google Scholar] [CrossRef] [PubMed]
  58. Shcherbakova, V.A.; Obraztsova, A.Y.; Laurinavichius, K.S.; Kotel’nikova, S.V.; Akimenko, V.K.; Navoa, M.K.; Kruz, M. Physiological properties of thermophilic methanosarcina isolated from active sludge of anaerobic digesters. Microbiology 1991, 60, 466–471. [Google Scholar]
  59. Gilichinsky, D.; Rivkina, E.; Shcherbakova, V.; Laurinavichuis, K.; Tiedje, J. Supercooled Water Brines within Permafrost-An Unknown Ecological Niche for Microorganisms: A Model for Astrobiology. Astrobiology 2003, 3, 331–341. [Google Scholar] [CrossRef] [PubMed]
  60. Deming, J.W. Psychrophiles and polar regions. Curr. Opin. Microbiol. 2002, 5, 301–309. [Google Scholar] [CrossRef]
  61. Marchant, D.R.; Head, J.W. Antarctic dry valleys: Microclimate zonation, variable geomorphic processes, and implications for assessing climate change on Mars. Icarus 2007, 192, 187–222. [Google Scholar] [CrossRef]
  62. Yakimov, M.M.; la Cono, V.; Spada, G.L.; Bortoluzzi, G.; Messina, E.; Smedile, F.; Arcadi, E.; Borghini, M.; Ferrer, M.; Schmitt-Kopplin, P.; et al. Microbial community of the deep-sea brine Lake Kryos seawater–brine interface is active below the chaotropicity limit of life as revealed by recovery of mRNA. Environ. Microbiol. 2015, 17, 364–382. [Google Scholar] [CrossRef] [PubMed]
  63. Chin, J.P.; Megaw, J.; Magill, C.L.; Nowotarski, K.; Williams, J.P.; Bhaganna, P.; Linton, M.; Patterson, M.F.; Underwood, G.J.C.; Mswaka, A.; et al. Solutes determine the temperature windows for microbial survival and growth. Proc. Natl. Acad. Sci. USA 2010, 107, 7835–7840. [Google Scholar] [CrossRef] [PubMed]
  64. Stevenson, A.; Cray, J.A.; Williams, J.P.; Santos, R.; Sahay, R.; Neuenkirchen, N.; McClure, C.D.; Grant, I.R.; Houghton, J.D.R.; Quinn, J.P.; et al. Is there a common water-activity limit for the three domains of life? ISME 2015, 9, 1333–1351. [Google Scholar] [CrossRef] [PubMed]
  65. Shcherbakova, V.; Rivkina, E.; Laurinavichuis, K.; Pecheritsina, S.; Gilchinsky, D. Physiological characteristics of bacteria isolated from water brines within permafrost. Int. J. Astrobiol. 2004, 3, 37–43. [Google Scholar] [CrossRef]
  66. Zehnder, A.; Brock, T. Methane formation and methane oxidation by methanogenic bacteria. J. Bacteriol. 1979, 137, 420–432. [Google Scholar] [PubMed]
  67. Boetius, A.; Ravenschlag, K.; Schubert, C.J.; Rickert, D.; Widdel, F.; Gieseke, A.; Amann, R.; Jёrgensen, B.B.; Witte, U.; Pfannkuche, O. A marine microbial consortium apparently mediating anaerobic oxidation of methane. Nature 2000, 407, 623–626. [Google Scholar] [CrossRef] [PubMed]
  68. Ettwig, K.F.; Shima, S.; van de Pas-Schoonen, V.; Katinka, T.; Kahnt, J.R.; Medema, M.H.; Op Den Camp, H.J.; Jetten, M.S.; Strous, M. Denitrifying bacteria anaerobically oxidize methane in the absence of Archaea. Environ. Microbiol. 2008, 10, 3164–3173. [Google Scholar] [CrossRef] [PubMed]
  69. Beal, E.J.; House, C.H.; Orphan, V.J. Manganese-and iron-dependent marine methane oxidation. Science 2009, 325, 184–187. [Google Scholar] [CrossRef] [PubMed]
  70. Karaevskaya, E.; Demchenko, L.; Demidov, N.; Rivkina, E.; Bulat, S.; Gilichinsky, D. Archaeal diversity in permafrost deposits of Bunger Hills Oasis and King George Island (Antarctica) according to the 16S rRNA gene sequencing. Microbiology 2014, 83, 398–406. [Google Scholar] [CrossRef]
  71. Krivushin, K.; Kondrashov, F.; Shmakova, L.; Tutukina, M.; Petrovskaya, L.; Rivkina, E. Two metagenomes from Late Pleistocene northeast Siberian permafrost. Genome Announc. 2015, 3, e01380-14. [Google Scholar] [CrossRef] [PubMed]
  72. Rivkina, E.; Laurinavichius, K.; McGrath, J.; Tiedje, J.; Shcherbakova, V.; Gilichinsky, D. Microbial life in permafrost. Adv. Space Res. 2004, 33, 1215–1221. [Google Scholar] [CrossRef] [PubMed]
  73. Rivkina, E.; Laurinavichus, K.; Gilichinsky, D.; Shcherbakova, V. Methane generation in permafrost sediments. Dokl. Biol. Sci. 2002, 383, 179–181. [Google Scholar] [CrossRef] [PubMed]

Share and Cite

MDPI and ACS Style

Shcherbakova, V.; Oshurkova, V.; Yoshimura, Y. The Effects of Perchlorates on the Permafrost Methanogens: Implication for Autotrophic Life on Mars. Microorganisms 2015, 3, 518-534. https://doi.org/10.3390/microorganisms3030518

AMA Style

Shcherbakova V, Oshurkova V, Yoshimura Y. The Effects of Perchlorates on the Permafrost Methanogens: Implication for Autotrophic Life on Mars. Microorganisms. 2015; 3(3):518-534. https://doi.org/10.3390/microorganisms3030518

Chicago/Turabian Style

Shcherbakova, Viktoria, Viktoria Oshurkova, and Yoshitaka Yoshimura. 2015. "The Effects of Perchlorates on the Permafrost Methanogens: Implication for Autotrophic Life on Mars" Microorganisms 3, no. 3: 518-534. https://doi.org/10.3390/microorganisms3030518

Article Metrics

Back to TopTop