V. A. Shcherbakova

Supercooled Water Brines Within Permafrost—An Unknown Ecological Niche for Microorganisms: A Model for Astrobiology

This study describes brine lenses (cryopegs) found in Siberian permafrost derived from ancient marine sediment layers of the Arctic Ocean. The cryopegs were formed and isolated from sediment ~100,000-120,000 years ago. They remain liquid at the in situ temperature of -10 degrees C as a result of their high salt content (170-300 g/L). [(14)C] Glucose is taken up by the cryopeg biomass at -15 degrees C, indicating microbial metabolism at low temperatures in this habitat. Furthermore, aerobic, anaerobic heterotrophs, sulfate reducers, acetogens, and methanogens were detected by most probable number analysis. Two psychrophilic microbes were isolated from the cryopegs, a Clostridium and a Psychrobacter. The closest relatives of each were previously isolated from Antarctica. The cryopeg econiche might serve as a model for extraterrestrial life, and hence is of particular interest to astrobiology.

Methanobacterium veterum sp. nov., from ancient Siberian permafrost

A methanogenic archaeon, strain MK4(T), was isolated from ancient permafrost after long-term selective anaerobic cultivation. The cells were rods, 2.0-8.0 microm long and 0.40-0.45 microm wide, and stained Gram-negative. Optimal growth was observed at 28 degrees C and pH 7.0-7.2 and in 0.05 M NaCl. The isolate used H(2) plus CO(2), methylamine plus H(2) and methanol plus H(2) as sources for growth and methanogenesis. Phylogenetic analysis of the 16S rRNA gene sequence of the strain showed close affinity with Methanobacterium bryantii (similarity >99 % to the type strain). On the basis of the level of DNA-DNA hybridization (62 %) between strain MK4(T) and Methanobacterium bryantii VKM B-1629(T) and phenotypic and phylogenetic differences, strain MK4(T) was assigned to a novel species of the genus Methanobacterium, Methanobacterium veterum sp. nov., with the type strain MK4(T) (=DSM 19849(T) =VKM B-2440(T)).

Methanobacterium arcticum sp. nov., a methanogenic archaeon from Holocene Arctic permafrost

A mesophilic, non-motile, hydrogenotrophic, rod-shaped methanogen, designated M2(T), was isolated from Holocene permafrost sediments of the Kolyma lowland in the Russian Arctic. Cells were 3-6 μm long and 0.45-0.5 μm wide. Strain M2(T) grew on H(2)/CO(2) and formate. Optimum conditions for growth were 37°C, pH 6.8-7.2 and 0.1 M NaCl. The DNA G+C content was 38.1 mol%. On the basis of 16S rRNA gene sequence comparison with known methanogens, strain M2(T) was affiliated with the genus Methanobacterium and was most closely related to Methanobacterium veterum MK4(T) and Methanobacterium bryantii DSM 863(T) (both 99 % 16S rRNA gene sequence similarity). However, no significant DNA-DNA relatedness was observed between strain M2(T) and these type strains. We propose that strain M2(T) represents a novel species, with the name Methanobacterium arcticum sp. nov., with type strain M2(T) (=DSM 19844(T) =VKM B-2371(T)).

Methane generation in permafrost sediments.

Discovery of viable microorganisms in permafrost [1–4] raised the problem of their metabolic status. It is uncertain whether or not the microorganisms stored in permafrost sediments remain metabolically active. The use of radioactively labeled compounds is one of a few methods of detection of metabolic processes in permafrost sediments. It was shown [5] that microbial lipids could be synthesized in permafrost sediments.

Clostridium tagluense sp. nov., a psychrotolerant, anaerobic, spore-forming bacterium from permafrost

A strictly anaerobic, Gram-positive, psychrotolerant, endospore-forming bacterium (strain A121(T)) was isolated from a permafrost sample collected in the Canadian High Arctic. Phylogenetic analysis of the 16S rRNA gene sequence of strain A121(T) showed its affiliation with the group of psychrophilic and psychrotolerant members of cluster I of the genus Clostridium, Clostridium bowmanii DSM 14206(T) being the closest relative (sequence similarity 98.5 %). Levels of DNA-DNA relatedness between strain A121(T) and the type strains of phylogenetically related species ranged from 33 to 52 %. Strain A121(T) grew in PY broth at temperatures between 4 and 28 degrees C (optimum 15-20 degrees C), at pH 6.0-8.0 (optimum pH 6.5-7.2) and in NaCl concentrations of 0-10.0 % (optimum 0-2.0 %). The strain utilized a narrow range of carbohydrates as sources of carbon and energy, including glucose, fructose, trehalose, maltose and starch; it also hydrolysed gelatin. Predominant fatty acids were C(16 : 1) cis9, C(16 : 1) cis9 DMA, C(16 : 0) and C(14 : 0). The DNA G+C content was 31.5 mol%. On the basis of its overall genotypic and phenotypic characteristics, strain A121(T) is classified within a novel species of the genus Clostridium, Clostridium tagluense sp. nov. The type strain is A121(T) (=VKM B-2369(T) =DSM 17763(T)).

Desulfovibrio arcticus sp. nov., a psychrotolerant sulfate-reducing bacterium from a cryopeg.

A psychrotolerant sulfate-reducing bacterium, designated B15(T), was isolated from supercooled water brine from within permafrost of the Varandey Peninsula, on the southern coast of the Barents Sea. Cells were Gram-negative, motile vibrions (3.0-4.0×0.4-0.5 µm) with a single polar flagellum. The isolate was positive for desulfoviridin as a bisulfite reductase. Strain B15(T) grew at -2 to 28 °C (optimum 24 °C) and with 0-2.0% NaCl (optimum 0.2%). The isolate used H(2) plus acetate, formate, ethanol, lactate, pyruvate and choline as electron donors and used sulfate, sulfite, thiosulfate, elemental sulfur, DMSO and Fe(3+) as electron acceptors. Pyruvate and lactate were not fermented in the absence of sulfate. The G+C content of genomic DNA was 55.2 mol%. Analysis of the 16S rRNA gene sequence showed that the isolate belonged to the genus Desulfovibrio. Its closest relatives were Desulfovibrio idahonensis CY1(T) (98.8% 16S rRNA gene sequence similarity) and Desulfovibrio mexicanus Lup1(T) (96.5%). On the basis of genotypic, phenotypic and phylogenetic characteristics, the isolate represents a novel species, for which the name Desulfovibrio arcticus sp. nov. is proposed; the type strain is B15(T) (=VKM B-2367(T)=DSM 21064(T)).

Sphaerochaeta associata sp. nov., a spherical spirochaete isolated from cultures of Methanosarcina mazei JL01.

An anaerobic, saccharolytic bacterial strain designated GLS2T was isolated from aggregates of the psychrotolerant archaeon Methanosarcina mazei strain JL01 isolated from arctic permafrost. Bacterial cells were non-motile, spherical, ovoid and annular with diameter 0.2-4 μm. They were chemoorganoheterotrophs using a wide range of mono-, di- and trisaccharides as carbon and energy sources. The novel isolate required yeast extract and vitamins for growth. The bacteria exhibited resistance to a number of β-lactam antibiotics, rifampicin, streptomycin and vancomycin. Optimum growth was observed between 30 and 34 °C, at pH 6.8-7.5 and with 1-2 g NaCl l- 1. Isolate GLS2T was a strict anaerobe but it tolerated oxygen exposure. On the basis of 16S rRNA gene sequence similarity, strain GLS2T was shown to belong to the genus Sphaerochaeta within the family Spirochaetaceae. Its closest relatives were Sphaerochaeta globosa BuddyT (99.3 % 16S rRNA gene sequence similarity) and Sphaerochaeta pleomorpha GrapesT (95.4 % similarity). The G+C content of DNA was 47.2 mol%. The level of DNA-DNA hybridization between strains GLS2T and BuddyT was 34.7 ± 8.8 %. Major polar lipids were phosphoglycolipids, phospholipids and glycolipids; major fatty acids were C14 : 0, C16 : 0, C16 : 0 3-OH, C16 : 0 dimethyl acetal (DMA), C16 : 1n8 and C16 : 1 DMA; respiratory quinones were not detected. The results of DNA-DNA hybridization, physiological and biochemical tests demonstrated genotypic and phenotypic differentiation of strain GLS2T from the four species of the genus Sphaerochaeta with validly published names that allowed its separation into a new lineage at the species level. Strain GLS2T therefore represents a novel species, for which the name Sphaerochaeta associata sp. nov. is proposed, with the type strain GLS2T ( = DSM 26261T = VKM B-2742T).

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

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.

Novel halotolerant bacterium from cryopeg in permafrost: Description of Psychrobacter muriicola sp. nov.

A novel halotolerant psychrotrophic gram-negative bacterium, strain 2pS, was isolated from lenses of water brine in Arctic permafrost (cryopeg). The optimal growth of the new strain was observed at 16–18°C; the maximal and minimal growth temperatures were 37°C and −2°C, respectively. The pH growth range was 5.8 to 8.5 (optimum 6.5–7.5) and the range of medium salinity was 0 to 100 g/l (optimum 3–8 g/l NaCl). The strain 2pS did not produce acid from carbohydrates and utilized acetate, yeast extract, pyruvate, glutarate, fumarate, caproate, heptanoate, butyrate, malate, DL-lactate, citrate, L-proline, L-tyrosine, butanol, and dulcitol as the sole carbon and energy sources. The major fatty acids of the cell wall at optimal growth temperature were C18:1ω7 and C18:1ω9. The G+C DNA base content was 46.0 mol.%. Phylogenetic analysis of the 16S rRNA gene sequences showed that the studied strain was the closest (97% similarity) to Psychrobacter nivimaris DSM 16093T, a halotolerant psychrotrophic bacterium isolated from the Arctic sea’s ice. Genotypic and phenotypic differences of the new bacterium from closely related species lead to the conclusion that strain 2pS belongs to a novel species of the genus Psychrobacter: Psychrobacter muriicola sp. nov.

Methanogenic Sarcina from an Anaerobic Microbial Community Degrading p-Toluene Sulfonate

The methanogenic strain MM isolated from an anaerobic microbial community degrading p-toluene sulfonate showed optimal values of temperature and pH for growth equal to 37°C and 6.3–6.9, respectively. The doubling times of the isolate grown on methanol, acetate, and methylamines under the optimal conditions were 8.8, 19.1, and 10.3–28.1 h, respectively. The growth of strain MM was observed only when the cultivation medium contained casamino acids or p-toluene sulfonate. The G+C content of the DNA of the isolate was 40.3 mol %. This, together with DNA–DNA hybridization data, allowed the new isolate to be identified as a strain of the species Methanosarcina mazei. The new isolate differed from the known representatives of this species in that it was resistant to alkylbenzene sulfonates and able to demethylate p-toluene sulfonate when grown on acetate.