Microbial populations in Antarctic permafrost: biodiversity, state, age, and implication for astrobiology.

Antarctic permafrost soils have not received as much geocryological and biological study as has been devoted to the ice sheet, though the permafrost is more stable and older and inhabited by more microbes. This makes these soils potentially more informative and a more significant microbial repository than ice sheets. Due to the stability of the subsurface physicochemical regime, Antarctic permafrost is not an extreme environment but a balanced natural one. Up to 10(4) viable cells/g, whose age presumably corresponds to the longevity of the permanently frozen state of the sediments, have been isolated from Antarctic permafrost. Along with the microbes, metabolic by-products are preserved. This presumed natural cryopreservation makes it possible to observe what may be the oldest microbial communities on Earth. Here, we describe the Antarctic permafrost habitat and biodiversity and provide a model for martian ecosystems.

Biogeochemical features of lipids in endolithic microbial communities in the Ross Desert (McMurdo Dry Valleys), Antarctica.

Endolithic microbial communities inhabiting porous rocks in the cold, dry mountainous regions of Antarctica have been studied extensively as examples of life's adaptations to extreme environments. Here, we examine hydrocarbons and fatty acids occurring in these communities in order to clarify their biogeochemical features with respect to source organisms, microbial activity, fossilization processes and the influence of Gondwanaland sediments. Unusually, long-chain (>C19) n-alkanes and anteiso-alkanes were often the major hydrocarbons in the samples. A suite of n-alkanoic acids (n-C9-n-C32) and long-chain anteiso-alkanoic acids (a-C20-a-C30) were found, along with short-chain iso- and anteiso-alkanoic acids, and n-alkenoic acids. The relationship between long-chain n-alkanoic acids (n-C20-n-C32) and long-chain anteiso-alkanoic acids suggests that these compounds probably originated from the same group of microorganisms, such as bacteria or endolithic lichens, under moderate pH conditions (pH 3-5). Relatively high trans/cis-C16:1 alkenoic acid ratios suggest the presence of unfavorable environmental conditions in the endolithic microbial habitat. Normal-alkenoic/alkanoic acid ratios may be a useful marker for the fossilization of endolithic microbial communities. Thermally matured triterpanes and steranes from fossilized associations on Mount Fleming strongly suggest the presence of Gondwanaland sediments formed during Devonian and Jurassic (400-180 million years ago).

Gene transfer to the desiccation-tolerant cyanobacterium Chroococcidiopsis.

The coccoid cyanobacterium Chroococcidiopsis dominates microbial communities in the most extreme arid hot and cold deserts. These communities withstand constraints that result from multiple cycles of drying and wetting and/or prolonged desiccation, through mechanisms which remain poorly understood. Here we describe the first system for genetic manipulation of Chroococcidiopsis. Plasmids pDUCA7 and pRL489, based on the pDU1 replicon of Nostoc sp. strain PCC 7524, were transferred to different isolates of Chroococcidiopsis via conjugation and electroporation. This report provides the first evidence that pDU1 replicons can be maintained in cyanobacteria other than Nostoc and Anabaena. Following conjugation, both plasmids replicated in Chroococcidiopsis sp. strains 029, 057, and 123 but not in strains 171 and 584. Both plasmids were electroporated into strains 029 and 123 but not into strains 057, 171, and 584. Expression of P(psbA)-luxAB on pRL489 was visualized through in vivo luminescence. Efficiencies of conjugative transfer for pDUCA7 and pRL489 into Chroococcidiopsis sp. strain 029 were approximately 10(-2) and 10(-4) transconjugants per recipient cell, respectively. Conjugative transfer occurred with a lower efficiency into strains 057 and 123. Electrotransformation efficiencies of about 10(-4) electrotransformants per recipient cell were achieved with strains 029 and 123, using either pDUCA7 or pRL489. Extracellular deoxyribonucleases were associated with each of the five strains. Phylogenetic analysis, based upon the V6 to V8 variable regions of 16S rRNA, suggests that desert strains 057, 123, 171, and 029 are distinct from the type species strain Chroococcidiopsis thermalis PCC 7203. The high efficiency of conjugative transfer of Chroococcidiopsis sp. strain 029, from the Negev Desert, Israel, makes this a suitable experimental strain for genetic studies on desiccation tolerance.

Chains of magnetite crystals in the meteorite ALH84001: evidence of biological origin.

The presence of magnetite crystal chains, considered missing evidence for the biological origin of magnetite in ALH84001 [Thomas-Keprta, K. L., Bazylinski, D. A., Kirschvink, J. L., Clemett, S. J., McKay, D. S., Wentworth, S. J., Vali, H., Gibson, E. K., Jr., & Romanek, C. S. (2000) Geochim. Cosmochim. Acta 64, 4049-4081], is demonstrated by high-power stereo backscattered scanning electron microscopy. Five characteristics of such chains (uniform crystal size and shape within chains, gaps between crystals, orientation of elongated crystals along the chain axis, flexibility of chains, and a halo that is a possible remnant of a membrane around chains), observed or inferred to be present in magnetotactic bacteria but incompatible with a nonbiological origin, are shown to be present. Although it is unlikely that magnetotactic bacteria were ever alive in ALH84001, decomposed remains of such organisms could have been deposited in cracks in the rock while it was still on the surface on Mars.

Metabolic activity of permafrost bacteria below the freezing point.

Metabolic activity was measured in the laboratory at temperatures between 5 and -20 degrees C on the basis of incorporation of (14)C-labeled acetate into lipids by samples of a natural population of bacteria from Siberian permafrost (permanently frozen soil). Incorporation followed a sigmoidal pattern similar to growth curves. At all temperatures, the log phase was followed, within 200 to 350 days, by a stationary phase, which was monitored until the 550th day of activity. The minimum doubling times ranged from 1 day (5 degrees C) to 20 days (-10 degrees C) to ca. 160 days (-20 degrees C). The curves reached the stationary phase at different levels, depending on the incubation temperature. We suggest that the stationary phase, which is generally considered to be reached when the availability of nutrients becomes limiting, was brought on under our conditions by the formation of diffusion barriers in the thin layers of unfrozen water known to be present in permafrost soils, the thickness of which depends on temperature.

Ionizing-radiation resistance in the desiccation-tolerant cyanobacterium Chroococcidiopsis.

The effect of X-ray irradiation on cell survival, induction, and repair of DNA damage was studied by using 10 Chroococcidiopsis strains isolated from desert and hypersaline environments. After exposure to 2.5 kGy, the percentages of survival for the strains ranged from 80 to 35%. In the four most resistant strains, the levels of survival were reduced by 1 or 2 orders of magnitude after irradiation with 5 kGy; viable cells were recovered after exposure to 15 kGy but not after exposure to 20 kGy. The severe DNA damage evident after exposure to 2.5 kGy was repaired within 3 h, and the severe DNA damage evident after exposure to 5 kGy was repaired within 24 h. The increase in trichloroacetic acid-precipitable radioactivity in the culture supernatant after irradiation with 2.5 kGy might have been due to cell lysis and/or an excision process involved in DNA repair. The radiation resistance of Chroococcidiopsis strains may reflect the ability of these cyanobacteria to survive prolonged desiccation through efficient repair of the DNA damage that accumulates during dehydration.

Soil temperatures and stability of ice-cemented ground in the McMurdo Dry Valleys, Antarctica.

Year-round temperature measurements at 1600 m elevation during 1994 in the Asgard Range Antarctica, indicate that the mean annual frost point of the ice-cemented ground, 25 cm below the surface, is -21.7 +/- 0.2 degrees C and the mean annual frost point of the atmosphere is -27.5 +/- 1.0 degrees C. The corresponding mean annual temperatures are -24.9 degrees C and -23.3 degrees C. These results imply that there is a net flux of water vapour from the ice to the atmosphere resulting in a recession of the ice-cemented ground by about 0.4-0.6 mm yr-1. The level of the ice-cemented permafrost is about 12 cm below the level of dry permafrost. The summer air temperatures would have to increase about 7 degrees C for thawing temperatures to just reach the top of the subsurface ice. Either subsurface ice at this location is evaporating over time or there are sporadic processes that recharge the ice and maintain equilibrium over long timescales.

Characterization of viable bacteria from Siberian permafrost by 16S rDNA sequencing.

Viable bacteria were found in permafrost core samples from the Kolyma-Indigirka lowland of northeast Siberia. The samples were obtained at different depths; the deepest was about 3 million years old. The average temperature of the permafrost is -10 degrees C. Twenty-nine bacterial isolates were characterized by 16S rDNA sequencing and phylogenetic analysis, cell morphology, Gram staining, endospore formation, and growth at 30 degrees C. The majority of the bacterial isolates were rod shaped and grew well at 30 degrees C; but two of them did not grow at or above 28 degrees C, and had optimum growth temperatures around 20 degrees C. Thirty percent of the isolates could form endospores. Phylogenetic analysis revealed that the isolates fell into four categories: high-GC Gram-positive bacteria, beta-proteobacteria, gamma-proteobacteria, and low-GC Gram-positive bacteria. Most high-GC Gram-positive bacteria and beta-proteobacteria, and all gamma-proteobacteria, came from samples with an estimated age of 1.8-3.0 million years (Olyor suite). Most low-GC Gram-positive bacteria came from samples with an estimated age of 5,000-8,000 years (Alas suite).

Geochemical characteristics of organic compounds in a permafrost sediment core sample from northeast Siberia, Russia.

We studied total organic carbon (TOC), hydrocarbons and fatty acids in a permafrost sediment core sample (well 6-90, length 32.0 m, 1.5-2.5 Ma BP) from northeast Siberia (approximately 70 degrees N, 158 degrees E), Russia, to elucidate their geochemical features in relation to source organisms and paleoenvironmental conditions. Long-chain n-alkanes and n-alkanoic acids (>C19) were most predominant hydrocarbons and fatty acids, respectively, so organic matter in the sediment core was derived mainly from vascular plants and, to a much smaller extent, from bacteria. Low concentrations of unsaturated fatty acids revealed that organic matter in the sediment core was considerably degraded during and/or after sedimentation. The predominance of vascular plant components, the major ionic components of nonmarine sources, and geological data strongly implied that the sediment layers were formed in shallow lacustrine environments, such as swamp with large influences of tundra or forest-tundra vegetation. Also, no drastic changes in paleoenvironmental conditions for biological activity or geological events, such as sea transgressions or ice-sheet influences, occurred at the sampling site approximately 100 km from the coast of the East Siberian Sea during the late Pliocene an early Pleistocene periods.

A primitive cyanobacterium as pioneer microorganism for terraforming Mars.

The primitive characteristics of the cyanobacterium Chroococcidiopsis suggest that it represents a very ancient type of the group. Its morphology is simple but shows a wide range of variability, and it resembles certain Proterozoic microfossils. Chroococcidiopsis is probably the most desiccation-resistant cyanobacterium, the sole photosynthetic organism in extreme arid habitats. It is also present in a wide range of other extreme environments, from Antarctic rocks to thermal springs and hypersaline habitats, but it is unable to compete with more specialized organisms. Genetic evidence suggests that all forms belong to a single species. Its remarkable tolerance of environmental extremes makes Chroococcidiopsis a prime candidate for use as a pioneer photosynthetic microorganism for terraforming of Mars. The hypolithic microbial growth form (which lives under stones of a desert pavement) could be used as a model for development of technologies for large-scale Martian farming.

Extreme environments and exobiology.

Ecological research on extreme environments can be applied to exobiological problems such as the question of life on Mars. If life forms (fossil or extant) are found on Mars, their study will help to solve fundamental questions about the nature of life on Earth. Extreme environments that are beyond the range of adaptability of their inhabitants are defined as "absolute extreme". Such environments can serve as terrestrial models for the last stages of life in the history of Mars, when the surface cooled down and atmosphere and water disappeared. The cryptoendolithic microbial community in porous rocks of the Ross Desert in Antarctica and the microbial mats at the bottom of frozen Antarctic lakes are such examples. The microbial communities of Siberian permafrost show that, in frozen but stable communities, long-term survival is possible. In the context of terraforming Mars, selected microorganisms isolated from absolute extreme environments are considered for use in creation of a biological carbon cycle.

Cytology of long-term desiccation in the desert cyanobacterium Chroococcidiopsis (Chroococcales).

Young and old cultures (up to 66 months) of two Chroococcidiopsis sp. strains isolated from the Negev desert, Israel, were examined by epifluorescence and electron microscopy. In old cultures, cell viability and autofluorescence were lower than in young cultures. An increase was seen with age in the polysaccharide content of the sheaths of nanocytes and nanocyte mother cells, and a decrease of phycobiliproteins was also seen. In the oldest cultures most of the cells were dead and in various stages of degeneration. Single living cells were scattered among the dead ones. No resting cells were formed in the oldest cultures, but many cell groups showed highly electron-dense sheaths and, in the cytoplasm, ribosomes and glycogen. These changes in cell structure may have a role in preventing water loss from the cell.

Biologically active substances produced by antarctic cryptoendolithic fungi.

NASA: Researchers report results of laboratory studies of over 200 microbial strains of fungi, algae, cyanobacteria, and heterotrophic bacteria collected in the Ross Desert region of Antarctica. All of the 35 fungal strains produced substances that inhibited the growth of cyanobacteria and algae. The inhibitory effect of the biologically active substance was evident in crushed cell extract but less in spent broth.^ieng

Long-term productivity in the cryptoendolithic microbial community of the Ross Desert, Antarctica.

Annual gross productivity of the lichen-dominated cryptoendolithic community was calculated from a computer analysis of photosynthetic response based on laboratory measurements of CO2 exchange and three years (1985-1988) of field nanoclimate data. Photosynthetic optimum increased from -3 to 2 degrees C between irradiance levels of 100 and 1500 micromoles photons m-2 s-1, while the upper compensation point rose from 1 to 17 degrees C. The mean yearly total time available for metabolic activity (temperature above -10 degrees C and moisture present) was 771.3 h for horizontal rock, 421.5 h for northeast-oriented sloped rock, and 1042.2 h for a small depression in horizontal rock (the characteristic site of occasional lichen apothecia). The calculated mean gross productivity value for a horizontal rock was 1215 mg C m-2 y-1, and net photosynthetic gain was 606 mg C m-2 y-1. Net ecosystem productivity (annual accretion of cellular biomass) estimated from long-term events amounted to only about 3 mg C m-2 y-1. The difference between these two values may represent the long-term metabolic costs of the frequent dehydration-rehydration and freezing-thawing cycles or of overwintering, and may account for the leaching of organic substances to the rock. The yearly gross productivity of the cryptoendolithic microbial community of the entire Ross Desert area was estimated at approximately 120,000-180,000 kg C. Of this, 600-900 kg C is in microbial biomass, and much of the rest is soluble compounds that leach into the rocks and possibly percolate to the valleys, providing a source of organic matter for lakes, rivers, and soils.

Terraforming Mars: dissolution of carbonate rocks by cyanobacteria.

One of the most difficult tasks in terraforming Mars is the release into the atmosphere of CO2 bound by the surface of Mars. Even if a sufficiently dense CO2 atmosphere can be created by appropriate technology, the maintenance of CO2 concentration remains a problem. As Mars lacks plate tectonics as well as active volcanism, an Earth-like carbon cycle cannot be reproduced there. We suggest that Matteia sp., a lime-boring cyanobacterium isolated from Negev desert rocks, be used to dissolve carbonate rocks both for initial release of CO2 and in design of a Martian carbon cycle.

Novel long-chain anteiso-alkanes and anteiso-alkanoic acids in Antarctic rocks colonized by living and fossil cryptoendolithic microorganisms.

Saponified extracts of rock samples colonized by cryptoendolithic microbial communities from the McMurdo Dry Valleys of Southern Victoria Land, Antarctica, were separated into hydrocarbon and fatty acid fractions by silica gel column chromatography. Hydrocarbons and methyl esters of fatty acids were analyzed by capillary gas chromatography-mass spectrometry. Unusually, a suite of long-chain anteiso-alkanes (a-C20 to a-C30) and anteiso-alkanoic acids (a-C20 to a-C30) were detected in many samples, together with straight-chain, branched and/or cyclic and acyclic isoprenoid compounds. These novel compounds are probably derived from unidentified heterotrophic bacteria or symbiotic processes in a unique microbial community in the Antarctic cold desert and suggest the occurrence of a special biosynthetic pathway. Long-chain anteiso-alkanes are probably formed through microbial decarboxylation of corresponding anteiso-alkanoic acids. They may serve as new biomarkers in environmental and geochemical studies.

History of water on Mars: a biological perspective.

We divide the history of water on the Martian surface into four epochs based upon the atmospheric temperature and pressure. In Epoch 1, during which a primordial CO2 atmosphere was actively maintained by impact and volcanic recycling, we presume the mean annual temperature to have been above freezing, the pressure to have exceeded one atmosphere, and liquid water to have been widespread. Under such conditions, similar to early Earth, life could have arisen and become abundant. After this initial period of recycling, atmospheric CO2 was irreversibly lost due to carbonate formation and the pressure and temperature declined. In Epoch II, the mean annual temperature fell below freezing but peak temperatures would have exceeded freezing. Ice covered lakes, similar to those in the McMurdo Dry Valleys of Antarctica could have provided a habitat for life. In Epoch III, the mean and peak temperatures were below freezing and there would have been only transient liquid water. Microbial ecosystems living in endolithic rock "greenhouses" could have continued to survive. Finally, in Epoch IV, the pressure dropped to near the triple point pressure of water and liquid water could no longer have existed on the surface and life on the surface would have become extinct.

From Siberia to Mars

Because Mars is so similar to Earth, planetary scientists looking for answers to questions like these often use analogous environments on Earth to help them design future Mars missions. Such terrestrial sites, however remote, are still much more accessible than Mars. Field studies in such places give us a chance to test and refine instruments and procedures, develop overall concepts and collect baseline data to compare with actual results from Mars. Perhaps the best terrestrial analogue to the martian permafrost lies in northeastern Siberia. Freezing conditions have persisted here for over 3 million years. Although young by martian standards, these are among the oldest continuously frozen localities on Earth. They also hold something remarkable: not only organic residues, but also large numbers of viable bacteria (up to 100 million per gram of frozen soil), preserved for 3 million years in ice.

Water relations, thallus structure and photosynthesis in Negev Desert lichens.

The role of lichen thallus structure in water relations and photosynthesis was studied in Ramalina maciformis (Del.) Bory and Teloschistes lacunosus (Rupr.) Sav. Water-vapour adsorption and photosynthesis are dependent upon thallus integrity and are significantly lower in crushed thalli. Cultured phycobiont (Trebouxia sp.) cells are capable of photosynthesis over the same relative humidity range (> 80% RH) as are intact lichens. Thus, water-vapour adsorption by the thallus and physiological adaptation of the phycobiont contribute to the ability of these lichens to photosynthesize in an arid environment. Despite differences in their anatomical structure and water-uptake characteristics, their CO2 incorporation is similar. The two lichens use liquid water differently and they occupy different niches.

Water relations and photosynthesis in the cryptoendolithic microbial habitat of hot and cold deserts.

Two cryptoendolithic microbial communities, lichens in the Ross Desert of Antarctica and cyanobacteria in the Negev Desert, inhabit porous sandstone rocks of similar physical structure. Both rock types adsorb water vapor by physical mechanisms unrelated to biological processes. Yet the two microbial communities respond differently to water stress: cryptoendolithic lichens begin to photosynthesize at a matric water potential of -46.4 megaPascals (MPa) [70% relative humidity (RH) at 8 degrees C], resembling thallose desert lichens. Cryptoendolithic cyanobacteria, like other prokaryotes, photosynthesize only at very high matric water potentials [> -6.9 MPa, 90% RH at 20 degrees C].