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.

a b s t r a c tThe predicted Exigobacterium sibiricum bacterirhodopsin gene was amplified from an ancient Siberian permafrost sample. The protein bacteriorhodopsin from Exiguobacterium sibiricum (ESR) encoded by this gene was expressed in Escherichia coli membrane. ESR bound all-trans-retinal and displayed an absorbance maximum at 534 nm without dark adaptation. The ESR photocycle is characterized by fast formation of an M intermediate and the presence of a significant amount of an O intermediate. Proteoliposomes with ESR incorporated transport protons in an outward direction leading to medium acidification. Proton uptake at the cytoplasmic surface of these organelles precedes proton release and coincides with M decay/O rise of the ESR.

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As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration.

Filamentous fungi in 36 samples of Antarctic permafrost sediments were studied. The samples collected during the Russian Antarctic expedition of 2007-2009 within the framework of the Antarctic Permafrost Age Project (ANTPAGE) were recovered from different depths in ice-free oases located along the perimeter of the continent. Fungal diversity was determined by conventional microbiological techniques combined with a culture-independent method based on the analysis of internal transcribed spacer (ITS2) sequences in total DNA of the samples. The study revealed a rather low fungal population density in permafrost, although the diversity found was appreciable, representing more than 26 genera. Comparison of the data obtained by different techniques showed that the culture-independent method enabled the detection of ascomycetous and basidiomycetous fungi not found by culturing. The molecular method failed to detect members of the genera Penicillium and Cladosporium that possess small-sized spores known to have a high resistance to environmental changes.

54 strains of viable green algae and 26 strains of viable cyanobacteria were recovered from 128 and 56 samples collected from Siberian and Antarctic permafrost, respectively, with ages from modern to a few million years old. Although species of unicellular green algae belonged to Chlorococcales were subdominant inside permafrost, green algae Pedinomonas sp. were observed in Antarctic permafrost. Filamentous cyanobacteria of Oscillatoriales, Nostocales were just found in Siberian permafrost. Algal biomass in the permanently frozen sediments, expressed as concentration of chlorophyll a, was 0.06–0.46 μg g−1. The number of viable algal cells varied between <102 and 9×103 cfu g−1, but the number of viable bacterial cells was usually higher from 102 to 9.2×105 cfu g−1. Frozen but viable permafrost algae have preserved their morphological characteristics and photosynthetic apparatus in the dark permafrost. In the laboratory, they restored their photosynthetic activity, growth and development in favourable conditions at positive temperatures and with the availability of water and light. The discovery of ancient viable algae within permafrost reflects their ability to tolerate long-term freezing. In this study, the tolerance of algae and cyanobacteria to freezing, thawing and freezing–drying stresses was evaluated by short-term (days to months) low-temperature experiments. Results indicate that viable permafrost microorganisms demonstrate resistance to such stresses. Apart from their ecological importance, the bacterial and algal species found in permafrost have become the focus for novel biotechnology, as well as being considered proxies for possible life forms on cryogenic extraterrestrial bodies.

Permafrost and ice are important components of cryogenic planets and other bodies, which are abundant in the universe. Earth is one of many planets of the cold type. Earth's permafrost and ice provide an opportunity to test hypotheses that could be applied in the search for possible ecosystems and potential life on extraterrestrial cryogenic planets. Permafrost sediments in polar and alpine regions are natural ecosystems with a unique feature of low‐temperature preservation of the biological material and its genetic information. Therefore, permafrost studies allow reconstruction of the events that occurred in the Cenozoic and the prediction of possible life that might have been preserved before the effect of anthropogenic factors on these ecosystems, and could be found within ice or permafrost on other cryogenic planets.

Permafrost microbes may be metabolically active in microscopic layers of liquid brines, even in ancient soil. Metagenomics can help discern whether permafrost microbes show adaptations to this environment. Thirty-three metagenome-assembled genomes (MAGs) were obtained from six depths (3.5 m to 20 m) of freshly-cored permafrost from the Siberia Kolyma-Indigirka Lowland region. These soils have been continuously frozen for ∼20,000 to 1,000,000 years. Eight of these MAGs were ≥80% complete with <10% contamination and were taxonomically identified as Aminicenantes , Atribacteria , Chloroflexi , and Actinobacteria within bacteria and Thermoprofundales within archaea. MAGs from these taxa have previously been obtained from non-permafrost environments and have been suggested to show adaptations to long-term energy-starvation, but they have never been explored in ancient permafrost. The permafrost MAGs had higher proportions of clusters of orthologous genes (COGs) from ‘Energy production and conversion’ and ‘Carbohydrate transport and metabolism’ than their non-permafrost counterparts. They also contained genes for trehalose synthesis, thymine metabolism, mevalonate biosynthesis and cellulose degradation that were less prevalent in non-permafrost genomes. Many of these genes are involved in membrane stabilization and osmotic stress responses, consistent with adaptation to the anoxic, high ionic strength, cold environments of permafrost brine films. Our results suggest that this ancient permafrost contains DNA in high enough quality to assemble MAGs from microorganisms with adaptations to subsist long-term freezing in this extreme environment. Importance Permafrost around the world is thawing rapidly. Many scientists from a variety of disciplines have shown the importance of understanding what will happen to our ecosystem, commerce, and climate when permafrost thaws. The fate of permafrost microorganisms is connected to these predicted rapid environmental changes. Studying ancient permafrost with culture independent techniques can give a glimpse into how these microorganisms function in these extreme low temperature and energy conditions. This will aid understanding of how they will change with the environment. This study presents genomic data from this unique environment aged ∼20,000 to 1,000,000-years-old.