Biomarker and stable isotope characterization of coastal pond-derived organic matter, McMurdo Dry Valleys, Antarctica

Small coastal ponds that contain photosynthetic microbial mat communities represent an extreme environment where a potentially significant source of labile organic carbon can be found within the McMurdo Dry Valleys, Antarctica. To distinguish coastal pond-derived organic matter from other sources of organic matter in the Dry Valleys, bulk organic carbon, nitrogen, and sulfur isotope signatures and phospholipid fatty acid (PLFA) profiles of benthic microbial mats located at two sites--Hjorth Hill coast and Garwood Valley--were investigated. The average isotope values at Hjorth Hill coast and Garwood Valley are, respectively, -10.9 per thousand and -10.2 per thousand for delta(13) C, 3.7 per thousand and -1.3 per thousand for delta(15)N, and 8.1 per thousand and 16.7 per thousand for delta(34)S. Microbial mats from all ponds are dominated by monounsaturated PLFAs (indicative of Gram-negative bacteria) and polyunsaturated PLFAs (indicative of microeukaryotes). Biomarkers specific to aerobic prokaryotes, eukaryotes, and photoautotrophic microeukaryotes, as well as sulfur-reducing bacteria, are present in all samples. Benthic mats at Garwood Valley are thicker and more laminated, have a higher biomass, and have a greater carbon and nitrogen content, which suggests greater productivity than mats at Hjorth Hill coast. Greater productivity is supported, as well, by higher dissolved oxygen contents likely derived from heightened photosynthetic productivity. More productivity at Garwood Valley likely results from a larger influx of terrestrial surface waters together with a concomitant nutrient loading.     

Distribution and stable isotopic composition of amino acids from fungal peptaibiotics: assessing the potential for meteoritic contamination

The presence of nonprotein α-dialkyl-amino acids such as α-aminoisobutyric acid (α-AIB) and isovaline (Iva), which are considered to be relatively rare in the terrestrial biosphere, has long been used as an indication of the indigeneity of meteoritic amino acids. However, recent work showing the presence of α-AIB and Iva in peptides produced by a widespread group of filamentous fungi indicates the possibility of a terrestrial biotic source for the α-AIB observed in some meteorites. We measured the amino acid distribution and stable carbon and nitrogen isotopic composition of four α-AIB-containing fungal peptides and compared this data to similar meteoritic measurements. We show that the relatively simple distribution of the C(4) and C(5) amino acids in fungal peptides is distinct from the complex distribution observed in many carbonaceous chondrites. We also identify potentially diagnostic relationships between the stable isotopic compositions of pairs of amino acids from the fungal peptides that may aid in ruling out fungal contamination as a source of meteoritic amino acids.     

Amino acids from ion-irradiated nitrile-containing ices

Solid CH(3)CN and solid H(2)O + CH(3)CN were ion irradiated near 10 K to initiate chemical reactions thought to occur in extraterrestrial ices. The infrared spectra of these samples after irradiation revealed the synthesis of new molecules. After the irradiated ices were warmed to remove volatiles, the resulting residual material was extracted and analyzed. Both unhydrolyzed and acid-hydrolyzed residues were examined by both liquid and gas chromatographic-mass spectral methods and found to contain a rich mixture of products. The unhydrolyzed samples showed HCN, NH(3), acetaldehyde (formed by reaction with background and atmospheric H(2)O), alkyamines, and numerous other compounds, but no amino acids. However, reaction products in hydrolyzed residues contained a suite of amino acids that included some found in carbonaceous chondrite meteorites. Equal amounts of D- and L-enantiomers were found for each chiral amino acid detected. Extensive use was made of (13)C-labeled CH(3)CN to confirm amino acid identifications and discriminate against possible terrestrial contaminants. The results reported here show that ices exposed to cosmic rays can yield products that, after hydrolysis, form a set of primary amino acids equal in richness to those made by other methods, such as photochemistry.     

Survival of amino acids in micrometeorites during atmospheric entry

The delivery of amino acids by micrometeorites to the early Earth during the period of heavy bombardment could have been a significant source of the Earth's prebiotic amino acid inventory provided that these organic compounds survived atmospheric entry heating. To investigate the sublimation of amino acids from a micrometeorite analog at elevated temperature, grains from the CM-type carbonaceous chondrite Murchison were heated to 550 degrees C inside a glass sublimation apparatus (SA) under reduced pressure. The sublimed residue that had collected on the cold finger of the SA after heating was analyzed for amino acids by HPLC. We found that when the temperature of the meteorite reached approximately 150 degrees C, a large fraction of the amino acid glycine had vaporized from the meteorite, recondensed onto the end of the SA cold finger, and survived as the rest of the grains heated to 550 degrees C. alpha-Aminoisobutryic acid and isovaline, which are two of the most abundant non-protein amino acids in Murchison, did not sublime from the meteorite and were completely destroyed during the heating experiment. Our experimental results suggest that sublimation of glycine present in micrometeorite grains may provide a way for this amino acid to survive atmospheric entry heating at temperatures > 550 degrees C; all other amino acids apparently are destroyed.     

Biochemical constraints in a protobiotic earth devoid of basic amino acids: the "BAA(-) world"

It has been hypothesized in this journal and elsewhere, based on surveys of published data from prebiotic synthesis experiments and carbonaceous meteorite analyses, that basic amino acids such as lysine and arginine were not abundant on prebiotic Earth. If the basic amino acids were incorporated only rarely into the first peptides formed in that environment, it is important to understand what protobiotic chemistry is possible in their absence. As an initial test of the hypothesis that basic amino acid negative [BAA(-)] proteins could have performed at least a subset of protobiotic chemistry, the current work reports on a survey of 13 archaeal and 13 bacterial genomes that has identified 61 modern gene sequences coding for known or putative proteins not containing arginine or lysine. Eleven of the sequences found code for proteins whose functions are well known and important in the biochemistry of modern microbial life: lysine biosynthesis protein LysW, arginine cluster proteins, copper ion binding proteins, bacterial flagellar proteins, and PE or PPE family proteins. These data indicate that the lack of basic amino acids does not prevent peptides or proteins from serving useful structural and biochemical functions. However, as would be predicted from fundamental physicochemical principles, we see no fossil evidence of prebiotic BAA(-) peptide sequences capable of interacting directly with nucleic acids.     

Stability of amino acids and their oligomerization under high-pressure conditions: implications for prebiotic chemistry

The polymerization of amino acids leading to the formation of peptides and proteins is a significant problem for the origin of life. This problem stems from the instability of amino acids and the difficulty of their oligomerization in aqueous environments, such as seafloor hydrothermal systems. We investigated the stability of amino acids and their oligomerization reactions under high-temperature (180-400°C) and high-pressure (1.0-5.5?GPa) conditions, based on the hypothesis that the polymerization of amino acids occurred in marine sediments during diagenesis and metamorphism, at convergent margins on early Earth. Our results show that the amino acids glycine and alanine are stabilized by high pressure. Oligomers up to pentamers were formed, which has never been reported for alanine in the absence of a catalyst. The yields of peptides at a given temperature and reaction time were higher under higher-pressure conditions. Elemental, infrared, and isotopic analyses of the reaction products indicated that deamination is a key degradation process for amino acids and peptides under high-pressure conditions. A possible NH(3)-rich environment in marine sediments on early Earth may have further stabilized amino acids and peptides by inhibiting their deamination.     

Analysis of underivatized amino acids in geological samples using ion-pairing liquid chromatography and electrospray tandem mass spectrometry

The capability of detecting biomarkers, such as amino acids, in chemically complex field samples is essential to establishing the knowledge required to search for chemical signatures of life in future planetary explorations. However, due to the complexities of in situ investigations, it is important to establish a new analytical scheme that utilizes a minimal amount of sample preparation. This paper reports the feasibility of a novel and sensitive technique, which has been established to quantitate amino acids in terrestrial crust samples directly without derivatization using volatile ion-pairing liquid chromatography and tandem mass spectrometry equipped with an electrospray ionization source. Adequate separation of 20 underivatized amino acids was achieved on a C(18) capillary column within 26 min with nonafluoropentanoic acid (NFPA) as ion-pairing reagent. Each amino acid was identified from its retention time as well as from its characteristic parent-to-daughter ion transition. Using tandem mass spectrometry as a detection technique allows co-elution of some amino acids, as it is more specific than traditional spectrophotometric methods. In the present study, terrestrial samples collected from 3 different locations were analyzed for their water-extractable free amino acid contents, following the removal of metal and organic interferences via ion exchange procedures. This is the first time that amino acids in geological samples were directly determined quantitatively without complicated derivatization steps. Depending on the amino acid, the detection limits varied from 0.02 to 5.7 pmol with the use of a 1 microl sample injection loop.     

Adsorption of amino acids (ALA, CYS, HIS, MET) on zeolites: fourier transform infrared and Raman spectroscopy investigations

Minerals adsorb more amino acids with charged R-groups than amino acids with uncharged R-groups. Thus, the peptides that form from the condensation of amino acids on the surface of minerals should be composed of amino acid residues that are more charged than uncharged. However, most of the amino acids (74%) in today's proteins have an uncharged R-group. One mechanism with which to solve this paradox is the use of organophilic minerals such as zeolites. Over the range of pH (pH 2.66-4.50) used in these experiments, the R-group of histidine (His) is positively charged and neutral for alanine (Ala), cysteine (Cys), and methionine (Met). In acidic hydrothermal environments, the pH could be even lower than those used in this study. For the pH range studied, the zeolites were negatively charged, and the overall charge of all amino acids was positive. The conditions used here approximate those of prebiotic Earth. The most important finding of this work is that the relative concentrations of each amino acid (X=His, Met, Cys) to alanine (X/Ala) are close to 1.00. This is an important result with regard to prebiotic chemistry because it could be a solution for the paradox stated above. Pore size did not affect the adsorption of Cys and Met on zeolites, and the Si/Al ratio did not affect the adsorption of Cys, His, and Met. ZSM-5 could be used for the purification of Cys from other amino acids (Student-Newman-Keuls test, p<0.05), and mordenite could be used for separation of amino acids from each other (Student-Newman-Keuls test, p<0.05). As shown by Fourier transform infrared (FT-IR) spectra, Ala interacts with zeolites through the [Formula: see text] group, and methionine-zeolite interactions involve the COO, [Formula: see text], and CH(3) groups. FT-IR spectra show that the interaction between the zeolites and His is weak. Cys showed higher adsorption on all zeolites; however, the hydrophobic Van der Waals interaction between zeolites and Cys is too weak to produce any structural changes in the Cys groups (amine, carboxylic, sulfhydryl, etc.); thus, the FT-IR and Raman spectra are the same as those of solid Cys.     

Analysis of carbonaceous biomarkers with the Mars Organic Analyzer microchip capillary electrophoresis system: carboxylic acids

The oxidizing surface chemistry on Mars argues that any comprehensive search for organic compounds indicative of life requires methods to analyze higher oxidation states of carbon with very low limits of detection. To address this goal, microchip capillary electrophoresis (μCE) methods were developed for analysis of carboxylic acids with the Mars Organic Analyzer (MOA). Fluorescent derivatization was achieved by activation with the water soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) followed by reaction with Cascade Blue hydrazide in 30 mM borate, pH 3. A standard containing 12 carboxylic acids found in terrestrial life was successfully labeled and separated in 30 mM borate at pH 9.5, 20 °C by using the MOA CE system. Limits of detection were 5-10 nM for aliphatic monoacids, 20 nM for malic acid (diacid), and 230 nM for citric acid (triacid). Polyacid benzene derivatives containing 2, 3, 4, and 6 carboxyl groups were also analyzed. In particular, mellitic acid was successfully labeled and analyzed with a limit of detection of 300 nM (5 ppb). Analyses of carboxylic acids sampled from a lava tube cave and a hydrothermal area demonstrated the versatility and robustness of our method. This work establishes that the MOA can be used for sensitive analyses of a wide range of carboxylic acids in the search for extraterrestrial organic molecules.     

Survival of rock-colonizing organisms after 1.5 years in outer space

Cryptoendolithic microbial communities and epilithic lichens have been considered as appropriate candidates for the scenario of lithopanspermia, which proposes a natural interplanetary exchange of organisms by means of rocks that have been impact ejected from their planet of origin. So far, the hardiness of these terrestrial organisms in the severe and hostile conditions of space has not been tested over extended periods of time. A first long-term (1.5 years) exposure experiment in space was performed with a variety of rock-colonizing eukaryotic organisms at the International Space Station on board the European EXPOSE-E facility. Organisms were selected that are especially adapted to cope with the environmental extremes of their natural habitats. It was found that some-but not all-of those most robust microbial communities from extremely hostile regions on Earth are also partially resistant to the even more hostile environment of outer space, including high vacuum, temperature fluctuation, the full spectrum of extraterrestrial solar electromagnetic radiation, and cosmic ionizing radiation. Although the reported experimental period of 1.5 years in space is not comparable with the time spans of thousands or millions of years believed to be required for lithopanspermia, our data provide first evidence of the differential hardiness of cryptoendolithic communities in space.     

Microbial influences on local carbon isotopic ratios and their preservation in carbonate

Analysis of the carbon isotopic composition of carbonates is a valuable tool for studying microbial processes and looking for evidence of life. Microbial fixation of CO2 and conversion of organic carbon to CO2 can produce measurable delta 13C shifts in a microbial mat environment. Diffusion modeling demonstrates that substantial isotopic shifts can develop within the mat and in the diffusion boundary layer in the fluid when CO2 fixation is rapid and prolonged for several hours. Carbonates that precipitate during rapid CO2 fixation can preserve these microbially produced isotopic shifts. However, continued precipitation during intervals when respiration dominates or after the cessation of active microbial growth commonly dilutes autotrophic isotopic signatures. Thus, preserved isotopic signatures rarely reflect the magnitude of isotopic shifts within the mat. Interpretation of observed isotopic shifts in microbial mat carbonate depends on fully characterizing ambient delta 13C and eliminating other origins for isotopic shifts. The carbon isotopic composition of reservoirs can vary substantially, both on Earth and on other planets. Characterizing the reservoir composition and any changes through time is critical to evaluating microbially induced shifts. In addition, careful evaluation of non-microbial causes for shifts in isotopic composition is essential for a reliable interpretation. Complicating processes include recrystallization, calcite precipitation over extended periods of time, variable precipitation rates and water chemistry, and mixing of carbonates having different isotopic signatures.     

Amino acid distribution in meteorites: diagenesis, extraction methods, and standard metrics in the search for extraterrestrial biosignatures

The relative abundance of the protein amino acids has been previously investigated as a potential marker for biogenicity in meteoritic samples. However, these investigations were executed without a quantitative metric to evaluate distribution variations, and they did not account for the possibility of interdisciplinary systematic error arising from inter-laboratory differences in extraction and detection techniques. Principal component analysis (PCA), hierarchical cluster analysis (HCA), and stochastic probabilistic artificial neural networks (ANNs) were used to compare the distributions for nine protein amino acids previously reported for the Murchison carbonaceous chondrite, Mars meteorites (ALH84001, Nakhla, and EETA79001), prebiotic synthesis experiments, and terrestrial biota and sediments. These techniques allowed us (1) to identify a shift in terrestrial amino acid distributions secondary to diagenesis; (2) to detect differences in terrestrial distributions that may be systematic differences between extraction and analysis techniques in biological and geological laboratories; and (3) to determine that distributions in meteoritic samples appear more similar to prebiotic chemistry samples than they do to the terrestrial unaltered or diagenetic samples. Both diagenesis and putative interdisciplinary differences in analysis complicate interpretation of meteoritic amino acid distributions. We propose that the analysis of future samples from such diverse sources as meteoritic influx, sample return missions, and in situ exploration of Mars would be less ambiguous with adoption of standardized assay techniques, systematic inclusion of assay standards, and the use of a quantitative, probabilistic metric. We present here one such metric determined by sequential feature extraction and normalization (PCA), information-driven automated exploration of classification possibilities (HCA), and prediction of classification accuracy (ANNs).     

Aspartic acid racemization and age-depth relationships for organic carbon in Siberian permafrost

We have analyzed the degree of racemization of aspartic acid in permafrost samples from Northern Siberia, an area from which microorganisms of apparent ages up to a few million years have previously been isolated and cultured. We find that the extent of aspartic acid racemization in permafrost cores increases very slowly up to an age of approximately 25,000 years (around 5 m in depth). The apparent temperature of racemization over the age range of 0-25,000 years, determined using measured aspartic acid racemization rate constants, is -19 degrees C. This apparent racemization temperature is significantly lower than the measured environmental temperature (-11 to -13 degrees C) and suggests active recycling of D-aspartic acid in Siberian permafrost up to an age of around 25,000 years. This indicates that permafrost organisms are capable of repairing some molecular damage incurred while in a "dormant" state over geologic time.     

Substrate limitation for methanogenesis in hypersaline environments

Motivated by the increasingly abundant evidence for hypersaline environments on Mars and reports of methane in its atmosphere, we examined methanogenesis in hypersaline ponds in Baja California Sur, Mexico, and in northern California, USA. Methane-rich bubbles trapped within or below gypsum/halite crusts have δ13C values near -40‰. Methane with these relatively high isotopic values would typically be considered thermogenic; however, incubations of crust samples resulted in the biological production of methane with similar isotopic composition. A series of measurements aimed at understanding the isotopic composition of methane in hypersaline systems was therefore undertaken. Methane production rates, as well as the concentrations and isotopic composition of the particulate organic carbon (POC), were measured. Methane production was highest from microbial communities living within gypsum crusts, whereas POC content at gypsum/halite sites was low, generally less than 1% of the total mass. The isotopic composition of the POC ranged from -26‰ to -10‰. To determine the substrates used by the methanogens, 13C-labeled methylamines, methanol, acetate, and bicarbonate were added to individual incubation vials, and the methane produced was monitored for 13C content. The main substrates used by the methanogens were the noncompetitive substrates, the methylamines, and methanol. When unlabeled trimethylamine (TMA) was added to incubating gypsum/halite crusts in increasing concentrations, the isotopic composition of the methane produced became progressively lower; the lowest methane δ13C values occurred when the most TMA was added (1000 μM final concentration). This decrease in the isotopic composition of the methane produced with increasing TMA concentrations, along with the high in situ methane δ13C values, suggests that the methanogens within the crusts are operating at low substrate concentrations. It appears that substrate limitation is decreasing isotopic fractionation during methanogenesis, which results in these abnormally high biogenic methane δ13C values.     

Testing the potential for computational chemistry to quantify biophysical properties of the non-proteinaceous amino acids

Although most proteins of most living organisms are constructed from the same set of 20 amino acids, all indications are that this standard alphabet represents a mere subset of what was available to life during early evolution. However, we currently lack an appropriate quantitative framework with which to test the qualitative hypotheses that have been offered to date as explanations for nature's "choices." Specifically, although many indices have been developed to describe the 20 standard amino acids, few or no comparable data extend to prebiotically plausible alternatives because of the costly and time-consuming bench experiments that would be required. Computational chemistry (specifically quantitative structure property relationship methods) offers a potentially fast, cost-effective remedy for this knowledge gap by predicting such molecular properties in silico. Thus, we investigated the use of various freely accessible programs to predict three key amino acid properties (hydrophobicity, charge, and size). We assessed the accuracy of these predictions by comparisons with experimentally determined counterparts for appropriate test data sets. In light of these results, and factors of software accessibility and transparency, we suggest a method for further computational assessments of prebiotically plausible amino acids. The results serve as a starting point for future quantitative analysis of amino acid alphabet evolution.     

Investigation of low-energy proton effects on aptamer performance for astrobiological applications

Biochips are promising instruments for the search for organic molecules in planetary environments. Nucleic acid aptamers are powerful affinity receptors known for their high affinity and specificity, and therefore are of great interest for space biochip development. A wide variety of aptamers have already been selected toward targets of astrobiological interest (from amino acids to microorganisms). We present a first study to test the resistance of these receptors to the constraints of the space environment. The emphasis is on the effect of cosmic rays on the molecular recognition properties of DNA aptamers. Experiments on beam-line facilities have been conducted with 2 MeV protons and fluences much higher than expected for a typical mission to Mars. Our results show that this irradiation process did not affect the performances of DNA aptamers as molecular recognition tools.     

FeS/S/FeS(2) redox system and its oxidoreductase-like chemistry in the iron-sulfur world

The iron-sulfur world (ISW) theory is an intriguing prediction regarding the origin of life on early Earth. It hypothesizes that life arose as a geochemical process from inorganic starting materials on the surface of sulfide minerals in the vicinity of deep-sea hot springs. During the last two decades, many experimental studies have been carried out on this topic, and some interesting results have been achieved. Among them, however, the processes of carbon/nitrogen fixation and biomolecular assembly on the mineral surface have received an inordinate amount of attention. To the present, an abiotic model for the oxidation-reduction of intermediates participating in metabolic pathways has been ignored. We examined the oxidation-reduction effect of a prebiotic FeS/S/FeS(2) redox system on the interconversion between several pairs of α-hydroxy acids and α-keto acids (i.e., lactate/pyruvate, malate/oxaloacetate, and glycolate/glyoxylate). We found that, in the absence of FeS, elemental sulfur (S) oxidized α-hydroxy acids to form corresponding keto acids only at a temperature higher than its melting point (113°C); in the presence of FeS, such reactions occurred more efficiently through a coupled reaction mechanism, even at a temperature below the phase transition point of S. On the other hand, FeS was shown to have the capacity to reversibly reduce the keto acids. Such an oxidoreductase-like chemistry of the FeS/S/FeS(2) redox system suggests that it can determine the redox homeostasis of metabolic intermediates in the early evolutionary phase of life. The results provide a possible pathway for the development of primordial redox biochemistry in the iron-sulfur world. Key Words: Iron-sulfur world-FeS/S/FeS(2) redox system-Oxidoreductase-like chemistry. Astrobiology 11, 471-476.     

Bacterial community in ancient Siberian permafrost as characterized by culture and culture-independent methods

The microbial composition of ancient permafrost sediments from the Kolyma lowland of Northeast Eurasia was examined through culture and culture-independent approaches. These sediments have been continuously frozen for 5,000 to 2-3 million years. A total of 265 Bacteria 16S rRNA gene sequences were amplified from the permafrost total-community genomic DNA and screened by amplified ribosomal 16S rRNA restriction analysis. Members of three major lineages were found: gamma-Proteobacteria (mostly Xanthomonadaceae), Actinobacteria, and Firmicutes. We also determined partial 16S rRNA gene sequences of 49 isolates from a collection of 462 aerobes isolated from these sediments. The bacteria included Actinomycetales (Arthrobacter and Microbacteriaceae); followed by the Firmicutes (Exiguobacterium and Planomicrobium); the Bacteroidetes (Flavobacterium); the gamma-Proteobacteria (Psychrobacter); and the alpha-Proteobacteria (Sphingomonas). Both culture and culture-independent approaches showed the presence of high and low G+C Gram-positive bacteria and gamma-Proteobacteria. Some of the 16S rRNA gene sequences of environmental clones matched those of Arthrobacter isolates. Two-thirds of the isolates grew at -2.5 degrees C, indicating that they are psychroactive, and all are closely related to phylogenetic groups with strains from other cold environments, mostly commonly from Antarctica. The culturable and non-culturable microorganisms found in the terrestrial permafrost provide a prototype for possible life on the cryogenic planets of the Solar System.     

Application of the Mars Organic Analyzer to nucleobase and amine biomarker detection

The Mars Organic Analyzer (MOA), a portable microfabricated capillary electrophoresis instrument being developed for planetary exploration, is used to analyze a wide variety of fluorescamine-labeled amine-containing biomarker compounds, including amino acids, mono and diaminoalkanes, amino sugars, nucleobases, and nucleobase degradation products. The nucleobases cytosine and adenine, which contain an exocyclic primary amine, were effectively labeled, separated, and detected at concentrations <500 nM. To test the general applicability of the MOA for biomarker detection, amino acids and mono- and diamines were extracted from bacterial cells using both hydrolysis and sublimation followed by analysis. The extrapolated limit of detection provided by the valine biomarker was approximately 4 x 10(3) cells per sample. Products of an NH(4)CN polymerization that simulate a prebiotic synthesis were also successfully isolated via sublimation and analyzed. Adenine and alanine/serine were detected with no additional sample cleanup at 120 +/- 13 microM and 4.1 +/- 1 microM, respectively, corresponding to a reaction yield of 0.04% and 0.0003%, respectively. This study demonstrates that the MOA provides sensitive detection and analysis of low levels of a wide variety of amine-containing organic compounds from both biological and abiotic sources.     

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.