Search for extraterrestrial life using chiral molecules: mandelate racemase as a test case

We have investigated an enzymatic racemization reaction as a marker for extraterrestrial life, which resulted in a change in optical rotation of a mandelic acid over time, as measured by polarimetry. Mandelate racemase was active in aqueous buffer in a temperature range between 0 degrees C and 70 degrees C and also in concentrated ammonium salt solutions and water-in-oil microemulsions in a temperature range between -30 degrees C and 60-70 degrees C; however, the enzyme was not active in several organic cryosolvents. Thus, we have demonstrated that concentrated ammonium salt solutions and water-in-oil microemulsions, both of which are able to form on extraterrestrial planets and moons in the presence of liquid water, are suitable media for enzyme reactions at subzero temperatures. Kinetic data for the mandelate racemase reaction obtained by polarimetry, while reproducible and internally consistent, differed significantly from several sets of data obtained previously by other methods such as chromatography and hydrogen-deuterium exchange. However, we conclude that reactions yielding a polarimetric signal, such as the racemizations employed in this work, are suitable mechanisms by which to utilize a change in chirality over time as a tool to detect signs of life.     

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.     

Temperature and moisture conditions for life in the extreme arid region of the Atacama desert: four years of observations including the El Niño of 1997-1998

The Atacama along the Pacific Coast of Chile and Peru is one of the driest and possibly oldest deserts in the world. It represents an extreme habitat for life on Earth and is an analog for life in dry conditions on Mars. We report on four years (September 1994-October 1998) of climate and moisture data from the extreme arid region of the Atacama. Our data are focused on understanding moisture sources and their role in creating suitable environments for photosynthetic microorganisms in the desert surface. The average air temperature was 16.5 degrees C and 16.6 degrees C in 1995 and 1996, respectively. The maximum air temperature recorded was 37.9 degrees C, and the minimum was -5.7 degrees C. Annual average sunlight was 336 and 335 W m(-2) in 1995 and 1996, respectively. Winds averaged a few meters per second, with strong f?hn winds coming from the west exceeding 12 m s(-1). During our 4 years of observation there was only one significant rain event of 2.3 mm, which occurred near midnight local time. We suggest that this event was a rainout of a heavy fog. It is of interest that the strong El Ni?o of 1997-1998 brought heavy rainfall to the deserts of Peru, but did not bring significant rain to the central Atacama in Chile. Dew occurred at our station frequently following high nighttime relative humidity, but is not a significant source of moisture in the soil or under stones. Groundwater also does not contribute to surface moisture. Only the one rain event of 2.3 mm resulted in liquid water in the soil and beneath stones for a total of only 65-85 h over 4 years. The paucity of liquid water under stones is consistent with the apparent absence of hypolithic (under-stone) cyanobacteria, the only known primary producers in such extreme deserts.     

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.     

Subfreezing activity of microorganisms and the potential habitability of Mars' polar regions

The availability of water-ice at the surface in the Mars polar cap and within the top meter of the high-latitude regolith raises the question of whether liquid water can exist there under some circumstances and possibly support the existence of biota. We examine the minimum temperatures at which liquid water can exist at ice grain-dust grain and ice grain-ice grain contacts, the minimum subfreezing temperatures at which terrestrial organisms can grow or multiply, and the maximum temperatures that can occur in martian high-latitude and polar regions, to see if there is overlap. Liquid water can exist at grain contacts above about -20 degrees C. Measurements of growth in organisms isolated from Siberian permafrost indicate growth at -10 degrees C and metabolism at -20 degrees C. Mars polar and high-latitude temperatures rise above -20 degrees C at obliquities greater than ~40 degrees, and under some conditions rise above 0 degrees C. Thus, the environment in the Mars polar regions has overlapped habitable conditions within relatively recent epochs, and Mars appears to be on the edge of being habitable at present. The easy accessibility of the polar surface layer relative to the deep subsurface make these viable locations to search for evidence of life.     

Implications of subzero metabolic activity on long-term microbial survival in terrestrial and extraterrestrial permafrost

The survival of microorganisms over extended time frames in frozen subsurface environments may be limited by chemical (i.e., via hydrolysis and oxidation) and ionizing radiation-induced damage to chromosomal DNA. In an effort to improve estimates for the survival of bacteria in icy terrestrial and extraterrestrial environments, we determined rates of macromolecular synthesis at temperatures down to -15°C in bacteria isolated from Siberian permafrost (Psychrobacter cryohalolentis K5 and P. arcticus 273-4) and the sensitivity of P. cryohalolentis to ionizing radiation. Based on experiments conducted over ≈400 days at -15°C, the rates of protein and DNA synthesis in P. cryohalolentis were <1 to 16 proteins cell(-1) d(-1) and 83 to 150 base pairs (bp) cell(-1) d(-1), respectively; P. arcticus synthesized DNA at rates of 20 to 1625?bp cell(-1) d(-1) at -15°C under the conditions tested. The dose of ionizing radiation at which 37% of the cells survive (D(37)) of frozen suspensions of P. cryohalolentis was 136?Gy, which was ～2-fold higher (71?Gy) than identical samples exposed as liquid suspensions. Laboratory measurements of [(3)H]thymidine incorporation demonstrate the physiological potential for DNA metabolism at -15°C and suggest a sufficient activity is possible to offset chromosomal damage incurred in near-subsurface terrestrial and martian permafrost. Thus, our data imply that the longevity of microorganisms actively metabolizing within permafrost environments is not constrained by chromosomal DNA damage resulting from ionizing radiation or entropic degradation over geological time.     

Microorganisms metabolizing on clay grains in 3-km-deep Greenland basal ice

We have discovered > 10(8) microbial cells/cm3 attached to clay grains in the bottom 13 m of the GISP2 (Greenland Ice Sheet Project) ice core. Their concentration correlates with huge excesses of CO2 and CH4. We show that Fe-reducing bacteria produce most of the excess CO2 and methanogenic archaea produce the excess CH4. The number of attached cells per clay grain is proportional to grain perimeter rather than to area, which implies that nutrients are accessed at grain edges. We conclude that Fe-reducing microbes immobilized on clay surfaces metabolize via "shuttle" molecules that transport electrons to grain edges, where they reduce Fe(III) ions at edges to Fe(II) while organic acid ions are oxidized to CO2. Driven by the concentration gradient, electrons on Fe(II) ions at grain edges "hop" to Fe(III) ions inward in the same edges and oxidize them. The original Fe(III) ions can then attach new electrons from shuttle molecules at the edges. Our mechanism explains how Fe-reducers can reduce essentially all Fe(III) in clay minerals. We estimate that the Fe(III) in clay grains in the GISP2 silty ice can sustain Fe-reducing bacteria at the ambient temperature of -9 degrees C for approximately 10(6) years. F420 autofluorescence imaging shows that > 2.4% of the cells are methanogens, which account for the excess methane.     

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.     

Climate model studies of synchronously rotating planets

M stars constitute 75% of main sequence stars though, until recently, their star systems have not been considered suitable places for habitable planets to exist. In this study the climate of a synchronously rotating planet around an M dwarf star is evaluated using a three-dimensional global atmospheric circulation model. The presence of clouds and evaporative cooling at the surface of the planet result in a cooler surface temperature at the subsolar point. Water ice forms at the polar regions and on the dark side, where the minimum temperature lies between -30 degrees C and 0 degrees C. As expected, rainfall is extremely high on the starlit side and extremely low on the dark side. The presence of a dry continent causes higher temperatures on the dayside, and allows accumulation of snow on the nightside. The absence of any oceans leads to higher day-night temperature differences, consistent with previous work. The present study reinforces recent conclusions that synchronously rotating planets within the circumstellar habitable zones of M dwarf stars should be habitable, and therefore M dwarf systems should not be excluded in future searches for exoplanets.     

On laboratory simulation and the effect of small temperature oscillations about the freezing point and ice formation on the evaporation rate of water on Mars

We report measurements of the evaporation rate of water under Mars-like conditions (CO2 atmosphere at 7 mbar and approximately 0 degrees C) in which small temperature oscillations about the freezing point repeatedly formed and removed a thin layer of ice. We found that the average evaporation at 2.7 +/- 0.5 degrees C without an ice layer (corrected for the difference in gravity on Earth and on Mars) was 1.24 +/- 0.12 mm/h, while at -2.1 +/- 0.3 degrees C with an ice layer the average evaporation rate was 0.84 +/- 0.08 mm/h. These values are in good agreement with those calculated for the evaporation of liquid water and ice when it is assumed that evaporation only depends on diffusion and buoyancy. Our findings suggest that such differences in evaporation rates are entirely due to the temperature difference and that the ice layer has little effect on evaporation rate. We infer that the formation of thin layers of ice on pools of water on Mars does not significantly increase the stability of water on the surface of Mars.     

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.     

Using electrical impedance spectroscopy to detect water in planetary regoliths

We present data in examination of the utility of electrical impedance spectroscopy measurements for in situ surveys to determine the water content, distribution, and phase in unconsolidated planetary regolith. We conducted calibration experiments under conditions relevant to Mars: the concentration of electrolytes in solution was varied up to 1 M to simulate the effects of unsaturated dissolved minerals and brines. We also varied the water content of heterogeneous water/sand mixtures, made with these electrolytic solutions from 0.01 wt% to 10 wt%. Tests were performed at temperatures from +25 degrees C to -65 degrees C. Conductivity and dielectric permittivity calculated from the impedance measurements indicate an expected dependence on electrolyte concentration and relative independence from electrolyte type for both liquid water and water ice. Conductivity and calculated dielectric relaxation times for these aqueous solutions agree with existing data in the literature. The relative permittivity for heterogeneous water/sand mixtures is dominated by polarization effects for the electrode configuration used. However, the characteristic orientational relaxation of ice is still visible. The conductivity retains the strong dependence on electrolyte concentration, and the permittivity is still not affected by electrolyte type. A "universal" curve between conductivity and water content establishes detectability limits of <0.01 wt% and approximately 0.3 wt% for water/sand mixtures containing liquid water and ice, respectively.     

Subcritical water extractor for Mars analog soil analysis

Abstract Technologies that enable rapid and efficient extraction of biomarker compounds from various solid matrices are a critical requirement for the successful implementation of in situ chemical analysis of the martian regolith. Here, we describe a portable subcritical water extractor that mimics multiple organic solvent polarities by tuning the dielectric constant of liquid water through adjustment of temperature and pressure. Soil samples, collected from the Yungay region of the Atacama Desert (martian regolith analogue) in the summer of 2005, were used to test the instrument's performance. The total organic carbon was extracted from the samples at concentrations of 0.2-55.4 parts per million. The extraction data were compared to the total organic carbon content in the bulk soil, which was determined via a standard analytical procedure. The instrument's performance was examined over the temperature range of 25-250 degrees C at a fixed pressure of 20.7 MPa. Under these conditions, water remains in a subcritical fluid state with a dielectric constant varying between approximately 80 (at 25 degrees C) and approximately 30 (at 250 degrees C).     

The PROCESS experiment: exposure of amino acids in the EXPOSE-E experiment on the international space station and in laboratory simulations

To understand the chemical behavior of organic molecules in the space environment, amino acids and a dipeptide in pure form and embedded in meteorite powder were exposed in the PROCESS experiment in the EXPOSE-E facility mounted on the European Technology Exposure Facility (EuTEF) platform on board the International Space Station (ISS). After exposure to space conditions for 18 months, the samples were returned to Earth and analyzed in the laboratory for reactions caused by solar UV and cosmic radiation. Chemical degradation and possible racemization and oligomerization, the main reactions caused by photochemistry in the vacuum ultraviolet domain (VUV, wavelength range 100-200?nm for photon energy from 6.2 to 12.4?eV) were examined in particular. The molecules were extracted and derivatized by silylation and analyzed by gas chromatograph coupled to a mass spectrometer (GC-MS) to quantify the rate of the degradation of the compounds. Laboratory exposure in several wavelength ranges from UV to VUV was carried out in parallel in the Cologne Deutsches Zentrum für Luft- und Raumfahrt (DLR) Center and Centre de biophysique moléculaire (CBM) laboratories. The results show that resistance to irradiation is a function of the chemical nature of the exposed molecules and the wavelengths of the UV light. The most altered compounds were the dipeptide, aspartic acid, and aminobutyric acid. The most resistant were alanine, valine, glycine, and aminoisobutyric acid. Our results also demonstrate the protective effect of meteorite powder, which reemphasizes the importance of exogenic contribution to the inventory of prebiotic organics on early Earth.     

Evaluation of spontaneous baroreflex response after 28 days head down tilt bedrest

The spontaneous baroreflex response was evaluated during supine rest and head up tilt (60 degrees) before and immediately after a 28 day 6 degrees HDT bedrest in 6 healthy adult men (age 30-42 years). Sequences of 3 or more beats where RR-interval and systolic blood pressure changed in the same direction were used to evaluate baroreflex response slope (BRS). Prior to bedrest, the mean BRS and RR-interval were 18.0 +/- 3.9 ms/mm Hg and 926 +/- 61 ms at rest and 10.5 +/- 2.5 ms/mm Hg and 772 +/- 63 ms during the first 10 min of 60 degrees tilt. Following bedrest, these values changed to 15.6 +/- 2.7 ms/mm Hg and 780 +/- 53 ms at rest, and to 6.5 +/- 1.2 ms/mm Hg and 636 +/- 44 ms during tilt. Thus, (1) the spontaneous baroreflex can be evaluated in human subjects during experiments of orthostatic stress; (2) the baroreflex slope was reduced on going from supine to the head up tilt position; and (3) 28 days of bedrest reduced the spontaneous baroreflex slope.     

Active polar gullies on Mars and the role of carbon dioxide

The detection of geologically recent channels and gullies on Mars has been interpreted as evidence for recent water activity on this arid and cold planet. The presence of active water would have considerable implications for the history of Mars and for the potential for an active near-surface biota; however, water is not the only substance that can flow and may actually be one of the least likely candidates when the modern permafrost environment is considered. Here, a key site of recent gully and channel development on Mars is reported at 71 degrees S in Sisyphi Cavi, within the annual zone of polar CO2 ice and snow accumulation. Superposition of channel features over and/or through the defrosting CO2 snowpack shows that the channels are active at the present day and probably have fluid flows every spring during the annual defrosting. In itself, this is a significant observation as active fluid flows of any nature have not yet been proven on Mars. However, the ambient temperature at the time of gully activity appears to require a role for CO2 in the formation of the channels, rather than water. A model is proposed for gas-lubricated flow in the channels based on avalanching of CO2 snowpack and clastic debris. If similar mechanisms are responsible for all the recent gullies on Mars, then perhaps no near-surface astrobiological targets are available.     

A facility for long-term Mars simulation experiments: the Mars Environmental Simulation Chamber (MESCH)

We describe the design, construction, and pilot operation of a Mars simulation facility comprised of a cryogenic environmental chamber, an atmospheric gas analyzer, and a xenon/mercury discharge source for UV generation. The Mars Environmental Simulation Chamber (MESCH) consists of a double-walled cylindrical chamber. The double wall provides a cooling mantle through which liquid N(2) can be circulated. A load-lock system that consists of a small pressure-exchange chamber, which can be evacuated, allows for the exchange of samples without changing the chamber environment. Fitted within the MESCH is a carousel, which holds up to 10 steel sample tubes. Rotation of the carousel is controlled by an external motor. Each sample in the carousel can be placed at any desired position. Environmental data, such as temperature, pressure, and UV exposure time, are computer logged and used in automated feedback mechanisms, enabling a wide variety of experiments that include time series. Tests of the simulation facility have successfully demonstrated its ability to produce temperature cycles and maintain low temperature (down to -140 degrees C), low atmospheric pressure (5-10 mbar), and a gas composition like that of Mars during long-term experiments.     

The structure of resting bacterial populations in soil and subsoil permafrost

The structure of individual cells in microbial populations in situ of the Arctic and Antarctic permafrost was studied by scanning and transmission electron microscopy methods and compared with that of cyst-like resting forms generated under special conditions by the non-spore-forming bacteria Arthrobacter and Micrococcus isolated from the permafrost. Electron microscopy examination of microorganisms in situ revealed several types of bacterial cells having no signs of damage, including "dwarf" curved forms similar to nanoforms. Intact bacterial cells in situ and frozen cultures of the permafrost isolates differed from vegetative cells by thickened cell walls, the altered structure of cytoplasm, and the compact nucleoid, and were similar in these features to cyst-like resting forms of non-spore-forming "permafrost" bacterial strains of Arthrobacter and Micrococcus spp. Cyst-like cells, being resistant to adverse external factors, are regarded as being responsible for survival of the non-spore-formers under prolonged exposure to subzero temperatures and can be a target to search for living microorganisms in natural environments both on the Earth and on extraterrestrial bodies.     

Cyanobacterial emergence at 2.8 gya and greenhouse feedbacks

Apparent cyanobacterial emergence at about 2.8 Gya coincides with the negative excursion in the organic carbon isotope record, which is the first strong evidence for the presence of atmospheric methane. The existence of weathering feedbacks in the carbonate-silicate cycle suggests that atmospheric and oceanic CO2 concentrations would have been high prior to the presence of a methane greenhouse (and thus the ocean would have had high bicarbonate concentrations). With the onset of a methane greenhouse, carbon dioxide concentrations would decrease. Bicarbonate has been proposed as the preferred reductant that preceded water for oxygenic photosynthesis in a bacterial photosynthetic precursor to cyanobacteria; with the drop of carbon dioxide level, Archean cyanobacteria emerged using water as a reductant instead of bicarbonate (Dismukes et al., 2001). Our thermodynamic calculations, with regard to this scenario, give at least a tenfold drop in aqueous CO2 levels with the onset of a methane-dominated greenhouse, assuming surface temperatures of about 60 degrees C and a drop in the level of atmospheric carbon dioxide from about 1 to 0.1 bars. The buildup of atmospheric methane could have been triggered by the boost in oceanic organic productivity that arose from the emergence of pre-cyanobacterial oxygenic phototrophy at about 2.8-3.0 Gya; high temperatures may have precluded an earlier emergence. A greenhouse transition timescale on the order of 50-100 million years is consistent with results from modeling the carbonate-silicate cycle. This is an alternative hypothesis to proposals of a tectonic driver for this apparent greenhouse transition.     

An examination of the carbon isotope effects associated with amino acid biosynthesis

Stable carbon isotope ratios (delta(13)C) were determined for alanine, proline, phenylalanine, valine, leucine, isoleucine, aspartate (aspartic acid and asparagine), glutamate (glutamic acid and glutamine), lysine, serine, glycine, and threonine from metabolically diverse microorganisms. The microorganisms examined included fermenting bacteria, organotrophic, chemolithotrophic, phototrophic, methylotrophic, methanogenic, acetogenic, acetotrophic, and naturally occurring cryptoendolithic communities from the Dry Valleys of Antarctica. Here we demonstrated that reactions involved in amino acid biosynthesis can be used to distinguish amino acids formed by life from those formed by nonbiological processes. The unique patterns of delta(13)C imprinted by life on amino acids produced a biological bias. We also showed that, by applying discriminant function analysis to the delta(13)C value of a pool of amino acids formed by biological activity, it was possible to identify key aspects of intermediary carbon metabolism in the microbial world. In fact, microorganisms examined in this study could be placed within one of three metabolic groups: (1) heterotrophs that grow by oxidizing compounds containing three or more carbon-to-carbon bonds (fermenters and organotrophs), (2) autotrophs that grow by taking up carbon dioxide (chemolitotrophs and phototrophs), and (3) acetoclastic microbes that grow by assimilation of formaldehyde or acetate (methylotrophs, methanogens, acetogens, and acetotrophs). Furthermore, we demonstrated that cryptoendolithic communities from Antarctica grouped most closely with the autotrophs, which indicates that the dominant metabolic pathways in these communities are likely those utilized for CO(2 )fixation. We propose that this technique can be used to determine the dominant metabolic types in a community and reveal the overall flow of carbon in a complex ecosystem.