A preliminary survey of non-lichenized fungi cultured from the hyperarid Atacama Desert of Chile

The Atacama Desert is one of the driest environments on Earth, and has been so for over 200,000 years. Previous reports have suggested that surprisingly low numbers of culturable bacteria, counted as biomass or species diversity, are present in Atacama sands collected from the most hyperarid regions. In previous studies, the presence of eukaryotic organisms was not discussed. In this report, we describe a method of direct plating onto rich media that resulted in culturing a range of fungi from Atacama samples. All fungi identified in this preliminary survey are spore-forming saprobes that are readily dispersed by wind, a likely mechanism that accounts for their presence in the central Atacama Desert.     

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.     

The microbial habitability of weathered volcanic glass inferred from continuous sensing techniques

Basaltic glasses (hyaloclastite) are a widespread habitat for life in volcanic environments, yet their interior physical conditions are poorly characterized. We investigated the characteristics of exposed weathered basaltic glass from a surface outcrop in Iceland, using microprobes capable of continuous sensing, to determine whether the physical conditions in the rock interior are hospitable to microbial life. The material provided thermal protection from freeze-thaw and rapid temperature fluctuations, similar to data reported for other rock types. Water activity experiments showed that at moisture contents less than 13% wet weight, the glass and its weathering product, palagonite, had a water activity below levels suitable for bacterial growth. In pore spaces, however, these higher moisture conditions might be maintained for many days after a precipitation event. Gas exchange between the rock interior and exterior was rapid (< 10 min) when the rocks were dry, but when saturated with water, equilibration took many hours. During this period, we demonstrated the potential for low oxygen conditions within the rock caused by respiratory stimulation of the heterotrophic community within. These conditions might exist within subglacial environments during the formation of the rocks or in micro-environments in the interior of exposed rocks. The experiments showed that microbial communities at the site studied here could potentially be active for 39% of the year, if the depth of the community within the outcrop maintains a balance between access to liquid water and adequate protection from freezing. In the absence of precipitation, the interior of weathered basaltic glass is an extreme and life-limiting environment for microorganisms on Earth and other planets.     

Degradation of cyanobacterial biosignatures by ionizing radiation

Primitive photosynthetic microorganisms, either dormant or dead, may remain today on the martian surface, akin to terrestrial cyanobacteria surviving endolithically in martian analog sites on Earth such as the Antarctic Dry Valleys and the Atacama Desert. Potential markers of martian photoautotrophs include the red edge of chlorophyll reflectance spectra or fluorescence emission from systems of light-harvesting pigments. Such biosignatures, however, would be modified and degraded by long-term exposure to ionizing radiation from the unshielded cosmic ray flux onto the martian surface. In this initial study into this issue, three analytical techniques--absorbance, reflectance, and fluorescence spectroscopy--were employed to determine the progression of the radiolytic destruction of cyanobacteria. The pattern of signal loss for chlorophyll reflection and fluorescence from several biomolecules is characterized and quantified after increasing exposures to ionizing gamma radiation. This allows estimation of the degradation rates of cyanobacterial biosignatures on the martian surface and the identification of promising detectable fluorescent break-down products.     

Hypersaline microbial systems of sabkhas: examples of life's survival in "extreme" conditions

Life and living systems need several important factors to establish themselves and to have a continued tradition. In this article the nature of the borderline situation for microbial life under heavy salt stress is analyzed and discussed using the example of biofilms and microbial mats of sabkha systems of the Red Sea. Important factors ruling such environments are described, and include the following: (1) Microbial life is better suited for survival in extremely changing and only sporadically water-supplied environments than are larger organisms (including humans). (2) Microbial life shows extremely poikilophilic adaptation patterns to conditions that deviate significantly from conditions normal for life processes on Earth today. (3) Microbial life adapts itself to such extremely changing and only ephemerally supportive conditions by the capacity of extreme changes (a) in morphology (pleomorphy), (b) in metabolic patterns (poikilotrophy), (c) in survival strategies (poikilophily), and (d) by trapping and enclosing all necessary sources of energy matter in an inwardly oriented diffusive cycle. All this is achieved without any serious attempt at escaping from the extreme and extremely changing conditions. Furthermore, these salt swamp systems are geophysiological generators of energy and material reservoirs recycled over a geological time scale. Neither energy nor material is wasted for propagation by spore formation. This capacity is summarized as poikilophilic and poikilotroph behavior of biofilm or microbial mat communities in salt and irradiationstressed environmental conditions of the sabkha or salt desert type. We use mainly cyanobacteria as an example, although other bacteria and even eukaryotic fungi may exhibit the same potential of living and surviving under conditions usually not suitable for life on Earth. It may, however, be postulated that such poikilophilic organisms are the true candidates for life support and survival under conditions never recorded on Planet Earth. Mars and some planets of other suns may be good candidates to search for life under conditions normally not thought to be favorable for the maintenance of life.     

Following the kinetics: iron-oxidizing microbial mats in cold icelandic volcanic habitats and their rock-associated carbonaceous signature

Icelandic streams with mean annual temperatures of less than 5 °C, which receive the cationic products of basaltic rock weathering, were found to host mats of iron-cycling microorganisms. We investigated two representative sites. Iron-oxidizing Gallionella and iron-reducing Geobacter species were present. The mats host a high bacterial diversity as determined by culture-independent methods. β-Proteobacteria, Actinobacteria, α-Proteobacteria, and Bacteroidetes were abundant microbial taxa. The mat contained a high number of phototroph sequences. The carbon compounds in the mat displayed broad G and D bands with Raman spectroscopy. This signature becomes incorporated into the weathered oxidized surface layer of the basaltic rocks and was observed on rocks that no longer host mats. The presence of iron-oxidizing taxa in the stream microbial mats, and the lack of them in previously studied volcanic rocks in Iceland that have intermittently been exposed to surface water flows, can be explained by the kinetic limitations to the extraction of reduced iron from rocks. This type of ecosystem illustrates key factors that control the distribution of chemolithotrophs in cold volcanic environments. The data show that one promising sample type for which the hypothesis of the existence of past life on Mars can be tested is the surface of volcanic rocks that, previously, were situated within channels carved by flowing water. Our results also show that the carbonaceous signatures of life, if life had occurred, could be found in or on these rocks.     

Evaluation of early Archean volcaniclastic and volcanic flow rocks as possible sites for carbonaceous fossil microbes

Sedimentary rocks have traditionally been the focus of the search for Archean microfossils; the Earth's oldest fossil bacteria are associated with carbonaceous matter in sedimentary cherts in greenstone belts in the eastern Pilbara block of Western Australia and Barberton greenstone belt of South Africa. Reports of possible fossils in a martian meteorite composed of igneous rock and the discovery of modern bacteria associated with basalts have stimulated a new look at Archean volcanic rocks as possible sites for fossil microbes. This study examines silicified volcaniclastic rocks, near-surface altered volcanic flow rocks, and associated stromatolite- like structures from the Archean Barberton greenstone belt to evaluate their potential for the preservation of carbonaceous fossils. Detrital carbonaceous particles are widely admixed with current-deposited debris. Carbonaceous matter is also present in altered volcanic flow rocks as sparse particles in silica veins that appear to be fed by overlying carbonaceous chert layers. Neither microfossils nor mat-like material was identified in the altered volcanic rocks or adjacent stromatolite-like structures. Ancient volcanic flow and volcaniclastic rocks are not promising sites for carbonaceous fossil preservation.     

A microbial oasis in the hypersaline Atacama subsurface discovered by a life detector chip: implications for the search for life on Mars

The Atacama Desert has long been considered a good Mars analogue for testing instrumentation for planetary exploration, but very few data (if any) have been reported about the geomicrobiology of its salt-rich subsurface. We performed a Mars analogue drilling campaign next to the Salar Grande (Atacama, Chile) in July 2009, and several cores and powder samples from up to 5?m deep were analyzed in situ with LDChip300 (a Life Detector Chip containing 300 antibodies). Here, we show the discovery of a hypersaline subsurface microbial habitat associated with halite-, nitrate-, and perchlorate-containing salts at 2?m deep. LDChip300 detected bacteria, archaea, and other biological material (DNA, exopolysaccharides, some peptides) from the analysis of less than 0.5?g of ground core sample. The results were supported by oligonucleotide microarray hybridization in the field and finally confirmed by molecular phylogenetic analysis and direct visualization of microbial cells bound to halite crystals in the laboratory. Geochemical analyses revealed a habitat with abundant hygroscopic salts like halite (up to 260?g kg(-1)) and perchlorate (41.13?μg g(-1) maximum), which allow deliquescence events at low relative humidity. Thin liquid water films would permit microbes to proliferate by using detected organic acids like acetate (19.14?μg g(-1)) or formate (76.06?μg g(-1)) as electron donors, and sulfate (15875?μg g(-1)), nitrate (13490?μg g(-1)), or perchlorate as acceptors. Our results correlate with the discovery of similar hygroscopic salts and possible deliquescence processes on Mars, and open new search strategies for subsurface martian biota. The performance demonstrated by our LDChip300 validates this technology for planetary exploration, particularly for the search for life on Mars.     

A bacterial enrichment study and overview of the extractable lipids from paleosols in the Dry Valleys, Antarctica: implications for future Mars reconnaissance

The Dry Valleys of Antarctica are one of the coldest and driest environments on Earth with paleosols in selected areas that date to the emplacement of tills by warm-based ice during the Early Miocene. Cited as an analogue to the martian surface, the ability of the Antarctic environment to support microbial life-forms is a matter of special interest, particularly with the upcoming NASA/ESA 2018 ExoMars mission. Lipid biomarkers were extracted and analyzed by gas chromatography--mass spectrometry to assess sources of organic carbon and evaluate the contribution of microbial species to the organic matter of the paleosols. Paleosol samples from the ice-free Dry Valleys were also subsampled and cultivated in a growth medium from which DNA was extracted with the explicit purpose of the positive identification of bacteria. Several species of bacteria were grown in solution and the genus identified. A similar match of the data to sequenced DNA showed that Alphaproteobacteria, Gammaproteobacteria, Bacteriodetes, and Actinobacteridae species were cultivated. The results confirm the presence of bacteria within some paleosols, but no assumptions have been made with regard to in situ activity at present. These results underscore the need not only to further investigate Dry Valley cryosols but also to develop reconnaissance strategies to determine whether such likely Earth-like environments on the Red Planet also contain life.     

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).     

Discovery of a new chert-permineralized microbiota in the Proterozoic Buxa Formation of the Ranjit window, Sikkim, northeast India, and its astrobiological implications

For the foreseeable future, the search for evidence of past life in rocks acquired from other planets will be constrained by the amount of sample available and by the fidelity of preservation of any fossils present. What amount of rock is needed to establish the existence of past life? To address this question, we studied a minute amount of rock collected from cherty dolomites of the Proterozoic Buxa Formation in the metamorphically altered tectonically active northeastern Himalaya. In particular, we investigated 2 small petrographic thin sections-one from each of 2 bedded chert horizons exposed in the Ranjit River stratigraphic section northwest of Rishi, Sikkim, India-that together comprise an area of approximately 5 cm(2) (about the size of a US postage stamp) and have a total rock weight of approximately 0.1 g. Optical microscopy, confocal laser scanning microscopy, and Raman spectroscopy and imagery demonstrate that each of the thin sections contains a rich assemblage of 3-dimensionally permineralized organic-walled microfossils. This study, the first report of Proterozoic microfossils in units of the Ranjit tectonic window, demonstrates that firm evidence of early life can be adduced from even a minuscule amount of fossil-bearing ancient rock.     

Mollusc-microbe mutualisms extend the potential for life in hypersaline systems

Metazoans in extreme environments have evolved mutualisms with microbes that extend the physical and chemical capabilities of both partners. Some of the best examples are bivalve molluscs in evaporite and hypersaline settings. Mollusc tissue is developmentally and evolutionarily amenable to housing vast numbers of symbiotic microbes. Documented benefits to the host are nutritional. Multiple postulated benefits to the microbes are related to optimizing metabolic performance at interfaces, where heterogeneity and steep gradients that cannot be negotiated by microbes can be spanned by larger metazoan hosts. A small cockle, Fragum erugatum, and its photosymbiotic microbes provide a remarkable example of a mutualistic partnership in the hypersaline reaches of Shark Bay, Western Australia. Lucinid bivalves and their endosymbiotic chemolithotrophic bacteria provide examples in which hosts span oxic/anoxic interfaces on behalf of their symbionts at sites of seafloor venting. Multiple lines of evidence underscore the antiquity of mutualisms and suggest that they may have played a significant role in life's first experiments above the prokaryotic grade of complexity. The study of metazoan-microbe mutualisms and their signatures in extreme environments in the geologic record will provide a significant augmentation to microbial models in paleobiology and astrobiology. There are strong potential links between mutualisms and the early history of life on Earth, the persistence of life in extreme environments at times of global crisis and mass extinction, and the possibilities for life elsewhere in the universe.     

Why Raman spectroscopy on Mars?--a case of the right tool for the right job

We provide a scientific rationale for the astrobiological investigation of Mars. We suggest that, given practical constraints, the most promising locations for the search for former life on Mars are palaeolake craters and the evaporite deposits that may reside within them. We suggest that Raman spectroscopy offers a promising tool for the detection of evidence of former (or extant) biota on Mars. In particular, we highlight the detection of hopanoids as long-lived bacterial cell wall products and photosynthetic pigments as the most promising targets. We further suggest that Raman spectroscopy as a fibre optic-based instrument lends itself to flexible planetary deployment.     

Fluorescence microscopy as a tool for in situ life detection

The identification of extant and, in some cases, extinct bacterial life is most convincingly and efficiently performed with modern high-resolution microscopy. Epifluorescence microscopy of microbial autofluorescence or in conjunction with fluorescent dyes is among the most useful of these techniques. We explored fluorescent labeling and imaging of bacteria in rock and soil in the context of in situ life detection for planetary exploration. The goals were two-fold: to target non-Earth-centric biosignatures with the greatest possible sensitivity and to develop labeling procedures amenable to robotic implementation with technologies that are currently space qualified. A wide panel of commercially available dyes that target specific biosignature molecules was screened, and those with desirable properties (i.e., minimal binding to minerals, strong autofluorescence contrast, no need for wash steps) were identified. We also explored the potential of semiconductor quantum dots (QDs) as bacterial and space probes. A specific instrument for space implementation is suggested and discussed.     

The simulated silicification of bacteria--new clues to the modes and timing of bacterial preservation and implications for the search for extraterrestrial microfossils

Evidence of microbial life on Earth has been found in siliceous rock formations throughout the geological and fossil record. To understand the mechanisms of silicification and thus improve our search patterns for evidence of fossil microbial life in rocks, a series of controlled laboratory experiments were designed to simulate the silicification of microorganisms. The bacterial strains Pseudomonas fluorescens and Desulphovibrio indonensis were exposed to silicifying media. The experiments were designed to determine how exposure time to silicifying solutions and to silicifying solutions of different Si concentration affect the fossilization of microbial biofilms. The silicified biofilms were analyzed using transmission electron microscopy (TEM) in combination with energy-dispersive spectroscopy. Both bacterial species showed evidence of silicification after 24 h in 1,000 ppm silica solution, although D. indonensis was less prone to silicification. The degree of silicification of individual cells of the same sample varied, though such variations decreased with increasing exposure time. High Si concentration resulted in better preservation of cellular detail; the Si concentration was more important than the duration in Si solution. Even though no evidence of amorphous silica precipitation was observed, bacterial cells became permineralized. High-resolution TEM analysis revealed nanometer-sized crystallites characterized by lattice fringe-spacings that match the (10-11) d-spacing of quartz formed within bacterial cell walls after 1 week in 5,000 ppm silica solution. The mechanisms of silicification under controlled laboratory conditions and the implication for silicification in natural environments are discussed, along with the relevance of our findings in the search for early life on Earth and extraterrestrial life.     

In situ microbial detection in Mojave Desert soil using native fluorescence

We report on the use of a portable instrument for microbial detection in the Mojave Desert soil and the potential for its use on Mars. The instrument is based on native fluorescence and employs four excitation wavelengths combined with four emission wavelengths. A soil dilution series in which known numbers of Bacillus subtilis spores were added to soil was used to determine the sensitivity of the instrument. We found that the fluorescence of the biological and organic components of the desert soil samples studied can be as strong as the fluorescence of the mineral component of these soils. Using the calibration derived from B. subtilis spores, we estimated that microbial content at our primary sampling site was 10(7) bacteria per gram of soil, a level confirmed by phospholipid fatty acid analysis. At a nearby site, but in a slightly different geological setting, we tested the instrument's ability to map out microbial concentrations in situ. Over a ～50 m diameter circle, soil microbial concentrations determined with the B. subtilis calibration indicate that the concentrations of microorganisms detected varies from 10(4) to 10(7) cells per gram of soil. We conclude that fluorescence is a promising method for detecting soil microbes in noncontact applications in extreme environments on Earth and may have applications on future missions to Mars.     

Impact disruption and recovery of the deep subsurface biosphere

Although a large fraction of the world's biomass resides in the subsurface, there has been no study of the effects of catastrophic disturbance on the deep biosphere and the rate of its subsequent recovery. We carried out an investigation of the microbiology of a 1.76 km drill core obtained from the ～35 million-year-old Chesapeake Bay impact structure, USA, with robust contamination control. Microbial enumerations displayed a logarithmic downward decline, but the different gradient, when compared to previously studied sites, and the scatter of the data are consistent with a microbiota influenced by the geological disturbances caused by the impact. Microbial abundance is low in buried crater-fill, ocean-resurge, and avalanche deposits despite the presence of redox couples for growth. Coupled with the low hydraulic conductivity, the data suggest the microbial community has not yet recovered from the impact ～35 million years ago. Microbial enumerations, molecular analysis of microbial enrichment cultures, and geochemical analysis showed recolonization of a deep region of impact-fractured rock that was heated to above the upper temperature limit for life at the time of impact. These results show how, by fracturing subsurface rocks, impacts can extend the depth of the biosphere. This phenomenon would have provided deep refugia for life on the more heavily bombarded early Earth, and it shows that the deeply fractured regions of impact craters are promising targets to study the past and present habitability of Mars.     

Magnetotactic bacteria on Earth and on Mars

Continued interest in the possibility of evidence for life in the ALH84001 Martian meteorite has focused on the magnetite crystals. This review is structured around three related questions: is the magnetite in ALH84001 of biological or non-biological origin, or a mixture of both? does magnetite on Earth provide insight to the plausibility of biogenic magnetite on Mars? could magnetotaxis have developed on Mars? There are credible arguments for both the biological and non-biological origin of the magnetite in ALH84001, and we suggest that more studies of ALH84001, extensive laboratory simulations of non-biological magnetite formation, as well as further studies of magnetotactic bacteria on Earth will be required to further address this question. Magnetite grains produced by bacteria could provide one of the few inorganic traces of past bacterial life on Mars that could be recovered from surface soils and sediments. If there was biogenic magnetite on Mars in sufficient abundance to leave fossil remains in the volcanic rocks of ALH84001, then it is likely that better-preserved magnetite will be found in sedimentary deposits on Mars. Deposits in ancient lakebeds could contain well-preserved chains of magnetite clearly indicating a biogenic origin.     

Serpentinization and its implications for life on the early Earth and Mars

Ophiolites, sections of ocean crust tectonically displaced onto land, offer significant potential to support chemolithoautotrophic life through the provision of energy and reducing power during aqueous alteration of their highly reduced mineralogies. There is substantial chemical disequilibrium between the primary olivine and pyroxene mineralogy of these ophiolites and the fluids circulating through them. This disequilibrium represents a potential source of chemical energy that could sustain life. Moreover, E (h)-pH conditions resulting from rock- water interactions in ultrabasic rocks are conducive to important abiotic processes antecedent to the origin of life. Serpentinization--the reaction of olivine- and pyroxene-rich rocks with water--produces magnetite, hydroxide, and serpentine minerals, and liberates molecular hydrogen, a source of energy and electrons that can be readily utilized by a broad array of chemosynthetic organisms. These systems are viewed as important analogs for potential early ecosystems on both Earth and Mars, where highly reducing mineralogy was likely widespread in an undifferentiated crust. Secondary phases precipitated during serpentinization have the capability to preserve organic or mineral biosignatures. We describe the petrology and mineral chemistry of an ophiolite-hosted cold spring in northern California and propose criteria to aid in the identification of serpentinizing terranes on Mars that have the potential to harbor chemosynthetic life.     

Using a mini-Raman spectrometer to monitor the adaptive strategies of extremophile colonizers in arid deserts: relationships between signal strength, adaptive strategies, solar radiation, and humidity

Abstract The survival strategies of one cyanobacteria colony and three terricolous lichen species from the hot subdesert of Tabernas, Spain, were studied along with topographical attributes of the area to investigate whether the protective strategies adopted by these pioneer soil colonizers are related to the environmental stressors under which they survive. A handheld Raman spectrometer was used for biomolecular characterization, while the microclimatic and topographic parameters were estimated with a Geographic Information System (GIS). We found that the survival strategies adopted by those organisms are based on different combinations of protective biomolecules, each with diverse ecophysiological functions, such as UV-radiation screening, free-energy quenching, antioxidants, and the production of different types and amounts of calcium oxalates. Our results show that the cyanobacteria community and each lichen species preferentially colonized a particular microhabitat with specific moisture and incident solar radiation levels and exhibited different adaptive mechanisms. In recent years, a number of studies have provided consistent results that suggest a link between the strategies adopted by those extremophile organisms and the microclimatic environmental parameters. To date, however, far too little attention has been paid to results from Raman analyses on dry specimens. Therefore, the results of the present study, produced with the use of our miniaturized instrument, will be of interest to future studies in astrobiology, especially due to the likely use of Raman spectroscopy at the surface of Mars. Key Words: Hot desert-Raman spectroscopy-Topography-Terricolous lichens-Cyanobacteria-Planetary exploration. Astrobiology 12, 743-753.