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Brines and evaporites: analogs for Martian life
Advances in Space Research 33 (2004) 1244–1246 www.elsevier.com/locate/asr
Brines and evaporites: analogs for Martian life R.L. Mancinelli *, T.F. Fahlen, R. Landheim, M.R. Klovstad SETI Institute/NASA – Ames Research Center, Mail Stop 239-12, Moffett Field, CA 94035, USA Received 24 March 2003; received in revised form 7 July 2003; accepted 14 August 2003
Abstract Data from recent Mars missions suggest that Mars almost certainly had abundant liquid water on its surface at some time in the past. As a result, Mars has emerged as a key solar system target that could have harbored some form of life in the past, and which could perhaps still possess remnants of life in brine-containing permafrost. As Mars lost its atmosphere it became cold and dry. Any remaining water on the surface may have formed saline brine pockets within the permafrost. These brine pockets may either be an ‘‘oasis’’ for an extant Martian biota, or the last refuge of an extinct Martian biota. Eventually, these brine pockets would have dried to form evaporites. Evaporites are deposits that result from the evaporation of saline water, which on earth represent primarily halite (NaCl), gypsum, (CaSO4 2H2 O), and anhydrite (CaSO4 ). Evaporites that contain bacterial and algal assemblages exist on earth today and are well known in the fossil record. The most likely organism type to survive in a brine or evaporite on earth is a halophile. The objective of this study was to determine the potential of microbes to survive in frozen evaporites. Washed mid-log phase and stationary phase cultures of Haloarcula-G (a species isolated by us during a previous study) and Halobacterium salinarum were either suspended in brine (25% NaCl solution), dried, and then exposed to )20 or )80 °C. For comparison, cultures of Deinococcus radiodurans, Escherichia coli, and Pseudomonas fluorescens were treated similarly, except they were resuspended in 0.5% NaCl solution. Also, to mimic a brine pocket samples of washed mid-log phase cells of each organism were placed in an aqueous solution of 25% NaCl, or in their respective nutrient medium containing 25% NaCl. Periodically, samples of the cells were removed and tested for survival. Data from these experiments suggest that halophiles survive better than non-halophiles under low temperature conditions. These observations would suggest that halophiles might survive in evaporites contained in permafrost. Ó 2004 COSPAR. Published by Elsevier Ltd. All rights reserved. Keywords: Astrobiology; Brines and evaporites; Analogs for Martian life; Halophiles; Osmophiles
1. Introduction A key question regarding life is, did water exist on the surface of Mars to allow life to originate and evolve? Current attempts to answer this question are based on interpretation of photographs from recent missions to Mars. The valley networks in the southern highlands suggest that water flowed across the Martian surface early in the history of the planet. Recently revealed gullies may be sites of present day near surface liquid water (Malin and Edgett, 2000; Christensen, 2003). Photographs of Holden Crater show apparent layers that may be remnants of former shorelines of a standing *
Corresponding author. Tel.: +1-650-604-6165; fax: +1-650-6041088. E-mail address: [email protected] (R.L. Mancinelli).
body of water on Mars (e.g., Parker et al., 2000). The high albedo feature observed near Schiaparelli Crater may be due to evaporites (e.g., Cabrol and Grin, 2001). Data collected during landed missions to Mars, as well as from analyses of the Martian meteorites suggest that all of the necessary chemical constituents for life were present on Mars during its early history (e.g., Banin and Mancinelli, 1995). Further, it has been suggested that the environmental conditions on early Mars would not preclude the origin and evolution of life on that planet (Mancinelli and Banin, 1995). A primary habitat for any life that may have evolved on Mars would have been water, which would certainly have contained various dissolved minerals. Mars is thought to have lost most of its atmosphere as time progressed. As this process continued the water on the planetÕs surface would have frozen and evaporated forming high saline
0273-1177/$30 Ó 2004 COSPAR. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.asr.2003.08.034
R.L. Mancinelli et al. / Advances in Space Research 33 (2004) 1244–1246
brine pockets and evaporite deposits. These brine pockets may be an oasis for an extant biota, or the last refuge for an extinct biota, because brine pockets would have a high osmolarity, the most likely organisms to survive in a brine pocket, or an evaporite are osmophiles or halophiles (Christensen, 2003). Further, data from recent missions suggest that evaporites should exist on Mars (Clark and Van Hart, 1981; Rothschild, 1990), and potential evaporite deposits have been discussed (e.g., Edgett and Parker, 1998; Cabrol and Grin, 2001). Organisms that inhabit brine pockets and evaporites must be able to withstand osmotic pressure. Many microorganisms respond to increases in osmolarity by accumulating substances, termed osmotica, in their cytosol. Osmotica protect organisms from cytoplasmic dehydration and desiccation (Brown, 1976; Yancey et al., 1982). With the exception of the Halobacteriaceae that use Kþ as their osmoticum (Larsen, 1967), glycine– betaine is the most abundant osmoticum in most prokaryotes (Galinski and Tr€ uper, 1982; Le Rudulier and Bouillard, 1983; Galinski, 1995). However, most of the organisms using the organic osmolytes are moderate halophiles and while they may be best suited for surviving the transition from a low saline to a moderately saline (15% salt) environment, they cannot survive once the transition is complete to a highly concentrated brine pocket or an evaporite where halophiles will have the selective advantage. Both Bacterial and Archaeal true halophiles will have the selective advantage, that is, those organisms able to grow in saline environments containing 15–30% salts to saturation (see Ventosa et al., 1998 for a review). In this study we have taken the first step toward understanding if any type of osmophile, or halophile could survive in a Martian brine pocket or evaporite. This study shows that non-halophilic, or osmophilic organisms cannot survive in a concentrated brine pocket. The next step is to broaden the range of organisms tested to include the non-Archaeal halophilic organisms. The data from this study indicate that non-osmophilic microbes, in this study represented by Escherichia coli, Pseudomonas fluorescens and Deinococcus radiodurans, cannot tolerate osmotic stress. Further, the data suggest that halophilic microbes can withstand simultaneous freezing and desiccation. The most likely organisms to survive in a brine pocket, or an evaporite are osmophiles or halophiles. The presence of osmophiles and halophiles has been documented in terrestrial systems, such as in gypsumhalite crusts in intertidal areas of the ocean (Rothschild et al., 1994), and in NaCl crystals (Oren, 2002). Additionally, studies have shown that organisms can metabolize in permafrost down to temperatures of )20 °C (Rivkina et al., 2000). Therefore, since organisms that live in salt exist on earth and organisms have been shown to live and metabolize in permafrost the next
1245
question to address is whether organisms can withstand freezing in salt. The objective of this study is to determine if halophiles can survive freeze-thaw cycles under dry conditions.
2. Materials and methods P. fluorescens (ATCC #17400), and D. radiodurans (ATCC #13939) were grown in nutrient broth supplemented with glucose, and E. coli (ATCC #23226) was grown in nutrient broth only. Halobacterium salinarum NRC-1 (ATCC #700922), and Haloarcula-G, isolated from a salt crystal (Mancinelli, 1998), were grown in nutrient rich medium as described by Oesterhelt and Stoeckenius (1974). E. coli, Haloarcula-G and H. salinarum were incubated at 37 °C. The D. radiodurans and P. fluorescens were incubated at 30 °C. Organisms were grown to mid-log phase and washed five times by centrifugation. Halophiles were washed in 25% NaCl solution, D. radiodurans, E. coli and P. fluorescens were washed in 0.5% NaCl solution. The final cell pellet was diluted with the appropriate wash medium to yield 107 microbes l11 , as determined from growth experiments in which colony forming units per ml were determined as a function of Klett units (Klett Summerson colorimeter). Sample aliquots of 20 ll were placed onto quartz discs and air dried. Sets of dried preparations of each organism were subjected to freezing at )20 or )80 °C for 144 days. During this time period samples were removed from the freezers, thawed by bringing them to 22 °C, and refrozen to mimic a freeze thaw cycle for a total of 10 cycles. The samples were removed and tested for viability 10 times, as described in the following section. The final samples were tested for viability after 144 days. Controls for each organism were treated similarly, except they were stored at room temperature. In addition, a sample of Haloarcula-G had been prepared similarly and stored dry at 22 °C for five years. Dried samples were placed into 200 ll of the appropriate medium. Samples were serially diluted ten-fold to extinction, and each dilution was tested in triplicate for viability using Molecular Probes Live/Dead Bac-Lite stain by microscopic examination, and cellular reproduction (i.e., growth on agar plates prepared from the appropriate medium for each organism). To mimic a brine pocket, mid-log phase cultures of each of the organisms were prepared and washed five times by centrifugation, placed in medium containing 25% NaCl, or saline solutions containing 25% NaCl. E. coli, Haloarcula-G and H. salinarum were incubated at 37 °C. The D. radiodurans and P. fluorescens were incubated at 30 °C. Samples were periodically tested for viability for up to two months as previously described
1246
R.L. Mancinelli et al. / Advances in Space Research 33 (2004) 1244–1246
in cold and dry environments. Collectively, these data suggest that the last vestiges of an extinct Martian biota, or an extant biota could be present in a cold, desiccating environment, perhaps in brine pockets within permafrost. Therefore, it is possible that the last organisms to survive on the Martian surface were osmophiles, and that any near surface extant life on Mars may be osmophiles.
References
Fig. 1. The number of viable organisms ml1 cell suspension recovered after being dried, dried and frozen at )20 °C, or dried and frozen at )80 °C for 3 months compared to the number of organisms in the original cell suspension.
using Molecular Probes Live/Dead Bac-Lite stain by microscopic examination, and cellular reproduction.
3. Results E. coli and P. fluorescens did not survive drying or freezing (Fig. 1). H. salinarum NRC-1 and Haloarcula-G can survive desiccation at 22 °C and 10 freeze thaw cycles at )20 and )80 °C for at least 144 days (Fig. 1). In addition, Haloarcula-G can survive desiccation at room temperature for at least five years (data not shown). D. radiodurans cannot survive desiccation for 144 days at room temperature, nor can it survive desiccation for 144 days at )20 °C (Fig. 1). But, D. radiodurans can survive desiccation and 10 freeze thaw cycles at )80 °C (Fig. 1). When placed in growth medium containing 25% NaCl, or saline solution containing 25% NaCl only the halophiles H. salinarum NRC-1 and Haloarcula-G survive.
4. Discussion and conclusions Although the Viking mission found no evidence of life on the Martian surface (reviewed by Klein, 1979; Mancinelli, 1998) the search for extant and extinct life on Mars continues. There is mounting evidence from missions to Mars that there was liquid water on the surface of the planet early in its history (e.g., Malin and Edgett, 2000). Additionally, there may be liquid water on the surface today resulting from melting snow fields. The data suggest that halophilic microbes can withstand simultaneous freezing and desiccation. It appears then that osmophiles in general are well adapted for survival
Banin, A., Mancinelli, R.L. Life on Mars? I. The chemical environment. Adv. Space Res. 15 (3), 163–170, 1995. Brown, A.D. Microbial water stress. Bacterial. Rev. 40, 803–846, 1976. Cabrol, N.A., Grin, E.A. Evolution of lacustrine environments on Mars and their significance: the case for the Brazos Lakes and East Terra Meridiani Basins as landing sites for Surveyor 2001, presented at LPSC, Houston, TX, USA, March, 2001. Christensen, P.R. Formation of recent martian gullies through melting of extensive water-rich snow deposits. Science 422, 45–47, 2003. Clark, B.C., Van Hart, D.C. The salts of Mars. Icarus 45, 370–378, 1981. Edgett, K.S., Parker, T.J. Bright aeolian dunes on Mars: Viking Orbiter observations, presented at LPSC, Houston, TX, USA, March, 1998. Galinski, E.A. Osmoadaptation in bacteria. Adv. Microb. Physiol. 37, 273–328, 1995. Galinski, E.A., Tr€ uper, H.G. Betaine, a compatible solute in the extremely halophilic phototrophic bacterium Ectothiorhodospira. Eur. J. Biochem. 149, 135–139, 1982. Klein, H.P. The Viking mission and the search for life on Mars. Rev. Geophys. Space Phys. 17, 1655–1662, 1979. Larsen, H. Biochemical aspects of extreme halophilism. Adv. Microb. Physiol. 1, 97–132, 1967. Le Rudulier, D., Bouillard, L. Glycine Betaine, an osmotic effector in Klebsiella pneumonia and other members of the Enterobacteriaceae. Appl. Environ. Microbiol. 46, 152–159, 1983. Malin, M.C., Edgett, K.S. Sedimentary rocks of early Mars. Science 290, 1927–1937, 2000. Mancinelli, R.L. Prospects for the evolution of life on Mars: Viking 20 years later. Adv. Space Res. 22 (3), 471–477, 1998. Mancinelli, R.L., Banin, A. Life on Mars? II. Physical restrictions. Adv. Space Res. 15 (3), 171–178, 1995. Oesterhelt, D., Stoeckenius, W. Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Method. Enzymoli. 31, 667–678, 1974. Oren, A. Halophilic Microorganisms and Their Environments. Kluwer Academic Publishers, Norwell, MA, USA, 2002. Parker, T.J., Clifford, S.M., Banerdt, W.B. Argyre Planitia and the Mars global hydrologic cycle, presented at LPSC, Houston, TX, USA, March, 2000. Rivkina, E.M., Friedmann, E.I., McKay, C.P., et al., Metabolic activity of permafrost bacteria below the freezing point, Appl. Environ. Microbiol. vol. 66, p. 3230, 2000. Rothschild, L.J., Giver, L.J., White, M.R., et al. Metabolic activity of microorganisms in gypsum-halite crusts. J. Phycol. 30, 431–438, 1994. Rothschild, L.J. Earth analogs for Martian life: microbes in evaporites, a new model system for life on Mars. Icarus 88, 246–260, 1990. Ventosa, A., Joaquin, J.N., Oren, A. Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Biol. Rev. 62 (2), 504–544, 1998. Yancey, P.H., Clark, M.E., Hand, S.C., et al. Living with water stress: evolution of osmolyte systems. Science 217 (1214–1216), 803–848, 1982.
Brines and evaporites: analogs for Martian life R.L. Mancinelli *, T.F. Fahlen, R. Landheim, M.R. Klovstad SETI Institute/NASA – Ames Research Center, Mail Stop 239-12, Moffett Field, CA 94035, USA Received 24 March 2003; received in revised form 7 July 2003; accepted 14 August 2003
Abstract Data from recent Mars missions suggest that Mars almost certainly had abundant liquid water on its surface at some time in the past. As a result, Mars has emerged as a key solar system target that could have harbored some form of life in the past, and which could perhaps still possess remnants of life in brine-containing permafrost. As Mars lost its atmosphere it became cold and dry. Any remaining water on the surface may have formed saline brine pockets within the permafrost. These brine pockets may either be an ‘‘oasis’’ for an extant Martian biota, or the last refuge of an extinct Martian biota. Eventually, these brine pockets would have dried to form evaporites. Evaporites are deposits that result from the evaporation of saline water, which on earth represent primarily halite (NaCl), gypsum, (CaSO4 2H2 O), and anhydrite (CaSO4 ). Evaporites that contain bacterial and algal assemblages exist on earth today and are well known in the fossil record. The most likely organism type to survive in a brine or evaporite on earth is a halophile. The objective of this study was to determine the potential of microbes to survive in frozen evaporites. Washed mid-log phase and stationary phase cultures of Haloarcula-G (a species isolated by us during a previous study) and Halobacterium salinarum were either suspended in brine (25% NaCl solution), dried, and then exposed to )20 or )80 °C. For comparison, cultures of Deinococcus radiodurans, Escherichia coli, and Pseudomonas fluorescens were treated similarly, except they were resuspended in 0.5% NaCl solution. Also, to mimic a brine pocket samples of washed mid-log phase cells of each organism were placed in an aqueous solution of 25% NaCl, or in their respective nutrient medium containing 25% NaCl. Periodically, samples of the cells were removed and tested for survival. Data from these experiments suggest that halophiles survive better than non-halophiles under low temperature conditions. These observations would suggest that halophiles might survive in evaporites contained in permafrost. Ó 2004 COSPAR. Published by Elsevier Ltd. All rights reserved. Keywords: Astrobiology; Brines and evaporites; Analogs for Martian life; Halophiles; Osmophiles
1. Introduction A key question regarding life is, did water exist on the surface of Mars to allow life to originate and evolve? Current attempts to answer this question are based on interpretation of photographs from recent missions to Mars. The valley networks in the southern highlands suggest that water flowed across the Martian surface early in the history of the planet. Recently revealed gullies may be sites of present day near surface liquid water (Malin and Edgett, 2000; Christensen, 2003). Photographs of Holden Crater show apparent layers that may be remnants of former shorelines of a standing *
Corresponding author. Tel.: +1-650-604-6165; fax: +1-650-6041088. E-mail address: [email protected] (R.L. Mancinelli).
body of water on Mars (e.g., Parker et al., 2000). The high albedo feature observed near Schiaparelli Crater may be due to evaporites (e.g., Cabrol and Grin, 2001). Data collected during landed missions to Mars, as well as from analyses of the Martian meteorites suggest that all of the necessary chemical constituents for life were present on Mars during its early history (e.g., Banin and Mancinelli, 1995). Further, it has been suggested that the environmental conditions on early Mars would not preclude the origin and evolution of life on that planet (Mancinelli and Banin, 1995). A primary habitat for any life that may have evolved on Mars would have been water, which would certainly have contained various dissolved minerals. Mars is thought to have lost most of its atmosphere as time progressed. As this process continued the water on the planetÕs surface would have frozen and evaporated forming high saline
0273-1177/$30 Ó 2004 COSPAR. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.asr.2003.08.034
R.L. Mancinelli et al. / Advances in Space Research 33 (2004) 1244–1246
brine pockets and evaporite deposits. These brine pockets may be an oasis for an extant biota, or the last refuge for an extinct biota, because brine pockets would have a high osmolarity, the most likely organisms to survive in a brine pocket, or an evaporite are osmophiles or halophiles (Christensen, 2003). Further, data from recent missions suggest that evaporites should exist on Mars (Clark and Van Hart, 1981; Rothschild, 1990), and potential evaporite deposits have been discussed (e.g., Edgett and Parker, 1998; Cabrol and Grin, 2001). Organisms that inhabit brine pockets and evaporites must be able to withstand osmotic pressure. Many microorganisms respond to increases in osmolarity by accumulating substances, termed osmotica, in their cytosol. Osmotica protect organisms from cytoplasmic dehydration and desiccation (Brown, 1976; Yancey et al., 1982). With the exception of the Halobacteriaceae that use Kþ as their osmoticum (Larsen, 1967), glycine– betaine is the most abundant osmoticum in most prokaryotes (Galinski and Tr€ uper, 1982; Le Rudulier and Bouillard, 1983; Galinski, 1995). However, most of the organisms using the organic osmolytes are moderate halophiles and while they may be best suited for surviving the transition from a low saline to a moderately saline (15% salt) environment, they cannot survive once the transition is complete to a highly concentrated brine pocket or an evaporite where halophiles will have the selective advantage. Both Bacterial and Archaeal true halophiles will have the selective advantage, that is, those organisms able to grow in saline environments containing 15–30% salts to saturation (see Ventosa et al., 1998 for a review). In this study we have taken the first step toward understanding if any type of osmophile, or halophile could survive in a Martian brine pocket or evaporite. This study shows that non-halophilic, or osmophilic organisms cannot survive in a concentrated brine pocket. The next step is to broaden the range of organisms tested to include the non-Archaeal halophilic organisms. The data from this study indicate that non-osmophilic microbes, in this study represented by Escherichia coli, Pseudomonas fluorescens and Deinococcus radiodurans, cannot tolerate osmotic stress. Further, the data suggest that halophilic microbes can withstand simultaneous freezing and desiccation. The most likely organisms to survive in a brine pocket, or an evaporite are osmophiles or halophiles. The presence of osmophiles and halophiles has been documented in terrestrial systems, such as in gypsumhalite crusts in intertidal areas of the ocean (Rothschild et al., 1994), and in NaCl crystals (Oren, 2002). Additionally, studies have shown that organisms can metabolize in permafrost down to temperatures of )20 °C (Rivkina et al., 2000). Therefore, since organisms that live in salt exist on earth and organisms have been shown to live and metabolize in permafrost the next
1245
question to address is whether organisms can withstand freezing in salt. The objective of this study is to determine if halophiles can survive freeze-thaw cycles under dry conditions.
2. Materials and methods P. fluorescens (ATCC #17400), and D. radiodurans (ATCC #13939) were grown in nutrient broth supplemented with glucose, and E. coli (ATCC #23226) was grown in nutrient broth only. Halobacterium salinarum NRC-1 (ATCC #700922), and Haloarcula-G, isolated from a salt crystal (Mancinelli, 1998), were grown in nutrient rich medium as described by Oesterhelt and Stoeckenius (1974). E. coli, Haloarcula-G and H. salinarum were incubated at 37 °C. The D. radiodurans and P. fluorescens were incubated at 30 °C. Organisms were grown to mid-log phase and washed five times by centrifugation. Halophiles were washed in 25% NaCl solution, D. radiodurans, E. coli and P. fluorescens were washed in 0.5% NaCl solution. The final cell pellet was diluted with the appropriate wash medium to yield 107 microbes l11 , as determined from growth experiments in which colony forming units per ml were determined as a function of Klett units (Klett Summerson colorimeter). Sample aliquots of 20 ll were placed onto quartz discs and air dried. Sets of dried preparations of each organism were subjected to freezing at )20 or )80 °C for 144 days. During this time period samples were removed from the freezers, thawed by bringing them to 22 °C, and refrozen to mimic a freeze thaw cycle for a total of 10 cycles. The samples were removed and tested for viability 10 times, as described in the following section. The final samples were tested for viability after 144 days. Controls for each organism were treated similarly, except they were stored at room temperature. In addition, a sample of Haloarcula-G had been prepared similarly and stored dry at 22 °C for five years. Dried samples were placed into 200 ll of the appropriate medium. Samples were serially diluted ten-fold to extinction, and each dilution was tested in triplicate for viability using Molecular Probes Live/Dead Bac-Lite stain by microscopic examination, and cellular reproduction (i.e., growth on agar plates prepared from the appropriate medium for each organism). To mimic a brine pocket, mid-log phase cultures of each of the organisms were prepared and washed five times by centrifugation, placed in medium containing 25% NaCl, or saline solutions containing 25% NaCl. E. coli, Haloarcula-G and H. salinarum were incubated at 37 °C. The D. radiodurans and P. fluorescens were incubated at 30 °C. Samples were periodically tested for viability for up to two months as previously described
1246
R.L. Mancinelli et al. / Advances in Space Research 33 (2004) 1244–1246
in cold and dry environments. Collectively, these data suggest that the last vestiges of an extinct Martian biota, or an extant biota could be present in a cold, desiccating environment, perhaps in brine pockets within permafrost. Therefore, it is possible that the last organisms to survive on the Martian surface were osmophiles, and that any near surface extant life on Mars may be osmophiles.
References
Fig. 1. The number of viable organisms ml1 cell suspension recovered after being dried, dried and frozen at )20 °C, or dried and frozen at )80 °C for 3 months compared to the number of organisms in the original cell suspension.
using Molecular Probes Live/Dead Bac-Lite stain by microscopic examination, and cellular reproduction.
3. Results E. coli and P. fluorescens did not survive drying or freezing (Fig. 1). H. salinarum NRC-1 and Haloarcula-G can survive desiccation at 22 °C and 10 freeze thaw cycles at )20 and )80 °C for at least 144 days (Fig. 1). In addition, Haloarcula-G can survive desiccation at room temperature for at least five years (data not shown). D. radiodurans cannot survive desiccation for 144 days at room temperature, nor can it survive desiccation for 144 days at )20 °C (Fig. 1). But, D. radiodurans can survive desiccation and 10 freeze thaw cycles at )80 °C (Fig. 1). When placed in growth medium containing 25% NaCl, or saline solution containing 25% NaCl only the halophiles H. salinarum NRC-1 and Haloarcula-G survive.
4. Discussion and conclusions Although the Viking mission found no evidence of life on the Martian surface (reviewed by Klein, 1979; Mancinelli, 1998) the search for extant and extinct life on Mars continues. There is mounting evidence from missions to Mars that there was liquid water on the surface of the planet early in its history (e.g., Malin and Edgett, 2000). Additionally, there may be liquid water on the surface today resulting from melting snow fields. The data suggest that halophilic microbes can withstand simultaneous freezing and desiccation. It appears then that osmophiles in general are well adapted for survival
Banin, A., Mancinelli, R.L. Life on Mars? I. The chemical environment. Adv. Space Res. 15 (3), 163–170, 1995. Brown, A.D. Microbial water stress. Bacterial. Rev. 40, 803–846, 1976. Cabrol, N.A., Grin, E.A. Evolution of lacustrine environments on Mars and their significance: the case for the Brazos Lakes and East Terra Meridiani Basins as landing sites for Surveyor 2001, presented at LPSC, Houston, TX, USA, March, 2001. Christensen, P.R. Formation of recent martian gullies through melting of extensive water-rich snow deposits. Science 422, 45–47, 2003. Clark, B.C., Van Hart, D.C. The salts of Mars. Icarus 45, 370–378, 1981. Edgett, K.S., Parker, T.J. Bright aeolian dunes on Mars: Viking Orbiter observations, presented at LPSC, Houston, TX, USA, March, 1998. Galinski, E.A. Osmoadaptation in bacteria. Adv. Microb. Physiol. 37, 273–328, 1995. Galinski, E.A., Tr€ uper, H.G. Betaine, a compatible solute in the extremely halophilic phototrophic bacterium Ectothiorhodospira. Eur. J. Biochem. 149, 135–139, 1982. Klein, H.P. The Viking mission and the search for life on Mars. Rev. Geophys. Space Phys. 17, 1655–1662, 1979. Larsen, H. Biochemical aspects of extreme halophilism. Adv. Microb. Physiol. 1, 97–132, 1967. Le Rudulier, D., Bouillard, L. Glycine Betaine, an osmotic effector in Klebsiella pneumonia and other members of the Enterobacteriaceae. Appl. Environ. Microbiol. 46, 152–159, 1983. Malin, M.C., Edgett, K.S. Sedimentary rocks of early Mars. Science 290, 1927–1937, 2000. Mancinelli, R.L. Prospects for the evolution of life on Mars: Viking 20 years later. Adv. Space Res. 22 (3), 471–477, 1998. Mancinelli, R.L., Banin, A. Life on Mars? II. Physical restrictions. Adv. Space Res. 15 (3), 171–178, 1995. Oesterhelt, D., Stoeckenius, W. Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Method. Enzymoli. 31, 667–678, 1974. Oren, A. Halophilic Microorganisms and Their Environments. Kluwer Academic Publishers, Norwell, MA, USA, 2002. Parker, T.J., Clifford, S.M., Banerdt, W.B. Argyre Planitia and the Mars global hydrologic cycle, presented at LPSC, Houston, TX, USA, March, 2000. Rivkina, E.M., Friedmann, E.I., McKay, C.P., et al., Metabolic activity of permafrost bacteria below the freezing point, Appl. Environ. Microbiol. vol. 66, p. 3230, 2000. Rothschild, L.J., Giver, L.J., White, M.R., et al. Metabolic activity of microorganisms in gypsum-halite crusts. J. Phycol. 30, 431–438, 1994. Rothschild, L.J. Earth analogs for Martian life: microbes in evaporites, a new model system for life on Mars. Icarus 88, 246–260, 1990. Ventosa, A., Joaquin, J.N., Oren, A. Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Biol. Rev. 62 (2), 504–544, 1998. Yancey, P.H., Clark, M.E., Hand, S.C., et al. Living with water stress: evolution of osmolyte systems. Science 217 (1214–1216), 803–848, 1982.