Bacterial Growth in Brines Formed by the Deliquescence of Salts Relevant to Cold Arid Worlds (2024)

2.1. Organisms

Salinotolerant bacterial isolates were obtained previously from the Great Salt Plains, Oklahoma, an environment rich in NaCl (Caton et al.,2004; Litzner et al.,2006) or from the epsomic lakes, Hot Lake, Washington, and Basque Lake, British Columbia, environments saturated with MgSO₄ (Kilmer et al.,2014; Crisler et al.,2019; Wilks et al.,2019). The isolates were obtained from enrichment cultures at 10% NaCl or 50% MgSO₄ (both w/v), with the BLE collection from Basque Lake additionally enriched at 4°C to select for psychrotolerant salinotolerant microbes. The primary organism studied here was Halomonas sp. str. HL12, a particularly robust organism with broad tolerances to salts. Parallel studies were performed on Halomonas sp. str. HL51, GSP63, and BLE7, Marinococcus sp. str. HL54, and Planococcus sp. str. HL20.

2.2. Growth media

Cultures were grown on SP medium (Caton et al.,2004), a eutrophic medium containing per liter: NaCl, 1 g; KCl, 2.0 g; MgSO₄·7H₂O, 1.0 g; CaCl₂·2H₂O, 0.36 g; NaHCO₃, 0.06 g; NaBr, 0.23 g; FeCl₃·6H₂O, 1.0 mg; trace minerals, 0.5 mL; tryptone, 5.0 g; yeast extract, 10.0 g; glucose, 1.0 g; final pH ∼7.0. Isolates were maintained on SP medium supplemented with 10% (w/v) NaCl. For dehydration/rehydration experiments, SP medium was prepared with 50% MgSO₄, 10% NaCl, 20% NaCl, or 20% NaClO₃ (all % w/v). Throughout this work, MgSO₄ is used to represent epsomite, MgSO₄·7H₂O.

2.3. Dehydration and rehydration of microbial cultures

Bacterial cultures were grown in SP medium supplemented with 50% (w/v) MgSO₄ until the culture reached mid-log phase. At a culture density of 0.2–0.4 OD units at 600 nm, rehydrated cultures could undergo several doublings before reaching maximum culture density (stationary phase), while sufficient cells were present to observe cultures in decline. Aliquots (50 μL) of culture were placed in a sterile small polypropylene cup (detached cap of a microcentrifuge tube; Fig. 1center) and dehydrated for ≥4 h at room temperature in a vacuum desiccator over fresh Drierite (WA Hammond; CaSO₄). Dehydration and rehydration patterns were determined gravimetrically by reaching constant weights. Cups (1–3) containing dehydrated culture evaporites were attached with adhesive to glass microscope slides (Fig. 1left). The slides were stood upright in a glass container (240 mL) having a continuous thread screw cap (Fig. 1right). The container was filled to a depth of ∼1 cm with the brine medium in which the cultures were grown and which was desiccated to deposit the evaporite minerals in the cups. When the containers were sealed, the growth medium brine reservoir maintained the RH corresponding to its a_w (RH/100 = a_w). The chambers were gently rocked side-to-side (2 rpm) at room temperature, which served to mix the headspace, thereby increasing the speed and consistency of deliquescent rehydration and aerating the subsequent microbial culture (Fig. 1center).

Open in a separate window

FIG. 1.

Images of the experimental apparatus used for the cultivation of microbes under deliquescing conditions. Left: Cup with desiccated culture evaporite. Center: View from above of an open container showing a drop of deliquescent microbial culture in a cup held above the reservoir of growth medium brine that controls the RH of the headspace. Right: Schematic drawing of the containers used for deliquescent rehydration experiments.

Manual rehydration was performed on some dried samples before being sealed in chambers with brine reservoirs, through the addition of sufficient sterile water to reach a final volume (50 μL) and salinity equal to that of the original culture aliquot before drying. Control experiments were performed to empirically determine the volume of water needed for rehydration, and the salinity and a_w of the resulting brine were measured. For deliquescent rehydration, dried sample cups were incubated in sealed chambers and thus wetted by exposure to the humidity produced in the headspace by the corresponding brine reservoir. Typically, desiccation takes place in ∼4–6 h, while deliquescent rehydration requires 12–18 h, so that each full cycle was approximately 24 h. The cell density of rehydrated cultures was determined by taking aliquots (5 μL) for serial dilution and standard plate counts on SP medium supplemented with 10% NaCl. Momentarily opening the chambers to take samples for growth curves only briefly disturbed headspace conditions.

2.4. Physicochemical measurements

A salinity refractometer (with automatic temperature compensation; Fisher) was used to measure the concentration of brines as percent salinity. Since the instrument is calibrated for NaCl solutions, standard curves were made to convert salinometer readings in the linear range to concentrations of MgSO₄ and NaClO₃. The water activity of each medium and brine was measured with an AqualLab Series 3 water activity meter (Decagon Devices, Pullman, WA). The instrument was calibrated with standard NaCl solutions and pure water and operated at room temperature. The RH in sealed chambers was measured by fitting the lid with the humidity/temperature probe of a 3-point NIST traceable HUMICAP HMT130 Humidity and Temperature Transmitter (Vaisala, Vantaa, Finland; accuracies at 25°C of ±0.2°C and ±1.5% RH at 90% RH). In every case, RH in the sealed chambers remained steady throughout incubation periods spanning weeks, at the level expected based on the a_w of the brine used for the reservoir.

Time-lapse videos (60 fps, 1080p) and still images were taken of brine droplets during rehydration and subsequent deliquescent rehydration with an iPhone 6 (Apple), SkyFlow software (KageDev), and a 20 × Macro lens (Anker, Guangdong, China). Observations of cells within crystal inclusions and on crystal surfaces were performed at 400 to 1000 × , after mounting ∼1 mm crystals under a cover slip in NVH high-viscosity immersion oil (Cargille Labs, Cedar Grove, NJ), using a Nikon ECLIPSE E800 microscope, fitted with CFI Apochromat objectives and a DS-Fi1 high-definition color camera head.

3.1. Deliquescent brines in the laboratory

The rate and extent of dehydration and rehydration of microbial cultures can affect cellular responses. We chose to dry small volumes of culture (50 μL) in a vacuum desiccator over Drierite. This avoided having to freeze cultures for lyophilization, potentially damaging cells. Further, desiccation was faster than drying in a Speed-Vac under conditions where cultures did not freeze. The evaporite minerals formed can be exsiccated more fully than done here, perhaps to kieserite, and this may affect cellular responses. Time-lapse video of the dehydration of salt droplets showed the expected formation of floating hopper crystals at the surface (Fig. 2) and subsequent settling and growth of crystals as the brine dried. Primary cubic halite crystals can form in the center of a ring of primary and secondary evaporites when dried (Fig. 3right). Desiccation of MgSO₄ (as epsomite) brine at room temperature under the same regime typically did not form the characteristic discrete spindly monoclinic crystals (Fig. 3left) observed after crystallization of supersaturated MgSO₄ solutions at lower temperatures (4°C). It is interesting to note that it was common for drops of MgSO₄ or NaClO₃ brine to instead form hemispheroids (Fig. 3center) before further crystallizing during desiccation, comparable to structures observed previously for perchlorate salts (Fischer et al.,2016). Aqueous solution was retained in the slushy center of the salt hemispheroids for days to weeks when exposed to room humidity (outside of a desiccator). Hygroscopic salts in the environment that retain moisture in hemispheroids would temporally extend conditions permissive for microbial survival and growth. During dehydration in the laboratory, cells within salt evaporite minerals could be entrapped within fluid inclusions in the salt crystals, in fissures and crevices in the crystals, more broadly between crystals in voids, and on crystals within the deposits.

Open in a separate window

FIG. 2.

Micrograph (at 20 × ) of hopper crystals formed at the surface of a saturated NaCl brine droplet (50 μL) as it dehydrates and begins crystallization at room temperature on a polypropylene surface.

Open in a separate window

FIG. 3.

Laboratory-grown evaporites of MgSO₄ and NaCl formed by dehydrating brine droplets (50 μL) on a polypropylene surface. Left: Monoclinic crystals of MgSO₄ formed by rapid cooling to 4°C of a warm supersaturated brine and air-drying. Center: Hemispheroid dome formed by air-drying saturated MgSO₄ brine at room temperature. Right: Primary cubic crystals and secondary crystal ring of NaCl formed during air-drying of saturated brine.

When brine evaporites were exposed to sufficient humidity, the salts rehydrated, at first forming a slurry and then a supersaturated solution. Over a period of several days, deliquescent brines approached the initial salt concentration of the medium. The humidity in the chambers during rehydration, and hence the equilibrium concentration of the deliquescent brines formed, was controlled by using reservoirs of the medium in which the cells were grown and which was dehydrated to deposit evaporite minerals. Supersaturation was more prevalent and longer lasting with MgSO₄ than with NaCl, while NaClO₃ was the most hygroscopic and deliquesced most rapidly. During rehydration at room temperature, evaporites typically deliquesced fully to liquid within 12–24 h. When held at their eutectic temperature of -4°C, MgSO₄ evaporites did not deliquesce to liquid over a period of 14 days, while NaClO₃ at -4°C deliquesced to liquid within a few days. For the current study, aliquots for growth measurements were taken once the dried salt was observed to have deliquesced entirely to liquid, even if still (super)saturated and not yet at its equilibrium salinity.

3.2. Microbial growth in deliquescent MgSO₄ brine

Halomonas sp. str. HL12 was grown to mid-log phase in SP medium supplemented with 50% MgSO₄ (a_w = 0.94), and aliquots of culture were dried in a vacuum desiccator. In one experiment, desiccated cultures were rehydrated by the manual addition of water to reconstitute the salinity (to 50% MgSO₄) of the initial medium, and the density of the resulting cultures was followed over time by using standard plate counts (Fig. 4). The cultures were mid-log phase when dehydrated, and once rehydrated, exponential growth resumed. Maximum culture density (stationary phase) was reached in about 24 h. Note that during the single cycle of dehydration and rehydration at the start of this experiment viable cell numbers were modestly reduced by less than half.

In a second experiment, desiccated cultures were rehydrated by using humidity alone to create deliquescent brines overnight (Fig. 4). Once the deliquescent liquid was formed, cells proliferated and grew rapidly, reaching maximum density in about 24 h. Cells grown in deliquescent liquids were morphologically similar at 1000 × to cells grown in dense brines. The loss of viability due to the initial dehydration-rehydration cycle could not be measured directly during deliquescence, since growth can begin in the salt slurry before the salts are fully rehydrated to a liquid that could be accurately sampled volumetrically. Growth responses similar to those of Halomonas sp. str. HL12 were obtained by using Bacillus sp. str. GSP63 and Marinococcus sp. str. HL54 (data not shown).

Not all salinotolerant microbes survived and proliferated as well as Halomonas sp. str. HL12 under deliquescent conditions. A representative and contrasting pattern is presented for Halomonas sp. str. BLE7 (Fig. 5). A similar pattern was observed for Halomonas sp. str. HL51 (data not shown). Desiccated cultures of Halomonas sp. str. BLE7 rehydrated through deliquescence did not proliferate robustly as Halomonas sp. str. HL12 had (Fig. 5). Instead, culture density remained relatively constant for days before declining. Thus, for Halomonas sp. str. HL51 and BLE7, survival was clear, but growth was not observed in the deliquescent liquid formed from MgSO₄ evaporites. Culture density fell relatively consistently after a few days.

3.3. Microbial growth in deliquescent brines of NaCl and NaClO₃

Deliquescence experiments were performed with NaCl and NaClO₃ using Halomonas sp. str. HL12. When cultures grown in SP medium supplemented with 10% NaCl (a_w = 0.92) were dried by vacuum desiccation and rehydrated by deliquescence, cells revived and began to grow, until near-maximum density (near-stationary phase) was reached in a few days (Fig. 6). When the salt concentration was increased to 20% NaCl, the deliquescent cultures did not resume growth, and cell numbers remained essentially unchanged (data not shown). Note that 20% NaCl has the lowest water activity (a_w = 0.85) of the brines tested in the current study. Cultures grown in SP medium supplemented with 20% NaClO₃ (a_w = 0.91), then desiccated and subsequently rehydrated by deliquescence, resumed rapid growth, reaching high density in a few days (Fig. 6). This is a particularly important observation, since (per)chlorate salts have the greatest potential for deliquescence on Mars. Sodium chlorate has the lowest eutectic temperature (-23°C) of the salts demonstrated here to support bacterial growth after deliquescent rehydration. Neither growth nor survival were observed in deliquescent liquids formed by perchlorate salt evaporites, which have even lower eutectic temperatures than NaClO₃.

Open in a separate window

FIG. 6.

Growth of Halomonas sp. str. HL12 after desiccation of cultures in SP medium supplemented with 50% (w/v) MgSO₄, 10% NaCl, or 20% NaClO₃, followed by rehydration through deliquescence. Means of triplicates ± SD. Note that deliquescent rehydration needed to proceed for 24 h before samples could be taken for plate counts.

3.4. Multiple cycles of dehydration and deliquescent rehydration

We previously demonstrated that our salinotolerant bacteria could survive several cycles of drying and rewetting, with a modest concomitant loss of viability with each cycle (Crisler et al.,2012). The current study demonstrated that Halomonas sp. str. HL12 cells survived and proliferated after several cycles of dehydration and rehydration, whether rehydration was through manual addition of water or deliquescence (Fig. 7). Cell survival was high after nine cycles of dehydration and manual rehydration. We have demonstrated here the survival and proliferation of bacteria through six cycles of desiccation and subsequent rehydration by deliquescence, with persistence through several additional cycles likely to occur if sufficient culture volume had remained in the cups for further sampling (Fig. 7). It must be noted that once rehydrated by deliquescence, cells will proliferate during the next dehydration phase. In certain experiments, growth was more pronounced and was particularly noticeable in those cultures rehydrated by deliquescence, since there is not a precise moment when the cultures were deemed rehydrated. Hence, there was an indeterminate period when growth could occur in the nascent deliquescent liquid (salt slurry) before the next cycle of desiccation.

Open in a separate window

FIG. 7.

Growth of Halomonas sp. str. HL12 cultures in SP medium supplemented with 50% (w/v) MgSO₄ through several cycles of desiccation and rehydration through manual addition of water or deliquescence by humidity. Means of triplicates ± SD. Note that deliquescent rehydration needed to proceed for 24 h before samples could be taken for plate counts.

• Adamiak J, Otlewska A, and Gutarowska B (2015) Halophilic microbial communities in deteriorated buildings. World J Microbiol Biotechnol31:1489–1499. [PubMed] [Google Scholar]
• Adamski JC, Roberts JA, and Goldstein RH (2006) Entrapment of bacteria in fluid inclusions in laboratory-grown halite. Astrobiology6:552–562. [PubMed] [Google Scholar]
• Al Soudi AF, Farhat O, Chen F, et al. (2017) Bacterial growth tolerance to concentrations of chlorate and perchlorate salts relevant to Mars. Int J Astrobiol16:229–235. [Google Scholar]
• Altheide T, Chevrier V, Nicholson C, et al. (2009) Experimental investigations of the stability and evaporation of sulfate and chloride brines on Mars. Earth Planet Sci Lett282:69–78. [Google Scholar]
• Amils R (2016) Lessons learned from thirty years of geomicrobiological studies of Río Tinto. Res Microbiol167:539–545. [PubMed] [Google Scholar]
• Bakermans C (2012) Psychrophiles: life in the cold. In Extremophiles: Microbiology and Biotechnology, edited by R Anitori. Horizon Scientific Press, Hethersett, UK, pp 53–76. [Google Scholar]
• Bruzewicz DA, Checco A, Ocko BM, et al. (2011) Reversible uptake of water on NaCl nanoparticles at relative humidity below deliquescence point observed by noncontact environmental atomic force microscopy. J Chem Phys134, doi: 10.1063/1.3524195. [PubMed] [CrossRef] [Google Scholar]
• Burch AY, Finkel OM, Cho JK, et al. (2013) Diverse microhabitats experienced by Halomonas variabilis on salt-secreting leaves. Appl Environ Microbiol79:845–852. [PMC free article] [PubMed] [Google Scholar]
• Carrier BL, Beaty DW, Meyer MA, et al. (2020) Mars extant life: what's next? Conference report. Astrobiology20:785–814. [PMC free article] [PubMed] [Google Scholar]
• Carter J, Poulet F, Bibring J-P, et al. (2013) Hydrous minerals on Mars as seen by the CRISM and OMEGA imaging spectrometers: updated global view. J Geophys Res Planets118:831–858. [Google Scholar]
• Caton TM, Witte LR, Ngyuen HD, et al. (2004) Halotolerant aerobic heterotrophic bacteria from the Great Salt Plains of Oklahoma. Microb Ecol48:449–462. [PubMed] [Google Scholar]
• Cesur RM, Ansari IM, Chen F, et al. (2019) Demonstration of bacterial growth in brines formed by the deliquescence of salts relevant to Mars. In Abstracts and Program, 119th Annual Meeting of the American Society for Microbiology. American Society for Microbiology, Washington,DC. [Google Scholar]
• Cesur RM, Ansari IM, Stewart CR, et al. (2021) Bacterial survival and growth in fluid inclusions and deliquescent brines of salt evaporites relevant to cold arid worlds [abstract 1243]. In 52nd Lunar and Planetary Science Conference. Lunar and Planetary Institute,Houston. [Google Scholar]
• Chevrier VF and Altheide TS (2008) Low temperature aqueous ferric sulfate solutions in the surface of Mars. Geophys Res Lett35, doi: 10.1029/2008GL035489. [CrossRef] [Google Scholar]
• Clark BC (1993) Geochemical components in martian soil. Geochim Cosmochim Acta57:4575–4581. [Google Scholar]
• Clark BC and Kounaves SP (2016) Evidence for the distribution of perchlorates on Mars. Int J Astrobiol15:311–318. [Google Scholar]
• Clark BC, Morris RV, McLennan SM, et al. (2005) Chemistry and mineralogy of outcrops at Meridiani Planum. Earth Planet Sci Lett240:73–94. [Google Scholar]
• Clarke A, Morris GJ, Fonseca F, et al. (2013) A low temperature limit for life on Earth. PLoS One8, doi: 10.1371/journal.pone.0066207. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Cofrade G, Mendiburu-Eliçabe M, and Romeo I (2019) Circular troughs in tessera terrain on Venus: morphometry, structural analysis and possible formation model. Planet Space Sci178, doi: 10.1016/j.pss.2019.104706. [CrossRef] [Google Scholar]
• Collins MA and Buick RK (1989) Effect of temperature on the spoilage of stored peas by Rhodotorula glutinus. Food Microbiol6:135–141. [Google Scholar]
• Crisler JD, Newville TM, Chen F, et al. (2012) Bacterial growth at the high concentrations of magnesium sulfate found in martian soils. Astrobiology12:98–106. [PMC free article] [PubMed] [Google Scholar]
• Crisler JD, Chen F, Clark BC, et al. (2019) Cultivation and characterization of the bacterial assemblage of epsomic Basque Lake, BC. Antonie Leeuwen112:1105–1119. [PMC free article] [PubMed] [Google Scholar]
• Cuthbertson L and Pearce DA (2017) Aeromicrobiology. In Psychrophiles: From Biodiversity of Biotechnology, 2nd ed., edited by R Margesin. Springer, Berlin, pp 41–56. [Google Scholar]
• Davila AF, Duport LG, Melchiorri R, et al. (2010) Hygroscopic salts and the potential for life on Mars. Astrobiology10:617–628. [PubMed] [Google Scholar]
• Davila AF, Hawes I, Ascaso C, et al. (2013) Salt deliquescence drives photosynthesis in the hyperarid Atacama Desert. Environ Microbiol Rep5:583–587. [PubMed] [Google Scholar]
• Dimmick RL, Chatigny MA, Wolochow H, et al. (1977) Evidence for propagation of aerobic bacteria in particles suspended in gaseous atmospheres. Life Sci Space Res15:41–45. [PubMed] [Google Scholar]
• Eigenbrode JL, Summons RE, Steele A, et al. (2018) Organic matter preserved in 3-billion-year-old mudstones at Gale crater, Mars. Science360:1096–1101. [PubMed] [Google Scholar]
• Elabed H, González-Tortuero E, Ibacache-Quiroga C, et al. (2019) Seawater salt-trapped Pseudomonas aeruginosa survives for years and gets primed for salinity tolerance. BMC Microbiol19, doi: 10.1186/s12866-019-1499-2. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Fendrihan S, Legat A, Pfaffenhuemer M, et al. (2006) Extremely halophilic archaea and the issue of long-term microbial survival. Rev Environ Sci Biotechnol5:203–218. [PMC free article] [PubMed] [Google Scholar]
• Fendrihan S, Musso M, and Stan-Lotter H (2009) Raman spectroscopy as a potential method for the detection of extremely halophilic archaea embedded in halite in terrestrial possible extraterrestrial samples. J Raman Spectrosc40:1996–2003. [PMC free article] [PubMed] [Google Scholar]
• Fendrihan S, Dornmayr-Pfaffenhuemer M, Gerbl FW, et al. (2012) Spherical particles of halophilic archaea correlate with exposure to low water activity—implications for microbial survival in fluid inclusions of ancient halite. Geobiology10:424–433. [PMC free article] [PubMed] [Google Scholar]
• Finkel OM, Burch AY, Lindow SE, et al. (2011) Geographical location determines the population structure in the phyllosphere microbial communities of a salt-excreting desert tree. Appl Environ Microbiol77:7647–7655. [PMC free article] [PubMed] [Google Scholar]
• Fischer E, Martínez GM, Elliot HM, et al. (2014) Experimental evidence for the formation of liquid saline water on Mars. Geophys Res Lett41:4456–4462. [PMC free article] [PubMed] [Google Scholar]
• Fischer E, Martínez GM, and Rennó NO (2016) Formation and persistence of brine on Mars: experimental simulations throughout the diurnal cycle at the Phoenix landing site. Astrobiology16:937–948. [PMC free article] [PubMed] [Google Scholar]
• Fonseca F, Meneghei J, Cenard S, et al. (2016) Determination of intracellular vitrification temperatures for unicellular microorganisms under conditions relevant for cryopreservation. PLoS One11, doi: 10.1371/journal.pone.0152939. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Fox-Powell MG and co*ckell CS (2018) Building a geochemical view of microbial salt tolerance: halophilic adaptation of Marinococcus in a natural magnesium sulfate brine. Front Microbiol9, doi: 10.3389/fmicb.2018.00739. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Gilichinsky D, Rivkina E, Shcherbakova V, et al. (2003) Supercooled water brines within permafrost—an unknown ecological niche for microorganisms: a model for astrobiology. Astrobiology3:331–341. [PubMed] [Google Scholar]
• Gilmore M, Treiman A, Helbert J, et al. (2017) Venus surface composition constrained by observation and experiment. Space Sci Rev212:1511–1540. [Google Scholar]
• Grant WD (2004) Life at low water activity. Philos Trans R Soc Lond B Biol Sci359:1249–1267. [PMC free article] [PubMed] [Google Scholar]
• Hallsworth JE (2019) Microbial unknowns at the saline limits for life. Nat Ecol Evol3:1503–1504. [PubMed] [Google Scholar]
• Hanley J, Chevrier VF, Berget DJ, et al. (2012) Chlorate salts and solutions on Mars. Geophys Res Lett39, doi: 10.1029/2012GL051239. [CrossRef] [Google Scholar]
• Hansen-Goos H and Wettlaufer JS (2010) Theory of ice premelting in porous media. Phys Rev E91, doi: 10.1103/PhysRevE.81.031604. [PubMed] [CrossRef] [Google Scholar]
• Hansen-Goos H, Thomson ES, and Wettlaufer JS (2014) On the edge of habitability and the extremes of liquidity. Planet Space Sci98:169–181. [Google Scholar]
• Harris RF (1981) Effect of water potential on microbial growth and activity. In Water Potential Relations in Soil Microbiology, SSSA Special Publication 9. Soil Science Society of America, Madison, WI, pp 23–95. [Google Scholar]
• Hecht MH, Kounaves SP, Quinn RC, et al. (2009) Detection of perchlorate and the soluble chemistry of martian soil at the Phoenix lander site. Science325:64–67. [PubMed] [Google Scholar]
• Heinz J, Schirmack J, Airo A, et al. (2018) Enhanced microbial survival in subzero brines. Astrobiology18:1171–1180. [PMC free article] [PubMed] [Google Scholar]
• Huby TJC, Clark DR, McKew BA, et al. (2020) Extremely halophilic archaeal communities are resilient to short-term entombment in halite. Environ Microbiol23:3370–3383. [PMC free article] [PubMed] [Google Scholar]
• Jakosky BM, Nealson KH, Bakermans C, et al. (2004) Subfreezing activity of microorganisms and the potential habitability of Mars' polar regions. Astrobiology3:343–350. [PubMed] [Google Scholar]
• Jänchen J, Feyh N, Szewzyk U, et al. (2016) Provision of water by halite deliquescence for Nostoc commune biofilms under Mars relevant surface conditions. Int J Astrobiol15:107–118. [Google Scholar]
• Johnston CG and Vestal JR (1991) Photosynthetic carbon incorporation and turnover in Antarctic cryptoendolithic microbial communities: are they the slowest-growing communities on Earth? Appl Environ Microbiol 57:2308–2311. [PMC free article] [PubMed] [Google Scholar]
• Junge K, Eicken H, and Deming JW (2004) Bacterial activity at -2 to -20 °C in arctic wintertime sea ice. Appl Environ Microbiol70:550–557. [PMC free article] [PubMed] [Google Scholar]
• Junge K, Eicken H, Swanson BD, et al. (2006) Bacterial incorporation of leucine into protein down to -20 °C with evidence for potential activity in sub-eutectic saline ice formations. Cryobiology52:417–429. [PubMed] [Google Scholar]
• Kilmer BR, Eberl TC, Chen F, et al. (2014) Molecular and phenetic characterization of the bacterial assemblage of Hot Lake, WA, an environment with high concentrations of magnesium sulphate, and its relevance to Mars. Int J Astrobiol13:69–80. [PMC free article] [PubMed] [Google Scholar]
• Lane LB (1925) Freezing points of glycerol and its aqueous solutions. Ind Eng Chem17, doi: 10.1021/ie50189a017. [CrossRef] [Google Scholar]
• Lange OL (1969) Experimentell-ökologische Untersuchungen an Flechten der Negev-Wüste. I. CO₂-Gaswechsel von Ramalina maciformis (Del.) Bory unter kontrollierten Bedingungen im Laboratorium. Flora (Jena) Abt B158:324–359. [Google Scholar]
• Limaye SS, Mogul R, Smith DJ, et al. (2018) Venus' spectral signatures and the potential for life in the clouds. Astrobiology18:1181–1198. [PMC free article] [PubMed] [Google Scholar]
• Litzner BR, Caton TM, and Schneegurt MA (2006) Carbon substrate utilization, antibiotic sensitivity, and numerical taxonomy of bacterial isolates from the Great Salt Plains of Oklahoma. Arch Microbiol185:286–296. [PubMed] [Google Scholar]
• Lowenstein TK, Schubert BA, and Timofeeff MN (2011) Microbial communities in fluid inclusions and long-term survival in halite. GSA Today21:4–9. [Google Scholar]
• Mader HM, Pettitt ME, Wadham JL, et al. (2006) Subsurface ice as a microbial habitat. Geology34:169–172. [Google Scholar]
• Mancinelli RL, Fahlen TF, Landheim R, et al. (2004) Brines and evaporites: analogs for martian life. Adv Space Res33:1244–1246. [Google Scholar]
• Marnocha CL, Chevrier VF, and Ivey DM (2011) Growth of sulfate-reducing bacteria in sulfate brines and the astrobiological implications for Mars [abstract 1604]. In 42nd Lunar and Planetary Science Conference. Lunar and Planetary Institute, Houston. [Google Scholar]
• Maus D, Heinz J, Schirmack J, et al. (2020) Methanogenic archaea can produce methane in deliquescence-driven Mars analog environments. Sci Rep10, doi: 10.1038/s41598-019-56267-4. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Mayol E, Arrieta JM, Jiménez MA, et al. (2017) Long-range transport of airborne microbes over the global tropical and subtropical ocean. Nat Comm8, doi: 10.1038/s41467-017-00110-9. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Mormile MR, Blesen MA, Gutierrez MC, et al. (2003) Isolation of Halobacterium salinarum retrieved directly from halite brine inclusions. Environ Microbiol5:1094–1102. [PubMed] [Google Scholar]
• Nair CPR and Unnikrishnan V (2020) Stability of the liquid water phase on Mars: a thermodynamic analysis considering martian atmospheric conditions and perchlorate brine solutions. ACS Omega5:9391–9397. [PMC free article] [PubMed] [Google Scholar]
• Nash TH III, Reiner A, Demmig-Adams B, et al. (1990) The effect of atmospheric desiccation and osmotic water stress on photosynthesis and dark respiration of lichens. New Phytol116:269–276. [Google Scholar]
• Norton CF and Grant WD (1988) Survival of halobacteria within fluid inclusions in salt crystals. J Gen Microbiol134:1365–1373. [Google Scholar]
• Nuding DL, Rivera-Valentín EG, Davis RD, et al. (2014) Deliquescence and efflorescence of calcium perchlorate: an investigation of stable aqueous solutions relevant to Mars. Icarus243:420–428. [Google Scholar]
• Ohno H, Iizuka Y, Horikawa S, et al. (2014) Potassium alum and aluminum sulfate micro-inclusions in polar ice from Dome Fuji, East Antarctica. Polar Sci8:1–9. [Google Scholar]
• Orevi T and Kashtan N (2021) Life in a droplet: microbial ecology in microscopic surface wetness. Front Microbiol12, doi: 10.3389/fmicb.2021.655459. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Orosei R, Lauro SE, Pettinelli E, et al. (2018) Radar evidence of subglacial liquid water on Mars. Science361:490–493. [PubMed] [Google Scholar]
• Pál B andKereszturi Á (2020) Annual and daily ideal periods for deliquescence at the landing site of InSight based on GCM model calculations. Icarus340, doi: 10.1016/j.icarus.2020.113639. [CrossRef] [Google Scholar]
• Palmer RJ and Friedmann EI (1990) Water relations and photosynthesis in the cryptoendolithic microbial habitat of hot and cold deserts. Microb Ecol19:111–118. [PubMed] [Google Scholar]
• Pointing SB and Belnap J (2012) Microbial colonization and controls in dryland systems. Nat Rev Microbiol10:551–562. [PubMed] [Google Scholar]
• Price PB and Sowers T (2004) Temperature dependence of metabolic rates for microbial growth, maintenance, and survival. Proc Nat Acad Sci USA101:4631–4636. [PMC free article] [PubMed] [Google Scholar]
• Primm KM, Gough RV, Chevrier VF, et al. (2017) Freezing of perchlorate and chloride brines under Mars-relevant conditions. Geochim Cosmochim Acta212:211–220. [Google Scholar]
• Qvit-Raz N, Jurkevitch E, and Belkin S (2008) Drop-size soda lakes: transient microbial habitats on a salt-secreting desert tree. Genetics178:1615–1622. [PMC free article] [PubMed] [Google Scholar]
• Rapin W, Ehlmann BL, Dromat G, et al. (2019) An interval of high salinity in ancient Gale crater lake on Mars. Nat Geosci12:889–895. [Google Scholar]
• Rebsdat S and Mayer D (2012) Ethylene glycol. In Ullmann's Encyclopedia of Industrial Chemistry, 5th ed, edited by W Gerhartz et al. Wiley-VCH, Weinheim, Germany, pp 531–546. [Google Scholar]
• Rivera-Valentín EG, Gough RV, Chevrier VF, et al. (2018) Constraining the potential liquid water environment at Gale crater, Mars throughout MSL's traverse [abstract 2752]. In 49th Lunar and Planetary Science Conference. Lunar and Planetary Institute,Houston. [PMC free article] [PubMed] [Google Scholar]
• Rivera-Valentín EG, Chevrier VF, Soto A, et al. (2020) Distribution and habitability of (meta)stable brines on present-day Mars. Nat Astron4:756–761. [PMC free article] [PubMed] [Google Scholar]
• Rivkina EM, Friedmann EI, McKay CP, et al. (2000) Metabolic activity of permafrost bacteria below the freezing point. Appl Environ Microbiol66:3230–3233. [PMC free article] [PubMed] [Google Scholar]
• Rollet A-P and Vuillard G (1956) Sur un novel hydrate de l'ammoniac. CR Acad Sci Paris243:383–386. [Google Scholar]
• Rothschild LJ, Giver LJ, White MR, et al. (1994) Metabolic activity of microorganisms in evaporites. J Phycol30:431–438. [PubMed] [Google Scholar]
• Rummel JD, Beaty DW, Jones MA, et al. (2014) A new analysis of Mars “special regions”: findings of the Second MEPAG Special Regions Science Analysis Group (SR-SAG2). Astrobiology14:887–968. [PubMed] [Google Scholar]
• Sakai A and Engelmann F (2007) Vitrification, encapsulation-vitrification and droplet-vitrification: a review. CryoLetters28:151–172. [PubMed] [Google Scholar]
• Schneegurt MA (2012) Media and conditions for the growth of halophilic and halotolerant bacteria and archaea. In Advances in Understanding the Biology of Halophilic Microorganisms, edited by RH Vreeland. Springer, Dordrecht, pp 35–58. [Google Scholar]
• Schulze-Makuch D and Irwin LN (2002) Reassessing the possibility of life on Venus: proposal for an astrobiology mission. Astrobiology2:197–202. [PubMed] [Google Scholar]
• Seager S, Petkowski JJ, Gao P, et al. (2021) The venusian lower atmosphere haze as a depot for desiccated microbial life: a proposed life cycle for persistence of the venusian aerial biosphere. Astrobiology21:1206–1223. [PubMed] [Google Scholar]
• Smith DJ, Timonen HJ, Jaffe DA, et al. (2013) Intercontinental dispersal of bacteria and archaea by transpacific winds. Appl Environ Microbiol79:1134–1139. [PMC free article] [PubMed] [Google Scholar]
• Sutter B, Quinn RC, Archer PD, et al. (2017) Measurements of oxychlorine species on Mars. Int J Astrobiol16:203–217. [Google Scholar]
• Thomas NH, Ehlmann BL, Meslin P-Y, et al. (2019) Mars Science Laboratory observations of chloride salts in Gale crater, Mars. Geophys Res Lett46:10754–10763. [PMC free article] [PubMed] [Google Scholar]
• Toner JD, Catling DC, and Light B (2014) The formation of supercooled brines, viscous liquids, and low-temperature perchlorate glasses in aqueous solutions relevant to Mars. Icarus233:36–47. [Google Scholar]
• Vreeland, R.H., Piselli, A.F.Jr., McDonnough, S., Meyers, S.S. (1998) Distribution and diversity of halophilic bacteria in a subsurface salt formation. Extremophiles2:321–331. [PubMed] [Google Scholar]
• Wang A, Ling Z, Freeman JJ, et al. (2012) Stability field and phase transition pathways of hydrous ferric sulfates in the temperature range 50°C to 5°C: implication for martian ferric sulfates. Icarus218:622–643. [Google Scholar]
• Wänke H, Brückner G, Dreibus G, et al. (2001) Chemical composition of rocks and soils at the Pathfinder site. Space Sci Rev96:317–330. [Google Scholar]
• Way MJ, Del Genio AD, Kiang NY, et al. (2016) Was Venus the first habitable world of our solar system?Geophys Res Lett43:8376–8383. [PMC free article] [PubMed] [Google Scholar]
• Wickramasinghe NC and Wickramasinghe JT (2008) On the possibility of microbiota transfer from Venus to Earth. Astrophys Space Sci317:133–137. [Google Scholar]
• Wierzchos J, Davila AF, Sanchez-Almazo IM, et al. (2012) Novel water source for endolithic life in the hyperarid core of the Atacama Desert. Biogeosci Discuss9:3071–3098. [Google Scholar]
• Wilks JM, Chen F, Clark BC, et al. (2019) Bacterial growth in saturated and eutectic solutions of magnesium sulphate and potassium chlorate with relevance to Mars and the ocean worlds. Int J Astrobiol18:502–509. [PMC free article] [PubMed] [Google Scholar]
• Wilson C, Caton TM, Buchheim JA, et al. (2004) DNA-Repair potential of Halomonas spp. from the Salt Plains Microbial Observatory of Oklahoma. Microb Ecol48:541–549. [PubMed] [Google Scholar]
• Zbeeb H, Joad MD, Zayed H, et al. (2020) Extreme bacterial growth tolerances to brines relevant to Mars. In Abstracts and Program, 120th Annual Meeting of the American Society for Microbiology. American Society for Microbiology, Washington,DC. [Google Scholar]
• Zorzano M-P, Mateo-Martí E, Prieto-Ballesteros O, et al. (2009) Stability of liquid saline water on present day Mars. Geophys Res Lett36, doi: 10.1029/2009GL040315. [CrossRef] [Google Scholar]