Introduction
Life as we know it is dependent on liquid water, which is conspicuously unstable on the present-day Martian surface due to low atmospheric pressures and freezing climate conditions. However, one potential outpost for putative Martian life could be through the availability of water in the form of brines formed by deliquescence (Maus et al., Reference Maus, Heinz, Schirmack, Airo, Kounaves and Wagner2020; Rivera-Valentín et al., Reference Rivera-Valentín, Chevrier, Soto and Martínez2020). Deliquescence is the process by which hygroscopic salts dissolve in water absorbed from the atmosphere. The conditions required for deliquescence are that the ambient relative humidity (RH) must exceed the deliquescent relative humidity (DRH) of the salt, and the ambient temperature must be above the eutectic temperature, which is the lowest temperature at which the salt-water mixture can melt to form a brine.
Brines have lower freezing points than pure water (Chevrier et al., Reference Chevrier, Rivera-Valentín, Soto and Altheide2020). This extends the availability of liquid water to sub-zero temperatures, which are prevalent on the Martian surface.
Several missions throughout the years, including the Phoenix lander (Hecht et al., Reference Hecht, Kounaves, Quinn, West, Young and Ming2009) and the Curiosity rover (Glavin et al., Reference Glavin, Freissinet, Miller, Eigenbrode, Brunner and Buch2013) have detected hygroscopic chlorine-containing salts, notably chlorides (Cl−) and perchlorates (ClO4 −), in the Martian regolith at distinct locations on the Red Planet (Rzymski et al., Reference Rzymski, Losiak, Heinz, Szukalska, Florek and Poniedziałek2024). It is also highly likely that intermediate forms of oxychlorides such as chlorates (ClO3 −) are present (Fernanders et al., Reference Fernanders, Gough, Chevrier, Schiffman, Ushijima and Martinez2022). High concentrations of chlorates, surpassing those of perchlorates, have indeed been detected in the EETA79001 Mars meteorite (ClO3 −/ClO4 − = ∼ 2) (Kounaves et al., Reference Kounaves, Carrier, O’Neil, Stroble and Claire2014).
Numerous studies have investigated the effects of these Mars-relevant salts on various types of microorganisms (Serrano et al., Reference Serrano, Alawi, deVera and Wagner2019; Waajen et al., Reference Waajen, Heinz, Airo and Schulze-Makuch2020; Cesur et al., Reference Cesur, Ansari, Chen, Clark and Schneegurt2022; Kloss et al., Reference Kloss, Doellinger, Gries, Soler, Lasch and Heinz2025), among which the halotolerant yeast Debaryomyces hansenii.
D. hansenii is a yeast frequently isolated from fermented products in brine and marine environments, including seawater and salt lakes (Almagro et al., Reference Almagro, Prista, Castro, Quintas, Madeira-Lopes and Ramos2000; Ramos, Reference Ramos, Gunde-Cimerman, Oren and Plemenitaš2005). This organism has evolved mechanisms to tolerate and thrive in high salt concentrations, highlighting its status as a halotolerant organism. It was observed to accumulate large amounts of intracellular Na+ without experiencing any impairing effects and survive in growth media with NaCl concentrations as high as ∼ 3.6 mol/L (corresponding to ∼ 4.0 mol/kg NaCl) (Musa et al., Reference Musa, Kasim, Nagoor Gunny and Gopinath2018). Moreover, enzymes in the TCA cycle are upregulated to meet the increased energy demands required for synthesising the osmolyte glycerol and for activating cation transporters to counteract heightened osmotic stress (Prista et al., Reference Prista, Loureiro-Dias, Montiel, García and Ramos2005).
D. hansenii has already been studied in the context of Martian habitability. Heinz et al. determined that it tolerates NaClO4 up to 2.5 mol/kg and NaClO3 up to 5.5 mol/kg (Heinz et al., Reference Heinz, Rambags and Schulze-Makuch2021) which are the highest microbial perchlorate and chlorate tolerances, respectively, reported to date. The enhanced tolerance to NaCl associated with lower water activity compared to NaClO4 suggests that the response to NaClO4 involves more than just osmotic stress. Specific stress responses to perchlorate were identified, with a notable focus on the upregulation of the glycosylation of cell wall proteins in the Golgi, an upregulation of pathways that synthesise cell wall components (such as chitin) and an upregulation of the calnexin cycle involved in protein folding. This may be attributed to specific properties of ClO4 − ions, such as chaotropicity (Heinz et al., Reference Heinz, Doellinger, Maus, Schneider, Lasch and Grossart2022). A chaotropic molecule perturbs the structure and stability of macromolecules, including proteins and membranes, by interfering with the hydrogen-bonding network and hydrophobic interactions (Salvi et al., Reference Salvi, de Los Rios and Vendruscolo2005).
D. hansenii has been subjected to and monitored under additional Mars-relevant stress factors such as sub-zero temperatures, low atmospheric pressure, regolith depth and UV irradiation (Fischer et al., Reference Fischer, Schulze-Makuch and Heinz2024). However, no studies have reported on the survival of D. hansenii on water provided by deliquescence. Similar work including the anaerobic methanogenic archaeon Methanosarcina soligelidi has been undertaken before, with methane production accounting as an indicator of metabolic activity (Maus et al., Reference Maus, Heinz, Schirmack, Airo, Kounaves and Wagner2020). Our study expands on these findings by exploring the limits of survival and growth of eukaryotic cells in Mars-relevant but insufficiently investigated salts such as chlorates, dispersed in Martian regolith simulant Mars Global Simulant (MGS-1) with the aim of providing new insights into the potential habitability of Mars.
Material and methods
Culture conditions
D. hansenii (strain HUT 7011, DSM No. 3428) was obtained from the German Collection of Microorganisms and Cell Culture of the Leibniz Institute (DSMZ). Cells were grown aerobically in liquid growth medium DSMZ #90 (3% malt extract, 0.3 % soya peptone) without shaking and incubated at the organism’s optimal growth temperature of 25 °C. Additionally the incubated samples contained either 2.5 mol/kg NaCl, 3 mol/kg NaClO3, 2 mol/kg NaClO4 or no additional salts (control samples). The chosen salt concentrations enabled a pre-adaptation of cells at comparable stress levels before starting the deliquescence experiment. Cell growth in liquid cultures was monitored spectrophotometrically by measuring the optical density at 600 nm (OD600). All growth experiments were performed in biological triplicates, with three separate samples inoculated for each salt condition.
Desiccation assay
Two mL of a cell suspension of D. hansenii grown in liquid growth media containing either 2.5 mol/kg NaCl, 3 mol/kg NaClO3, 2 mol/kg NaClO4 or no additional salts (control samples) were used to inoculate 4.5 g of Mars-simulating regolith supplemented with an additional amount of 0.5 g of either NaCl, NaClO3 or NaClO4 (or without additional salts for control samples). The supplementation with 0.5 g of salts (NaCl, NaClO3 or NaClO4) was required to establish a salt concentration within the regolith high enough to optimise the conditions required to initiate the deliquescence process. The regolith used was the Mars Global High-Fidelity Martian Dust Simulant MGS-1 (Cannon et al., Reference Cannon, Britt, Smith, Fritsche and Batcheldor2019) (Exolith Lab, Orlando, USA). MGS-1 was chosen as it is widely recognised as having the highest fidelity in terms of mineral, chemical, volatile, and spectral properties among currently available Martian global regolith simulants (Karl et al., Reference Karl, Cannon and Gurlo2022).
The samples consisting of a mixture of MGS-1, D. hansenii cells and the respective salt (or no salt for control samples) were placed in a low-pressure environment in a desiccator above the desiccant phosphorus pentoxide (P4O10) over 2 days. The yeast’s ability to withstand desiccation was measured. Since spectrophotometric recording was not feasible due to the presence of regolith particles, colony forming unit (CFU) counts on medium DSMZ #90 agar plates were used instead to determine the yeast’s survival. CFUs were recorded pre- and post-desiccation. Spatula tip-sized aliquots were taken from the samples, weighed and diluted in Phosphate Buffered Saline (PBS) at a ratio of 10 mL of PBS per 1 gram of regolith. The regolith-PBS mixture was then serially diluted, and 100 µL aliquots were plated on agar plates.
Water contents (w) of freshly inoculated samples were measured by placing a portion of the sample in 1.5 mL centrifuge tubes, which were weighed before (mwet) and after (mdry) desiccation in the desiccator. The water content was then calculated using Equation 1.
$${\rm{w}} = {{{{\rm{m}}_{{\rm{wet}}}} - {{\rm{m}}_{{\rm{dry}}}}} \over {{{\rm{m}}_{{\rm{wet}}}}}} \times 100$$
The water content and the determined CFU per ml of regolith-PBS mixture (CFU/mL) were subsequently used to calculate the CFU per gram of dry regolith using Equation 2 (assuming that 1 mL PBS ∼ 1 g PBS) in order to correctly compare CFU values for samples before and after desiccation.
$${\rm{CFU/g}}\;{\rm{dry}}\;{\rm{regolith}} = {{{\rm{CFU/mL}}} \over {1 - {w \over {100}}}}$$
The survival rate (S) in the desiccation assay is determined by calculating the ratio of the initial CFU/g of dry regolith (CFU/g dry regolith before) to the CFU/g dry regolith after the desiccation (CFU/g dry regolith desiccated):
$$S = {{{\rm{CFU/g}}\;{\rm{dry}}\;{\rm{regolit}}{{\rm{h}}_{{\rm{before}}}}} \over {{\rm{CFU/g}}\;{\rm{dry}}\;{\rm{regolit}}{{\rm{h}}_{{\rm{desiccated}}}}}} \times 100\;\% $$
All equations were also used in previous work by Fischer et al. (Fischer et al., Reference Fischer, Schulze-Makuch and Heinz2024).
Deliquescence experimental setup
A deliquescence experimental setup (DES) (Figure 1) was assembled to rehydrate the desiccated mixture of MGS-1, D. hansenii cells, growth media components and salts (except for salt-free control samples). The design of the setup was based on the concept described by Maus et al. (Maus et al., Reference Maus, Heinz, Schirmack, Airo, Kounaves and Wagner2020), but it was adapted to meet the specific requirements of the present experimental conditions: the sample, placed in a ø 50 mm petri dish, was positioned inside a larger ø 90 mm petri dish containing a saturated solution of potassium sulphate (K2SO4). The desiccated sample was not in direct contact with the K2SO4 solution. The larger petri dish was sealed and wrapped in parafilm, creating a closed system where the saturated K2SO4 solution and the desiccated sample shared the same headspace. The deliquescence relative humidity (DRH) of a saturated K2SO4 solution is temperature-dependent. The samples were incubated at approximately 25°C, at which the RH in the closed system reaches about 98% at equilibrium (Maus et al., Reference Maus, Heinz, Schirmack, Airo, Kounaves and Wagner2020).
Figure 1. Deliquescence experimental setup. The inner Petri dish contains Martian regolith simulant with salts (except for the salt-free control) and the model organism, placed within a larger petri dish containing saturated potassium sulphate solution. This configuration generates a high relative humidity (RH) environment, inducing the deliquescence of salts in the regolith.
Moreover, for comparison purposes, an additional DES setup was prepared for low RH control samples whereby the saturated K2SO4 solution was replaced with the desiccant phosphorus pentoxide (P4O10) in order to avoid any moisture in the headspace.
Salt solubility and solute concentration
Before inoculation, each salt-containing sample consisted of 4.5 g of MGS-1 regolith supplemented with 0.5 g of salt (NaCl, NaClO3, NaClO4 or no additional salt for control samples). Two mL of cell suspension containing the respective salt (or no additional salt) were added to the corresponding regolith-salt mixture. As a result, in the salt-containing samples the total salt content increased beyond the initial 0.5 g (10 wt %) due to the contribution from the cell suspension. After desiccation, the salt contents in the desiccated samples were calculated to be 15.1 wt % for NaCl, 20.5 wt % for NaClO3 and 18.3 wt % for NaClO4.
During the rehydration phase in the deliquescence experiment, the water content was measured for the calculation of CFU/g dry regolith as described above. The water content was also used to calculate the salt concentrations (in mol/kg) in the water absorbed during the deliquescence experiment, as they were expected to decrease with increasing water content.
Solubilities of the salts at 25 °C used for calculations are as follows: 36 g per 100 g of water (6.16 mol/kg) for NaCl (Haynes, Reference Haynes2014), 106 g per 100 g of water (9.96 mol/kg) (Seidell & Linke, Reference Seidell and Linke1952) for NaClO3 and 210 g per 100 g of water (17.15 mol/kg) for NaClO4 (Ullmann et al., Reference Ullmann, Gerhartz, Yamamoto, Campbell, Pfefferkorn and Rounsaville1985).
Results
Survival rate after desiccation
The survival rate of D. hansenii after desiccation was examined to determine whether the organism can survive dry periods, a major challenge in potential Martian habitats given the possible transient presence of liquid water. The survival rate was investigated via CFU counts as a measure of cell viability. No survival of D. hansenii was observed in desiccated NaClO4-supplemented samples. Desiccated NaCl or NaClO3-containing samples had a survival rate of D. hansenii of 21.9 % and 39.3 % respectively, while the survival rate in the control samples was 1.9 % (Table 1).
Table 1. Survival rate of D. hansenii after desiccation
Water content and solute concentration in the deliquescence experimental setup
The water content of the salt and control samples was monitored during the wetting process in the DES. The high RH environment allowed the hygroscopic salts in the samples to absorb moisture, forming a brine/regolith mixture over a period of a few days. All three salt-containing samples (NaCl, NaClO3 and NaClO4) showed an increase in water content (Figure 2a). The final water contents were 60 ± 8 wt %, 55 ± 6 wt %, and 60 ± 16 wt % for NaCl, NaClO3 and NaClO4, respectively. Meanwhile, the control samples exhibited a slight increase in water content as well, stabilising at around 7.9 ± 1.7 wt % throughout the 63-day experiment.
Figure 2. a) Water content (wt %) of NaCl, NaClO3, NaClO4 and salt-free samples measured over the 63-day experiment. NaCl, NaClO4, and NaClO3 absorbed water to a final content of approximately 55–60 wt %. Salt-free samples maintained a steady water content of ∼8 wt % throughout the experiment. Error bars represent the standard deviation in water content measurements across triplicates. b) Salt concentration (mol/kg) of NaCl (yellow), NaClO3 (blue) or NaClO4 (magenta)-containing samples over the 63-day experiment. The initial salt concentrations (Day 0) represent the saturation concentrations of each salt at room temperature. Solute concentrations decreased for all salt-containing samples to a final concentration of approximately 1.1–1.7 mol/kg. The dotted lines represent the highest solute concentration (mol/kg) of NaCl (yellow) (4.0), NaClO3 (blue) (5.5) or NaClO4 (magenta) (2.5) tolerated by D. hansenii as reported by Heinz et al. (Heinz et al., Reference Heinz, Rambags and Schulze-Makuch2021). c) Growth curves of D. hansenii during deliquescence-driven wetting in the DES after desiccation. Control samples are in black, NaCl in yellow and NaClO3 in blue (n = 3). Lower error bars are missing for several NaCl values in this logarithmic diagram due to their large size, which stems from one triplicate deviating notably from the other two. As a result, the full error bars cannot be shown.
The solute concentrations of the salts (NaCl, NaClO3 or NaClO4) in the absorbed water were calculated as described above and are illustrated in Figure 2b, providing insights on how salt concentrations in the brines evolved during the wetting process in the DES. It was observed that sufficient water was absorbed to fully dissolve the salt already before the first sampling after the start of the deliquescence experiments (i.e. 12, 14 and 8 days for NaCl, NaClO3 and NaClO4 samples, respectively). Overall, the solute concentrations for all salt samples decreased throughout the experiment due to continuous water absorption and were all below the maximum levels tolerated by D. hansenii by the end of the experimental period. The highest solute concentrations tolerated by D. hansenii have been reported as 4.0 mol/kg, 5.5 mol/kg and 2.5 mol/kg for NaCl, NaClO3 and NaClO4, respectively (Heinz et al., Reference Heinz, Rambags and Schulze-Makuch2021).
Growth patterns in the deliquescence experimental setup
Growth patterns of D. hansenii in the DES were monitored via CFU/g dry regolith (Figure 2c). NaCl samples entered exponential growth phase without a noticeable lag phase. NaClO3 samples initially exhibited a decline in CFU/g dry regolith which was followed by exponential growth phase culminating in a cell density that matched that of the NaCl samples by the end of the experiment.
Control samples also indicated an accelerated growth phase relative to the NaCl samples. Additional experiments were conducted using a modified DES whereby the saturated K2SO4 solution was replaced with the desiccant phosphorus pentoxide to eliminate any residual humidity. These samples were used as low RH controls. This setup was designed to assess whether the growth of D. hansenii in the salt-free control samples was tied to the ∼ 8% water content resulting from the absorption capabilities of MGS-1. In the low RH control samples, with moisture nearly eliminated, CFU values steadily declined.
Discussion
Our results indicate that D. hansenii does not survive desiccation in the presence of NaClO4. This indicates that the process of desiccation in a NaClO4-enriched environment imposed excessive stress on the cells. While NaCl, NaClO3 and NaClO4 all lower the water activity (aw) of a solution and induce increasing salt stress during evaporation of water, cells in the NaCl and NaClO3-containing samples were able to survive the desiccation process. The lack of survival in NaClO4-containing samples could be attributed to several properties of NaClO4. Due to its higher solubility at 25 °C (210 g/100 g water) (Ullmann et al., Reference Ullmann, Gerhartz, Yamamoto, Campbell, Pfefferkorn and Rounsaville1985) compared to NaCl (36 g/100 g water) (Haynes, Reference Haynes2014) and NaClO3 (106 g/100 g water) (Seidell & Linke, Reference Seidell and Linke1952), NaClO4 forms more concentrated brines during desiccation, resulting in lower water activity (aw < 0.5 (Toner & Catling, Reference Toner and Catling2016)) (Chevrier et al., Reference Chevrier, Hanley and Altheide2009), which is known to inhibit cellular processes and compromise microbial viability (Stevenson et al., Reference Stevenson, Cray, Williams, Santos, Sahay and Neuenkirchen2015), further exacerbating the stress on the organism. Additionally, the reduced water evaporation rate due to the reduced aw (Sears & Moore, Reference Sears and Moore2005; Mor et al., Reference Mor, Assouline, Tanny, Lensky and Lensky2018) coupled with the relatively low efflorescence relative humidity (the humidity level at which salts crystallise out of a solution) of NaClO4 (15-23 % at 25 °C) (Peng et al., Reference Peng, Chen and Tang2022), prolongs the cells’ exposure to a highly concentrated brine during the desiccation process. These harsh conditions, combined with the chaotropic (i.e. biomacromolecule-destabilising) nature of NaClO4 (Heinz et al., Reference Heinz, Rambags and Schulze-Makuch2021), likely contributed to the eventual death of D. hansenii in NaClO4-containing samples. Furthermore, studies have demonstrated that the presence of perchlorate accelerates the leaching of certain elements, including magnesium, sodium, sulphur, aluminium and chromium from MGS-1 (Rzymski et al., Reference Rzymski, Klimaszyk, Kasianchuk, Jakubiak, Proch and Niedzielski2023). The impact of these elevated elemental concentrations on D. hansenii remains uncertain. However, previous research suggests that chemicals such as hexavalent chromium (Cr(VI)), may interact with perchlorate to produce synergistic toxic effects in other organisms like planktons (Zhou et al., Reference Zhou, Du, Li, Qin, Li and Chen2021).
Interestingly, our results also suggest that D. hansenii not only survives desiccation but also thrives using only water absorbed via deliquescence in NaCl- and NaClO3-containing samples. The heightened tolerance of D. hansenii to NaCl is due to several salt stress responses that have been documented before, such as the upregulation of enzymes in the TCA cycle to meet the increased energy demands required for synthesising the osmolyte glycerol and activating cation transporters to counteract heightened osmotic stress (Prista et al., Reference Prista, Michán, Miranda and Ramos2016; Heinz et al., Reference Heinz, Doellinger, Maus, Schneider, Lasch and Grossart2022).
In contrast to NaCl samples, growth patterns in NaClO3 samples indicated an initial decline in CFU counts. This might be explained by the slightly higher solubility of NaClO3 compared to NaCl (but still substantially lower than NaClO4) resulting in lower aw directly after the start of the deliquescence experiment causing increased stress to cells which might result in retarded cell growth. Furthermore, chlorate remains chaotropic albeit to a lesser extent than perchlorates (Heinz et al., Reference Heinz, Rambags and Schulze-Makuch2021), which potentially adds an additional stress factor at elevated salt concentrations which might account for the initial lag phase. The observed growth in NaClO3-containing samples could be promising for potential life on Mars. The Martian meteorite EETA79001 already showed a higher ratio of ClO3 − to ClO4 − ions (Kounaves et al., Reference Kounaves, Carrier, O’Neil, Stroble and Claire2014). A study by Qu et al. (Qu et al., Reference Qu, Zhao, Cui, Yin, Jackson and Nie2022) suggests that ClO3 − ions are present on Mars in higher concentrations than previously believed. This is due to the preferential oxidation of Cl− ions to ClO3 − under hyperarid and Fe (hydr)oxides abundant conditions on Mars throughout the Amazonian period.
Control samples (with no salt addition) also demonstrated growth of D. hansenii with an absorbed water content of ∼ 8 wt %. Fischer et al. (Fischer et al., Reference Fischer, Schulze-Makuch and Heinz2024) also documented the growth of halotolerant organisms D. hansenii and Planococcus halocryophilus in salt-free MGS-1 samples at similar water content levels (∼ 9 wt %).
The growth observed in the salt-free control in our study contrasts with findings from similar studies. For instance, Maus et al. (Maus et al., Reference Maus, Heinz, Schirmack, Airo, Kounaves and Wagner2020) explored the survival and growth of methanogenic archaea under anaerobic conditions in a closed deliquescent system following desiccation. They examined the effects of various salts and regolith types on methane detection as an indicator of metabolic activity. Their study included a negative control using P-MRA regolith that was not supplemented with any hygroscopic salts to absorb water. In this negative control, methane detection and water absorption was non-significant, leading the authors to conclude that there was no significant growth under this condition.
Interestingly, in our low RH control experiments, when the K2SO4 solution in the DES was replaced by the desiccant phosphorus pentoxide, the minimal water availability (water content less than 1 %) led to decreasing CFU counts. This indicates that a minor amount of residual water content after desiccation was not responsible for the observed cell growth which is instead attributed to the water absorbed by pure MGS-1 at high RH conditions.
The composition of the Martian regolith analogues used here and in other studies varies and might be responsible for this particular water-absorbing characteristic of MGS-1. MGS-1 is a third-generation simulant considered one of the best analogues to Martian regolith due to its matching mineral composition and similar spectral properties (Karl et al., Reference Karl, Cannon and Gurlo2022). The results in this study suggest that MGS-1 exhibits some amount of hygroscopicity on its own without the addition of external salts. This might be due to the presence of soluble minerals such as epsomite (MgSO4 ·7 H2O) (Cannon et al., Reference Cannon, Britt, Smith, Fritsche and Batcheldor2019). Magnesium sulphates are widespread across the Martian surface (Shi et al., Reference Shi, Zhang, Zeng, Xin, Ju and Ling2024), with occurrences documented in regions such as Gale Crater (Chipera et al., Reference Chipera, Vaniman, Rampe, Bristow, Martínez and Tu2023) and is known to be hygroscopic (Vaniman et al., Reference Vaniman, Bish, Chipera, Fialips, William Carey and Feldman2004; Cesur et al., Reference Cesur, Ansari, Chen, Clark and Schneegurt2022), with a DRH of 85 % (Peng et al., Reference Peng, Chen and Tang2022). However, these minerals have substantially higher eutectic temperatures and DRH values – for instance, the eutectic temperature of magnesium sulphate solutions is -3.9 °C (269 K) (Chevrier et al., Reference Chevrier, Rivera-Valentín, Soto and Altheide2020). In contrast, more soluble salts such as NaCl, NaClO3 and NaClO4 exhibit much lower DRH and eutectic temperatures: NaCl has a DRH of 75 % and a eutectic temperature of -21 °C (252 K) (Drebushchak et al., Reference Drebushchak, Ogienko and Yunoshev2017; Chevrier et al., Reference Chevrier, Fitting, Elsenousy and Rivera-Valentín2022). NaClO3 has a DRH of 73 % and a eutectic temperature of -23 °C (250 K) (Hanley et al., Reference Hanley, Chevrier and Adams2012; Toner & Catling, Reference Toner and Catling2018). NaClO4 is even more hygroscopic, with a DRH of 40 % and a eutectic temperature of -37 °C (236 K) (Chevrier et al., Reference Chevrier, Hanley and Altheide2009; Gough et al., Reference Gough, Baustian, Wise and Tolbert2009). These lower DRH values enable these salts to absorb water from the atmosphere at much lower relative humidity levels, even as low as ∼ 40%, and to remain in liquid form at sub-zero temperatures. This suggests that the colder temperatures and lower relative humidity regimes on Mars might allow for water absorption by regolith only in the presence of the more adequate NaCl, NaClO3 and NaClO4 salts. Thus, the presence of these hygroscopic salts on Mars may play a critical role in facilitating deliquescence under Martian environmental conditions and potentially in promoting halotolerant life in these environments.
This study demonstrates the survival and growth of D. hansenii in deliquescence-driven brines. However, Martian brines would exist at sub-zero eutectic temperatures. Under such conditions, several limiting factors to biological activity exist, including reduced membrane fluidity, enzyme inactivation and a drop in physiological processes (Georlette et al., Reference Georlette, Blaise, Collins, D’Amico, Gratia and Hoyoux2004). Thus, any organism that potentially survives close to the surface of the planet is expected to be psychrophilic in addition to being halophilic. Psychrophilic organisms have evolved adaptations such as ‘cold-active’ enzymes, enhanced membrane fluidity via higher unsaturated lipid content, and the deployment of chaperone proteins to thrive at low temperatures (D’Amico et al., Reference D’Amico, Collins, Marx, Feller and Gerday2006). Growth of the bacterium Planococcus halocryophilus at -15 ºC – the lowest temperature recorded for growth – has been observed previously (Mykytczuk et al., Reference Mykytczuk, Foote, Omelon, Southam, Greer and Whyte2013). Metabolic activity of permafrost bacteria has been detected at temperatures as low as – 20 ºC (Rivkina et al., Reference Rivkina, Friedmann, McKay and Gilichinsky2000; Jakosky et al., Reference Jakosky, Nealson, Bakermans, Ley and Mellon2003). D. hansenii, however, seems not to be adapted to such conditions and its survival and growth dynamics in brines at sub-zero temperatures remains uncertain, calling for further research.
Conclusion
The findings of this work suggest that perchlorate dominated environments may pose a significant challenge for putative life on Mars, as the here tested organism D. hansenii did not survive desiccation in NaClO4 samples. On the other hand, according to our experimental results, deliquescence-driven NaCl and NaClO3 brines mixed with regolith could create potential niches for halotolerant life on the Red Planet. This warrants placing greater emphasis on chlorates and chloride salts in the context of potential Martian habitats, rather than on perchlorate salts, which have previously been the primary focus of research in this field.
Acknowledgements
This Research was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – 455070607. The authors declare no competing interest.
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