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Extraterrestrial environments encompass physicochemical conditions and habitats that are unknown on Earth, such as perchlorate-rich brines that can be at least temporarily stable on the martian surface. To better understand the potential for life in these cold briny environments, we determined the maximum salt concentrations suitable for growth (MSCg) of six different chloride and perchlorate salts at 25°C and 4°C for the extremotolerant cold- and salt-adapted bacterial strain Planococcus halocryophilus. Growth was measured through colony-forming unit (CFU) counts, while cellular and colonial phenotypic stress responses were observed through visible light, fluorescence, and scanning electron microscopy. Our data show the following: (1) The tolerance to high salt concentrations can be increased through a stepwise inoculation toward higher concentrations. (2) Ion-specific factors are more relevant for the growth limitation of P. halocryophilus in saline solutions than single physicochemical parameters like ionic strength or water activity. (3) P. halocryophilus shows the highest microbial sodium perchlorate tolerance described so far. However, (4) MSCg values are higher for all chlorides compared to perchlorates. (5) The MSCg for calcium chloride was increased by lowering the temperature from 25°C to 4°C, while sodium- and magnesium-containing salts can be tolerated at 25°C to higher concentrations than at 4°C. (6) Depending on salt type and concentration, P. halocryophilus cells show distinct phenotypic stress responses such as novel types of colony morphology on agar plates and biofilm-like cell clustering, encrustation, and development of intercellular nanofilaments. This study, taken in context with previous work on the survival of extremophiles in Mars-like environments, suggests that high-concentrated perchlorate brines on Mars might not be habitable to any present organism on Earth, but extremophilic microorganisms might be able to evolve thriving in such environments.

It is well known that dissolved salts can significantly lower the freezing point of water and thus extend habitability to subzero conditions. However, most investigations thus far have focused on sodium chloride as a solute. In this study, we report on the survivability of the bacterial strain Planococcus halocryophilus in sodium, magnesium, and calcium chloride or perchlorate solutions at temperatures ranging from +25°C to −30°C. In addition, we determined the survival rates of P. halocryophilus when subjected to multiple freeze/thaw cycles. We found that cells suspended in chloride-containing samples have markedly increased survival rates compared with those in perchlorate-containing samples. In both cases, the survival rates increase with lower temperatures; however, this effect is more pronounced in chloride-containing samples. Furthermore, we found that higher salt concentrations increase survival rates when cells are subjected to freeze/thaw cycles. Our findings have important implications not only for the habitability of cold environments on Earth but also for extraterrestrial environments such as that of Mars, where cold brines might exist in the subsurface and perhaps even appear temporarily at the surface such as at recurring slope lineae.It is well known that dissolved salts can significantly lower the freezing point of water and thus extend habitability to subzero conditions. However, most investigations thus far have focused on sodium chloride as a solute. In this study, we report on the survivability of the bacterial strain Planococcus halocryophilus in sodium, magnesium, and calcium chloride or perchlorate solutions at temperatures ranging from +25°C to −30°C. In addition, we determined the survival rates of P. halocryophilus when subjected to multiple freeze/thaw cycles. We found that cells suspended in chloride-containing samples have markedly increased survival rates compared with those in perchlorate-containing samples. In both cases, the survival rates increase with lower temperatures; however, this effect is more pronounced in chloride-containing samples. Furthermore, we found that higher salt concentrations increase survival rates when cells are subjected to freeze/thaw cycles. Our findings have important implications not only for the habitability of cold environments on Earth but also for extraterrestrial environments such as that of Mars, where cold brines might exist in the subsurface and perhaps even appear temporarily at the surface such as at recurring slope lineae.

Microorganisms living in sub-zero environments can benefit from the presence of dissolved salts, as they significantly increase the temperature range of liquid water by lowering the freezing point. However, high concentrations of salts can reduce microbial growth and survival, and can evoke a physiological stress response. It remains poorly understood how the physicochemical parameters of brines (e.g. water activity, ionic strength, solubility and hydration shell strength between the ions and the surrounding water molecules) influence the survival of microorganisms. We used the cryo− and halotolerant bacterial strain Planococcus halocryophilus as a model organism to evaluate the degree of stress different salts assert. Cells were incubated in liquid media at −15°C containing single salts at eutectic concentrations (CaCl2, LiCl, LiI, MgBr2, MgCl2, NaBr, NaCl, NaClO4 and NaI). Four of these salts (LiCl, LiI, MgBr2 and NaClO4) were also investigated at concentrations with a low water activity (0.635) and, separately, with a high ionic strength (8 mol/L). Water activity of all solutions was measured at −15°C. This is the first time that water activity has been measured for such a large number of liquid salt solutions at constant sub-zero temperatures (−15°C). Colony-Forming Unit (CFU) counts show that the survival of P. halocryophilus has a negative correlation with the salt concentration, molecular weight of the anion and anion radius; and a positive correlation with the water activity and anions’ hydration shell strength. The survival of P. halocryophilus did not show a significant correlation with the ionic strength, the molecular weight of the cation, the hydrated and unhydrated cation and hydrated anion radius, and the cations’ hydration bond length. Thus, the water activity, salt concentration and anion parameters play the largest role in the survival of P. halocryophilus in concentrated brines. These findings improve our understanding of the limitations of microbial life in saline environments, which provides a basis for better evaluation of the habitability of extraterrestrial environments such as Martian cryobrines.

(1) Background: Future missions to potentially habitable places in the Solar System require biochemistry-independent methods for detecting potential alien life forms. The technology was not advanced enough for onboard machine analysis of microscopic observations to be performed in past missions, but recent increases in computational power make the use of automated in-situ analyses feasible. (2) Methods: Here, we present a semi-automated experimental setup, capable of distinguishing the movement of abiotic particles due to Brownian motion from the motility behavior of the bacteria Pseudoalteromonas haloplanktis, Planococcus halocryophilus, Bacillus subtilis, and Escherichia coli. Supervised machine learning algorithms were also used to specifically identify these species based on their characteristic motility behavior. (3) Results: While we were able to distinguish microbial motility from the abiotic movements due to Brownian motion with an accuracy exceeding 99%, the accuracy of the automated identification rates for the selected species does not exceed 82%. (4) Conclusions: Motility is an excellent biosignature, which can be used as a tool for upcoming life-detection missions. This study serves as the basis for the further development of a microscopic life recognition system for upcoming missions to Mars or the ocean worlds of the outer Solar System.

It is well known that dissolved salts can significantly lower the freezing point of water and thus extend habitability to subzero conditions. However, most investigations thus far have focused on sodium chloride as a solute. In this study, we report on the survivability of the bacterial strain Planococcus halocryophilus in sodium, magnesium, and calcium chloride or perchlorate solutions at temperatures ranging from +25°C to −30°C. In addition, we determined the survival rates of P. halocryophilus when subjected to multiple freeze/thaw cycles. We found that cells suspended in chloride-containing samples have markedly increased survival rates compared with those in perchlorate-containing samples. In both cases, the survival rates increase with lower temperatures; however, this effect is more pronounced in chloride-containing samples. Furthermore, we found that higher salt concentrations increase survival rates when cells are subjected to freeze/thaw cycles. Our findings have important implications not only for the habitability of cold environments on Earth but also for extraterrestrial environments such as that of Mars, where cold brines might exist in the subsurface and perhaps even appear temporarily at the surface such as at recurring slope lineae.It is well known that dissolved salts can significantly lower the freezing point of water and thus extend habitability to subzero conditions. However, most investigations thus far have focused on sodium chloride as a solute. In this study, we report on the survivability of the bacterial strain Planococcus halocryophilus in sodium, magnesium, and calcium chloride or perchlorate solutions at temperatures ranging from +25°C to −30°C. In addition, we determined the survival rates of P. halocryophilus when subjected to multiple freeze/thaw cycles. We found that cells suspended in chloride-containing samples have markedly increased survival rates compared with those in perchlorate-containing samples. In both cases, the survival rates increase with lower temperatures; however, this effect is more pronounced in chloride-containing samples. Furthermore, we found that higher salt concentrations increase survival rates when cells are subjected to freeze/thaw cycles. Our findings have important implications not only for the habitability of cold environments on Earth but also for extraterrestrial environments such as that of Mars, where cold brines might exist in the subsurface and perhaps even appear temporarily at the surface such as at recurring slope lineae.