Can Halophilic and Psychrophilic Microorganisms Modify the Freezing/Melting Curve of Cold Salty Solutions? Implications for Mars Habitability

Abstract

We present the hypothesis that microorganisms can change the freezing/melting curve of cold salty solutions by protein expression, as it is known that proteins can affect the liquid-to-ice transition, an ability that could be of ecological advantage for organisms on Earth and on Mars. We tested our hypothesis by identifying a suitable candidate, the well-known psycrophile and halotolerant bacteria Rhodococcus sp. JG3, and analyzing its response in culture conditions that included specific hygroscopic salts relevant to Mars-that is, highly concentrated magnesium perchlorate solutions of 20 wt % and 50 wt % Mg(ClO₄)₂ at both end members of the eutectic concentration (44 wt %)-and subfreezing temperatures (263 K and 253 K). Using a combination of techniques of molecular microbiology and aqueous geochemistry, we evaluated the potential roles of proteins over- or underexpressed as important players in different mechanisms for the adaptability of life to cold environments. We recorded the changes observed by micro-differential scanning calorimetry. Unfortunately, Rhodococcus sp. JG3 did not show our hypothesized effect on the melting characteristics of cold Mg-perchlorate solutions. However, the question remains as to whether our novel hypothesis that halophilic/psychrophilic bacteria or archaea can alter the freezing/melting curve of salt solutions could be validated. The null result obtained after analyzing just one case lays the foundation to continue the search for proteins produced by microorganisms that thrive in very cold, high-saline solutions, which would involve testing different microorganisms with different salt components. The immediate implications for the habitability of Mars are discussed.

Keywords: Calorimetry; Chaotropism; Cold brines; Extremophiles; Mars; Melting/freeze point; Perchlorates.

FIG. 1.

(A) Experimental data for the 20 wt % solution (red points), together with the optical density at 600 nm of the cultures (blue points), after 10 days at 253 K and 263 K, plotted against the temperature of the liquidus point (T liquidus melting) recorded by calorimetry. (B) Variation of TH (K) as a function of the cell concentration in Rhodococcus after 10 days in 20 wt % Mg(ClO₄)₂, at 253 K (red circles) and 263 K (red squares). The blue diamond corresponds to the sterile solution. TH is normally calculated as the difference between T_onset freezing and T_onset congruent melting (Lee et al., 2018), but in the case of perchlorate, we used the T_onset incongruent melting (see Supplementary Fig. S3). TH, thermal hysteresis. Color images are available online.

FIG. 2.

(A) Relative survival rate of cells (%) after 10 days of cultivation in 15 mL of 20 wt % magnesium perchlorate at 263 K and 253 K, as determined by flow cytometry using propidium iodide as the staining agent. The shift was from 10⁵ cells/mL of initial concentration to 10³ cell/mL of survival. (B) Confocal microscopy images of cell cultures in perchlorate after 10 days at temperatures of 263 K and 253 K: (a) Rhodococcus sp. JG3 and (b) Escherichia coli living cells are highlighted in green, dead cells in red, and cells with compromised membranes (i.e., permeable to propidium iodide) in yellow. The images show a high proportion of dead Rhodococcus sp. JG3 cells, but still some living bacteria could be appreciated. Microscopy imagery revealed that the size and shape of E. coli seemed to be slightly modified after the exposure to the cold and salty stress conditions. Color images are available online.

FIG. 3.

Comparison of electrophoretic banding patterns of protein extracts between cultures of Rhodococcus sp. JG3 and Escherichia coli in LB at optimal temperature, with those in 20 wt % Mg(ClO₄)₂ cultivated at 263 K. Central lane (Protein marker) is the known pattern of a protein standard (Precision Plus Protein™ All Blue Prestained Protein Standards; Bio-Rad), which main molecular weights are indicated on the right (kDa). Black stars show examples of proteins in Rhodococcus sp. JG3 overexpressed at 263 K in 20 wt % Mg(ClO₄)₂. LB, Luria–Bertani broth.