Enhanced Microbial Survivability in Subzero Brines

Abstract

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.

Keywords: Brines; Halophile; Mars; Perchlorate; Subzero; Survival.

FIG. 1.

Bacterial growth curve of Planococcus halocryophilus in DMSZ growth media #92 + 10 wt% NaCl at 25°C. CFUs obtained as technical duplicates. Crosses mark the sampling times for inoculating of sample types ST 1 and ST 2. CFUs, colony forming units.

FIG. 2.

Survival rates of P. halocryophilus in eutectic Cl⁻ samples. Initial cell cultures were incubated for 4 days (ST 1) or 7 days (ST 2) at 25°C in growth medium containing 10 wt% NaCl before mixing them with the salt solution. CFUs were obtained as biological duplicates. Detection limit for CaCl₂ containing samples at 10³ CFU/mL results from the dilution factor of 3 that is necessary to decrease the Ca²⁺ concentration on the agar plate sufficiently for colony growth to occur.

FIG. 3.

(A) Survival rates of P. halocryophilus in ClO₄⁻ samples. Initial cell cultures were incubated for 7 days at 25°C in growth medium containing 10 wt% NaCl before mixing them with the salt solution (B). Effects of different preconditioning methods at −30°C. Before mixing them with the salt solution, the initial cell cultures were incubated for 7 days at 25°C in growth medium containing salts as indicated in the figure legend. CFUs were obtained in biological duplicates.

FIG. 4.

(A) Arrhenius-type plot for all Cl⁻ samples and NaClO₄, including slopes (S) for linear parts of the curves and molar concentrations (c), water activities (a_w), and ionic strengths (I) for all samples. (B) Slopes (S) of the steeper curve parts plotted as bar charts.

FIG. 5.

Survivability of P. halocryophilus during freeze/thaw cycles. Cells were incubated for 7 days at 25°C in growth medium containing either no additional salt (black circles) or 10 wt% NaCl (gray triangles) before subjecting them to freeze/thaw cycles. CFUs obtained from biological triplicates.