Survival of Halophiles at Mars-Simulated Conditions
A.A. Berezhnoy1, A.V. Bryanskaya2, A.S. Rozanov2, S.I. Baiborodin2, A.K. Pavlov3, S.E. Peltek2
1
Sternberg Astronomical Institute, Moscow State University, Russia; 2Institute of Cytology and Genetics, SB RAS,
Novosibirsk, Russia; 3 Ioffe Physical-Technical Institute, St. Petersburg, Russia. Contact: [email protected]
Introduction: Earth’s microorganisms can be delivered to Mars by impacts of meteoroids of Earth’s
origin and modern missions. Mars is not hospitable planet for Earth’s microbes due to low day and night
temperatures, high content of salts, high level of UV-radiation, and low content of organic species. To
study the possibility of survival of Earth’s microorganisms on Mars, we need to select the most suitable
types of them. Halophiles are one of the most interesting types of such microorganisms. Study of the
elemental composition of the Martian soils shows high concentrations of Cl, perchlorates, and solubable
sulfates. At Viking landing sites the content of perchlorates and organic carbon is estimated to be < 0.1%
and 0.7 – 6.5 ppm, respectively [1]. High abundance of perchlorates around 1% was discovered at
Phoenix landing site [2]. The aim of this study is to select bacterial and archeal strains most adapted to
Martian conditions. Results of previous experiments on survival at various NaCl concentrations and
freezing conditions allowed us to choose strains which are the most resistant to NaCl and low temperature
[3].
Methods: Bacterial (Halomonas sp. H8b, Halomonas sp. H12b, Salicola sp. H9b) and archeal
(Halorubrum sp. H3a, Halorubrum sp. H13a) strains were isolated from different salt lakes of Altai
region (Russia). Strains were grown in medium, which contained per liter 0-300 g NaCl, 5 g MgCl2, 1 g
KCl, 1 g CaCl2, 4 g tryptone, 2 g yeast extract, 10 ml of a trace metal solution, and 0-95 g MgSO4, 0-60 g
Na2SO4, 0-29 g NaClO4. To study survival at low temperatures strains were placed at -18 and -70 oC for 7
days during each freeze-thaw cycle. For exposure experiments cells were placed on solidified growth
medium of the same composition and incubated at 8, 22, 37 oC during 7 days. At least three exposure
experiments were performed. Cell numbers were estimated from CFU.
Results: In previous experiments [3] we found that halotolerant bacteria have the widest NaCl content
growth ranges. Obligatory halophilic archeal strains have smaller growth ranges and growth optimum at
200-300 g/L NaCl. Bacterial strains are more tolerant to different incubation temperatures. Studied strains
are able to grow at 8 oC.
Best behavior at high Na2SO4 and MgSO4 content is detected for Halomonas sp. H12b and Salicola
sp. H9b. At optimal NaCl content (200 g/L) the maximal NaClO4 content allowing grow of strains is 14
g/L.
The highest survival fraction after freeze-thaw cycles (1 and 2 cycles) at -18 and -70 oC is found for
optimal NaCl content and for experiments performed during summer period. Archeal strains were less
tolerant to freezing; the most significant mortality is detected at -70 °C, which was earlier demonstrated
for the halophilic archeobacterium Natronorubrum sp. [4]. Survival of Halorubrum sp. H7b was analyzed
at different temperatures (-10, -19, -28, -70 oC). Survival of this strain is higher for completely frozen
solution in comparison with partly frozen solution. Images obtained by electron microscope technique
show that low temperatures lead to destruction of cell membranes of strains which are just partly survived
after freeze-thaw cycles.
Discussion and Conclusions: Halomonas sp. Н12b and Halorubrum sp. Н13a strains are the most
resistant to wide range of extreme factors. Survival of studied halophiles at low NaClO4 content show that
presence of perchlorates in small amounts cannot be a limiting factor for survival of Earth’s
extremophiles at the subsurface layers of Martian regolith. Smaller decrease of survival of several strains
during second freeze-thaw cycle may be interpreted as possible evidence of existence of strains which are
able to grow after multiple freeze-thaw cycles at -70 oC typical for Martian nights. Experiments with
survival at different cooling temperatures show that partly frozen solutions are not optimal for survival of
microbes. Additional checking of this conclusion is possible by performing of planned experiments with
solutions having lower eutectic temperatures.
Acknowledgements. The financial support by Integration Projects 93 and 94 of the Siberian Branch of
RAS, RFBR 11-04-12093-ofi-m is gratefully acknowledged.
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