1. SURVIVAL OF HALOPHILES AT PRESENCE OF SULFATES AND PERCHLORATES A.V. Bryanskaya 1, A.A. Berezhnoy 2, A.S. Rozanov 1, S.E. Peltek 1, A.K. Pavlov 3 1 Institute of Cytology and Genetics, SB RAS, Novosibirsk, Russia 2 Sternberg Astronomical Institute, Moscow State University, Russia 3 Ioffe Physical-Technical Institute, St. Petersburg, Russia Contact: bal412003@mail.rubal412003@mail.ru

2. Search for life on Mars The electron microscope revealed chain structures in Martian meteorite ALH84001 Evidence of detection of remnants of Martian fossils – bacteria-like life forms? Meteorite ALH 84001 was delivered to Earth from Mars Exogenesys is hypothesis that proposes life on Earth was transferred from elsewhere in the Universe Early Mars has had dense atmosphere and liquid water on its surface.

3. Volatiles on Mars The surface of Mars with CO 2 frost. Viking image (NASA). Pure water is unable to exist on Martian surface now because it is too cold and the pressure is too low. However, salt solutions (for example, NaCl, MgSO 4, and CaCl 2 ) remain to be liquid until at -70 °C. 252 K273 K Potential local range Equator (T S = 218 K) ~2.3 - 3.4~3.7 - 5.51 - 10 Poles (T S = 155 K) ~6.5 - 9.7~7.9 - 11.83 - 20 Depth (km) to melting isotherm on Mars Sources of energy to support subsurface life are not so powerful as solar radiation at the surfaces of planets

4. Aim The aim of this study was to select bacterial and archeal strains most adapted to Martian conditions Martian extreme conditions: -dryness (water vapor content is about 0.01 % -low atmospheric pressure, just 0.05 bar - low night temperature, about -70 o C - high intensity of radiation, - low content of organic species, less than 10 ppm - high concentration of salts… Common-salt.jpg in bg.wikipedia.org

5. Previous experiments Strains Experiment Conditions Results Reference two up to 230 g/l NaCl, 50-80 o C, then 1 – 10 % Leuko et al., haloarchaeas 1 g/l yeast extract -60 o C for 6 hours survival 2002 two 200 g/l NaCl, 30 g/l MgSO 4 -70 o C and 0.3 – 1 % Weidler et al., haloarchaeas 3 g/l yeast extract, -196 o C survival 2002 Mars atmosphere five sulfate- 50-180 g/l MgSO 4, 37 o C reducing 50-170 g/l FeSO 4, for 10-80 % Marnocha et al., bacteria 100-480 g/l Fe 2 (SO 4 ) 3, five survival 2011 0.4 – 1.3 g/l proteins months non- 0.01 – 0.1 g/l glucose, desiccation, metabolism extremophilic 0.5 g/kg salts, 7 o C and 27 o C, and Pavlov et al., bacterium Mars atmosphere then -70 o C reproduction 2010

6. Why we choose halophiles? Earth’s microorganisms can be delivered to Mars by impacts of meteoroids of Earth’s origin and modern mission to Mars. To study of 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 microorganisms, because salt solutions on Mars could be more widely distributed through subsurface Martian soil in comparison with pure liquid water. The existence of salt solutions that could serve as media for organisms analogous to halophilic archaea at -23 °C and high salt concentrations on Mars has been widely discussed (Litchfield, 1998). Study of the elemental composition of the Martian soils shows high concentrations of chlorine (Taylor et al., 2010), perchlorates (Navarro- González et al., 2010), and solubable sulfates (Kounaves et al., 2010). At Viking landing sites the content of perchlorates and organic carbon is estimated to be < 0.1% and 0.7 – 6.5 ppm, respectively (Navarro-González et al., 2010). High abundance of perchlorates around 1% was discovered at Phoenix landing site (Hecht et al., 2009).

7. The location of microbes isolated from the salt lakes of Altai region

8. H4 Halorubrum sp H2 Halorubrum sp H11 Halorubrum sp H13 Halorubrum sp H3 Halorubrum sp H7 Halorubrum sp H1 Haloarcula sp Escherichia coli K12 H9 Salicola sp H8 Halomonas sp H6 Halomonas sp H12 Halomonas sp 98 100 51 98 99 100 29 58 61 0.05 Archaea Bacteria

9. Freezing and different NaCl content Bacterial (Halomonas sp. H8b, Halomonas sp. H12b, Salicola sp. H9b) and archeal (Halorubrum sp. H2a, Halorubrum sp. H3a, Halorubrum sp. H4a, Halorubrum sp. H7a, Halorubrum sp. H11a, Halorubrum sp. H13a) strains were isolated from different salt lakes of Altai region. Strains were grown in medium, which contained per liter 0-300 g NaCl, 5 g MgCl 2, 1 g KCl, 1 g CaCl 2, 4 g tryptone, 2 g yeast extract, and 10 ml of a trace metal solution, at 37 o C. For exposure experiments cells were suspended in a medium with the same NaCl concentration. Following treatments, cells were plated on solidified growth medium and incubated at 37 o C for several days. Cell numbers were estimated from CFU. Treatments were as follows: aliquots of cell suspensions were kept both at – 70 o C and - 18 o C for up to seven days. At least three exposure experiments were performed.

10. Ratio of the amount of survived microorganisms after freezing at -18 °C to that in the control sample versus different NaCl content.

11. Ratio of the amount of survived microorganisms after freezing at -70 °C to that in the control sample versus different NaCl content.

12. Range and optimum of growth of archaeal and bacterial strains under different content of NaCl Content of NaCl (g/l)Strain Best

13. Analysis of the results Bacterial strains were more tolerant to different incubation temperatures. Archeal strains were less tolerant to freezing The most significant mortality was detected at -70 °C, which was earlier demonstrated for the halophilic archeobacterium Natronorubrum sp. (Peeters et al., 2010). Judging from the results of our experiments, we can suggest that these are not halophilic archea but halotolerant bacteria that could be the analogs of Martian organisms, since they can survive wide mineralization ranges and low temperatures with the lowest decline of viability. For next experiments five cultures (H3a, H8b, H9b, H12b, H13a) were selected.

14. Survival at mixtures of NaCl and MgSO 4, Na 2 SO 4, NaClO 4 solutions 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. For exposure experiments cells were plated on solidified growth medium with different concentrations (0, 1, 7, 30, 50 %, where NaCl at 200 g/L was taken for 100 %) of NaClO 4, Na 2 SO 4, MgSO 4, respectively, and incubated at 37 o C for 7 days. Except mixture of NaCl with perchlorates or sulfates there are in 5 g MgCl 2, 1 g KCl, 1 g CaCl 2, 4 g tryptone, 2 g yeast extract, and 10 ml of a trace metal solution the medium. Cell numbers were estimated from CFU. At least three exposure experiments were performed.

15. Growing of microorganisms at different content of NaCl and Na 2 SO 4. The total content of salts is 200 g/L. The solution was incubated at 37 o C during 7 days.

16. Growing of microorganisms at different content of NaCl and MgSO 4. The total content of salts is 200 g/L. The solution was incubated at 37 o C during 7 days.

17. Growing of microorganisms at different content of NaCl and NaClO 4. The total content of salts is 200 g/L. The solution was incubated at 37 o C during 7 days.

18. Analysis Best behavior at high content of sulfates is detected for Halomonas sp. H12b and Salicola sp. H9b, probably, due to the fact that molar concentration of salts decreases with increasing content of sulfates while Halomonas sp. H12b and Salicola sp. H9b have optimal growth at lowest NaCl content (100 g/L) in comparison with other microorganisms. Best behavior during freezing is shown for bacterial strains H9b and H12b while archeal strains H3a and H13a grow well at presence of sodium perchlorate. For this reason we choose for next experiments best bacterial (Halomonas sp. Н12b) and archeal (Halorubrum sp. Н13a) strains.

19. Judging from the results of our experiments, we can suggest that halophilic archea and halotolerant bacteria could be the analogs of Martian organisms, since they can survive wide mineralization ranges and low temperatures with the lowest decline of viability.

20. The best microorganisms are Halomonas sp. H12b and Halorubrum sp. Н13a Next experiments: 1)growing of microorganisms al lower temperatures (+5, +8, and +25 o C, in our experiments +37 o C) 2)Growing of microorganisms at lower content of organic species (on Mars 7 ppm, in our experiments 4 000 ppm) 3)Survival of microbes at lower atmospheric pressure (in our experiments P = 1 bar)

21. Thank you for attention!