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SURVIVAL OF HALOPHILES AT PRESENCE OF SULFATES AND PERCHLORATES

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

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SURVIVAL OF HALOPHILES AT PRESENCE OF SULFATES AND PERCHLORATES

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  1. SURVIVAL OF HALOPHILES AT PRESENCE OF SULFATES AND PERCHLORATES A.V. Bryanskaya1, A.A. Berezhnoy2, A.S. Rozanov1, S.E. Peltek1, A.K. Pavlov3 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.ru

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

  3. Volatiles on Mars 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, MgSO4, and CaCl2) remain to be liquid until at -70 °C. The surface of Mars with CO2 frost. Viking image (NASA). Sources of energy to support subsurface life are not so powerful as solar radiation at the surfaces of planets Depth (km) to melting isotherm on Mars

  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 oC • - high intensity of radiation, • -low content of organic species, • less than 10 ppm • -high concentration of salts… Common-salt.jpg inbg.wikipedia.org

  5. Previous experiments Strains Experiment Conditions Results Reference two up to 230 g/l NaCl, 50-80 oC, then 1 – 10 % Leuko et al., haloarchaeas 1 g/l yeast extract -60 oC for 6 hours survival 2002 two 200 g/l NaCl, 30 g/l MgSO4 -70 oC and 0.3 – 1 % Weidler et al., haloarchaeas 3 g/l yeast extract, -196 oC survival 2002 Mars atmosphere five sulfate- 50-180 g/l MgSO4, 37oC reducing 50-170 g/l FeSO4, for 10-80 % Marnocha et al., bacteria 100-480 g/l Fe2(SO4)3 , five survival 2011 0.4 – 1.3 g/l proteinsmonths non- 0.01 – 0.1 g/l glucose, desiccation, metabolism extremophilic 0.5 g/kg salts, 7 oC and 27 oC, and Pavlov et al., bacterium Mars atmosphere then -70 oC 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 (Tayloret al., 2010), perchlorates (Navarro-Gonzálezet 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-Gonzálezet al., 2010). High abundance of perchlorates around 1% was discovered at Phoenix landing site (Hechtet al., 2009).

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

  8. Archaea H4Halorubrum sp 98 100 H2 Halorubrum sp H11 Halorubrum sp 99 H13 Halorubrum sp 100 H3 Halorubrum sp 98 H7 Halorubrum sp 51 H1 Haloarcula sp Escherichia coli K12 H9 Salicola sp H8 Halomonas sp 61 H6 Halomonas sp 58 H12 Halomonas sp 29 0.05 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 MgCl2, 1 g KCl, 1 g CaCl2, 4 g tryptone, 2 g yeast extract, and 10 ml of a trace metal solution, at 37oC. 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 37oC for several days. Cell numbers were estimated from CFU. Treatments were as follows: aliquots of cell suspensions were kept both at – 70 oC and - 18 oC 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 Strain Content of NaCl (g/l) Best Best Best Best Best

  13. Analysis of the results Bacterialstrainsweremoretolerant todifferentincubationtemperatures. Archealstrainswerelesstoleranttofreezing Themostsignificantmortalitywasdetectedat -70 °C, whichwasearlierdemonstratedforthehalophilicarcheobacteriumNatronorubrumsp. (Peeters et al., 2010). Judgingfromtheresultsofourexperiments, wecansuggestthatthesearenothalophilicarcheabuthalotolerantbacteriathatcouldbetheanalogsofMartianorganisms, sincetheycansurvivewidemineralizationrangesandlowtemperatureswiththelowestdeclineofviability. For next experiments five cultures (H3a, H8b, H9b, H12b, H13a) were selected.

  14. Survival at mixtures of NaCl and MgSO4, Na2SO4, NaClO4 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 NaClO4, Na2SO4, MgSO4, respectively, and incubated at 37 oC for 7 days. Except mixture of NaCl with perchlorates or sulfates there are in 5 g MgCl2, 1 g KCl, 1 g CaCl2, 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 Na2SO4. The total content of salts is 200 g/L. The solution was incubated at 37 oC during 7 days.

  16. Growing of microorganisms at different content of NaCl and MgSO4. The total content of salts is 200 g/L. The solution was incubated at 37 oC during 7 days.

  17. Growing of microorganisms at different content of NaCl and NaClO4. The total content of salts is 200 g/L. The solution was incubated at 37 oC 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. Н13aNext experiments: • growing of microorganisms al lower temperatures (+5, +8, and +25 oC, in our experiments +37 oC) • Growing of microorganisms at lower content of organic species (on Mars 7 ppm, in our experiments 4 000 ppm) • Survival of microbes at lower atmospheric pressure (in our experiments P = 1 bar)

  21. Thank you for attention!

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