survival of halophiles at presence of sulfates and perchlorates a.v. bryanskaya 1, a.a. berezhnoy 2,...
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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, Russia2 Sternberg Astronomical Institute, Moscow State University, Russia
3 Ioffe Physical-Technical Institute, St. Petersburg, Russia Contact: bal412003@mail.ru
Search for life on Mars
The electron microscope revealed chain structures in Martian meteorite ALH84001Evidence 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 atmosphereand liquid water on its surface.
Volatiles on Mars
The surface of Mars with CO2 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, MgSO4, and CaCl2) remain to be liquiduntil at -70 °C.
252 K 273 K Potential local
range
Equator (TS =
218 K) ~2.3 - 3.4 ~3.7 - 5.5 1 - 10
Poles (TS =
155 K) ~6.5 - 9.7 ~7.9 - 11.8 3 - 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
AimThe 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 in bg.wikipedia.org
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, 37oCreducing 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 proteins months
non- 0.01 – 0.1 g/l glucose, desiccation, metabolismextremophilic 0.5 g/kg salts, 7 oC and 27 oC, and Pavlov et al.,
bacterium Mars atmosphere then -70 oC reproduction 2010
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).
The location of microbes isolated from the salt lakes of Altai region
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
Freezing and different NaCl contentBacterial (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.
Ratio of the amount of survived microorganisms after freezing at -18 °C to that in the control sample versus different NaCl content.
0
0.2
0.4
0.6
0.8
1
0 50 100 150 200 250 300 350
Survival after
freezing
NaCl content, g/l
H12b
H8b
H9b
H3aH13a
H11a
H7aH4a
H2aNo growth
Ratio of the amount of survived microorganisms after freezing at -70 °C to that in the control sample versus different NaCl content.
0
0.2
0.4
0.6
0.8
1
0 50 100 150 200 250 300 350
Survival after
freezing
H12b
H9b
H8b
H13a H3a
H7aH4a
H11a
H2aNaCl
content, g/lNo growth
Range and optimum of growth of archaeal and bacterial strains under different content of NaCl
Content of NaCl (g/l)Strain
Best
Best
Best
Best
Best
Analysis of the resultsBacterial 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.
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.
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.
0
2
4
6
8
10
12
0 5 10 15 20 25 30Mass wt%, Na2SO4
H3aH8b
H12b
H9b
H13a
CFU, 106/ml
Mass wt%, NaCl100 95 90 85 80 75 70
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.
0
2
4
6
8
10
12
0 10 20 30 40 50
H8b
H9bH13a
H12bH3a
Mass wt%, MgSO4
CFU, 106/ml
Mass wt%, NaCl100 90 80 70 60 50
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.
0
2
4
6
8
10
12
0 1 2 3 4 5 6 7 8Mass wt %, NaClO4
CFU, 106/ml
H13a
H3a
H12b
H9bH8b
Mass wt%, NaCl
100 98 96 9294
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.
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.
The best microorganisms are Halomonas sp. H12b and Halorubrum sp. Н13a
Next experiments:
1) growing of microorganisms al lower temperatures (+5, +8, and +25 oC, in our experiments +37 oC)
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)
Thank you for attention!Thank you for attention!
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