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!