The extreme halophilic microorganisms, a

possible model for life on other planets

Dr. Sergiu Fendrihan

Res. Instit Plant Protection
Univeristy Vasile Goldis Arad
Romanian Bioresource Centre
 Living world
• The phylogentic tree of the living world
 Saline environments

The environments were live the extreme

halophyles are-hypersaline waters-lakes and
soda –saline lakes, solar salterns, halite-rock
salts, antropic hypersaline lakes, stromatolites,
saline soils, saline treated hides and skins,
intesive salted foods, in some Martian origin
meteorites.
 Halophilic organisms

In hyersaline waters, there are a lots of microorganisms and some organisms.

They can be
- Halotolerant 7-10% NaCl (Oren et al., 1999)
- Halophiles 10-20% conc salt and extreme halophiles.
- Those organisms are of many taxonomic units starting with the most
simples –Myoviridae viruses parazites to halophilic Archaea halophile,
viruses from family (Le Romancer et al. 2007). Archaea halophiles from
Halobacteriaceae family are spreaded in extremes saline evironments,
Photosynthetic halophilic bateria like cyanobacteria Halospirulina,
Aphanotece halophytica different Oscillatoria species (Das Sarma et Arora,
2001). There are too bacteria Halorhodospira (anaerobes) methanogenes
(Methanohalophilus halophilus) and Salinibacter (Anton et al, 2005).
In those environments there are halpphilic algae Asteromonas gracilis and
Dunaliella salina (Das Sarma et Arora 2001), si fungi like Wallemia
ichthyophaga, Candida famata, Gymnascella maris mortui (Gunde Cimerman
et al2003).Plants like Salicornia herbaceea live in soils with a big salt
concentration. Protozoars like the amoeba Tetramitus salina and ciliate
Fabrea salina live too in such environments. An crustacean very common in
hipesaline waters is Artemia salina.
 Halospirulina

Halorhodospira

Apahanotece

Wallemia

Artemia salina
Dunaliella salina
 History

The halophilic archaea are from Fillum Euryarchaeota,

Order Halobacteriales, Familly Halobacteriaceae
containing over 132 species in 37 genera, de actualizat
Were seen the first time on salted fish in 1880 by WG
Farlow and isolted by H Becker in 1912. The biologist
Klebahn isolated the Bacillus halobium ruber in 1919
and Sarcina morrhuae Another researcher studying the
group, is Elazari Volcani, which studied the
microorganisms from the Dead Sea and nominate the
genera Halobacterium and Halococcus.
 Halophyles environments

Flat salt surface

from evaporated
Great Salt Lake -USA
salt lake

Solar saltern salt production red

Salted lake colour is from presence of halophilic
archaea
 Salted fermented food
Salted fish

Salted hides
Meteorites of Matian origin with salt content
 Isolation of haloarchaea from rock salt

Following the researches of Norton , Grant and McGenity (1993 ) , Stan Lotter et
al (2001), Radax et al. (2001) and Fendrihan (2006), there are a lots of isolates
from rock salt which cannot be found in open environments. Some scientist think
that they live in fluid inclusion from geological eras.

Salt crystal with

rocksalt haloarhaea inside

The same details

Samples of boring haloarhaea prestained
cores of halite and embedded in salt
There are about 55 genera and 159 species of Halobacteriaceae
The most known are Halobacterium, Halococcus, Halorubrum,
Haloterrigena, Natronobacterium. They are in many extreme saline
waters, soils and others, where they resist due to theyr Na K pumps
energise by halorhodopsin, due to their metabolism, to their special
conformation of proteins and enzymes. The type genus is Halobacterium
with the species Halobacterium salinarum.
 Researches with Raman spectroscopy
We tested the Raman spectroscopy to detect the haloarchaea in
salt crystals in order to see if will possible to use this technique to
detection of life (organic substances) in Martian rocks taking into
account the halite minerals that can be found in Mars subsuface
and in sone Martian meteorites. In the second case a confocal
microscope BX40 (Olympus Corp., Japan) coupled to a Dilor XY
Raman spectrometer (Horiba Jobin Yvon, Longjumeau, France)
operated in the subtractive mode was used, the spectrometer
running with the Labspec 3.0 acquisition software.
Raman spectra of haloarhaea and without
 Exploration of Mars
In last decades the interest for Mars exploration is
growing, thats why there are a lots of researches and
project launched by Space Agencies.
 UV effect on haloarchea

Halococcus dombrowski H4 were growth in M2 culture media until it reached OD= 1.0
the he was centrifuged growth they were centrifugated at 5000 rpm in Eppendorf
minicentrifuge tubes, at 5000 rpm (2236 gs) 5 minutes with the rotor F 2402 of the
Beckman centrifuge, obtaining a thick pellet. For experiments were prepared 8
unpigmernted and 8 pigmented samples of concentrated haloarchaea, being adjusted
at OD (600nm)-1,5 diluted and mixed in about 1600 ul of TN buffer (233 g NaCl Sigma,
12,1 g Tris Base ICN Biochemicals, Millipore water was added at 1000 ml, pH adjusted
with conc. HCl at 7.4 4 M NaCl, Tris solution ) and aliquot of 40ul were deposed on
sterilized quartz disk of 11 mm diameter.and inserted in Tissue Culture Plate with lid
(Multiwell Primaria 24 well, Becton Dickinson Labware, USA) for drying, and obtaining
samples for UV irradiation with special UV lamp at different irradiation times.
Following irradiation, the samples were resuspended in 3,5M TN buffer 300ul each
and from this aliquots were use for cultivation on M2 solid culture media and cfu s
counting, 50 ul for LD test (LIVE DEAD BacLight Kit L-7012 (Molecular Probes USA),
100ul for the immunoassay experiments detection of CCD (Thymine Dimer Antibody
Gene Tex Inc, USA, and Goat Anti Mouse IgG antibodies Invitrogen Molecular Probes
USA), and 20ul for cultivation on liquid media in test tubes. The photos where taken
with a CCD camera of the fluorescence Microscope, and with a laser confocal
microscope allowing the quantification.
Equipment of irradiation used in
experiments
Aspect of culture and electonic microscope immge of H dombrowski H4
the strain used in experiments
 Culture
with and
without
pigments
Spectro
Culture photom Deposition
centrifugation of aliquots
in M2 eter on quart
disck
TN
LD Buffer
Kit
Survival
Cultiva analysis
tion Tissue
UV culture
irra
Molecular dati Drying plates with
analysis, on wells
immunologyy

Diagram of the experiments

Embedding in salt crystals

Crystals with haloarchaea and prestained

bacteria in fluid inclusions
 Quartz disks

Salt crystals with haloarchaea on quartz discs

Culture of H4 strain stained with LD Kit
1042 (Molecular Probes USA)
 Results

Preliminary results of survival tests using cultivation and flurorescence microscopy

showed that Hcc. dombrowskii, embedded in salt crystals, survived a dosis of 10
kJ/m2, which was about 20 fold higher than the dosis which was lethal to Hbt.
salinarum NRC-1 (in liquid medium). The influence of pigments on protection by UV
radiation is being investigated by inhibition of carotenoid synthesis of Hcc.
dombrowskii with diphenylamine and with glycerol inserted in culture media. For
sensitive detection of carotenoids in small samples, Raman spectrometry was
established with salt-embedded cells of Hcc. dombrowskii.
 Synoptic table with main result of ground experiments

Time energy Procent survival %

3600s 148,52 KJ 36,2

1800s 74,16 KJ 61,2

1020 s 42,02 KJ 62,6

765 s 31,52 KJ 68, 6

510 s 15,5 KJ 60, 6

255 s 10,5 KJ 66,2

 Space studies

Inernational Space Station and the trays prpared for the

experiments
NASA facilities
preparation of the
dispositive with
the samples for
EXPOSE
experiments
Launching
platform
3 4 19 (yellow) 20 (yellow) Disposal of
dark space dark space UV, space UV space the
pigmented not pigmented not
pigmented pigmented samples
7 8 23 (yellow) 24 (yellow)
dark space dark space, UV space UV space
pigmented not pigmented pigmented not
pigmented
11 12 27 28
dark space dark space UV space UV space
pigmented not pigmented pigmented not
pigmented
15 16 31 32
dark,space dark, space, UV space UV space
pigmented not pigmented not
pigmented pigmented

TRAY 1 Space conditions ADAPT 1 Halococcus dombrowski H4

under 110nm (neutral density filters 100%MgF2) no?
131 132 147 (yellow) 148 (yellow)
dark, Mars CO2 dark, Mars CO2 UV, Mars CO2 UV, Mars CO2
pigmented not pigmented pigmented Not pigmented
135 136 151 (yellow) 152(yellow)
dark, Mars CO2 dark, Mars CO2 UV, Mars CO2 UV, Mars CO2
pigmented Not pigmented pigmented not pigmented
139 140 155 156
dark, Mars CO2 dark, Mars CO2 UV, Mars CO2 UV, Mars CO2
pigmented Not pigmented pigmented not pigmented
143 144 159 160
dark, Mars CO2 dark, Mars CO2 UV, Mars CO2 UV, Mars CO2
pigmented Not pigmented pigmented not pigmented

TRAY 2 Mars Conditions ADAPT 1 Halococcus dombrowski

H4 under 200nm (neutral density filters Suprasil ) Mars CO2
 Conclusions

The extreme halophylic Archaea can resist to long time and big dosis of irradiation,
the survival was about 35%.
The carotenoids and bacteriorhodopsin looks like to not offer much protection in
time of space irradiation.
It looks like the mechanisms of repair of DNA can play a role in this case.
After about 2 years in International Space Station the survival was about 9 %, in the
case of halophylic archaea protected by a thin layer of salt .Further researches is
necessary to understand the inner and molecular mechanisms.