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Halophilic archaea on Earth
and in space: growth and
survival under extreme
conditions
rsta.royalsocietypublishing.org
Review
Cite this article: Oren A. 2014 Halophilic
archaea on Earth and in space: growth and
survival under extreme conditions. Phil. Trans.
R. Soc. A 372: 20140194.
http://dx.doi.org/10.1098/rsta.2014.0194
One contribution of 14 to a Theme Issue
‘Raman spectroscopy meets extremophiles on
Earth and Mars: studies for successful search of
life’.
Subject Areas:
astrobiology
Keywords:
Halobacteriaceae, anaerobic, perchlorate,
Mars, carotenoids, Raman spectroscopy
Author for correspondence:
Aharon Oren
e-mail: [email protected]
Aharon Oren
Department of Plant and Environmental Sciences, The Alexander
Silverman Institute of Life Sciences, The Edmond J. Safra Campus,
Givat Ram, Jerusalem 91904, Israel
Salts are abundant on Mars, and any liquid water that
is present or may have been present on the planet
is expected to be hypersaline. Halophilic archaea
(family Halobacteriaceae) are the microorganisms best
adapted to life at extremes of salinity on Earth. This
paper reviews the properties of the Halobacteriaceae
that may make the group good candidates for life
also on Mars. Many species resist high UV and
gamma radiation levels; one species has survived
exposure to vacuum and radiation during a space
flight; and there is at least one psychrotolerant species.
Halophilic archaea may survive for millions of years
within brine inclusions in salt crystals. Many species
have different modes of anaerobic metabolism, and
some can use light as an energy source using the
light-driven proton pump bacteriorhodopsin. They
are also highly tolerant to perchlorate, recently
shown to be present in Martian soils, and some
species can even use perchlorate as an electron
acceptor to support anaerobic growth. The presence of
characteristic carotenoid pigments (α-bacterioruberin
and derivatives) makes the Halobacteriaceae easy to
identify by Raman spectroscopy. Thus, if present on
Mars, such organisms may be detected by Raman
instrumentation planned to explore Mars during the
upcoming ExoMars mission.
1. Introduction
The ExoMars mission to Mars, planned to be launched
in 2018, will include Raman spectroscopy equipment
to probe the chemical properties of the environment
near the landing site [1]. As Raman spectroscopy is a
2014 The Author(s) Published by the Royal Society. All rights reserved.
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It has been stated that neither liquid brines nor liquid water are currently stable on Mars [2,3].
Still, liquid brines could be temporarily present in certain areas, and the existence of stable brines
in the Martian subsurface cannot be excluded [2]. There is even some evidence for the presence
of liquid water at shallow depths beneath the surface [4].
The observation of ‘recurring slope lineae’, what looks like seasonal flows on (relatively)
warm Martian slopes as observed by the Mars Reconnaissance Orbiter [5], revived the discussion
about the possible availability of liquid brines on present-day Mars. Temperatures on these
slopes during the warm season can be as high as 250–300 K. It was calculated that the observed
seasonality of the apparent flows on these slopes could be best explained by a solution with a
freezing temperature of approximately 223 K (approx. −50◦ C) [6].
Salts are considered to be an important component of the fine-grained regolith on Mars,
with (Mg,Na)SO4 , NaCl and other chlorides, and (Mg,Ca)CO3 as likely components [7].
The presence of hygroscopic salts such as NaCl, CaCl2 and MgCl2 may also lead to
the formation of droplets of liquid brine, at least locally and temporarily, as a result of
deliquescence [8,9].
Another, only recently recognized component of the salts on Mars is perchlorate. The source of
the formation of these perchlorate salts and the implications of their presence for the possibility
of life on the planet are discussed in §4e. Perchlorate salts of sodium and magnesium are
highly hygroscopic, and they can absorb moisture and form approximately 1 mm large liquid
spheroids at temperatures as low as 225 K [10], thus increasing the availability of liquid water
at least locally.
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2. Salts and brines on Mars
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sensitive technique for the detection of certain biological molecules such as carotenoid pigments,
the ExoMars mission will provide new possibilities to search for specific biomarkers as an
indication of present or past life on Mars.
If life exists today or has existed on Mars in earlier periods when conditions on the planet
were more conductive to life, it is tempting to speculate that such life was based on salt-requiring
and/or salt-tolerant microorganisms. Life depends on the presence of liquid water, and liquid
water can exist at the temperatures prevailing on Mars only when the freezing temperature
becomes sufficiently depressed by dissolved salts. It is becoming increasingly clear that chloride
and sulfate salts are abundantly present on Mars. A search for halophilic microorganisms
may therefore be a major goal in the quest for the discovery of life on Mars, now or in
the past.
Life in hypersaline environments on Earth is extremely diverse, as many different types of
microorganisms (and a few macroorganisms as well) are adapted to exist in brines, often even up
to NaCl saturation. The halophiles par excellence are the members of the family Halobacteriaceae,
which belongs to the Euryarchaeota phylum of the archaea. They are by no means the only
microorganisms able to survive and even to thrive at the highest salinities, but they are the most
abundant ones and also the most versatile ones. Moreover, they contain specific carotenoids and
other molecules that can be used as biomarkers that can be detected with great sensitivity using
Raman spectroscopy.
This review paper first summarizes the evidence for the existence of salts and brines on
Mars, and then explores different hypersaline environments on Earth as possible analogues that
simulate the Martian environment to different extents as a potential biotope. The properties of
the halophilic archaea family Halobacteriaceae are then presented with special emphasis on those
features that may be relevant for survival and even growth on Mars. Finally, the applications
of Raman spectroscopy for the detection of halophilic archaea, and some other halophilic
microorganisms as well, in extreme environments on Earth and in laboratory simulation
experiments are discussed in view of the possibility to search for similar biomarkers by the
ExoMars Lander in the near future.
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3. Model systems on Earth for the study of life on Mars
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rsta.royalsocietypublishing.org Phil. Trans. R. Soc. A 372: 20140194
Space exploration programmes, including the search for life on Mars and other planets, have
greatly benefitted from the exploration of the most extreme environments on Earth as model
systems, simulating at least to some extent the kind of conditions to which microorganisms
on Mars may be exposed. Terrestrial model systems were selected to represent different
combinations of extreme conditions: wet and hypersaline, based on dominance of NaCl, CaSO4
or MgSO4 (thus including both kosmotropic-stabilizing and chaotropic-destabilizing ions [11]),
dry and hypersaline, cold and hypersaline, and acidic and hypersaline.
Wet and hypersaline NaCl-based extreme environments used as model systems for the study
of life on Mars include continental saline environments such as evaporites of gypsum, halite and
other materials in the alkaline Wadi Natrun, Egypt, the Salar de Atacama and other evaporite sites
in the Atacama Desert, and Tunisian sabkhas [12,13]. The study of endoevaporitic cyanobacteria
and other microorganisms within gypsum and halite evaporites in the lagoons of Baja California,
Mexico [14] also belongs to this category. Permian (260 Ma) and Miocene (6 Ma) gypsumpermineralized microfossils from New Mexico and Italy, respectively, as well as recent gypsum
deposits from Australia, Mexico and Peru containing microfossils of diatoms, cyanobacteria and
other microorganisms were also studied as model systems for the search for life on Mars. These
studies included Raman spectroscopy among the methods used [15].
As Martian soils may contain magnesium sulfate as the dominant salt, it is also interesting
to explore life in terrestrial environments rich in epsomite (MgSO4 · 7H2 O). One of these
environments is the soil of the Great Salt Plains of Oklahoma. About one-third of the isolates
collected from the site grew in the presence of 2 M MgSO4 , and these included representatives
of the bacterial genera Halomonas, Staphylococcus and Halobacillus [16]. No epsomite-tolerant
members of the archaea were retrieved during this study. Another magnesium-sulfate-rich
environment is Hot Lake, WA, USA, which recently became an object of study once more, after
having been neglected for many years. A molecular and phenetic characterization of the bacterial
assemblage of Hot Lake was made in view of its potential importance as a model system for
life on Mars. All isolates able to grow in the presence of 1.7 M NaCl or 2 M MgSO4 belonged to
the bacteria, and included both Gram-positive and Gram-negative types. The most frequently
cultured species belonged to the genera Halomonas, Idiomarina, Marinobacter, Marinococcus,
Nesterenkonia, Nocardiopsis and Planococcus. No archaea were isolated during this study, and
a parallel culture-independent, 16S rRNA gene-based study showed very low abundance of
archaea [17].
The possibility for halophilic life to persist under conditions of extreme aridity was extensively
investigated in the Atacama Desert, Chile, which is one of the driest places on our planet.
In certain sites, halite evaporites are found, which are inhabited by unicellular cyanobacteria
morphologically similar to Chroococcidiopsis, but phylogenetically closer to Halothece [18]. Further
Atacama Desert studies, including the use of Raman spectroscopy to search for specific
biomarkers, are discussed in §5.
In view of the low temperatures prevailing on Mars, investigations of the microbiology of cold
and hypersaline environments on Earth are also of interest. However, only very few such studies
have been performed thus far. There exist a few hypersaline lakes in Antarctica. One of these,
Deep Lake located in the Vestfold Hills, is the source of Halorubrum lacusprofundi [19], an archaeon
whose properties are described in §4d. A recent study of the brines of an ice-sealed Antarctic lake
(Lake Vida, Victoria Valley, East Antarctica) with a salinity of 200 showed presence of a diverse
community of bacteria displaying a low level of radiolabelled leucine at the in situ temperature
of −13◦ C [20].
The possibility that some Martian soils may not only be saline, but also acidic makes it
worthwhile to search for analogous salty and acidic sites on our planet. Such sites are very rare.
There are a number of small ephemeral acidic and hypersaline lakes in Western Australia that
can serve as model systems for Mars-related studies [21]. The most acidic of these lakes included
in a survey had pH values of 2.8, 3.0, 4.2 and 4.3, with 83, 160, 214 and 40 ppt total dissolved
3
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Halophilic life on planet Earth is extremely diverse. We find microorganisms adapted to life
at high salt concentrations, even up to NaCl saturation, in all three domains of life: archaea,
bacteria and eukarya. Functionally, halophiles can perform nearly all processes known at low salt
concentrations: oxygenic and anoxygenic photosynthesis, respiration using molecular oxygen or
other electron acceptors such as nitrate and sulfate, fermentation and chemoautotrophy [24]. To
osmotically adjust the intracellular environment with the external medium, different strategies
have evolved. One strategy is based on the accumulation of high concentrations of K+ and Cl−
ions within the cell. This option is used by a few groups only: the Halobacteriaceae (archaea)
and Salinibacter, an extremely halophilic member of the Bacteroidetes (bacteria) found in the
same habitats where members of the Halobacteriaceae thrive. This ‘salt-in’ strategy requires a
far-going adaptation of the intracellular enzymatic machinery to the constant presence of molar
concentrations of KCl. A different, and far more widespread strategy, used by most other groups
of halophilic and halotolerant microorganisms, is the biosynthesis and/or accumulation from the
environment of simple, generally electroneutral organic ‘compatible’ solutes used to provide the
necessary osmotic balance [24,25].
This review explores the suitability of archaea of the family Halobacteriaceae as candidates
to grow and/or survive on Mars, now or in the past. The family contains those microorganisms
growing up to the highest salt concentrations and better adapted to life at high salt than any other
known microbial group. As of February 2014, the family was composed of 47 genera with a total
of 165 species. Basically, the Halobacteriaceae are aerobic chemoheterotrophic microorganisms
that obtain their energy by the oxidation of simple organic compounds using molecular oxygen
as the electron acceptor for respiration [26]. At first sight, organisms with such a mode of
metabolism are not very promising candidates for extraterrestrial life. However, many of the
halophilic archaea that inhabit the hypersaline brines worldwide have a surprising metabolic
versatility, and/or have unusual properties enabling their survival and possibly even growth
under the kind of conditions prevailing on Mars. These extreme halophiles are therefore excellent
models for astrobiology studies, even to the extent that DasSarma termed these organisms as
‘exophiles’ [27]. Their importance for the study of life on Mars has been realized since the last
years of the twentieth century [28].
The following paragraphs summarize the different properties that make the Halobacteriaceae
relevant model organisms for studies on life on Mars and also possibly elsewhere in the universe.
(a) Diverse modes of energy generation in the absence of molecular oxygen
Although the Halobacteriaceae generally grow as aerobic heterotrophs, many members of the
family possess different modes of energy generation and even anaerobic growth under anaerobic
conditions [29,30]. One such way is the use of light energy instead of chemical energy obtained
by aerobic respiration. Many members of the family can produce the membrane-bound purple
protein bacteriorhodopsin, a 25 kDa protein that carries a retinal moiety bound as a Schiff base
to one of its lysine residues. Bacteriorhodopsin acts as a light-driven proton pump. Following
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4. The Halobacteriaceae as model organisms and the properties that may make
them suitable for life on Mars
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salts, respectively. Using bacteria-specific primers, a number of 16S rRNA sequences were
retrieved from these lakes, belonging to different groups: mainly Proteobacteria, Bacteroidetes
and Actinobacteria [22]. No reports exist yet on the isolation of specialized acidophilic halophiles
from these lakes. Another saline and acidic environment suggested as a suitable Mars analogue
is the endolithic community existing in the acidic salt (natrojarosite) deposits of Rio Tinto, Spain,
whose waters have a pH of approximately 1.0. In a culture-independent molecular study, small
subunit rRNA sequences of both prokaryotic and eukaryotic phototrophic microorganisms were
retrieved from these acidic salt deposits [23].
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A halophilic archaeon isolated from evaporites in Baja California, Mexico and originally
designated ‘Haloarcula isolate G’ was exposed to high radiation in space. It was dried on quartz
discs and flown for 15 days in orbit in 1994 in the BIOPAN facility, a small retrievable capsule
developed by the European Space Agency for exposure of biological samples to conditions of
outer space. After exposure to 104 kJ m−2 radiation in high vacuum, part of the cells (about
one out of 107 –108 cells, when compared with a laboratory control experiment) had retained its
viability, as shown by colony counts on agar plates and growth in dilutions in liquid culture [33].
A comparative study using the archaeal ‘Haloarcula isolate G’ and Halobacterium salinarum NRC-1
and different non-halophiles (Deinococcus radiodurans, Escherichia coli, Pseudomonas fluorescens),
testing survival after desiccation and subsequent exposure to −20 or −80◦ C, showed that the
halophiles survived better under dry and low-temperature conditions than the non-halophiles.
The two halophilic archaea survived desiccation and 10 freeze–thaw cycles at −20 and −80◦ C
for at least 144 days, whereas E. coli and P. fluorescens did not survive any of the treatments [34].
A later taxonomic study identified ‘isolate G’ not as a member of the genus Haloarcula, but instead
belonging to the genus Halorubrum. Together with similar strains obtained from Western Australia
and from Naxos, Greece, it was described as a new species, Halorubrum chaoviator, ‘the traveller
of the void’, referring to the exposure of the type strain to conditions of outer space [35].
The 1994 BIOPAN experiment was not the only occasion at which members of the
Halobacteriaceae were sent into space. There have been at least two other missions that included
survival experiments with halophilic archaea. During the EXPOSE-E mission on the Space Shuttle
Atlantis, launched in February 2008, Halococcus dombrowskii was exposed for 559 days to the space
environment, and Halorubrum chaovator was sent once more into space in November 2008 with
the EXPOSE-R mission with the Russian Progress 31-P [36]. Unfortunately, no results of these
experiments have yet been published.
There have been numerous studies to assess the resistance of halophilic archaea to UV
radiation, and some of these were specifically intended to investigate the possibility for such
organism to survive on Mars. Cells of H. dombrowskii, an isolate obtained from a salt mine [37],
and other members of the family (Hbt. salinarum NRC-1, Haloarcula japonica) embedded in a thin
layer of laboratory-grown halite showed no loss of viability (assessed using the LIVE/DEAD kit
dyes) after exposure to 21 kJ m−2 , and they resumed growth in liquid medium with lag phases of
12 days or more after exposure to 148 kJ m−2 of radiation supplied by a ‘Mars UV simulator lamp’.
The D37 (dose of 37% survival) was greater than or equal to 400 kJ m−2 . However, in liquid culture,
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(b) Survival in the space environment exposed to high radiation and high vacuum
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absorption of a photon (maximum absorbance at 570 nm), the molecule undergoes a complex
photocycle during which the Schiff base is sequentially protonated and deprotonated. Protons
are taken up from the cytoplasm, and during deprotonation released to the external medium.
A proton gradient is thus established, which can be used to drive the formation of adenosine
triphosphate. Anaerobic light-driven growth is thus possible, but organic substrates are still
needed as carbon source [29]. Photoautotrophic growth is not possible.
Many members of the Halobacteriaceae can replace oxygen with alternative electron acceptors
for respiration. The ability to reduce nitrate and grow by denitrification is widespread within
the group. Other electron acceptors supporting growth by anaerobic respiration in many species
are trimethylamine N-oxide, dimethylsulfoxide, fumarate and even perchlorate and chlorate (see
§4e). Fermentative growth is possible in a few species of Halobacteriaceae only. Members of the
genus Halobacterium, but as far as is known not of any of the other genera of the family, can
grow anaerobically by fermentation of the amino acid arginine [29]. One species, Halorhabdus
tiamatea, a non-pigmented extremely halophilic archaeon isolated from Shaban Deep, a deep-sea,
hypersaline anoxic basin on the bottom of the Red Sea, prefers an anaerobic or microaerophilic
lifestyle over respiration with oxygen [31]. The mode of fermentation used by this species is not
yet clear, but analysis of its genome showed presence of an L-lactate dehydrogenase, which may
participate in the process [32].
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When halite crystals are formed during the evaporation of salt-saturated brines, brine inclusions
often remain within the crystals. Halophilic archaea (and other microorganisms as well) that were
present in the brine can thus become entrapped within the growing crystals. Many experiments in
the past two decades have shown that members of the Halobacteriaceae can retain their viability
within halite crystals for periods of thousands and possibly of millions of years [25,37]. Several
species were isolated from underground salt deposits. One such isolate is H. dombrowskii, obtained
from Permian salt from an Austrian salt mine and named in honour of Heinz Dombrowski
who pioneered isolation of microorganisms from ancient material such as salt deposits in the
1960s [49]. Other examples are Halococcus salifodinae, also from Permian rock salt in Austria, and
Halosimplex carlsbadense from Permian salt in New Mexico.
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(c) Longevity of dormant cells of Halobacteriaceae entrapped within salt crystals
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the D37 was only approximately 1 kJ m−2 [38]. A comparison of the survival of the haloarchaea
Natrialba magadii (an alkaliphilic species) and Haloferax volcanii exposed to vacuum UV radiation
with the radiation-resistant bacterium D. radiodurans showed that Nab. magadii survived exposure
to high vacuum for 1 h better than Deinococcus. Following doses of synchrotron radiation up to
150 J m−2 (57.6–200 nm), the two archaea survived as well as Deinococcus, but at higher doses, the
archaea performed less well [39,40].
One of the factors that may contribute to the high degree of resistance of halophilic archaea
to high levels of ionizing radiation, in addition to the presence of multiple copies of the genome
within each cell, is the high intracellular concentration of halide ions (chloride, possibly bromide
as well) that act as ‘chemical chaperones’. The intracellular salts may protect the cells against
radiation damage by scavenging reactive oxygen species, as shown in a study with Hbt. salinarum
NRC-1 [41].
A number of research efforts have targeted the mechanisms and the enzymes responsible for
the UV resistance of Halobacteriaceae. When exposed to UV-C (254 nm, up to 500 J m−2 ), the
nucleotide excision repair genes uvrA, uvrB and uvrC were upregulated in Halococcus hamelinensis,
an isolate from living stromatolites in Shark Bay, Western Australia. When incubated in the light,
a 20-fold increase in photolyase phr2 was also observed [42].
The uvr genes and phr2 were also identified as relevant to excision repair after UV-induced
damage in Hbt. salinarum NRC-1, an organism that withstands up to 110 J m−2 UV radiation [43].
Different repair mechanisms were identified in this organism: photoreactivation, d(CTAG)
methylation-directed mismatch repair, four oxidative damage repair enzymes and two proteases
for eliminating damaged proteins [44]. This organism is also highly resistant to desiccation, high
vacuum and 60 Co gamma radiation (5 kGy at least), and double-strand breaks in the DNA are
efficiently repaired. Among the factors implicated in its radiation resistance are the carotenoid
pigment bacterioruberin as well as the intracellular salts [45]. Mutant studies using Hbt. salinarum
NRC-1 showed the homologous recombination protein Mre11 to be essential for recovery
following UV-C exposure, but the Rad50 protein is not essential for the Mre11-dependent repair
of DNA double-strand breaks [46].
Another factor that may contribute to the high radiation resistance of Hbt. salinarum NCR-1,
and possibly of other members of the family as well, is the high intracellular concentration
of manganese ions found in manganese–antioxidant complexes. Mutants resistant to increased
doses of ionizing radiation (60% survival at 17 Gy, when compared with 5 Gy for the parent strain)
contained increased intracellular concentrations of manganese (15–30 µM). However, deletion
mutants lacking superoxide dismutase and catalase showed no decreased survival after exposure
to ionizing radiation [47].
Not all members of the Halobacteriaceae, however, are equally resistant to exposure to UV
radiation. Natronorubrum strain HG-1, an isolate from a hypersaline lake the Kulunda Steppe,
Altai, Russia, cannot survive for more than a few hours in a simulated Martian environment
with UV exposure similar to that at the surface of Mars, low temperatures (+4, −20, −80◦ C) and
desiccation [48].
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Little is known about the growth and adaptation mechanisms of halophilic archaea to life at low
temperatures. The best studied cold environment that harbours a community of Halobacteriaceae
is Deep Lake, located in the Vestfold Hills, Antarctica. This hypersaline (210–280 g l−1 salts)
lake never freezes as a result of the high salinity, not even at the minimum recorded water
temperature of −20◦ C. Water temperatures above 0◦ C are observed only in the top few metres
during the summer months, and the highest measured temperature is +11.5◦ C. It was estimated
that no more than six generations of archaea can develop annually at the in situ temperatures [54].
Halorubrum lacusprofundi was isolated from this lake. Its optimal growth is found at 31–37◦ C,
but slow growth was still recorded at 4◦ C, a temperature too low for the other members
of the family [19]. The ability to synthesize unsaturated diether lipids, which are otherwise
seldom found in the archaea, may be one key adaptation of Hrr. lacusprofundi to life at low
temperatures [55]. A recent metagenomic study of the archaeal community inhabiting Deep
Lake showed that the lake is populated, in addition to Hrr. lacusprofundi, by three other types of
Halobacteriaceae: a Halohasta sp., a Halobacterium sp. and a type designated DL31, not belonging
to any of the currently recognized genera. The complete genomes of all four types could be
reconstructed from the metagenomic data [54], but except for Hrr. lacusprofundi no other cultured
halophilic cold-tolerant haloarchaea have yet been described.
(e) Perchlorate reduction
The wet chemistry laboratory of the Phoenix Lander found high concentrations of perchlorate
in Martian soils near its landing site: leachates of soil samples contained 0.4–0.6% perchlorate
by mass. Martian soils are thus highly oxidizing [56]. The perchlorate is probably formed by
atmospheric interactions between ozone and volatile chlorine compounds [57,58]. Perchlorate
salts are highly deliquescent, and therefore they may promote the formation of droplets or films
of liquid water, thereby enhancing the possibilities for life [57]. On the other hand, perchlorates
are strong oxidizers, and their presence may be harmful to life forms.
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(d) Adaptation to low temperatures
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Many discussions have been devoted to the question whether indeed such isolates represent
organisms that grew many million years ago and have remained viable in a dormant state since
the time they became entrapped within halite crystals. It is not easy to prove that the organisms
have not grown in situ more recently following local dissolution of some of the salt [50]. But
evidence is accumulating that halophilic archaea may indeed survive for millions of years, and
then can either be revived as colonies on agar plates or be identified based on 16S rRNA gene
sequences. It is not possible to discuss all the published data within the framework of this brief
review paper.
Recent studies of cores from Death Valley, CA containing layers of halite deposited 10–35 ka
ago and from Saline Valley, CA (salt layers dated 75 and 150 ka) have greatly contributed to our
insights into the longevity of halophilic archaea within salt crystals that serve as repositories for
microbial life. Microscopic examination shows the prokaryotic cells entrapped in brine inclusions
within the salt crystals to be smaller than their modern counterparts grown in nutrient-rich
media [51]. Reduction in cell size is commonly found as a reaction to prolonged starvation. Still,
it must be asked how such cells may have survived for so many years in the absence of a supply
of organic carbon and energy sources. A partial answer to this question is that not only do cells
of Halobacteriaceae become entrapped within the growing salt crystals, but also cells of the alga
Dunaliella, generally found in salt-saturated brines together with the archaea. Dunaliella cells are
large and contain huge amounts of glycerol produced to provide osmotic stabilization for this
alga, which in contrast to the archaea excludes salt from its cytoplasm. The organic material of a
single Dunaliella cell may suffice as a carbon and energy source to support survival of halophilic
archaea for prolonged periods [52,53].
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The first pioneering studies of the detection of carotenoids of members of the Halobacteriaceae
by resonance Raman spectroscopy were published by Marshall et al. [63]. In view of the finding
of halite and sulfate evaporites on Mars, they provided the first Raman spectra of bacterioruberin
carotenoids and of bacteriorhodopsin, using Hbt. salinarum, Halococcus morrhuae and Natrinema
pallidum as model organisms. To test the suitability of Raman spectroscopy for the detection
of extremely halophilic archaea embedded in halite in terrestrial and possibly extraterrestrial
samples as well, Fendrihan et al. [64] embedded different species of Halobacteriaceae in growing
halite crystals. Strong bands of carotenoids (bacterioruberin) were detected as well as signals
derived from peptide bonds and nucleic acids. Raman spectroscopy was also used to probe
colonies of Hbt. salinarum on agar plates. Pigmentation was found not to be uniform, but instead,
rings of denser and less dense pigmentation were observed [65].
A detailed study of the Raman bands of bacterioruberin was made, combined with a study of
the Raman spectrum of salinixanthin, the carotenoid acyl glycoside of Salinibacter, the extremely
halophilic representative of the bacteria often found in the same salt-saturated habitat in which
members of the Halobacteriaceae thrive [66]. Small, hand-held Raman spectrometers have also
been successfully tested for the detection of these pigments [67].
Raman spectroscopy can sensitively detect other biomarkers in high-salt, low water activity
environments as well. Examples are chlorophyll a, different carotenoids and scytonemin in
colonies of cyanobacteria (Gloeocapsa, Nostoc) in gypsum and other evaporites in the High
Arctic [68] and within halite from hyperarid areas of the Atacama Desert, Chile [69]. Here, the
use of hand-held instruments in the field was combined with a Raman microscope to obtain highresolution measurements in the laboratory. Both hand-held and laboratory-size instruments were
used to probe the coloured layers of different types of cyanobacteria and of purple sulfur bacteria
within the gypsum crust that develops on the bottom of saltern evaporation ponds in Eilat, Israel,
at a salinity of approximately 200 g l−1 [67].
Pigments are not the only possible biomarkers to indicate presence of halophilic and
other microorganisms using Raman spectroscopy. Simple organic biomolecules such as amino
acids (glycine, alanine, serine) trapped within fluid inclusions inside halite crystals can be
detected [70]. Most halophilic and halotolerant microorganisms (but not including members of
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5. Raman spectroscopy as a tool to detect halophilic archaea and other
halophilic microorganisms
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However, perchlorate can be used as an electron acceptor for anaerobic respiration by some
prokaryotes, bacteria as well as archaea. Early studies with anaerobic enrichment cultures in
the presence of 5 mM sodium perchlorate and salt concentrations up to 70–110 g l−1 salt yielded
growth with reduction of perchlorate [59]. The organisms that developed in these cultures
were not further characterized. Pure cultures of the archaeon Haloferax denitrificans and the
bacterium Halomonas halodenitrificans reduced perchlorate as well as nitrate [60]. Use of the
halophilic archaeon Haloferax mediterranei was even proposed for the bioremediation of high-salt
waste-water containing chlorate and perchlorate [61].
Halophilic archaea are able to tolerate far higher concentrations of perchlorate than the
approximately 5 mM used in the above-described experiments. All Halobacteriaceae species
tested grew well in NaCl-based media containing 0.4 M sodium perchlorate, and Hfx. mediterranei
even grow weakly in the presence of 0.6 M. But, at the highest tolerated perchlorate
concentrations, cells were swollen and distorted. The ability to use perchlorate as an electron
acceptor for anaerobic respiration, supporting growth in the absence of molecular oxygen,
was shown for several species (Hfx. mediterranei, Hfx. denitrificans, Hfx. gibbonsii, Haloarcula
marismortui, Har. vallismortis). Species growing anaerobically on perchlorate generally could also
use chlorate as an electron acceptor [62]. These results show that, rather than being toxic, presence
of perchlorate can be favourable for the development of halophilic archaea in the absence of
molecular oxygen, provided that a suitable electron donor and energy source is also available.
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Whether organisms similar to the halophilic archaea of the family Halobacteriaceae indeed live
on Mars, or may have lived there in earlier times when the climate there was more conducive to
life, is still unknown. As shown above, they make good candidates for organisms that can adapt
to the Martian environment. It has been speculated before whether recent or ancient halobacteria
may be present entrapped within salt on Mars [74].
Raman spectroscopy is an excellent technique to detect halophilic archaea, thanks to the
presence of specific carotenoids and other potential biomarkers such as the retinal protein
bacteriorhodopsin. NaCl does not have Raman signals, so biomarkers entrapped within halite
crystals can be detected without interference by the crystal matrix.
A Raman spectrometer will be included in the instrumentation planned to land on Mars in 2018
with the ExoMars mission, and therefore different model geological and biogeological specimens
have been tested as reference material, including evaporites colonized by carotenoid-containing
microorganisms [1]. We will have to wait and see whether or not the ExoMars Lander, if indeed
it will successfully land on Mars, will detect similar biomarkers there. If it will, then our current
speculations about possible present-day or past halophilic life on that planet may well become
confirmed by solid data.
Funding statement. This study was supported by grant nos. 1103/10 and 343/13 from the Israel Science
Foundation, and was performed in the framework of the activities of the Minerva Center on the Emergence &
Evolution of Early Life under Extreme Planetary Conditions.
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