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Phil. Trans. R. Soc. A (2010) 368, 3059–3065
doi:10.1098/rsta.2010.0100
I N T RO D U C T I O N
Raman spectroscopic approach to analytical
astrobiology: the detection of key geological and
biomolecular markers in the search for life
1. Prologue
The compilation of this Theme Issue of Philosophical Transactions of the Royal
Society on the theme of Raman spectroscopic analysis of materials from extreme
environments is timely for several reasons, perhaps the most significant of these
being the realization in the last decade that this technique can provide unique
information about the limits of survival of biological organisms in terrestrial
locations, where hitherto the prospects of finding life were thought to be minimal.
The year 2009 marked the bicentenary of the birth of Charles Darwin and the
sesquicentenary of the publication of his monumental work, On the Origin of
Species by Natural Selection in November 1859. This book ran to six editions and
it would be superfluous to comment here on the astounding debates that raged
in the second half of the nineteenth century involving all aspects of arts and
sciences and religion. Although today we associate Darwin’s work with evolution,
it is interesting that this was only mentioned in the final and sixth edition,
which also contained his first recognition of and counter argument against the
claims then being made by theologians. Although Darwin himself did not have
knowledge then of the extreme-tolerant, ‘extremophilic’, organisms that today we
regard as ubiquitous, the analogy with the survival by natural selection of those
organisms that were best suited to survive predatory attacks or environmental
changes independently proposed by Alfred Russell Wallace and Darwin is quite
similar in evolutionary terms to the birds, beetles and lizards that caused so
much wonder from Darwin’s observations in the Galapagos Islands in the Beagle
expedition of the 1830s.
It is clear from the analytical data that have been obtained in recent years that
the strategies being adopted by organisms in stressed environments, examples
of which are tabulated in table 1, in life-threatening scenarios are critical for
their survival and evolutionary behaviour. The key factors that have emerged
suggest that the synthesis of particular suites of protective biochemicals that
act as protectants and repair agents when damage has occurred, often in multifunctional roles, is critical for survival in these ‘limits of life’ situations. The
One contribution of 12 to a Theme Issue ‘Raman spectroscopic approach to analytical astrobiology:
the detection of key geological and biomolecular markers in the search for life’.
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This journal is © 2010 The Royal Society
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H. G. M. Edwards
Table 1. Terrestrial extremes for the survival of extremophilic organisms.
temperature
pressure
radiation
underground depth
acidic pH
basic pH
longest in space
saltiest
smallest
Pyrolobus fumarii (Volcano Island, Italy) 113◦ C
Chroococcidiopsis sp. (Mars Oasis, Antarctica) −15◦ C
1200 bar
Deinococcus radiodurans 5 Mrad
(5000 times the fatal dose for humans)
3.2 km
pH 0.0
pH 13.0
Bacillus subtilis 6 yr NASA satellite
Haloarcula 30% salt
Picoplankton 0.1 mm
correlation of the extreme terrestrial survival by Archaean cyanobacterial colonies
that have had to adjust and evolve from a primitive and hostile Earth some
3.8 Gyr ago with the stresses of possible similar colonizations on our neighbouring
planets and their satellites is now at the forefront of multidisciplinary research
efforts to understand and evaluate these possibilities using unmanned planetary
rovers and accompanying sensor instrumentation.
The recognition of extinct or extant life signatures in the terrestrial geological
record is fundamentally dependent upon the understanding of both the structural
morphology and chemical composition of relict biomaterials; the identification
of cyanobacterial colonies that have adapted biogeologically their mineral
matrices in early evolutionary processes is a fundamental step in the acquisition
of analytical data from remote planetary probes designed for life-detection
experiments, particularly on Mars and on the planetary satellite moons Europa
and Titan. A key factor in the assessment of early life signatures is the
molecular presence of chemicals designed to protect the emerging organisms from
the damaging effect of radiation exposure and of desiccation and temperature
changes; in this respect the non-destructive capability of Raman spectroscopy
to delineate the interfacial interactions between substrates and endolithic
biology is now deemed an essential part of the ExoMars life-detection suite
of instrumentation planned by the European Space Agency (ESA) in the
Aurora programme. A description of the scientific basis for the biogeological
discrimination offered by Raman spectroscopy between organic and inorganic
moieties in specimens from terrestrial Mars analogue sites is a recurrent theme
in the original research papers presented in this Theme Issue.
The search for life signatures in early evolutionary processes informs the
need for the addressing of basic questions about the origins of life arising
from a prebiotic world (Edwards 2007). In our terrestrial geological history, the
recognition of the presence of fossilized relict cyanobacterial organisms through
their morphological traces is a key factor in the ‘top-down’ approach to an
understanding of the early biological colonization and the prebiological chemistry
of the evolving geosphere.
Cyanobacterial colonization of geological substrates has been identified in our
earliest terrestrial geological formations, extending from some 3.8 Gyr, and it is
clear that an understanding of the survival strategies and protection mechanisms
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Introduction
3061
being adopted by these organisms can provide some important clues as to the
conditions that were operating on early Earth and in their adaptation to the
evolutionary conditions (Schopf et al. 2002; Brasier et al. 2004).
The survival of cyanobacterial extremophiles in stressed terrestrial
environments is fundamentally dependent upon their synthesis of a specialized
suite of protective biochemicals in response to desiccation, low-wavelength
high-energy radiation insolation, extremes of pH, temperature and pressure
and high concentrations of toxic heavy metal ions (Cockell & Knowland 1999;
Wynn-Williams & Edwards 2000a,b). Our understanding of the roles of these
chemical protectants reveals a sophistication in design for specific functions;
for example, the ultraviolet radiation screening effectiveness of scytonemin in
cyanobacterial sheaths, which selectively absorbs in the UVA, UVB and UVC
regions of the electromagnetic spectrum, while permitting the transmission of
the longer wavelength photosynthetically active radiation that is necessary for
the operation of chlorophyll in photosynthetic colonies. Other protectants, such
as trehalose, which is synthesized in Antarctic cyanobacterial mats colonizing saltrich lakes, are dualistic in their roles; trehalose is used as a water-replacement
intracellular molecule, which also combats osmotic stress caused by abnormally
large concentrations of soluble salts such as calcium chloride, and a range of
carotenoids serve as light-harvesting pigments and also as cellular DNA damage
repair agents.
It is generally accepted that life started on Earth around 3800 Myr, when
the environmental conditions on the surface were drastically different from the
current situation. At that time, it is predicted that Mars had similar conditions
to those pertaining on Earth and this has led to the idea that life could also
have appeared on Mars (Davis & McKay 1996; McKay 1997; Cockell et al.
2000; Holm & Andersson 2005). As the external conditions were changing on
early Mars, therefore as on Earth, organisms needed to adapt to new habitats
and colonize even the most extreme niches using a range of response strategies.
Those organisms that were able to survive in such hostile environments can be
considered to have left signatures of their presence on Mars; it is fundamentally
important, therefore, to analyse our terrestrial analogues of possible Mars
organisms (Raulin & McKay 2002; Simoneit 2004; Delaye & Lazcano 2005).
Cold deserts, such as Antarctica or the Arctic, have been proposed as
the closest Martian analogues on Earth (Wynn-Williams 2000). The extreme
low temperatures, highly dry atmospheres, strong UV radiation, low nutrient
availability or long periods without sunlight have not been an obstacle for the
adaptation and proliferation of many different types of micro-organisms. The
survival adaptations of micro-organisms in these environments extend to both
inorganic and organic strategies (Jorge Villar et al. 2003, 2005; Edwards et al.
2005), especially involving the use of a wide range of radiation protective pigments
(Mueller et al. 2005; Jorge Villar et al. 2006).
2. What can we learn from astrobiology?
Astrobiology has been described as the only scientific discipline that has no formal
evidence for its own existence; that still awaits the analytical data that will emerge
from remote planetary probes and eventually exploration of our Solar System and
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H. G. M. Edwards
beyond. The key to this question must address initially the compounds that are
to be identified in meteorites and cosmic material harvested on Earth and their
relevance to prebiotic synthesis elsewhere. Clearly, the material found on Earth
resulting from meteoritic infall has been subjected to the stresses of atmospheric
entry following aeons of exposure to the low temperatures and high-energy
radiation in deep space. We are currently unable to assess the influence of these
conditions upon the chemistry of organic compounds contained within meteorites,
but recent experiments performed under project STONE, a ESA-sponsored effort
resulting in the first artificial meteorite experiment designed to understand the
alteration of geological and biological materials subjected to a space environment
and their subsequent atmospheric re-entry, have made significant progress in this
direction (Brack et al. 2006; Cockell et al. 2007). However, it should be recognized
that the origin of meteorites formed from ejecta resulting from impact events on
planetary surfaces could have involved conditions and processes that would have
altered the residual chemical signatures of any organisms that could have existed
on the planetary surface prior to the impact collision. It is believed therefore that
the presence or otherwise of key organic biomolecular signatures in meteorites or
cosmic debris can only represent one part of the astrobiological scenario and that
a major thrust would necessitate the examination of the geological record of the
original planet. This is the current thinking behind the use of novel analytical
instrumental techniques for the determination of chemical life signatures in the
robotic search for life on planetary surfaces prior to human exploration, as
exemplified by the NASA and ESA Aurora missions.
Raman spectroscopy is being adopted by ESA as a part of an analytical
instrumentation suite for Mars surface exploration, including the ExoMars
mission scheduled for 2018. The particular characteristics of Raman spectroscopy
that make this technique a valuable instrument for the unambiguous
identification of either biological or geological signatures (Jorge Villar &
Edwards 2006) are primarily those of non-destructive analysis of specimens,
ease of sample examination that does not involve any physical or chemical
surface preparation and an ability to collect data remotely via an optical
fibre. A crucial part of the analytical procedures will rest with the
interpretation of spectral signatures and comparison with laboratory databases of
key biochemicals.
The miniaturization of Raman spectrometers involves the consideration of
several parameters that have an effect on the spectrum and for the subsequent
identification of both organic and inorganic materials. The effects of laser
wavelength, spectral resolution, laser power or the selection of a particular
wavenumber range have been studied in our laboratory and their impact and
consequences for the observed spectral analysis have been assessed.
Different laser wavelengths have different capabilities for the detection of
specific biomolecules and minerals and some compounds could not be detected;
furthermore, the changes in relative intensities of the characteristic Raman bands
related to laser excitation make the unambiguous identification of some molecules
difficult, which compromised several databases. Fluorescence and resonance
scattering also have an important effect on the observed Raman spectrum
in terms of band intensities. A high laser power can induce subtle molecular
changes in either mineral and organic molecules and too low a laser power would
increase significantly the time necessary for the spectrum accumulation to be
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Introduction
3063
effected. If a restricted wavenumber region is demanded in the miniaturized
instrument, the priority for the identification of bio- or geo-signatures has then
to be selected consequent upon the more limited wavenumber region of the
Raman spectrum.
Spectroscopic and microscopic data have demonstrated the various protective
and adaptive mechanisms and strategies developed by Arctic extremophile microorganims for their survival in cold desert environments. The use of dark basaltic
rocks as a protective niche for endolithic and chasmolithic communities and the
development of specialized pigments as survival strategies by psychrophilic snow
algae are exemplified in this scenario.
It has been shown (Jorge Villar et al. 2003, 2005, 2006) that in stressed
conditions micro-organisms protect themselves through the colonization of light
rocks, by living close to the surface, either inside a porous matrix (endolith)
or inside a crack (chasmolith), where light and water can be easily supplied.
However, it has now been shown that a dark basaltic rock containing endolithand chasmolith-like communities can also play a protective role and that
the restricted light availability in such an environment is not a deterrent
for colonization. By using in situ Raman spectroscopy combined with high
resolution microscopic and elemental analyses using scanning electron microscopy
(SEM), the location and chemical characteristics of these communities have
been identified non-destructively and for the first time it has been shown that
photosynthetic organisms living in terrestrial dark basaltic rocks can survive and
even thrive in such extreme environments (Jorge Villar et al. 2006). The Raman
spectra demonstrate the presence of radiation protective scytonemin, a range of
carotenoids, accessory photosynthetic pigments such as c-phycocyanin as well as
chlorophyll.
In another example, the strategies adopted by snow algae collected from a
permanent snow field in the Murchison Fjord area of Spitzbergen (80◦ N, Norway)
have been examined; this scenario would be relevant for Mars polar life detection
remote-sensing expeditions. Again, a combination of Raman spectroscopy and
SEM techniques have been used for a comprehensive study of the protective
strategies adopted by surface-dwelling micro-organisms on glaciers and snow
fields, and pigment and other radiation protective compounds such as scytonemin,
carotenoids and an anthraquinone similar to emodin have been identified.
Although specifically there is no single terrestrial Mars analogue site,
and indeed this would have been too narrow to define, given the broad
range of geological niches that have been identified on Mars by the latest
NASA rovers, Spirit and Opportunity, the examination of a range of closely
related extremophilic scenarios on Earth is of potential relevance to the
diverse situations that may be expected on Mars; in this context, a study
is currently being undertaken on ancient terrestrial rocks that harbour
morphological structures of fossilized early Earth organisms to identify the
presence of residual protective chemicals in geological niche sites that would
be indicative of early life signatures (Pullan et al. 2008) that could perhaps
be indicative of first-pass screening experiments on Mars carried out by
planetary surface rover vehicles. The motto of the Royal Air Force, Per Ardua
ad Astra, is descriptive of the terrestrial analytical work that is essential
for potential astrobiological data to be understood from remote planetary
searches for life signals, extinct or extant, and towards that objective the
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H. G. M. Edwards
compilation of papers in this Theme Issue is dedicated: Raman spectroscopy
of extreme environments. A good representation is thereby afforded of the
versatility of this analytical technique for the detection of life signatures from
materials in terrestrial extreme environments encompassing hot and cold deserts,
Arctic and Antarctic regions, deep-sea smokers, glacial sediments, meteorites,
volcanic lava flows and strongly acidic locations that will be a foundation
for the appreciation of future experiments being undertaken remotely on
Mars missions.
Howell G. M. Edwards
Centre for Astrobiology and Extremophiles Research,
School of Life Sciences, University of Bradford,
Bradford BD7 1DP, UK
E-mail address: [email protected]
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