Download Why choose Raman spectroscopy for the exploration of Mars?

Spectroscopy
Why choose Raman spectroscopy
for the exploration of Mars?
by Craig P. Marshall, Vibrational Spectroscopy Facility, School of Chemistry, The University of Sydney
and Candace M. Coyle, Department of Chemistry, Circle, The University of Texas at San Antonio.
Raman spectroscopy is well recognised as a powerful analytical tool for compositional
and structural elucidation information pertaining to material in the solid, liquid or
gaseous state. Significantly, both macro- and micro- capabilities are non-destructive,
which has made this technique unique for many applications when material is scarce
or very valuable.
AS A RESULT, application of Raman
spectroscopy has increased over the last
20 years across many areas, including
chemistry, mineralogy, geology, art
and archaeology, forensic sciences and
material sciences, such as polymers and
biomaterials.
To some extent however, Raman
spectroscopy is still viewed as a laboratory
or research technique. Nevertheless, in
recent years several systems have been
specifically developed for field-based
applications. Recent advances in laser
sources, optical elements, spectrometers
and detectors have significantly led to
the development of robust, compact,
and miniaturised Raman systems.
Consequently, the potential use
of Raman spectroscopy in planetary
exploration as part of a rover or lander
instrumentation package, particularly
in the exploration of Mars, is now being
recognised. NASA and ESA currently
consider Raman spectroscopy, either
separately or in combination with laser
induced breakdown spectroscopy (LIBS)
or fluorescence, as a fundamental
next generation instrument for the
characterisation of mineralogical and
organic material during the exploration
of Mars. While instrumentation for
robotic missions is probably the most
important consideration for Mars
exploration, it is also important to note
Figure 1: A comparison of the
chemical structures of β-carotene and
bacterioruberin carotenoids.
β-carotene
OH
OH
OH
OH
26
bacterioruberin
that Raman applications adding to the
knowledge of Mars also cover other
aspects such as the study of potential
terrestrial Martian analogues.
Figure 2: Universal Tree of Life displaying
the relation between the three branches
of life, Bacteria, Archaea, and Eucarya.
Raman spectroscopy for
detecting life on Mars
Microbial life, whether extinct or
existing on Mars, would produce
biomolecules that might be preserved
and detectable in Martian rocks.
Therefore it is crucial to construct a
database of biosignatures for microbial
life on Earth to facilitate the detection
of biosignatures on Mars (and possibly
beyond) and eliminate false-positive
detection (either of earth microbes
contaminating Mars, or of organic
molecules that do not have a biological
origin).
Examples of potential biomolecules
are materials with molecular structures
that define their functionality with such
functionality being fundamental to all
organisms. Furthermore, it is desirable
to target organic materials that are
clearly distinguishable from abiogenic
compounds, such as polycyclic aromatic
hydrocarbons (PAHs) that are widely
distributed throughout the cosmos.
Potential biomarker compounds include:
chlorophyll (degrades to porphyrins),
carotenoids/retinal protein complexes
(degrade to isoprenoids), hopanoids
(derived from degraded cell membranes
of bacterio-hopanetretol), and steroids
(derived from degraded eukaryote
cell membranes and walls). Each of
these biomolecules and their degraded
products are readily detected by Raman
spectroscopy.
Raman spectroscopy of
carotenoids
Carotenoids are π-electron-conjugated
carbon-chain molecules and are similar
to polyenes with regard to their structure
and optical properties. Distinguishing
features of these molecules are a linear,
chain-like conjugated carbon backbone
consisting of alternating carbon single
(C-C) and double bonds (C=C) with
varying numbers of conjugated double
bonds, and a varying number of attached
methyl side groups. An example of the
molecular structure of β-carotene and
bacterioruberin which are the most
important carotenoids in cyanobacteria
and halophilic archaea respectively is
shown in Figure 1.
Carotenoids are strongly coloured as
they have an allowed π-π* transition
which occurs in the visible region. This
colour is strongly dependent on the
number of conjugated double bonds in
the main linear chain. The red shifting
of this π-π* absorption band indicates an
increase in the conjugation length which
is reflected in its colour, ranging from
yellow, orange to red respectively. For
example, β-carotene has 11 conjugated
double bonds and is orange in colour,
while bacterioruberin, which has 13
conjugated double bonds, is red (Figure
1). Significantly, for Raman spectroscopy
when the wavelength of laser excitation
coincides with this allowed π-π* (S0-S2)
electronic transition of carotenoids, their
resonance Raman spectra are obtained.
Materials Australia - September/October 2006
Spectroscopy
Figure 4: Microbial mat containing filamentous cyanobacteria (left), and the Obsidian Creek collection site and waterway (right).
Mars analogues
Life consists of three domains of life
that is, bacteria, archaea, and eucarya
(Figure 2). It has become apparent that
life is predominantly microbial and
the greatest diversity is found within
the bacteria and archaea. In most
interpretations, all the organisms near
the base of the universal tree of life are
extremophiles. Current interest in the
remote exploration of Martian sites
of possible astrobiological significance
is convolved with the realisation that
Mars and Earth have similar geological
histories and that in the early ages, Mars
was a wetter and warmer planet than
it is now. Conceivably, if extremophilic
life arose on early Earth under the
same conditions, it could have likewise
potentially evolved and diversified on
early Mars.
Terrestrial analogues for Mars can be
defined as settings on our planet where
Figure 3: Stacked resonance Raman
spectra of bacterioruberin and
bacteriorhodopsin acquired from
Halobacterium salinarum. Collection
parameters for both spectra are 514 nm
excitation, 10 s exposure,
5 accumulations, and 1.2 mW laser power
at the sample on an InVia Reflex Renishaw
Raman spectrometer.
geological features, biological attributes,
or combinations thereof, offer potential
comparison with possible Martian
counterparts. There are several potential
locations for past or present life on
Mars, yet not necessarily exclusively. For
example, regions where water existed for
a significant period of time (palaeolakes
and water-cut channels), hypersaline
brines or evaporate deposits indicative
of salt mineral deposition in water,
permafrosts, hydrothermal regions,
impact craters which are another possible
hydrothermal source, and lake sites from
catastrophic outflows.
Two examples pertaining to specific
relevance in this context will be
discussed: hypersaline micro-organisms
and thermophilic microbes from modern
hydrothermal settings.
Hypersaline environments halophilic archaea
Recently, halite and sulfate evaporate
rocks have been discovered on Mars
by the NASA Mars rovers, Spirit and
Opportunity. This suggests that brine
pools may have been relatively common
on the surface of Mars thus, providing
regions of high salt concentration. It is
reasonable to propose that halophilic
micro-organisms could have potentially
flourished in these conditions. Therefore,
modern terrestrial salt basin and cultured
salt-tolerant microbes are good analogues
for conditions under which life might
have evolved on Mars. If so, biomolecules
found in micro-organisms adapted to
high salinity and basic pH environments
on Earth may be reliable biomarkers for
detecting life on Mars.
Materials Australia - September/October 2006
Halophilic archaea are chemoorganotrophs that belong to the class
Euryarchaeota.
These microbes are
often the predominant micro-organism
present in salt lakes, pools of evaporating
seawater, solar salterns and other
hypersaline environments with salt
concentrations as high as halite
saturation. Significantly, extremely
halophilic archaea have been noted
for their bright red or purple colour.
The pigments responsible for these
colours consist of isoprenoid-derived
or retinal-protein compounds. The
pigment responsible for the purple
colour is a retinal-protein complex,
bacteriorhodopsin, while the isoprenoidderived pigment, bacterioruberin, gives
rise to a bright red colour.
Bacteriorhodopsin converts the energy
of green light (500 - 650 nm) into an
electrochemical proton gradient, which
in turn is used for ATP production by
ATP-synthases. Bacterioruberin is a
ubiquitous and abundant red pigment
in moderately to extremely halophilic
archaea. This red pigment, located in the
membrane of halobacteria, not only plays
a role in the photoprotection system, but
is also important for the adaptation of
membrane fluidity to changing osmotic
conditions.
Both pigments have been readily
detected through Raman spectroscopy,
each generating distinctive Raman
spectra (Figure 3). In the resonance
Raman spectra, three prominent
bands occur at 1,505 and 1,536 cm-1,
1,152 and 1,199 cm-1, and 1,000 and
Continued on page 28
27
Spectroscopy
a.
b.
Figure 5: Photomicrographs of the
microbial mats at Obsidian Creek reveal
the presence of the following:
a. Chloroflexus and Oscillatoria
(magenta) and Synechococcus (green);
b. Chloroflexus, Coccobacillus (red) and
Diatoms (yellow).
Continued from page 27
1,007 cm-1 which are associated with
C=C stretching, C-C stretching, and C-CH bending modes respectively. The C=C
stretching mode is an important marker
band and it is well known that this
frequency correlates inversely with the
extent of conjugation length of the linear,
chain-like conjugated carbon backbone
of the carotenoid, owing to electronphonon coupling. Thus, the occurrence
and spatial distribution of preserved
pigments in hypersaline environments
of Mars should be detectable in situ by
non-destructive Raman techniques, as
on Earth.
Hydrothermal settings –
Yellowstone National Park
microbes
It has been proposed that localised
hydrothermal regions at shallow depths
(less than 500 m) created by volcanic
activity may support a limited ecology
similar to terrestrial hyperthermophilic
organisms. Yellowstone National Park
(YNP) supports a considerable diversity
of bacterial and archaeal communities
in numerous hot springs. Hydrothermal
systems, including geysers and hot
springs, are regions in Earth’s crust
where hot fluids circulate at shallow
depths. The Yellowstone volcanic field
28
centred in YNP is one of the largest silicic
volcanic systems in the world. Hence,
this locality is ideally suited for the
exploration of Mars in a hydrothermal
context. Documentation of biomolecules
found in these thermophilic microorganisms on Earth may serve as reliable
biomarkers for detecting life on Mars.
Hot spring thermophiles from
Obsidian Creek (Norris Geyser Basin)
in YNP were investigated using Raman
spectroscopy.
Geothermally-heated
Obsidian Creek flows 0.6 km south
of Grizzly Lake and communities
of microbes form films or layers of
mats on the creek bed. Typical water
temperatures range from 60-90oC and
pH from 5.50 - 6.50. These conditions
are idea for cyanobacterial communities
chiefly composed of Oscillatoria sp.
(filamentous cyanobacteria) which
forms green mats (Figure 4). Microscopic
analyses of these mat materials (Figure
5) reveal the presence of several microorganisms including Oscillatoria sp.
(filamentous green cyanobacteria),
Chloroflexus sp. (filamentous orange
nonsulfur bacteria), Coccobacillus sp.
(pink bacteria), Synechococcus sp.
(rod-shaped green cyanobacteria),
Klebsormidium sp. (filamentous green
micro-algae), and diatoms (algae
frustule). The absorption spectra
(UV-Vis) exhibit spectral bands
indicative of various carotenoids (410,
430, 478 nm) associated with these
organisms, as well as Chlorophyll a
(664 nm) (Figure 6).
Two representative resonances of
Raman spectra of photosynthetic
bacteria and eukaryotic micro-algae
are also shown in Figure 6. Notably,
the Raman spectra contain spectral
features similar to halophilic archaea
near 1,005, 1,155, and 1,520 cm-1 which
are strongly resonant-enhanced due to
carotenoid pigments. The differences
between the cyanobacteria and algae
are small but distinct. The distinct
changes within the spectra is the change
in wavenumber location of the C=C (ν1)
band. The wavenumber location of
the ν1 band is strongly dependent on
the length of the carotenoid chain9-12.
The wavenumber location of ν1 band
acquired from the resonance Raman
spectra of the cyanobacteria and algae
follow the trend. Significantly, the
1,523 cm-1 corresponds with the
structural conjugation of β-carotene,
while the 1,516 cm-1 suggests a longer
chain-length similar to lycopene.
Moreover, the distinct correspondence
between the absorption spectral bands
and the wavenumber associated with
the ν1 band is clear (410 and 430/478
nm with 1,523 and 1,516 cm-1,
respectively).
Significantly, this work is the first
study using Raman spectroscopy
for the chemical characterisation of
thermophilic microbes inhabiting these
modern hydrothermal systems. The
data generated will be used to establish
a spectral database of biomarker
compounds associated with the
cyanobacteria and micro-algae found
in hydrothermal settings serving as
potential Martian analogue systems.
Figure 6: Stacked resonance Raman
and UV-Vis absorption spectra of
photosynthetic bacteria and algae
acquired from Obsidian Creek.
Collection parameters for both spectra
are 514 nm excitation, 15 s exposure, 10
accumulations, and 2 mW laser power at
the sample using a SPEX 1877 triplemate
spectrometer coupled with a Roper
Scientific CCD.
Summary
There is a need for a scientific
instrument to characterise in situ
the microstructure and inorganic
composition of potential microbial
habitats on Mars with concurrent
analysis of molecular components of
organic material and biomolecules.
Raman spectroscopy fulfils this
requirement. Moreover, the viability
of Raman spectroscopy for the
detection of molecular biosignatures
namely, carotenoids from extant
Martian analogue haloarchaea and
hot spring microbes, has been clearly
demonstrated.
Acknowledgements
CPM would like to thank financial support from
the Australian Research Council for fellowship and
grant. The authors would like to acknowledge the
Department of the Interior, Yellowstone National
Park, and Research Permit #YELL-2006-SCI-5558.
CMC would like to thank Christie Hendrix and
Christine Smith in the Yellowstone Center for
Resources for their continued support and helpful
discussions.
Materials Australia - September/October 2006