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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