JOURNAL OF RAMAN SPECTROSCOPY J. Raman Spectrosc. 2007; 38: 1352–1361 Published online 3 July 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jrs.1775
Morphological biosignatures from relict fossilised sedimentary geological specimens: a Raman spectroscopic study Howell G. M. Edwards,1∗ Susana E. Jorge Villar,1,2 Derek Pullan,3 Michael D. Hargreaves,1 Beda A. Hofmann4 and Frances Westall5 1
Centre for Astrobiology and Extremeophile Research, University Analytical Centre, School of Life Sciences, University of Bradford, Bradford BD7 1DP, UK 2 Area de Geodinamica Interna, Facultad de Humanidades y Educacion, Calle Villadiego s/n, 09001 Burgos, Spain 3 Space Research Centre, Department of Physics and Astronomy, University of Leicester, University Road, Leicester LE1 7RH, UK 4 Natural History Museum, Bernastrasse 15, CH-3005 Bern, Switzerland 5 Centre de Biophysique Biomoleculaire, CNRS, Rue Charles Sadron, Orleans cedex 245071, France Received 29 November 2006; Accepted 17 April 2007
Morphological biosignatures (features related to life) and associated terrestrial sedimentary structures that provide possible sampling targets for the remote astrobiological exploration of planets have been analysed using Raman spectroscopic techniques. The spectral data from a suite of samples comprising cryptochasmoendoliths, preserved microbial filaments and relict sedimentary structures comprise a preliminary database for the establishment of key Raman biosignatures. This will form the basis for the evaluation of prototype miniaturised instrumentation for the proposed ESA ExoMars mission scheduled for 2013. The Raman spectral biosignatures of carotenoids and scytonemin, organic biomolecules characteristic of the cyanobacterial colonisation of geological matrices and biogeologically modified minerals are also identifiable in the sedimentary specimen materials. The results of this study demonstrate the basis of the molecular recognition of extinct and extant exobiology that will feed into the elemental structural analyses of morphological structures provided by associated SEM, XRD and laser-induced breakdown spectroscopy (LIBS) techniques on robotic analytical landers. Copyright 2007 John Wiley & Sons, Ltd.
KEYWORDS: Mars analogues; Raman spectroscopy; spectral biosignatures; biogeological modification; ExoMars instrumentation
INTRODUCTION The origin and evolution of life on Earth some 3.5 Gya, termed the ‘Early Eden’ hypothesis,1 and the similarities between the planetologies of early Earth and Mars2 have focused attention on the astrobiological search for relict and extant life signatures on Mars.3,4 The preservation of morphological fossils and stromatolites in the terrestrial geological record and the identification of putative fossilised biological structures in the ALH 84001 SNC Martian meteorite5 have occasioned much vigorous and controversial debate in the literature. A critical factor in the search for evidence of early terrestrial life has been the analytical characterisation of Ł Correspondence
to: Howell G. M. Edwards, Centre for Astrobiology and Extremeophile Research, University Analytical Centre, School of Life Sciences, University of Bradford, Bradford BD7 1DP, UK. E-mail:
[email protected]
Copyright 2007 John Wiley & Sons, Ltd.
carbonaceous deposits in the geological record consequent upon the discrimination between the biogenic and abiogenic origins of carbons and kerogens.6,7 This is a particularly difficult problem to address because of the insufficient information existing about the survivability of biological molecular structures with geological time spans. However, recent analytical spectroscopic data from Raman studies of the protective biomolecules that have been synthesised by early biological organisms such as cyanobacteria that exist in niche geological environments,8 – 11 currently representing ‘limits of life’ situations, has provided another approach to the established analogues for terrestrial and astrobiological life-detection scenarios.12 – 15 Specific molecular structural groups represented by scytonemin, carotenoids, phycocyanins, chlorophylls, depsides, hopanoids and mycosporine amino acids provide unique and key biomarkers for the presence of extremophilic bacterial organisms and colonies; furthermore, these molecules have
Raman spectra of fossilised sedimentary geological specimens
been detected in geological matrices even when the biology has become extinct.16 – 20 In this respect, therefore, the analytical protocols for the life-detection monitoring of potential astrobiological extraterrestrial habitats will be focused on the spectral biosignatures of these radiation protectants, lightharvesting accessory pigments, cryogenic protectants and anti-desiccants.21 – 24 The selection of a miniaturised combined Raman/laserinduced breakdown spectroscopy (LIBS) spectrometer for the Pasteur life-detection instrumentation suite on the ExoMars/AURORA project by the European Space Agency25 is a major commitment to the application of novel enabling analytical technologies to the search for life on Mars scheduled in the 2013 mission. An essential requirement of the selected miniaturised Raman/LIBS instrument is the provision of molecular spectroscopic data from biomolecules resident in the Mars planetary surface and subsurface geological record. To this end, we have required an archived, well-characterised suite of biogeological specimens which have been subjected to molecular, elemental and microscopic analysis using multiple instrumental techniques.26 Here we describe for the first time the detailed Raman spectroscopic analysis of some highly important geological morphological structures; from the molecular analysis of these materials, the presence of biological and biogeological signatures will be correlated within an astrobiological context to the proposed search for life on Mars using remote Raman instrumentation on a robotic lander, exemplified by the ExoMars mission under the AURORA/Pasteur project banner.25
EXPERIMENTAL Samples All the specimens selected for this study exhibit morphological features related to the biological colonisation of rock substrates. They were all obtained from terrestrial Martian analogue field sites, including high-latitude extreme environments, tertiary (¾10–39 Ma) crater lake/hydrothermal deposits and Archaean (¾3.45 Ga) volcaniclastic sediments. The samples comprise the following three morphological categories: fossilised microbial filaments, endolithic microbial communities and ancient relict sedimentary structures containing microbial fossils. Observable features range in size between sub-millimetres to a few centimetres, and the preservation states on different surfaces of each specimen range from pristine (fresh) to degraded (weathered). This range of specimens provide an opportunity for the evaluation of Raman spectroscopic techniques for the molecular analysis of relict or extant biological colonies in several geological environments through the spectral signatures of targeted biochemicals that have been produced in their protective survival strategies.
Copyright 2007 John Wiley & Sons, Ltd.
Microbial filaments in freshwater limestone, Hainsfarth, Ries Crater, Germany (GSPARC 140 – the sample code refers to the Planetary Analogues Field Studies Network curated collection) The Ries and Steinheim craters of the Jurassic Alb plateau in Southern Germany represent well-studied examples of terrestrial impact structures.25,26 Near-shore crater lake carbonates at these sites are exposed at Buschelberg near ¨ ¨ Hainsfarth (48° 57.150 N, 10° 38.10 E), 2.5 km east of Ottingen and have been extensively studied.27 The carbonate sequence is >8 m thick and contains Cladophorites (green algae) with minor stromatolites and carbonate sands composed of gastropods and ostracods. This specimen represents a combination of wellpreserved (fresh) and weathered calcified remnants of Cladophorites cemented in a dolomite matrix (Fig. 1). The tubular morphology is ascribed to an accumulation of carbonate veneers around the original Cladophorites threads, which have been oxidised leaving a void. This, together with sinter-like crusts on associated algal constructions within the bioherm, suggests temporal vadose conditions prevailed where evaporation has led to the impregnation of dolomitised carbonate into the biofilms. Although the visible calcified tubes are abiogenic, their morphology resulting from mineralisation over a Cladophorites substrate classifies these features as a visible biosignature.
Endolithic colonisation of orthoquartzite, McMurdo Dry Valley, Victoria Land, Antarctica (GSPARC 114) The Dry Valleys of Southern Victoria Land, Antarctica, extend across an area of 5000 km2 and lie between 76° 300 S and 160–164° E. Geomorphologically, they are a system of gouged glacial valleys with a predominant east–west trend. During summer air temperatures range between
Figure 1. Tubular weathered abiogenic calcified deposits of Cladophorites in a dolomite matrix: Ries Crater, Hainsfarth, Southern Germany. This figure is available in colour online at www.interscience.wiley.com/journal/jrs.
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15 and 0 ° C and fall to 60 ° C in the winter. The Upper Devonian orthoquartzites of the Beacon Sandstone Formation outcrop throughout the Dry Valleys and contain well-studied examples of cryptoendolithic lichens and microalgal communities.28 – 29 Specimens of exfoliated orthoquartzite containing cryptoendolithic colonies were collected by the British Antarctic Survey (BAS) in 1995 from the Ross Desert McMurdo Dry Valleys at Linnaeus Terrace (77° 360 S, 161° 050 E, elevation 1600 m). The photosynthetic cryptolichens form a benchmark for organisms at these ‘limits of life’ situations. Over time, oxalic acid secretions from the lichens dissolve the intergranular cement of the host rock, leading to bioweathering and exfoliation of the rock surface.30 Communities typically occur as distinct layers within the rock fabric (Fig. 2). The upper (near surface) black stratum is commonly 1 mm thick and close to the exfoliation interface. Below this layer is a white zone between 1 and 4 mm thickness where the lichens have mobilised iron compounds and leached the rock of iron-bearing minerals, concentrating them in a red coloured zone above and below the white, iron-depleted zone. A green algal layer is typically found within this iron-depleted zone.21
Chasmolithic colonisation, dolomitic marble, McMurdo Dry Valleys, Victoria Land, Antarctica (GSPARC 194) In other parts of the McMurdo Dry Valley system, crystalline rocks contain examples of chasmolithic cyanobacteria.31 Some specimens were collected by the late David WynnWilliams (BAS) during a field expedition from a talus slope in the vicinity of the Long Term Ecological Research (LTER) site on Andrews Ridge near Lake Hoare, Taylor Valley (77° 380 S, 162° 520 E). Our study sample (Fig. 3) is a weathered
Figure 2. Exfoliated orthoquartzite containing cryptoendolithic colonies: Linnaeus Terrace, McMurdo Dry Valleys, Ross Desert, Antarctica. This figure is available in colour online at www.interscience.wiley.com/journal/jrs.
Copyright 2007 John Wiley & Sons, Ltd.
Figure 3. Weathered dolomite marble colonised by Chroococcidiopsis: LTER site, Andrews Ridge, Lake Hoare, Taylor Valley, Antarctica. This figure is available in colour online at www.interscience.wiley.com/journal/jrs.
marble colonised by Chroococcidiopsis, a desiccation-resistant, radiation-resistant cyanobacterium. Microbial growth occurs along fracture planes on both fresh and internal weathered, and the chasmolithic colonies are distinct due to their blue-green colouration, penetrating several centimetres into the rock.
Halotrophic colonisation, gypsum (var. selenite), Haughton Crater, Devon Island (GSPARC 44) The Haughton Crater meteorite impact structure is located in the western region of Devon Island in the Canadian High Arctic (75° 220 N, 89° W) and was formed during the late Eocene (¾39 Ma).32 Surface mapping confirms a crater of approximately 24 km in diameter formed from the impact of an asteroid or comet which penetrated target rocks comprising thick carbonate sequence (¾1.8 km) underlain by Precambrian granites and gneisses. Allochthonous polymict impact breccia dominates the central portion of the crater (¾10 km diameter), which is largely composed of target rock clasts from the carbonate sequence plus basement gneisses. Evidence for impact-induced hydrothermal activity is well preserved within the structure including sulfate mineralisation and mobilisation.33 Microbial colonisation within the selenitic gypsum sulphate deposits has recently been described.34,35 The gypsum specimen studied here (Fig. 4) is in the form of selenite, in which the microbial colonies inhabit the interlaminar spaces and appear up to a few centimetres from the external margins. The clarity of the selenite crystals provides a spectroscopic ‘window’ through which it is possible to observe the microbial communities at successive levels. Two species of halotrophic cyanobacteria have been identified,36 Gloeocapsa alpine (Nageli) Brand and Nostoc commune Vaucher.
J. Raman Spectrosc. 2007; 38: 1352–1361 DOI: 10.1002/jrs
Raman spectra of fossilised sedimentary geological specimens
Figure 4. Halotrophic colonisation of gypsum (var. selenite) by Nostoc and Gloeocapsa: Haughton meteorite impact crater, Devon Osland, Arctic Canada. This figure is available in colour online at www.interscience.wiley.com/journal/jrs.
Figure 5. Banded chert: Kitty’s Gap, Coppin Gap, Panorama Formation, Pilbara Craton, western Australia. This figure is available in colour online at www.interscience.wiley.com/journal/jrs.
Relict sedimentary structures
domical stromatolites from the Panorama formation near North Pole Dome was acquired by one of us (Westall) in 2000, before the site was considered for protection.
Two Early-Mid Archaean samples (¾3.45 Ga volcaniclastic sediments from the Pilbara, Western Australia, and a 3.2. Ga Banded Iron Formation (BIF) from the Barberton Greenstone Belt, South Africa) represent an epoch when Earth and Mars possibly experienced similar conditions.37
Banded chert, Kitty’s Gap, Pilbara, Australia (GSPARC 190) The oldest part of the Pilbara craton of Western Australia lies to the east within the 3.72 Ga to 2.85 Ga granite-greenstone terrain and includes the Warrawoona Group of volcanics and cherts. The Panorama Formation of the Warrawoona Group is characterised by volcaniclastic sediments ranging from rhyolitic to dacitic in composition towards the top of the sequence. These sediments are thought to have formed in a shallow marine to subaerial (intertidal) environment and the sediments were rapidly silicified owing to the high silica content of the sea and pore water and a contribution from the rapid silification of biogenic organic remains within the sediments.38 Radiometric studies date these rocks at 3.446 š 5 Ma.39 The study specimen (Fig. 5) of black and white laminated chert from the Coppin Gap locality rocks of the Panorama Formation exposed at Kitty’s Gap (120° 50 E, 20° 530 S) exhibits well-preserved macroscopic flaser-linsen bedding and crossbedding structures and micro-laminae on fresh surfaces.
Raman spectroscopy Raman spectra were excited using a Bruker FT-Raman system incorporating an IFS 66 spectrometer and FRA 106 Raman module attachment operating at 1064 nm in the near infrared with a Nd3C /YAG laser in microscopic and macroscopic modes. For the former, the spectral footprint was about 8 µm using a 100ð lens objective whereas the latter mode of operation collected radiation from a spectral footprint of about 100 µm. Typical laser powers were 50 mW to avoid
Stromatolitic chert, North Pole Dome, Trendall, Pilbara, Australia (GSPARC 159) Well-preserved coniform and columnar stromatolites in silicified carbonate Panorama formation sediments (3446 Ga) occur in the North Pole Dome area and contain microbial mediation in their formation.40 A specimen (Fig. 6) of these
Copyright 2007 John Wiley & Sons, Ltd.
Figure 6. Stromatolitic chert, silicified carbonate deposits: North Pole Dome, Trendall, Pilbara Craton, Western Australia. This figure is available in colour online at www.interscience.wiley.com/journal/jrs.
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sample deterioration and some 2000 spectral scans were accumulated with a spectral resolution of 4 cm1 . Each spectrum required approximately 60 min scan time and several replicates were examined in each spectral region of interest. A Renishaw InVia Reflex Raman microscope operating confocally at laser wavelengths of 514.5 and 785 nm with a spectral footprint of 2 µm using a 100ð lens objective gave spectra at 2 cm1 resolution with up to 25 scans accumulated using laser powers of several milliwatts. Each spectrum required about 5 min total scan time, and several replicates were examined. Calibration was effected using internal laser wavelengths and also by reference to standard Raman bands of minerals occurring in the specimens. Key biochemicals produced by the extremophilic organisms and biologically modified substrates were identified using our Raman databases and literature values. This is especially important for the characterisation of carotenoids, whose C C and C–C bands represent good biomarkers of the protective survival strategies operating in the systems.
RESULTS AND DISCUSSION Filamentous limestone: GSPARC 140–420 This sample, collected in Hainsfarth, the Riess crater (Germany), is a limestone formed in an aqueous environment.41,42 The sample consisted of calcified tubular microbial filamentous morphology (Fig. 1). Three tubes, each of approximately 100 µm diameter, have been analysed in a transverse section to study the mineralogical distribution and to detect the presence of organic compounds. About 20–30 sites were examined in each tube in concentric and radial distribution patterns. One of the tubes evidenced a distribution of calcite (1086, 712, 281 and 156 cm1 ) in the central area with a dolomite sheath (1098, 725, 300 and 178 cm1 ) found externally around the calcite comprising the ‘body’ of the tube. Gypsum (1008, 669, 618, 492 and 413 cm1 ) appears to be heterogeneously distributed in both the calcite and dolomite zones of this particular tube but gypsum was not found in all the examined tubes. A second tube shows the presence of calcite in the central area but it also appears in the mid-section and in the external areas of the tube together with dolomite; gypsum is absent in this tube. A third tube examined gave the signatures of only dolomite and neither calcite nor gypsum was found. Several different black spots found in the tubes, which can be observed visually in Fig. 1 have been analysed with both 785 and 514.5 nm laser excitation; a typical spectrum achieved of a black spot in the second tube (Fig. 7) shows broad Raman bands that are characteristic of degraded organic compounds which are as yet unidentified. Bands characteristic of carotenoids near 1520 and 1155 cm1 are visible in this spectrum.
Copyright 2007 John Wiley & Sons, Ltd.
Figure 7. Raman spectrum (514.5 nm excitation, wavenumber range 150–1800 cm1 ) of a black spot in a calcite tube from the Ries Crater sample (Fig. 1), showing the broad spectral features characteristic of aromatic organic components and the presence of a carotenoid with bands at 1520 and 1155 cm1 .
Endolithic specimen: GSPARC 114 Here, the surface crust shows the characteristic features of haematite, goethite and quartz, whereas the cyanobacterial zone gives Raman signatures of carotenoids, chlorophyll and calcium oxalate monohydrate (Fig. 8); the latter compound, with Raman bands at 1494, 1463, 908 and 506 cm1 , is a metabolic by-product of the reaction between oxalic acid with calcareous material in the rock substrate. A visual identifier of this type of colonisation is the colour depletion in the colonised zone arising from the movement of iron oxide to the surface crust; in this process, the iron oxide is partially converted to goethite, which results in the colour change observed at the surface of cryptoendolithic specimens.11
Marble chasmolith: GSPARC 194–435 The rock substrate is composed mainly of dolomite (1098, 724, 338, 300 and 174 cm1 ). The black organic stratum shows Raman signatures at 1603, 1590, 1551, 1435, 1384, 1358, 1324, 1285, 1245, 1167, 1096, 984, 909, 887, 777, 753, 676, 658, 574, 306 and 272 cm1 unambiguously identifiable as scytonemin and signatures at 1508, 1155 and 1001 cm1 assigned to a carotenoid, probably astaxanthine or decapreno-betacarotene. Particularly interesting is the presence of calcite, in the form of a white dust, which appears only in the black and reddish zones, identified by its Raman signatures at 1086, 713, 281 and 156 cm1 . The reddish band shows a quartz signature (206 and 464 cm1 ), and bands at 956, 919, 881, 856 and 824 cm1 are unambiguously assigned to olivine. The presence of olivine in a marble rock can be ascribed to detritic particles, the product of wind or water transport and deposited in a rock crack or in a weathered surface depression. Signatures of 1512, 1155 and 1003 cm1 indicate the presence of a carotenoid, which could be echinenone;
J. Raman Spectrosc. 2007; 38: 1352–1361 DOI: 10.1002/jrs
Raman spectra of fossilised sedimentary geological specimens
Figure 8. Raman spectra (785 nm excitation, wavenumber range 150–1800 cm1 ) of cryptoendolithic orthoquartzite (Fig. 2): upper spectrum, surface crust with bands characteristic of haematite, goethite and quartz; lower spectrum, cryptoendolithic colony zone with bands characteristic of chlorophyll, beta-carotene, scytonemin and calcium oxalate monohydrate (whewellite).
whereas the bands at 1327, 988 and 742 cm1 have been assigned to chlorophyll. The greenish stratum in this specimen shows broad signatures of scytonemin. A second organic compound, with broad bands at 1544, 1500, 1436, 1356, 1316, 1306 and 1154 cm1 , also appears but its identification is still a matter for conjecture. In the 1500 cm1 broad band, a shoulder around 1515 cm1 , which, together with the signatures at 1154 and 1003 cm1 , has been assigned to beta-carotene. Raman bands from the substratum dolomite also appear in all the spectra. In the blue-green zone, two carotenes have been identified: the first with Raman signatures at 1520, 1156 and 1005 cm1 (zeaxanthine) and a second carotenoid with bands at 1515, 1154 and 1002 cm1 (beta-carotene). Bands at 1630, 1580, 1498, 1367, 1327, 1308, 1047, 739 and 661 cm1 can be assigned to the photosynthetic accessory pigment cphycocyanin (Fig. 9); here, the Raman signature of dolomite is also seen to be very closely associated with the carotenoid.
Figure 9. Raman spectrum of chasmolithic colonised zone in dolomitic marble (Fig. 3) (514.5 nm excitation, wavenumber range 200–1700 cm1 ) showing bands characteristic of c-phycocyanin, zeaxanthin and beta-carotene in addition to the dolomite matrix signature at 1094 cm1 .
(a)
Selenite: Haughton Crater: GSPARC 44 The presence of halotrophic cyanobacterial colonies some 5–10 cm below the upper surface of a gypsum crystal (selenite) were identified as type 1 (Nostoc) and type 2 (Gloeocapsa) species from the Raman band signatures exhibited with 785 nm excitation as shown in Fig. 10. Figure 10(a) shows the Raman spectrum composed of scytonemin and carotenoid signatures, whereas Fig. 10(b) gives parietin (an accessory radiation protective photopigment), carotenoid and chlorophyll. This specimen is of particular interest since it demonstrates that two survival strategies are being adopted by the cyanobacterial extremophiles at similar depths and locations within the same crystal matrix.
Copyright 2007 John Wiley & Sons, Ltd.
(b)
Figure 10. Raman spectra of halotrophic cyanobacterial inclusions in selenitic gypsum from a meteorite impact crater (Fig. 4) (785 nm excitation, wavenumber range 100–1800 cm1 ): (a) type 1 (Nostoc), showing scytonemin and a carotenoid signatures; (b) type 2 (Gloeocapsa), showing parietin, carotenoid and chlorophyll signatures.
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Banded chert, Pilbara, Kitty’s Gap: GSPARC 190-X There is little spectroscopic difference observed between the black and white band compositions. Both areas have quartz and carbon Raman signatures, although the white zones show relatively less carbon than the black analogoues. Raman band signatures at 1314 and 1598 cm1 are observed but the signatures at 1314 cm1 (sp3 carbon) in this sample are usually stronger than the signature at 1598 cm1 (sp2 carbon). Rutile (447 and 610 cm1 ) and anatase (141, 194, 262, 394, 513 and 637 cm1 ) are also present in several spectra and frequently appear together, as small nodules in the black and white zones. Goethite, with Raman bands at 246, 299, 386 and 551 cm1 , haematite (225, 295, 411, 504 and 615 cm1 ), calcite (156, 281, 712 and 1086 cm1 ) and graphite with a band centred at 1581 cm1 sp2 and another stronger one at 1368 cm1 sp3 have also been identified at several sites in the black and white zones; an example is shown in Fig. 11. Several black particles randomly scattered in the white or black areas show the weaker signatures of an organic compound at 1723, 1611, 1453, 1380, 1364, 1340, 1307, 1245, 1229, 1215, 1178, 1151, 1110 and 983 cm1 ; a broad, stronger feature centred at 666 cm1 is assigned to magnetite, which seems to be always related with the organic signatures (Fig. 12), along with that of alpha-quartz with a characteristic band at 465 cm1 . The width of the bands in all the Raman spectra collected from this organic compound are always very broad and are indicative of extensive biological degradation having occurred.
Stromatolitic chert, Trendall, Pilbara: GSPARC 159 The Trendall stromatolite presents an alteration of grey and white coloured silica bands and grey carbonated bands with irregularly distributed ochre-coloured regions (Fig. 6).
Figure 11. Raman spectrum of Kitty’s Gap chert (Fig. 5), showing the mineral components typical of the banded zones (514.5 nm excitation, wavenumber range 100–1700 cm1 ): carbon (C), dolomite (D), haematite (H) and goethite (G) features can be identified.
Copyright 2007 John Wiley & Sons, Ltd.
Figure 12. Raman spectrum of a black particle in the specimen of Kitty’s Gap chert (Fig. 5) (514.5 nm excitation, wavenumber range 100–1800 cm1 ) showing strong bands assigned to magnetite at 666 cm1 and quartz at 465 cm1 with weaker, broader features ascribed to degraded organic compounds.
Organic biosignatures are found in these orange-red ‘ochre’ areas. In the white silicaceous strata, the Raman spectrum of only quartz with Raman bands at 128, 106, 264, 354, 393, 402, 464, 696, 808, 1064, 1081, 1161 and 1227 cm1 has been identified, whereas in the grey silicaceous regions quartz appears together with graphite (1601 and 1307 cm1 ). Dolomite, CaMgCO3 2 , is identified as the carbonate mineral in this specimen by its Raman bands at 1098, 724, 300 and 175 cm1 ; other minerals identified in association with this dolomite are quartz, carbon (1610, 1320 cm1 ), goethite (250, 300, 387, 478 and 548 cm1 ), haematite (224, 297, 407, 500, 611 and 650 cm1 ). Rutile has also been found (606, 447 cm1 ). The difference between the grey and the orangered coloured carbonated areas is ascribed to the haematite and goethite concentrations and these minerals are observed to be more abundant in the orange-red areas. Organic biosignatures were detected in the spectra from small black and blue particles (estimated diameter 5 µm) in the geological matrix of this specimen. In the black particles with 514.5 nm laser excitation, the Raman bands at 1516, 1155 and 1004 cm1 permit the identification of betacarotene whereas the signature at 1535, 1144 and 1006 cm1 are assigned to a shorter chain carotenoid, possibly zeaxanthine (Fig. 13). The Raman bands at 1714, 1629, 1605, 1593, 1554, 1520, 1435, 1384, 1323, 1282, 1243, 1172, 1084, 983, 887, 838, 753, 678, 539 and 450 cm1 can be unambiguously assigned to scytonemin (Fig. 14). The presence of dolomite and goethite minerals can also be observed with bands at 1098, 387 and 300 cm1 . In several of these particles, the scytonemin UV-screening pigment is associated spectroscopically with the carotenes. In the spectra collected from light-blue coloured particles, a third carotene with Raman bands at 1530, 1144 and 1007 cm1 (violaxanthin) appears together with the signatures at 1592, 1485, 1451, 1307, 1216, 1194, 1108, 953, 847,
J. Raman Spectrosc. 2007; 38: 1352–1361 DOI: 10.1002/jrs
Raman spectra of fossilised sedimentary geological specimens
Figure 13. Raman spectra of a black particle in Trendall, Pilbara stromatolitic chert (Fig. 6) (514.5 nm excitation, wavenumber range 200–1800 cm1 ) showing the presence of zeaxanthin (lower spectrum) and a mixture of zeaxanthin and beta-carotene (upper spectrum).
Figure 14. As for Fig. 13, (but 785 nm excitation, wavenumber range 100–1800 cm1 ) showing the presence of scytonemin and a carotenoid, probably astaxanthin, with weaker features attributed to dolomite and goethite.
779, 746, 679, 640, 594 and 483 cm1 assigned to a porphyrin (Fig. 15). The identification of three types of carotenes, two of them associated with scytonemin and the third related with a porphyrin, indicates the presence of several relict micro-organisms: one micro-organism with carotene and scytonemin, a second one with carotene at 1517–1157 cm1 (both occurring in the black particles) and another light-blue coloured relict micro-organism with carotene (1530–1144 cm1 ) and a porphyrin, which has presumably been produced from chlorophyll degradation.
Comparison between the Raman spectra of the relict Archaean cherts at Kitty’s Gap and Trendall Although there are several minor spectral differences, the organic material identified in the black particles in the Kitty’s
Copyright 2007 John Wiley & Sons, Ltd.
Figure 15. Raman spectrum of a blue particle in Trendall, Pilbara stromatolitic chert (Fig. 6) (785 nm excitation, wavenumber range 100–1800 cm1 ) showing a carotenoid C (violaxanthin) and a porphyrin.
Gap chert is similar to the pigment found in the blue spots in the Trendall Pilbara stromatolitic chert and we infer that it is a degradation product of porphyrins and chlorophylls. In the Raman spectra achieved of the blue spots in the Trendall sample, a carotenoid appears with bands at 1529, 1143 and 1006 cm1 , which is absent in the analyses of the black spots of the Kitty’s Gap sample (Figs 11 and 5). The doublet at 1606 and 1591 cm1 in the Trendall sample appears now as a broad band centred at 1611 cm1 , which also shows a shoulder at 1585 cm1 . The medium intensity band at 1451 in the Trendall blue spot spectrum appears as a broad band with medium relative intensity at 1453 in the Kitty’s Gap specimen but the weaker signature at 1429 cm1 is no longer visible. Several important differences between the organic signatures from Trendall and Kitty’s Gap cherts are observable in the 1400–1300 cm1 wavenumber region. The strong band at 1339 and the medium intensity signature at 1308 cm1 in the Trendall stromatolite blue spot spectra appear as broad and strong signatures in the Kitty’s Gap specimen and a shoulder is visible, centred at 1294 on the 1307 cm1 band; the signature at 1370 cm1 has now disappeared and has been replaced with two medium intensity bands at 1380 and 1362 cm1 . In the wavenumber region 1200–900 cm1 there are also some visible differences; in the black and white Kitty’s Gap chert a succession of broad signatures at 1229, 1214, 1180, 1152, 1111 and 983 cm1 are observable, whereas the bands at 1214, 1180, 1152 and 1111 cm1 are closely similar to those occurring at 1216, 1187, 1157 and 1108 cm1 in the Trendall stromatolite spectra. However, the medium intensity signatures at 1194 and 953 cm1 in the Trendall sample are no longer visible in the Raman spectra collected from the Kitty’s Gap chert. In the 900–200 cm1 region, the spectra achieved from the Kitty’s Gap sample show, apart from signatures of quartz,
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a broad band at 666 cm1 and, in some spectra, also a weak signature at 513 cm1 , both assignable to magnetite, Fe3 O4 . The degraded organic compounds in the Kitty’s Gap specimen always appear related to a significant magnetite signal. It is possible that the presence of the magnetite could therefore be related to the organism activity and may even be assisting in the conservation of the relict organic compounds.
CONCLUSIONS The observation of Raman spectra from biomaterials and minerals in a range of specimens from extremophile niche environments that have been identified as possible terrestrial analogues for Martian sites demonstrates the versatility of the Raman technique for biogeological exploration. A particular advantage is the ability to obtain precise molecular information about the organic and inorganic composition from real samples without the necessity of their pretreatment or preparation. In several cases, the chemical signatures of the organic components have been identified from materials that are apparently abiological, which indicates that the technique is capable of identifying the presence of relict organisms as well as living colonies. The construction of a Raman spectroscopic database of key biomolecular bands and features arising from the biogeological modification of rocks will be a vital part of the automatic recognition process being envisaged for remote robotic Mars landers and rovers that will include miniaturised Raman spectroscopic instrumentation as part of their analytical and life-detection package. To this end, this article provides a useful base for the commencement of this database initiation. Clearly, while the specimens described here are not exhaustive from the Mars analogue point of view, it is important that as many terrestrial extremophilic niches as possible are analysed in this way to the strategies being adopted for the survival of stressed organisms. This will better facilitate our attempt to understand the types of organisms that may have once existed on Mars, the recognition of their spectral signatures and information on the selection of possible sampling or landing sites for extraterrestrial exploration of planetary surfaces and subsurfaces in the search for signs of extinct or extant life.
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