Identification of beta-carotene in an evaporitic matrix--evaluation of Raman spectroscopic analysis for astrobiological research on Mars.

Since evaporitic rocks on the Martian surface could (or still can) serve as potential habitats for microbial life on Mars, there is a reasonable possibility that these rocks may sustain molecular remnants as evidence for the presence of extinct or extant living organisms on Mars and that beta-carotene could be a suitable biomarker. In this paper, Raman microspectrometry was tested as a nondestructive method of determining the lowest detectable beta-carotene content in experimentally prepared evaporitic matrices--namely, gypsum, halite and epsomite. Two excitation wavelengths were compared--514.5 nm, because of the resonance Raman enhancement in the carotenoid analysis, and 785 nm, as a more universal wavelength now much used in the detection of biomolecules terrestrially. Mixtures were measured directly as well as with a laser beam penetrating the crystals of gypsum and epsomite. We have obtained beta-carotene signals at the 0.1 to 10 mg kg(-1) level--the number of registered beta-carotene Raman bands differed depending on the particular mineral matrix and the excitation wavelength. Concentrations of beta-carotene of about one order of magnitude higher were identified when analysed through single crystals of gypsum and epsomite, respectively.

Analyzing carotenoids of snow algae by Raman microspectroscopy and high-performance liquid chromatography.

We tested the potential of Raman microspectroscopy to determine carotenoid pigments - both primary (lutein, beta-carotene) and secondary (astaxanthin) carotenoids - in the different species and life-cycle stages of snow algae from the order Chlamydomonadales (Chlorophyta). We compared the performance of Raman spectrometry to a reference method of biological pigment analysis, high-performance liquid chromatography (HPLC). The three main carotenoid Raman bands of the astaxanthin-rich red cysts were located at 1520, 1156 and 1006 cm-1. The shifts (orange aplanozygotes and green motile cells with flagella) in the position of the ν1(CC) Raman band of the polyenic chain is consistent with the expected changes in the ratios of the various carotenoid pigments. Flagellated green cells commonly contain lutein as a major carotenoid, together with minor amounts of ß­carotene and varying amounts of antheraxanthin, violaxanthin and neoxanthin. Aplanozygotes contain mixtures of both primary and secondary carotenoids. In most cases, the ν1(CC) band is an overlapping set of bands, which is due to the signal of all carotenoid pigments in the sample, and a deconvolution along with the band position shifts (mainly ν1) could be used to characterize the mixture of carotenoids. However, the ability of Raman spectroscopy to discriminate between structurally slightly differing carotenoid pigments or several carotenoids in an admixture in an unknown biological system remains limited.

Potential and limits of Raman spectroscopy for carotenoid detection in microorganisms: implications for astrobiology.

In this paper, it is demonstrated how Raman spectroscopy can be used to detect different carotenoids as possible biomarkers in various groups of microorganisms. The question which arose from previous studies concerns the level of unambiguity of discriminating carotenoids using common Raman microspectrometers. A series of laboratory-grown microorganisms of different taxonomic affiliation was investigated, such as halophilic heterotrophic bacteria, cyanobacteria, the anoxygenic phototrophs, the non-halophilic heterotrophs as well as eukaryotes (Ochrophyta, Rhodophyta and Chlorophyta). The data presented show that Raman spectroscopy is a suitable tool to assess the presence of carotenoids of these organisms in cultures. Comparison is made with the high-performance liquid chromatography approach of analysing pigments in extracts. Direct measurements on cultures provide fast and reliable identification of the pigments. Some of the carotenoids studied are proposed as tracers for halophiles, in contrast with others which can be considered as biomarkers of other genera. The limits of application of Raman spectroscopy are discussed for a few cases where the current Raman spectroscopic approach does not allow discriminating structurally very similar carotenoids. The database reported can be used for applications in geobiology and exobiology for the detection of pigment signals in natural settings.

Detection of pigments of halophilic endoliths from gypsum: Raman portable instrument and European Space Agency's prototype analysis.

A prototype instrument, under development at the University of Leicester, for the future European Space Agency (ESA) ExoMars mission, was used for the analysis of microbial pigments within a stratified gypsum crust from a hypersaline saltern evaporation pond at Eilat (Israel). Additionally, the same samples were analysed using a miniaturized Raman spectrometer, featuring the same 532 nm excitation. The differences in the position of the specific bands, attributed to carotenoid pigments from different coloured layers, were minor when analysed by the ESA prototype instrument; therefore, making it difficult to distinguish among the different pigments. The portable Delta Nu Advantage instrument allowed for the discrimination of microbial carotenoids from the orange/green and purple layers. The purpose of this study was to complement previous laboratory results with new data and experience with portable or handheld Raman systems, even with a dedicated prototype Raman system for the exploration of Mars. The latter is equipped with an excitation wavelength falling within the carotenoid polyene resonance region. The ESA prototype Raman instrument detected the carotenoid pigments (biomarkers) with ease, although further detailed distinctions among them were not achieved.

Raman spectrometric discrimination of flexirubin pigments from two genera of Bacteroidetes.

Flexirubins are specific polyene pigments produced by several genera of Bacteroidetes. Colonies and cell extracts of Flavobacterium johnsoniae and Flexibacter elegans have been investigated by Raman spectroscopy to show that this fast and non-destructive technique can be used to differentiate these pigments from carotenoids and to compare the flexirubin content of the two microorganisms. The presence or absence of certain distinguishing features in the CH combination band region at 2500-2750 cm(-1) can assist in the discrimination between the two flexirubins investigated. Raman spectroscopy is thus a suitable tool not only to detect flexirubin pigments in bacterial cells, but also to further characterize the pigments present in members of the Bacteroidetes genera that are rich in flexirubins.

Feasibility of Raman microspectroscopic identification of biomarkers through gypsum crystals.

The miniaturized Raman spectrometer is considered to be a candidate instrument for the Pasteur payload (the ExoMars mission scheduled for 2018). This mission will, for the first time, combine mobility and access to subsurface locations where organic molecules might be well preserved. Evaporitic crystals are among the potential protected habitats that have been postulated. Various concentrations of biomarkers (beta-carotene, glycine and phthalic acid) dispersed in a gypsum matrix were analyzed through transparent mineral (gypsum) plates of different thicknesses. By doing so, conditions were simulated in which biomarkers were trapped within evaporitic crystals. Using a long-working distance objective, all studied concentrations of biomarkers mixed in gypsum powder were detected. The characteristic Raman bands were easily observable for a 10% mixture of all chosen biomarkers not only through a 3.3 mm plate and but even through a 5.2 mm plate. It was possible to detect key Raman bands of 1% phthalic acid/gypsum mixture and 1% beta-carotene/gypsum mixture even through a 5.2 mm gypsum plate. The 1% beta-carotene/gypsum mixture was still clearly distinguishable through an 8.5mm gypsum crystal due to the known resonance Raman effect of the molecule.

Investigation of biomolecules trapped in fluid inclusions inside halite crystals by Raman spectroscopy.

Raman spectroscopy was tested for the identification of biomolecules (glycine, L-alanine, ß-alanine, L-serine, and γ-aminobutyric acid) trapped in fluid inclusions inside halite model crystals. The investigated biomolecules represent important targets for future astrobiological missions. We know from terrestrial conditions that organic molecules and microorganisms can be sealed within fluid inclusions and can survive intact even for hundreds of millions of years. Raman spectroscopy is currently being miniaturized for future extraterrestrial planetary exploration (ExoMars 2018). Raman spectroscopy has shown the ability to detect investigated aminoacids nondestructively without any sample preparation, in short measurement times, and in relatively low concentrations. The number of registered Raman bands of investigated aminoacids and their intensity clearly correlate with the given concentration of biomolecules within fluid inclusions.

Raman spectroscopic identification of phthalic and mellitic acids in mineral matrices.

Raman spectroscopy has many advantages for planetary exploration, and the Raman technique is currently being developed for future space missions. The Raman microspectroscopic study of organic acids (phthalic and mellitic acids) in experimentally prepared mixtures with halite and gypsum was performed using near infrared 785 nm and visible 514 nm excitation wavelengths. Gypsum and halite matrices were chosen as analogues of Martian minerals. Carboxylic acids mixed with mineral powders were also investigated through a UV-transparent crystal of gypsum and halite (approximately 2 mm, resp. 5 mm thick), thereby creating a type of artificial inclusion that could potentially be present in Martian minerals. The detection limit of phthalic acid mixed in mineral matrices and analyzed through crystals was 1 wt% using both excitation wavelengths. A Raman mellitic acid signal was obtained at a concentration as low as 1 wt% in halite matrix, and at a concentration of 5 wt% when analyzed through halite crystal. In the case of mellitic acid mixed with gypsum and for analyses through a gypsum crystal, the detection limit is 5 wt% using both excitation wavelengths.

Raman spectroscopic identification of usnic acid in hydrothermal minerals as a potential Martian analogue.

Raman spectroscopy using 785 nm excitation was tested as a nondestructive method for determining the presence of the potential biomarker, usnic acid, in experimentally prepared mineral matrices. Investigated samples consisting of usnic acid mixed with powdered hydrothermal minerals, gypsum and calcite were studied. Various concentrations of usnic acid in the mineral matrix were studied to determine the detection limits of this biomarker. Usnic acid was mixed with gypsum (respectively, calcite) and covered by a UV-transparent crystal of gypsum (CaSO(4) x 2 H(2)O), thereby creating artificial inclusions similar to those which could be present in Martian minerals. A Raman usnic acid signal at the concentration level as low as 1 g kg(-1) was obtained in the powdered mineral matrix and 5 g kg(-1) when analyzed through the monocrystal. The number of registered usnic acid key Raman bands was dependent on the particular mineral matrix. If a similar concentration of usnic acid could persist in Martian samples, then Raman spectroscopy will be able to identify it. Obtained results will aid both in situ Raman analyses on Mars and on Earth.