Inclusions in evaporitic minerals sometimes contain remnants of microorganisms or biomarkers, which can be considered as traces of life. Raman spectroscopy with resonance enhancement is one of the best analytical methods to search for such biomarkers in places of interest for astrobiology, including the surface and near subsurface of planet Mars. Portable Raman spectrometers are used as training tools for detection of biomarkers. Investigations of the limits and challenges of detecting biomolecules in crystals using Raman spectroscopy is important because natural occurrences often involve mineral assemblages as well as their fluid and solid inclusions. A portable Raman spectrometer with 532 nm excitation was used for detection of carotenoid biomarkers: salinixanthin of Salinibacter ruber (Bacteroidetes) and α-bacterioruberin of Halorubrum sodomense (Halobacteria) in laboratory-grown artificial inclusions in compound crystals of several chlorides and sulfates, simulating entrapment of microorganisms in evaporitic minerals. Crystals of halite (NaCl), sylvite (KCl), arcanite (K2SO4) and tschermigite ((NH4)Al(SO4)2·12H2O) were grown from synthetic solutions that contained microorganisms. A second crystalline layer of NaCl or K2SO4 was grown subsequently so that primary crystals containing microorganisms are considered as solid inclusions. A portable Raman spectrometer with resonance enabling excitation detected signals of both carotenoid pigments. Correct positions of diagnostic Raman bands corresponding to the specific carotenoids were recorded.
Cell suspensions of the haloarchaea Halorubrum sodomense and Halobacterium salinarum and the extremely halophilic bacterium Salinibacter ruber (Bacteroidetes) in saturated solutions of chlorides and sulfates (NaCl, KCl, MgSO4·7H2O, K2SO4, and (NH4)Al(SO4)2·12H2O) were left to evaporate to produce micrometric inclusions in laboratory-grown crystals. Raman spectra of these pinkish inclusions were obtained using a handheld Raman spectrometer with green excitation (532 nm). This portable instrument does not include any microscopic tool. Acceptable Raman spectra of carotenoids were obtained in the range of 200-4000 cm-1. This detection achievement was related to the mode of illumination and collection of scattered light as well as due to resonance Raman enhancement of carotenoid signals under green excitation. The position of diagnostic Raman carotenoid bands corresponds well to those specific carotenoids produced by a given halophile. To our best knowledge, this is the first study of carotenoids included in the laboratory in crystalline chlorides and sulfates, using a miniature portable Raman spectrometer. Graphical abstract ᅟ.
Nicotine has a profound influence on the carotenoid metabolism in halophilic Archaea of the class Halobacteria. In a study of Halobacterium salinarum, Haloarcula marismortui and Halorubrum sodomense, using different analytical techniques to monitor the production of different carotenoids as a function of the presence of nicotine, we showed that the formation of α-bacterioruberin was inhibited in all. In Hbt. salinarum, addition of nicotine led to a significant change in the color of the culture due to the accumulation of lycopene, in addition to the formation of bisanhydrobacterioruberin which does not differ in color from α-bacterioruberin. Very little or no lycopene was formed in Har. marismortui and in Hrr. sodomense; instead bisanhydrobacterioruberin was the only major carotenoid found in nicotine-amended cultures. The findings are discussed in the framework of the recently elucidated biochemical pathway for the formation of the different carotenoid pigments encountered in the Halobacteria.
We explored the use of Raman spectroscopy to simultaneously monitor the presence of different biomarkers (carotenoids, elemental sulfur) within single cells of the purple sulfur photosynthetic bacteria Allochromatium vinosum and A. warmingii. Raman microspectrometry using excitation at 532 nm allowed the detection of different carotenoids. Raman signals of elemental sulfur appeared soon after feeding starved cells with sulfide. Raman spectroscopy is thus a convenient and sensitive technique to qualitatively and semiquantitatively assess the presence of different compounds of interest within single bacterial cells.
Raman spectroscopy is a rapid nondestructive technique providing spectroscopic and structural information on both organic and inorganic molecular compounds. Extensive applications for the method in the characterization of pigments have been found. Due to the high sensitivity of Raman spectroscopy for the detection of chlorophylls, carotenoids, scytonemin, and a range of other pigments found in the microbial world, it is an excellent technique to monitor the presence of such pigments, both in pure cultures and in environmental samples. Miniaturized portable handheld instruments are available; these instruments can be used to detect pigments in microbiological samples of different types and origins under field conditions.
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.
We explored the use of Raman spectroscopy to detect organic osmotic solutes as biomarkers in the moderately halophilic heterotrophic bacterium Halomonas elongata grown in complex medium (accumulation of glycine betaine) and in defined medium with glucose as carbon source (biosynthesis of ectoine), and in the anoxygenic phototrophic Ectothiorhodospira marismortui known to synthesize glycine betaine in combination with minor amounts of trehalose and N-α-carbamoyl glutamineamide. We tested different methods of preparation of the material: lyophilization, two-phase extraction of water-soluble molecules, and perchlorate extraction. Raman signals of glycine betaine and ectoine were detected; perchlorate extraction followed by desalting the extract on an ion retardation column gave the best results. Lyophilized cells of E. marismortui showed strong signals of carotenoid pigments, and glycine betaine could be detected only after perchlorate extraction and desalting. The data presented show that Raman spectroscopy is a suitable tool to assess the mode of osmotic adaptation used by halophilic microorganisms.
