Comparative analysis of cyanobacterial communities in gypsum outcrops: insights from sites in Israel and Poland.

Today, the biodiversity of endolithic microbial colonisations are only partly understood. In this study, we used a combination of molecular community metabarcoding using the 16S rRNA gene, light microscopy, CT-scan analysis, and Raman spectroscopy to describe gypsum endolithic communities in 2 sites-southern Poland and northern Israel. The obtained results have shown that despite different geographical areas, climatic conditions, and also physical features of colonized gypsum outcrops, both of these sites have remarkably similar microbial and pigment compositions. Cyanobacteria dominate both of the gypsum habitats, followed by Chloroflexi and Pseudomonadota. Among cyanobacteria, Thermosynechococcaceae were more abundant in Israel while Chroococcidiopsidaceae in Poland. Interestingly, no Gloeobacteraceae sequences have been found in Poland, only in Israel. Some of the obtained 16S rRNA gene sequences of cyanobacteria matched previously detected sequences from endolithic communities in various substrates and geographical regions, supporting the hypothesis of global metacommunity, but more data are still needed. Using Raman spectroscopy, cyanobacterial UV-screening pigments-scytonemin and gloeocapsin have been detected alongside carotenoids, chlorophyll a and melanin. These pigments can serve as potential biomarkers for basic taxonomic identification of cyanobacteria. Overall, this study provides more insight into the diversity of cyanobacterial endolithic colonisations in gypsum across different areas.

Effects of nicotine on the biosynthesis of carotenoids in halophilic Archaea (class Halobacteria): an HPLC and Raman spectroscopy study.

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.

Using a portable Raman spectrometer to detect carotenoids of halophilic prokaryotes in synthetic inclusions in NaCl, KCl, and sulfates.

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.

Raman spectroscopy of microbial pigments.

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.

Evaluation of pore-fracture microstructure of gypsum rock fragments using micro-CT.

This study utilized X-ray micro-computed tomography (micro-CT) to investigate weathered gypsum rocks which can or do serve as a rock substrate for endolithic organisms, focusing on their internal pore-fracture microstructure, estimating porosity, and quantitative comparison between various samples. Examining sections and reconstructed 3D models provides a more detailed insight into the overall structural conditions within rock fragments and the interconnectivity in pore networks, surpassing the limitations of analyzing individual 2D images. Results revealed diverse gypsum forms, cavities, fractures, and secondary features influenced by weathering. Using deep learning segmentation based on the U-Net models within the Dragonfly software enabled to identify and visualize the porous systems and determinate void space which was used to calculate porosity. This approach allowed to describe what type of microstructures and cavities is responsible for the porous spaces in different gypsum samples. A set of quantitative analysis of the detected void and modeled networks provided a needed information about the development of the pore system, connectivity, and pore size distribution. Comparison with mercury intrusion porosimetry showed that both methods consider different populations of pores. In our case, micro-CT typically detects larger pores (> 10 µm) which is related to the effective resolution of the scanned images. Still, micro-CT demonstrated to be an efficient tool in examining the internal microstructures of weathered gypsum rocks, with promising implications particularly in geobiology and microbiology for the characterization of lithic habitats.

Glycine betaine is the main organic osmotic solute in a stratified microbial community in a hypersaline evaporitic gypsum crust.

We have examined the organic osmotic solutes content within the stratified microbial communities in an evaporitic gypsum crust found in an evaporation pond (~194 g/l total dissolved salts) of the salterns of the Israel Salt Company, Eilat. We extracted intracellular solutes from the upper three pigmented layers of the crust: a yellow-orange layer dominated by unicellular cyanobacteria, a green layer with filamentous cyanobacteria, and a layer colored red-purple by purple sulfur bacteria; dense communities of heterotrophic bacteria were present in all layers. The solutes were analyzed by Raman spectroscopy, (1)H and (13)C nuclear magnetic resonance, and HPLC. All layers contained glycine betaine as the only detectable osmotic solute; ectoine and other solutes known to be produced by many halophilic and halotolerant prokaryotes were not found. In this first attempt to assess the osmotic solute content within complex natural communities of halophilic microorganisms, the predominant role of glycine betaine as an osmolyte was established. Most heterotrophic bacteria cannot produce glycine betaine but preferentially use it when it is supplied. Presence of glycine betaine produced by the photoautotrophic members of the community, therefore, may relieve the heterotrophs from the need to synthesize other compounds at a high-energy cost.

Scytonin in gypsum endolithic colonisation: First Raman spectroscopic detection of a new spectral biosignature for terrestrial astrobiological analogues and for exobiological mission database extension.

Microbial colonisations of gypsum from Eastern Poland (Badenian, Middle Miocene age) were investigated by Raman microspectrometry with a rarely used excitation 445 nm excitation. Zones of microbial colonisation in selenitic gypsum endolithic outcrops comprise algae and cyanobacteria, which commonly contain the photosynthetic and protective pigments carotenoids, scytonemin and gloeocapsin. Diagnostic bands differing from those of scytonemin have been identified in black colonies in gypsum outcrops at Chotel Czierwony (Poland). Raman spectral signatures of scytonin are reported here for the first time in two endolithic specimens identified by the band wavenumbers predicted from DFT calculations. The strong or medium strong intensity Raman bands observed at 1603, 1585, 1559, 1435, and 1424 cm-1. Other weaker bands were located at 1676 (sh), 1660 (sh), 1649, 1399, 1362, 1342, 1320, 1294, 1272, 1259, and 1052 cm-1. The first observation of the Raman spectrum of scytonin in the cyanobacterial colonisation of gypsum facilitates the inclusion of this new biomolecular signature in the library of unique Raman spectra of biological pigments invaluable for detection of traces of life in frame of the planetary missions.

Gypsum endolithic phototrophs under moderate climate (Southern Sicily): their diversity and pigment composition.

In this study, we used microscopic, spectroscopic, and molecular analysis to characterize endolithic colonization in gypsum (selenites and white crystalline gypsum) from several sites in Sicily. Our results showed that the dominant microorganisms in these environments are cyanobacteria, including: Chroococcidiopsis sp., Gloeocapsopsis pleurocapsoides, Gloeocapsa compacta, and Nostoc sp., as well as orange pigmented green microalgae from the Stephanospherinia clade. Single cell and filament sequencing coupled with 16S rRNA amplicon metagenomic profiling provided new insights into the phylogenetic and taxonomic diversity of the endolithic cyanobacteria. These organisms form differently pigmented zones within the gypsum. Our metagenomic profiling also showed differences in the taxonomic composition of endoliths in different gypsum varieties. Raman spectroscopy revealed that carotenoids were the most common pigments present in the samples. Other pigments such as gloeocapsin and scytonemin were also detected in the near-surface areas, suggesting that they play a significant role in the biology of endoliths in this environment. These pigments can be used as biomarkers for basic taxonomic identification, especially in case of cyanobacteria. The findings of this study provide new insights into the diversity and distribution of phototrophic microorganisms and their pigments in gypsum in Southern Sicily. Furthemore, this study highlights the complex nature of endolithic ecosystems and the effects of gypsum varieties on these communities, providing additional information on the general bioreceptivity of these environments.

Use of Raman spectroscopy for identification of compatible solutes in halophilic bacteria.

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.

Critical evaluation of portable Raman spectrometers: From rock outcrops and planetary analogs to cultural heritage - A review.

This review focuses on the progress made and recent developments regarding the use of portable Raman spectrometric devices in the geosciences as well as with scientific testing of objects of cultural heritage. By utilizing different types of Raman spectrometric devices - from the modular; through systems operating with optical heads mounted via optical fibers; to the compact, lightweight, and handheld - are herein described and critically evaluated. The wide range of applications under indoor and closed environments (museums, collections, religious buildings, etc.) are reviewed, and the more commonly used for characterization of art in the broadest sense, as well as fully outdoor investigations (e.g., on rocky outcrops; of the minerals, microbiological colonization, etc.), and including extreme Earth environments and projects with exo-planetary research potential. The rapid acquisition of relevant spectroscopic data and the non-destructiveness of the analyses and diagnostics are greatly appreciated by art historians and conservators on the one hand, as well as by mineralogists, geoscientists, microbiologists on the other. The various environments investigated and the broad range of compounds that can be detected by obtaining their Raman spectra have demonstrated the vast potential of portable, and especially handheld, Raman spectrometer devices for on-site applications.

Fast outdoor screening and discrimination of carotenoids of halophilic microorganisms using miniaturized Raman spectrometers.

Eight miniaturized Raman spectrometers were used to perform a fast outdoor screening and discrimination of carotenoids of a series of halophilic and non-halophilic microorganisms on a set of eight lyophilized samples, each containing high concentrations of a specific dominant carotenoid pigment. Raman spectra were acquired using different excitations (532, 785, sequentially shifted excitation of 785 and 853, and 1064 nm), based on the model of each Raman spectrometer, in order to ascertain the feasibility of individual wavelengths. The wavenumber positions of diagnostic Raman bands of carotenoids were observed for the different carotenoid species. Characteristic carotenoid Raman bands of the pigment bacterioruberin were reported (using the 532 nm excitation) at 1504-1509 cm-1, salinixanthin at 1510-1513 cm-1, spirilloxanthin at 1509-1513 cm-1, decaprenoxanthin at 1519 cm-1, ß-carotene at 1526 cm-1, and sarcinaxanthin at 1526-1528 cm-1. A 532 nm excitation consistently provided best results due to the significant resonance signal enhancement (both quantitative and qualitative carotenoid detection). Good results were also obtained using the sequentially shifted excitation combining two lasers in the near infrared spectral region, and similarly good results were acquired using a standard 1064 nm excitation. The least suitable was a 785 nm excitation, with the carotenoid Raman signal almost always weaker compared to major fluorescence signal arising from other types of pigments or biomolecules in the samples. A thorough light shielding was essential in order to acquire good quality data. This study shows that miniaturized Raman spectrometers, some even equipped with longer wavelength excitation, are able to detect different carotenoid pigments under non-laboratory conditions in a fast way, and discriminate between them, to a certain degree. The implications of this type of research are especially useful in astrobiology, where the searching, detection and discrimination of biomarkers such as carotenoids is receiving significant attention.

Raman spectra of pure biomolecules obtained using a handheld instrument under cold high-altitude conditions.

A handheld Raman spectrometer (Ahura First Defender) was tested for the unambiguous identification of biomolecules (pure amino acids, carboxylic acids, saccharides and trehalose) in the solid state under outdoor conditions (including moderate climate conditions as well as cold temperatures and high altitudes). The biomolecules investigated represent important objects of interest for future exobiological missions. Repetitive measurements carried out under identical instrumental setups confirmed the excellent reliability of the Raman spectrometer. Raman bands are found at correct wavenumbers +/-3 cm(-1) compared with reference values. This testing represents the first step in a series of studies. In a preliminary, challenging investigation to determine the detection limit for glycine dispersed in a powdered gypsum matrix, 10% was the lowest content confirmed unambiguously. Clearly there is a need to investigate further the detection limits of Raman spectroscopic analyses of biomolecules in more complex samples, to demonstrate the usefulness or disqualify the use of this technique for more realistic outdoor situations, such as eventual future missions to Mars.

Raman spectroscopic study of the uranyl sulphate mineral zippeite: low wavenumber and U-O stretching regions.

The uranyl sulphate mineral zippeite was studied by Raman spectroscopy. The phase purity of the sample was initially checked by X-ray powder diffraction and its chemical composition was defined by electron microprobe (wavelength dispersive spectroscopy, WDS) analysis. The Raman spectroscopy research focused on the low wavenumber and uranyl stretching vibration regions. Vibration bands down to 50 cm(-1) were tentatively assigned. The U-O bond lengths were calculated based on empirical relations. Inferred values are consistent with those obtained from the crystal structure analysis of synthetic zippeite. Number of bands was interpreted on the basis of factor group analysis.

Evaluation of handheld and portable Raman spectrometers with different laser excitation wavelengths for the detection and characterization of organic minerals.

Organic minerals occur rather rarely in some types of peat bogs, sedimentary geological environments, and hydrothermal veins. Commonly, calcium oxalates are produced by several plants, terpenoids are often associated with conifers. Because of the organic precursor, these minerals, from the smallest group of the mineralogical system, are sometimes considered as biomarkers. Potential detection of these compounds has high relevance in the fields of exobiology or geobiology. Here we show the potential of four portable Raman spectrometers, using different excitation wavelengths and technologies (operating at 532, 785, and 1064nm together with an advanced spectrometer using the sequentially shifted excitation (SSE) technology), for the rapid and non-destructive identification of these phases. For the organic minerals investigated here, the most intense Raman bands are generally detected at the expected wavenumber positions ±1-4cm-1 in the region 100-2000cm-1 in the spectra obtained from all spectrometers. Additionally, two spectrometers (the 532nm instrument and the SSE) are capable of detecting Raman bands in the higher wavenumber shift region of 2000-3500cm-1, allowing the more detailed characterization and differentiation of the related phases. From this work, and on the basis of the experimental data obtained, it is clear that the longer laser excitation wavelengths are more preferable for organic minerals identification due to the better mitigation of fluorescence emission. In contrast, the Raman spectrometer equipped with the shortest excitation wavelength (532nm) gives a significantly higher spectral resolution and a more detailed discrimination of the Raman bands, provided that the conditions of general lower level of fluorescence emission are met. The results presented in the current study complement the knowledge on minerals and biomarkers of relevance for Martian environments which have been measured with mobile Raman spectrometers. The outcome creates a solid base towards the use of lightweight mobile Raman systems that can be used outdoors and on terrestrial outcrops. Moreover, these results and conclusions are of use for the further development of dedicated spectrometers destined for the instrumental suites on planetary rovers, in the frame of the forthcoming exobiology focused missions to Mars to be launched by NASA and ESA.

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.

Comparison of Miniaturized Raman Spectrometers for Discrimination of Carotenoids of Halophilic Microorganisms.

We present a comparison of the performance of four miniature portable Raman spectrometers for the discrimination of carotenoids in samples of carotene-producing microorganisms. Two spectrometers using a green laser allowing to obtain Resonance Raman (or pre-Resonance Raman) signals, one instrument with a 785 nm laser, and a recently developed Portable Sequentially Shifted Excitation Raman spectrometer (PSSERS) were used for identifying major pigments of different halophilic (genera Halobacterium, Halorubrum, Haloarcula, Salinibacter, Ectothiorhodospira, Dunaliella) and non-halophilic microorganisms (Micrococcus luteus, Corynebacterium glutamicum). Using all the tested instruments including the PSSERS, strong carotenoids signals corresponding to the stretching vibrations in the polyene chain and in-plane rocking modes of the attached CH3 groups were found at the correct positions. Raman spectra of carotenoids can be obtained from different types of microbiological samples (wet pellets, lyophilized culture biomass and pigment extracts in organic solvents), and can be collected fast and without time-consuming procedures.

Detection of carotenoids of halophilic prokaryotes in solid inclusions inside laboratory-grown chloride and sulfate crystals using a portable Raman spectrometer: applications for Mars exploration.

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.

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.

Unleaded gasoline as a significant source of Pb emissions in the Subarctic.

After the phasing out of leaded gasoline, Pb emissions to the atmosphere dramatically decreased, and other sources became more significant. The contribution of unleaded gasoline has not been sufficiently recognized; therefore, we evaluated the impact of Pb from unleaded gasoline in a relatively pristine area in Subarctic NE Norway. The influence of different endmembers (Ni slag and concentrate from the Nikel smelter in Russia, PM10 filters, and traffic) on the overall Pb emissions was determined using various environmental samples (snow, lichens, and topsoils) and Pb isotope tracing. We found a strong relationship between Pb in snow and the Ni smelter. However, lichen samples and most of the topsoils were contaminated by Pb originating from the current use of unleaded gasoline originating from Russia. Historical leaded and recent unleaded gasoline are fully distinguishable using Pb isotopes, as unleaded gasoline is characterized by a low radiogenic composition (206Pb/207Pb = 1.098 and 208Pb/206Pb = 2.060) and remains an unneglectable source of Pb in the region.

Raman spectroscopy as tool for the characterization of thio-polyaromatic hydrocarbons in organic minerals.

Benzothiophene and dibenzothiophene have been studied by Raman microspectroscopy using a 785 nm excitation wavelength. The spectra obtained have been compared with the previously measured spectra of idrialite, a complex natural mineral composed entirely of cata-condensed polyaromatic hydrocarbons (PAHs), usually containing a thiophenic or aliphatic five-membered ring. For comparison, the Raman spectra of 2,3-benzofluorene crystals have been obtained for the first time. Some of the bands in the idrialite spectra are attributed to specific vibrational modes of thiophene or fluorene-type PAHs, especially in the region below 1000 cm(-1). These modes at 495, 705 and 750 cm(-1) along with C-H or C-H(2) stretching modes around 3000 cm(-1) can be then used to distinguish such groups of PAHs in complicated organic mineral mixtures like idrialite.