Exploring Rock-inhabiting Microbes in Atacama Desert's Gypcrete: Raman Spectroscopy Unveils the Biomolecular Adaptations

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This study delves into the biomolecular composition of endolithic phototrophic microbes thriving within these gypcretes. Using advanced Raman spectroscopy techniques, including Raman imaging, complemented by microscopic and 3D microscopic observations, we unveil new insights into the adaptive strategies of gypsum-inhabiting algae. Our Raman imaging results provide a detailed chemical map of photoprotective and photosynthetic pigments associated with microbial colonization. This map reveals a significant gradient in pigment composition, highlighting a critical survival mechanism for algae and cyanobacteria in this polyextreme environment. Intriguingly, we detected carotenoid signals not only in the algae-colonized layer but also deeper within the gypsum matrix, indicating pigment migration following cell disruption. In addition, we conducted an in-depth analysis of individual algal cells from the Trebouxiae family, noting their color variations from green to orange and describing the spectral differences in detail. This investigation identified in-vivo pigments (carotenoids, chlorophyll) and lipids at the cellular level, offering a comprehensive view of the molecular adaptations enabling life in one of Earth's most extreme habitats. Biological sciences/Biophysics Biological sciences/Plant sciences Earth and environmental sciences/Biogeochemistry Earth and environmental sciences/Ecology Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Preandean zone of the Atacama Desert occurs in northeastern area of the desert in altitudes higher that the core parts (i.e., ~ 3000m a.s.l.). As a result, the area belongs to the Earths most intensively sun irradiated locations (Rondanelli et al., 2015 ). It includes high level of UV radiation that is reported for high-altitude cloudless Andean regions, with very low values of relative humidity (RH) and low-ozone column (UV index 43.3, see Cabrol et al., 2014 ). The yearly precipitation at the studied site is reported to be 27.1 mm (Morillas et al., 2015 ), while mean annual RH recorded by Wierzchos et al. ( 2015 ) is 16.5%, which is one of the lowest values measured within the Atacama Desert (Azua-Bústos et al., 2015 ). Together with the high evapotranspiration rate of 2920 mm year − 1 recorded in nearby Salar de Atacama basin, the region can be characterized as hyperarid with aridity index (AI) of 0,009 and create polyextreme abiotic stress upon the biota with which they have to cope. Raman spectroscopy is known to be a powerful tool for nondestructive in-vivo characterization of biomolecules associated with extremophilic phototrophs, with emphasis on pigment analysis (e.g., Edwards et al., 2005 , Villar et al., 2005 , Marshall et al., 2007 , Vítek et al., 2010 , Jehlička et al., 2014 ). The technique has been applied for analysis of gypsum inhabiting endolithic communities from the Atacama Desert (Vítek et al., 2013 ), including Raman imaging applied to the algae from gypcrete in Cordon de Lila area (Wierzchos et al., 2015 , Vítek et al., 2016 ). Authors describe the gradient of the carotenoid Raman signal intensity distribution, with its enhancement close to the surface, which was interpreted as an adaptation mechanism to excessive solar irradiation. Here, while studying the same geomicrobiological system, we focused on the detailed in-vivo spectral characterization of algal cells visually differing in color, imaging of carotenoids, including zones outside the colonization zone. Microalgae are known to contain lipid storage bodies, which can form a significant portion of the algal mass and can be detected by Raman spectroscopy (Samek et al., 2010 ). Algal lipids are of special importance from a biotechnological point of view (Schenk et al., 2008 ). They are considered an important source of biofuels that do not compete for land area with other demands, i.e. food production. Various algal strains were examined to obtain information about lipid composition using Raman spectroscopy, e.g. Botryococcus sudeticus , Chlamydomonas sp., Trachydiscus minutus (Samek et al., 2010 ). Here, both pigment and lipid composition was monitored using Raman spectroscopy. Results Raman Spectroscopy – pigments Typical Raman spectra obtained on the algal cells are presented in Fig. 2 . Two types of cells are compared – orange-green (Fig. 2 A) and red-orange without visible green pigmentation (Fig. 2 B). The Raman spectroscopy shows differences in pigment composition (Fig. 2 C, Fig. 3 ). Carotenoid Raman features dominate the both spectra with the in-phase ν 1 (C = C) and ν 2 (C–C) stretching vibrations of the polyene chain located at 1523–1526 cm − 1 and 1158–1159 cm − 1 , respectively. The feature of medium intensity occurs around 1009 cm − 1 , corresponding to the in-plane rocking modes of the CH 3 groups attached to the polyene chain (Gill et al., 1970 , Merlin, 1985 ). The small shifts of the two strong Raman bands within the wavenumber range mentioned above were observed between the two types of cells, with the higher values detected in orange-green cells. Simultaneously, clear corroborative chlorophyll features were detected in these cells, with major bands located at 746, 918, and 1329 cm − 1 . Significantly higher Raman intensity of carotenoid spectral features were detected within red-orange cell in the uppermost zone of colonization, with substantially lower chlorophyll intensity compared to the green or orange-green cells from the lower parts. It is evident from the Raman spectra at Fig. 2 C. The gradient of Raman intensity carotenoids is confirmed by Raman imaging of the whole thin section from the cross-cut gypcrete sample (Fig. 3 ). Raman spectroscopy - carotenoid distribution within gypcrete Carotenoid signal as obtained by resonance Raman imaging produce specific zonation within the crosscut through the gypcrete (Fig. 3 ). Within the colonization zone ~ 1–2 mm beneath the surface, we see the gradient of ν 1 (C = C) band intensity, with the most intensive Raman signal occuring at the uppermost parts of the colony (umbrella-like pattern). Interestingly, we see signal of lower intensity, but clearly detectable, attributed to carotenoids several mm beneath the visible colonization by algal cells. The signal come clearly from carotenoids dispersed in the gypsum matrix. Raman Spectroscopy - lipids In Raman spectra, lipids are characterized by couple of corroborative Raman bands in the range 1000–1800 cm − 1 (Czamara et al., 2015 ). As demonstrated by Samek et al. ( 2010 , 2011 ), the degree of fatty acid unsaturation can be determined in-vivo based on Raman spectroscopic record. The authors successfully verified the Raman spectroscopic approach with GC-MS. In order to obtain an information about lipid unsaturation, intensity ratio of the band located around 1657 cm − 1 , assigned to C = C stretching vibrational mode and the band at 1445 cm − 1 due to scissoring of CH 2 is calculated. The ratio of the two bands can be used as indicator of lipid unsaturation (Samek et al., 2010 ; 2011 ; Wu et al., 2011 ). The contribution of carotenoid Raman signal to the band located at 1445 cm − 1 has to be taken into account. Within the β-carotene standard, the intensity of the band at 1445 cm − 1 is 1/33 of the intensity of the ν(C = C) band. After correction of carotenoid contribution to the band around 1445 cm − 1 , the intensity ratio of unsaturated/saturated band (I 1657 /I 1445 ) was calculated for the algae studied here. Generally, accepted parameter for expressing the unsaturation of lipids and derivatives is the iodine value (IV). Originally, it was defined as grams of iodine added to 100 g of fat by titration (Pomeranz and Meloan, 1987 ). The iodine value can be related to the I 1657 /I 1445 ratio obtained by Raman spectroscopy and is proportional to the number of double bonds in the lipid structure (Samek et al., 2010 , 2011 ). Here, the both bands assigned to lipids were detected as weak features in the spectral record obtained on algae in gypsum dominated by strong carotenoid features (Fig. 4 ). The I 1657 /I 1445 ratio was calculated for algae with negligible chlorophyll Raman signal as chlorophyll a Raman features also contribute to the spectral region around 1445 cm − 1 and, hence, may potentially artificially affect the obtained value. The obtained value of I 1657 /I 1445 after correction of carotenoid contribution was 1.26. Applying the calibration curve obtained by Samek et al. ( 2011 ) for three algal species, i.e. Botryococcus sudeticus , Chlamydomonas sp., Trachydiscus minutus , the iodine value corresponds to IV ≈ 150. Discussion The umbrella-like pattern of carotenoid distribution as detected by Raman imaging (Fig. 3 ) was presented also in the former studies of the same material (see Wierzchos et al., 2015 , Vítek et al., 2016 , 2020 ). It is interpreted as a biomolecular adaptation of algae to excessive solar radiation, that occurs in preandean region of the Atacama Desert (Rondanelli et al., 2015 ). We hypothesize, that lower positioned cells benefit from the shielding provided by the cells from higher positions. We do not expect, that the algal cells located at the uppermost parts may, on the other hand, benefit from the photosynthetic activity of cells located below that have denser chloroplasts through the cytoplasm (Wierzchos et al., 2015 ) and higher chlorophyll content as described here. Role of carotenoids Carotenoids are pigments involved in photosynthesis, photoprotection, and membrane stabilization, and they are characterized by a long conjugated C = C bond system composed of isoprenoid units. One important role of xanthophyll carotenoids in very high PAR conditions is dissipation of excess excitation energy in the xanthophyll cycle, which is considered a key photoprotective mechanism in higher plants and algae (Adams and Demmig-Adams, 1992 ; Lunch et al., 2013 ). Thus, it prevents the formation of singlet oxygen ( 1 O 2 ), by rapidly quenching chlorophyll triplet states. Moreover, there is an important role of carotenoids to rapidly direct scavenge of singlet oxygen if any is formed (Anderson and Robertson, 1960 , Krinsky, 1979 , Siefermann-Harms, 1987 , Telfer et al., 2008 ). The radical scavenging ability of carotenoids was reported to be more effective with higher conjugation of the polyene structure (Saito et al., 1997). Role of lipids Employment of Raman spectroscopy for assessment of algal lipids increased in recent years, benefiting from its nondestructive, label-free nature and possibility to examine single cells (Samek et al., 2010 , 2011 , Wu et al., 2011 , Challagula et al., 2016). Application of coherent anti-stokes Raman scattering microscopy for imaging of the algal lipid bodies was presented by Cavonius et al. ( 2015 ). As described by Samek et al. ( 2010 , 2011 ), the Raman spectroscopy can be applied for the determination of iodine value as applied here and presented in “Results”. Lipids can be produced by algae and stored in the form of subcellular structures called lipid bodies, lipid droplets, oil bodies, oil globules or oleosomes (Huang, 1996 , Murphy 2001 ). The terms are dependent on particular community and reflect the same structure. In this study we use the term lipid body. The major role of lipid bodies is in lipid storage prior to the dormancy period or when subjected to stressful environmental conditions (Arakawa-Kobayashi and Kanaseki, 2004 , Hu et al., 2008 ). Triacylglycerols, that form the basis of the lipid bodies, contain twice more energy compared to starch or protein per weight, hence represent an effective carbon and energy storage in eukaryotic cells in general (Hu et al., 2008 , Goold et al., 2015 ). It was reported by Li et al. ( 2012 ), that Chlamydomonas mutant pgd 1, containing less amount of storage lipids readily lost its viability under nitrogen-deprived conditions. Another study reports, that nitrogen starvation-induced formation of lipid bodies and conversion of membrane lipid acyl groups to triacylglycerol in green algae Chlorella sp. (Goncalves et al. ( 2013 ). Nevertheless, there are variety of functions beyond the energy storage (Goold et al., 2015 ). It was reported, that triacylglicerides in the lipid bodies can serve as a temporary depot for acyl chains. These are removed from membrane structures under adverse conditions like osmotic stress, freezing, heat stress etc. to compensate changes in bilayer structures (Goold et al., 2015 , Legeret et al., 2016 ). When environmental conditions change again, the acyl chains are released from lipid bodies and used for membrane lipids synthesis and assembly again. In this way, the cell bypass the need for de novo fatty acid synthesis (Goold et al., 2015 ). It could require more energy consumption which in the extreme environmental conditions like the studied zone of the Atacama Desert may be a crucial factor. Another role in cellular physiology are hypothesized based on proteomics of lipid bodies (Moellering and Benning, 2010 , Nguyen et al., 2011 ). These are cell signalling, protection, molecular transport etc. (Goold et al., 2015 ). It was also demonstrated, that the algal lipid content can be stimulated by manipulating the solar light spectrum with color paints dissolved in water (Seo et al., 2014 , Ramanna et al., 2017 ). Conclusions A novel insight into adaptation strategy of endolithic, gypsum-inhabiting algae from the polyextreme environment of the Atacama Desert is unveiled, providing fresh insights into their survival mechanisms. Carotenoids were discovered beneath the microbially colonized zone within the gypsum matrix, suggesting migration of these pigments following cell disruption. Comprehensive Raman spectroscopic analysis revealed distinct differences in carotenoid and chlorophyll composition between green-orange and orange algal cells of the Trebouxiae family, cohabiting the gypcrete rock. For the first time, in-vivo chemical detection of lipids within these algal cells has been achieved. Methods Study site and samples The sampling zone (23° 53′ S, 068° 08′ W; 2720 m a.s.l.) was located within the southern edge of the Salar de Atacama basin (Fig. 1 A) in the north–south-trending depression of the Cordon de Lila range, in northern Chile. This depression is mostly covered by volcanic material, but large gypsum outcrops can be found in several locations (Fig. 1 B). In the field, gypsum deposits were assessed for endolithic colonization by visual inspection for microbial pigments present in fractured samples. These pigments indicate the presence of cryptoendolithic (occupying pore spaces beneath the rock surface) and hypoendolithic (colonizing the undermost layer of the rock, sensu Wierzchos et al. (2011, 2015 ). microbial colonization, forming horizons beneath the surface and close to the bottom of the gypsum deposits. The gypsum rock fragments were collected in hyperarid conditions with air relative humidity (RH) of 10% and air temperature (T) of 28°C. They were stored in dry and dark conditions at room temperature until analysis. Gypsum formations at the studied site called gypcrete (according to the nomenclature of Horta 1980 ) occur as hard layer deposits on the soil surface, interbedded between layers of ignimbrites and sometimes filling cracks on these rocks. Abundant colonization can be recognized visually in the field by green-to-orange pigmentation after cracking open the gypcretes. The cryptoendolithic habitat appears beneath the 0.5–5-mm-thin surface layer. The typical zonation was revealed to be due to orange (higher position) and green (lower position) algae, with cyanobacteria appearing together within the green layer. Moreover, cyanobacteria were found to occupy a hypoendolithic habitat, e.g., at the bottom position close to the contact with the soil. For a detailed characterization of the gypsum formations and the microbial colonization by chlorophototrophic biota, see the comprehensive study by Wierzchos et al. ( 2015 ). Optical microscopy and 3D surface microscopy A Zeiss AxioImager D1 microscope (Carl Zeiss, Oberkochen, Germany) was employed to obtain optical images of algal cells, equipped with a Plan-Apo 609/1.4 Zeiss oil-immersion objective. In addition, we have used digital microscope Keyence VHX − 900F (Keyence, UK) with 100x magnification objective to scan 3D surface structure of the algal cell embedded in gypsum samples. Analyses of single microalgal cells by Raman spectroscopy Microalgae cells colonizing cryptoendolithic habitat of gypcrete, were examined using point Raman analysis on an InVia spectrometer (Renishaw, Wotton-under-Edge, UK) equipped with a Leica confocal microscope. A 785 nm laser line was employed as a universal excitation wavelength capable of detecting variety of pigments and other biomolecules, including lipids. The instrument was calibrated to a silicon Raman band at 520.5 cm − 1 . The point analysis was undertaken employing 50x magnification objective, 2s – 5s exposure time was set, accumulated 10 times. The laser power between 15–30 mW at source was used. The analyses were performed on transects of the gypcrete substrate stored in dark conditions at 20°C until analysis. The Raman spectroscopy technique was chosen for its ability to analyze small amounts of biological material in situ . Raman imaging and Raman data processing The same InVia spectrometer (Renishaw, Wotton-under-Edge, UK) equipped with a Leica confocal microscope was used in point-to-point scanning mode for the Raman imaging. The instrument was calibrated to a silicon Raman band at 520.5 cm -1 . For imaging, an Ar laser line at 514.5 nm, with 10 mW power at the source, and a 1 s exposure time accumulated 1x times was employed at each point. Benefiting from the resonance Raman effect, a strong signal of carotenoids was obtained within the Raman imaging using a relatively short exposure time. As a result, a relatively large area was scanned at a high spatial resolution. The laser was focused using a 5 × magnification Leica objective (NA = 0.12). Single spectra (averaged from 7 neighbor spectra) were extracted from the zones of interest to show the spectral differences. The Raman imaging data were acquired using Wire 3.4 (Renishaw). The subsequent data processing workflow was provided by ImageLab software, version 3.20 (Epina GmbH Retz, Austria). Spikes (due to cosmic rays) were detected and removed using the following parameters: spike half-width − 3; threshold − 1. Next, the spectra were smoothed out using the Savitzky-Golay polynomial function, window: 7. Then, the baseline was corrected using the Eilers algorithm using the following parameters: smoothness − 10000; asymmetry − 0.002; iterations − 7. Suspicious pixels (data without spectral noise) were masked and corresponded mainly to the oversaturated signal of epoxide used for sample preparation. Declarations Competing interests The authors declare that they have no conflicts of interest. Author Contribution P.V. contributed to the sample collection, measurements, data processing, writing and editing of the text; C.A. contributed to the project management, writing and editing; O.A. contributed to the area selection, sampling, interpretation of the data and text editing; J.W contributed to the project management, area selection, sample collection, data interpretation, writing and editing. Acknowledgement PV thanks to the Czech Science Foundation (project number 22-29315S) and to the project of the Ministry of Education, Youth and Sports of the Czech Republic (AdAgriF; CZ.02.01.01/00/22_008/0004635). Authors are thankful for financial support by grant PID2021-124362NB-I00 from MCIN/AEI/10.13039/501100011033/FEDER, UE. We are grateful to Prof. Miloš Barták for his help with capturing 3D-microscopic images and the support provided by the CARP infrastructure (CzechPolar-I and II, LM2010009 andLM2015078). Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Adams, W.W. III & Demmig-Adams, B. Operation of the xanthophyll cycle in higher plants in response to diurnal changes in incident sunlight. Planta 186, 390–398 (1992). Anderson, I.C. & Robertson, D.S. Role of carotenoids in protecting chlorophyll from photodestruction. Plant Physiology 35, 531–534 (1960). Arakawa-Kobayashi, S. & Kanaseki, T. A study of lipid secretion from the lichen symbionts, ascomycetous fungus Myelochroa leucotyliza and green alga Trebouxia sp. J. Struct. Biol. 146, 401–415 (2004). Azua-Bústos, A., Caro-Lara, L. & Vicuňa, R. Discovery and microbial content of the driest site of the hyperarid Atacama Desert, Chile. 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Cite Share Download PDF Status: Published Journal Publication published 13 Oct, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 18 Jul, 2024 Reviews received at journal 12 Jul, 2024 Reviews received at journal 12 Jul, 2024 Reviewers agreed at journal 02 Jul, 2024 Reviewers agreed at journal 02 Jul, 2024 Reviewers invited by journal 02 Jul, 2024 Editor assigned by journal 02 Jul, 2024 Editor invited by journal 24 Jun, 2024 Submission checks completed at journal 21 Jun, 2024 First submitted to journal 20 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4611340","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":321642973,"identity":"e329e8c2-14ee-45a7-a8a0-a15c2525666c","order_by":0,"name":"Petr Vitek","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIie3QvQrCMBDA8QsH51LpmiLoK0QKfoAP0+LgJDg6BgSnYtf6Fn2EloAuPoCDoC7uIoiKg9VWx9hRMP8hZOiPXA/AZPrFEIBJgHpx74D4TvBF3ILwEgRy4svSRCztdB/BZhCGKj2Nbrzerkh2vOiIQmzGcBhGyaJfm8+42w0S5FxDnAmSswM1jFnQwmrA/XjtkXa6jFSuGRkItM852e4IPA2xEYnFoDxBFqF1eb4CBImeuE4kDs1oRW6tKp//4k8cqSGULewYjDcNO1T7k3XvZRtTqXZjeaIYhE1fp+6NT+/Z72U+NplMpn/rAQKlRwm+WhnDAAAAAElFTkSuQmCC","orcid":"","institution":"Global Change Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Petr","middleName":"","lastName":"Vitek","suffix":""},{"id":321642974,"identity":"fde1e698-0ac5-46da-83c8-c755621021ff","order_by":1,"name":"Ascaso Carmen","email":"","orcid":"","institution":"Museo Nacional de Ciencias Naturales","correspondingAuthor":false,"prefix":"","firstName":"Ascaso","middleName":"","lastName":"Carmen","suffix":""},{"id":321642975,"identity":"4a3bfe3a-3d4b-490b-b8f4-117a8977709d","order_by":2,"name":"Octavio Artieda","email":"","orcid":"","institution":"University of Extremadura","correspondingAuthor":false,"prefix":"","firstName":"Octavio","middleName":"","lastName":"Artieda","suffix":""},{"id":321642976,"identity":"28c31892-9a20-4678-bee9-beb7c97fb7db","order_by":3,"name":"Jacek Wierzchos","email":"","orcid":"","institution":"Museo Nacional de Ciencias Naturales","correspondingAuthor":false,"prefix":"","firstName":"Jacek","middleName":"","lastName":"Wierzchos","suffix":""}],"badges":[],"createdAt":"2024-06-20 11:14:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4611340/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4611340/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-75526-7","type":"published","date":"2024-10-13T15:57:49+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60022721,"identity":"7efc113b-0f63-4c84-907c-66439842276c","added_by":"auto","created_at":"2024-07-10 16:35:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1480903,"visible":true,"origin":"","legend":"\u003cp\u003eSampling site close to the southern edge of Salar de Atacama (yellow star on the map (A) is formed by volcanic rocks with zones of gypcrete outcrops (B). Green-to-red layers of endolithic colonization can be found 1-3mm below the surface (C). In (D) a slide cut from the gypcrete with intensively colored colonization and high magnification image with algal cells is depicted. Scale bar in the magnified image in D = 20mm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4611340/v1/9cbfcbbff30ebbf06321a8c0.png"},{"id":60022722,"identity":"51556a92-6a0e-4d84-a315-b54e80e4d772","added_by":"auto","created_at":"2024-07-10 16:35:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":443138,"visible":true,"origin":"","legend":"\u003cp\u003e3D microscopic images and Raman spectra from the algae: A) an image of orange-green algal cells embedded in gypsum, B) an image of orange algal cells in gypsum, C) Raman spectra obtained at the two types of cells (regions of interest magnified below), obviously differing in chlorophyll Raman features (major features indicated by *) and slight shift in carotenoid n(C=C) band. Note that green spectrum corresponds to orange-green cells in A) while the red spectrum corresponds to orange cells in B). Scale bars = 20mm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4611340/v1/21594ecd99afa62ab9e979c9.png"},{"id":60023702,"identity":"7a8bfa0e-9a9d-4e7c-8e55-0f3ae83f12c9","added_by":"auto","created_at":"2024-07-10 16:43:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":649966,"visible":true,"origin":"","legend":"\u003cp\u003eRaman imaging of the cryptoendolithoc colonization in crosscut of gypcrete. A) The rainbow scale correspond to the intensity of carotenoid C=C band. The averaged spectra extracted from the zones of various intensity are presented: colonized zone in B), gypcrete matrix with carotenoid signal in C). Pixels in pink color at the upper part were deleted and represent Raman signal from the resin used for the sample preparation, that caused dan oversaturation of the detector.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4611340/v1/6a923643f910dfa2d64bb766.png"},{"id":60022719,"identity":"9c97062f-150a-49a8-8537-979242ec4ca8","added_by":"auto","created_at":"2024-07-10 16:35:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":65829,"visible":true,"origin":"","legend":"\u003cp\u003eRaman spectroscopic features of lipids at 1657 cm\u003csup\u003e-1\u003c/sup\u003e due to C=C stretching (indicator of unsaturation) and 1445 cm\u003csup\u003e-1\u003c/sup\u003e assigned to CH\u003csub\u003e2\u003c/sub\u003e scissoring in lipids (indicator of lipid saturation). The later is at the similar position as a weak Raman features of carotenoids and chlorophyll.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4611340/v1/533fc2587c02a488ba4e67be.png"},{"id":66597865,"identity":"c8e1d881-3309-40ac-9ea7-9918ad4baff7","added_by":"auto","created_at":"2024-10-14 16:11:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2809462,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4611340/v1/0fe34f35-25e6-4ef7-80e6-20bf20282abd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring Rock-inhabiting Microbes in Atacama Desert's Gypcrete: Raman Spectroscopy Unveils the Biomolecular Adaptations","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePreandean zone of the Atacama Desert occurs in northeastern area of the desert in altitudes higher that the core parts (i.e., ~\u0026thinsp;3000m a.s.l.). As a result, the area belongs to the Earths most intensively sun irradiated locations (Rondanelli et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). It includes high level of UV radiation that is reported for high-altitude cloudless Andean regions, with very low values of relative humidity (RH) and low-ozone column (UV index 43.3, see Cabrol et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The yearly precipitation at the studied site is reported to be 27.1 mm (Morillas et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), while mean annual RH recorded by Wierzchos et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) is 16.5%, which is one of the lowest values measured within the Atacama Desert (Azua-B\u0026uacute;stos et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Together with the high evapotranspiration rate of 2920 mm year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e recorded in nearby Salar de Atacama basin, the region can be characterized as hyperarid with aridity index (AI) of 0,009 and create polyextreme abiotic stress upon the biota with which they have to cope.\u003c/p\u003e \u003cp\u003eRaman spectroscopy is known to be a powerful tool for nondestructive in-vivo characterization of biomolecules associated with extremophilic phototrophs, with emphasis on pigment analysis (e.g., Edwards et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Villar et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Marshall et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, V\u0026iacute;tek et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Jehlička et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The technique has been applied for analysis of gypsum inhabiting endolithic communities from the Atacama Desert (V\u0026iacute;tek et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), including Raman imaging applied to the algae from gypcrete in Cordon de Lila area (Wierzchos et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, V\u0026iacute;tek et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Authors describe the gradient of the carotenoid Raman signal intensity distribution, with its enhancement close to the surface, which was interpreted as an adaptation mechanism to excessive solar irradiation. Here, while studying the same geomicrobiological system, we focused on the detailed \u003cem\u003ein-vivo\u003c/em\u003e spectral characterization of algal cells visually differing in color, imaging of carotenoids, including zones outside the colonization zone.\u003c/p\u003e \u003cp\u003eMicroalgae are known to contain lipid storage bodies, which can form a significant portion of the algal mass and can be detected by Raman spectroscopy (Samek et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Algal lipids are of special importance from a biotechnological point of view (Schenk et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). They are considered an important source of biofuels that do not compete for land area with other demands, i.e. food production. Various algal strains were examined to obtain information about lipid composition using Raman spectroscopy, e.g. \u003cem\u003eBotryococcus sudeticus\u003c/em\u003e, \u003cem\u003eChlamydomonas\u003c/em\u003e sp., \u003cem\u003eTrachydiscus minutus\u003c/em\u003e (Samek et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHere, both pigment and lipid composition was monitored using Raman spectroscopy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRaman Spectroscopy \u0026ndash; pigments\u003c/h2\u003e \u003cp\u003eTypical Raman spectra obtained on the algal cells are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Two types of cells are compared \u0026ndash; orange-green (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and red-orange without visible green pigmentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The Raman spectroscopy shows differences in pigment composition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Carotenoid Raman features dominate the both spectra with the in-phase ν\u003csub\u003e1\u003c/sub\u003e(C\u0026thinsp;=\u0026thinsp;C) and ν\u003csub\u003e2\u003c/sub\u003e(C\u0026ndash;C) stretching vibrations of the polyene chain located at 1523\u0026ndash;1526 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1158\u0026ndash;1159 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. The feature of medium intensity occurs around 1009 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, corresponding to the in-plane rocking modes of the CH\u003csub\u003e3\u003c/sub\u003e groups attached to the polyene chain (Gill et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1970\u003c/span\u003e, Merlin, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). The small shifts of the two strong Raman bands within the wavenumber range mentioned above were observed between the two types of cells, with the higher values detected in orange-green cells. Simultaneously, clear corroborative chlorophyll features were detected in these cells, with major bands located at 746, 918, and 1329 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSignificantly higher Raman intensity of carotenoid spectral features were detected within red-orange cell in the uppermost zone of colonization, with substantially lower chlorophyll intensity compared to the green or orange-green cells from the lower parts. It is evident from the Raman spectra at Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC. The gradient of Raman intensity carotenoids is confirmed by Raman imaging of the whole thin section from the cross-cut gypcrete sample (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRaman spectroscopy - carotenoid distribution within gypcrete\u003c/h2\u003e \u003cp\u003eCarotenoid signal as obtained by resonance Raman imaging produce specific zonation within the crosscut through the gypcrete (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Within the colonization zone\u0026thinsp;~\u0026thinsp;1\u0026ndash;2 mm beneath the surface, we see the gradient of ν\u003csub\u003e1\u003c/sub\u003e(C\u0026thinsp;=\u0026thinsp;C) band intensity, with the most intensive Raman signal occuring at the uppermost parts of the colony (umbrella-like pattern). Interestingly, we see signal of lower intensity, but clearly detectable, attributed to carotenoids several mm beneath the visible colonization by algal cells. The signal come clearly from carotenoids dispersed in the gypsum matrix.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRaman Spectroscopy - lipids\u003c/h2\u003e \u003cp\u003eIn Raman spectra, lipids are characterized by couple of corroborative Raman bands in the range 1000\u0026ndash;1800 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Czamara et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). As demonstrated by Samek et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), the degree of fatty acid unsaturation can be determined \u003cem\u003ein-vivo\u003c/em\u003e based on Raman spectroscopic record. The authors successfully verified the Raman spectroscopic approach with GC-MS. In order to obtain an information about lipid unsaturation, intensity ratio of the band located around 1657 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, assigned to C\u0026thinsp;=\u0026thinsp;C stretching vibrational mode and the band at 1445 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e due to scissoring of CH\u003csub\u003e2\u003c/sub\u003e is calculated. The ratio of the two bands can be used as indicator of lipid unsaturation (Samek et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The contribution of carotenoid Raman signal to the band located at 1445 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e has to be taken into account. Within the β-carotene standard, the intensity of the band at 1445 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is 1/33 of the intensity of the ν(C\u0026thinsp;=\u0026thinsp;C) band. After correction of carotenoid contribution to the band around 1445 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the intensity ratio of unsaturated/saturated band (I\u003csub\u003e1657\u003c/sub\u003e/I\u003csub\u003e1445\u003c/sub\u003e) was calculated for the algae studied here. Generally, accepted parameter for expressing the unsaturation of lipids and derivatives is the iodine value (IV). Originally, it was defined as grams of iodine added to 100 g of fat by titration (Pomeranz and Meloan, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). The iodine value can be related to the I\u003csub\u003e1657\u003c/sub\u003e/I\u003csub\u003e1445\u003c/sub\u003e ratio obtained by Raman spectroscopy and is proportional to the number of double bonds in the lipid structure (Samek et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Here, the both bands assigned to lipids were detected as weak features in the spectral record obtained on algae in gypsum dominated by strong carotenoid features (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The I\u003csub\u003e1657\u003c/sub\u003e/I\u003csub\u003e1445\u003c/sub\u003e ratio was calculated for algae with negligible chlorophyll Raman signal as chlorophyll \u003cem\u003ea\u003c/em\u003e Raman features also contribute to the spectral region around 1445 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and, hence, may potentially artificially affect the obtained value. The obtained value of I\u003csub\u003e1657\u003c/sub\u003e/I\u003csub\u003e1445\u003c/sub\u003e after correction of carotenoid contribution was 1.26. Applying the calibration curve obtained by Samek et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) for three algal species, i.e. \u003cem\u003eBotryococcus sudeticus\u003c/em\u003e, \u003cem\u003eChlamydomonas\u003c/em\u003e sp., \u003cem\u003eTrachydiscus minutus\u003c/em\u003e, the iodine value corresponds to IV\u0026thinsp;\u0026asymp;\u0026thinsp;150.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe umbrella-like pattern of carotenoid distribution as detected by Raman imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) was presented also in the former studies of the same material (see Wierzchos et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, V\u0026iacute;tek et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It is interpreted as a biomolecular adaptation of algae to excessive solar radiation, that occurs in preandean region of the Atacama Desert (Rondanelli et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). We hypothesize, that lower positioned cells benefit from the shielding provided by the cells from higher positions. We do not expect, that the algal cells located at the uppermost parts may, on the other hand, benefit from the photosynthetic activity of cells located below that have denser chloroplasts through the cytoplasm (Wierzchos et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and higher chlorophyll content as described here.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRole of carotenoids\u003c/h2\u003e \u003cp\u003eCarotenoids are pigments involved in photosynthesis, photoprotection, and membrane stabilization, and they are characterized by a long conjugated C\u0026thinsp;=\u0026thinsp;C bond system composed of isoprenoid units. One important role of xanthophyll carotenoids in very high PAR conditions is dissipation of excess excitation energy in the xanthophyll cycle, which is considered a key photoprotective mechanism in higher plants and algae (Adams and Demmig-Adams, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Lunch et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Thus, it prevents the formation of singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e), by rapidly quenching chlorophyll triplet states. Moreover, there is an important role of carotenoids to rapidly direct scavenge of singlet oxygen if any is formed (Anderson and Robertson, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1960\u003c/span\u003e, Krinsky, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1979\u003c/span\u003e, Siefermann-Harms, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1987\u003c/span\u003e, Telfer et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The radical scavenging ability of carotenoids was reported to be more effective with higher conjugation of the polyene structure (Saito et al., 1997).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRole of lipids\u003c/h2\u003e \u003cp\u003eEmployment of Raman spectroscopy for assessment of algal lipids increased in recent years, benefiting from its nondestructive, label-free nature and possibility to examine single cells (Samek et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Wu et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Challagula et al., 2016). Application of coherent anti-stokes Raman scattering microscopy for imaging of the algal lipid bodies was presented by Cavonius et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). As described by Samek et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), the Raman spectroscopy can be applied for the determination of iodine value as applied here and presented in \u0026ldquo;Results\u0026rdquo;.\u003c/p\u003e \u003cp\u003eLipids can be produced by algae and stored in the form of subcellular structures called lipid bodies, lipid droplets, oil bodies, oil globules or oleosomes (Huang, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Murphy \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The terms are dependent on particular community and reflect the same structure. In this study we use the term lipid body. The major role of lipid bodies is in lipid storage prior to the dormancy period or when subjected to stressful environmental conditions (Arakawa-Kobayashi and Kanaseki, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Hu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Triacylglycerols, that form the basis of the lipid bodies, contain twice more energy compared to starch or protein per weight, hence represent an effective carbon and energy storage in eukaryotic cells in general (Hu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Goold et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). It was reported by Li et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), that \u003cem\u003eChlamydomonas\u003c/em\u003e mutant \u003cem\u003epgd\u003c/em\u003e1, containing less amount of storage lipids readily lost its viability under nitrogen-deprived conditions. Another study reports, that nitrogen starvation-induced formation of lipid bodies and conversion of membrane lipid acyl groups to triacylglycerol in green algae \u003cem\u003eChlorella\u003c/em\u003e sp. (Goncalves et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Nevertheless, there are variety of functions beyond the energy storage (Goold et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). It was reported, that triacylglicerides in the lipid bodies can serve as a temporary depot for acyl chains. These are removed from membrane structures under adverse conditions like osmotic stress, freezing, heat stress etc. to compensate changes in bilayer structures (Goold et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Legeret et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). When environmental conditions change again, the acyl chains are released from lipid bodies and used for membrane lipids synthesis and assembly again. In this way, the cell bypass the need for de novo fatty acid synthesis (Goold et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). It could require more energy consumption which in the extreme environmental conditions like the studied zone of the Atacama Desert may be a crucial factor. Another role in cellular physiology are hypothesized based on proteomics of lipid bodies (Moellering and Benning, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Nguyen et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These are cell signalling, protection, molecular transport etc. (Goold et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). It was also demonstrated, that the algal lipid content can be stimulated by manipulating the solar light spectrum with color paints dissolved in water (Seo et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Ramanna et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eA novel insight into adaptation strategy of endolithic, gypsum-inhabiting algae from the polyextreme environment of the Atacama Desert is unveiled, providing fresh insights into their survival mechanisms.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eCarotenoids were discovered beneath the microbially colonized zone within the gypsum matrix, suggesting migration of these pigments following cell disruption.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eComprehensive Raman spectroscopic analysis revealed distinct differences in carotenoid and chlorophyll composition between green-orange and orange algal cells of the Trebouxiae family, cohabiting the gypcrete rock.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eFor the first time, in-vivo chemical detection of lipids within these algal cells has been achieved.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStudy site and samples\u003c/h2\u003e \u003cp\u003eThe sampling zone (23\u0026deg; 53\u0026prime; S, 068\u0026deg; 08\u0026prime; W; 2720 m a.s.l.) was located within the southern edge of the Salar de Atacama basin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) in the north\u0026ndash;south-trending depression of the Cordon de Lila range, in northern Chile. This depression is mostly covered by volcanic material, but large gypsum outcrops can be found in several locations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In the field, gypsum deposits were assessed for endolithic colonization by visual inspection for microbial pigments present in fractured samples. These pigments indicate the presence of cryptoendolithic (occupying pore spaces beneath the rock surface) and hypoendolithic (colonizing the undermost layer of the rock, sensu Wierzchos et al. (2011, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). microbial colonization, forming horizons beneath the surface and close to the bottom of the gypsum deposits. The gypsum rock fragments were collected in hyperarid conditions with air relative humidity (RH) of 10% and air temperature (T) of 28\u0026deg;C. They were stored in dry and dark conditions at room temperature until analysis.\u003c/p\u003e \u003cp\u003eGypsum formations at the studied site called gypcrete (according to the nomenclature of Horta \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1980\u003c/span\u003e) occur as hard layer deposits on the soil surface, interbedded between layers of ignimbrites and sometimes filling cracks on these rocks. Abundant colonization can be recognized visually in the field by green-to-orange pigmentation after cracking open the gypcretes. The cryptoendolithic habitat appears beneath the 0.5\u0026ndash;5-mm-thin surface layer. The typical zonation was revealed to be due to orange (higher position) and green (lower position) algae, with cyanobacteria appearing together within the green layer. Moreover, cyanobacteria were found to occupy a hypoendolithic habitat, e.g., at the bottom position close to the contact with the soil. For a detailed characterization of the gypsum formations and the microbial colonization by chlorophototrophic biota, see the comprehensive study by Wierzchos et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOptical microscopy and 3D surface microscopy\u003c/h2\u003e \u003cp\u003eA Zeiss AxioImager D1 microscope (Carl Zeiss, Oberkochen, Germany) was employed to obtain optical images of algal cells, equipped with a Plan-Apo 609/1.4 Zeiss oil-immersion objective.\u003c/p\u003e \u003cp\u003eIn addition, we have used digital microscope Keyence VHX \u0026minus;\u0026thinsp;900F (Keyence, UK) with 100x magnification objective to scan 3D surface structure of the algal cell embedded in gypsum samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnalyses of single microalgal cells by Raman spectroscopy\u003c/h2\u003e \u003cp\u003eMicroalgae cells colonizing cryptoendolithic habitat of gypcrete, were examined using point Raman analysis on an \u003cem\u003eInVia\u003c/em\u003e spectrometer (Renishaw, Wotton-under-Edge, UK) equipped with a Leica confocal microscope. A 785 nm laser line was employed as a universal excitation wavelength capable of detecting variety of pigments and other biomolecules, including lipids. The instrument was calibrated to a silicon Raman band at 520.5 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The point analysis was undertaken employing 50x magnification objective, 2s \u0026ndash; 5s exposure time was set, accumulated 10 times. The laser power between 15\u0026ndash;30 mW at source was used.\u003c/p\u003e \u003cp\u003eThe analyses were performed on transects of the gypcrete substrate stored in dark conditions at 20\u0026deg;C until analysis. The Raman spectroscopy technique was chosen for its ability to analyze small amounts of biological material \u003cem\u003ein situ\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRaman imaging and Raman data processing\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe same InVia spectrometer (Renishaw, Wotton-under-Edge, UK) equipped with a Leica confocal microscope was used in point-to-point scanning mode for the Raman imaging. The instrument was calibrated to a silicon Raman band at 520.5 cm\u003csup\u003e-1\u003c/sup\u003e. For imaging, an Ar laser line at 514.5 nm, with 10 mW power at the source, and a 1 s exposure time accumulated 1x times was employed at each point. Benefiting from the resonance Raman effect, a strong signal of carotenoids was obtained within the Raman imaging using a relatively short exposure time. As a result, a relatively large area was scanned at a high spatial resolution. The laser was focused using a 5 \u0026times; magnification Leica objective (NA\u0026thinsp;=\u0026thinsp;0.12). Single spectra (averaged from 7 neighbor spectra) were extracted from the zones of interest to show the spectral differences. The Raman imaging data were acquired using Wire 3.4 (Renishaw).\u003c/p\u003e \u003cp\u003eThe subsequent data processing workflow was provided by ImageLab software, version 3.20 (Epina GmbH Retz, Austria). Spikes (due to cosmic rays) were detected and removed using the following parameters: spike half-width \u0026minus;\u0026thinsp;3; threshold \u0026minus;\u0026thinsp;1. Next, the spectra were smoothed out using the Savitzky-Golay polynomial function, window: 7. Then, the baseline was corrected using the Eilers algorithm using the following parameters: smoothness \u0026minus;\u0026thinsp;10000; asymmetry \u0026minus;\u0026thinsp;0.002; iterations \u0026minus;\u0026thinsp;7. Suspicious pixels (data without spectral noise) were masked and corresponded mainly to the oversaturated signal of epoxide used for sample preparation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eP.V. contributed to the sample collection, measurements, data processing, writing and editing of the text; C.A. contributed to the project management, writing and editing; O.A. contributed to the area selection, sampling, interpretation of the data and text editing; J.W contributed to the project management, area selection, sample collection, data interpretation, writing and editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003ePV thanks to the Czech Science Foundation (project number 22-29315S) and to the project of the Ministry of Education, Youth and Sports of the Czech Republic (AdAgriF; CZ.02.01.01/00/22_008/0004635). Authors are thankful for financial support by grant PID2021-124362NB-I00 from MCIN/AEI/10.13039/501100011033/FEDER, UE. We are grateful to Prof. Miloš Bart\u0026aacute;k for his help with capturing 3D-microscopic images and the support provided by the CARP infrastructure (CzechPolar-I and II, LM2010009 andLM2015078).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdams, W.W. III \u0026amp; Demmig-Adams, B. Operation of the xanthophyll cycle in higher plants in response to diurnal changes in incident sunlight. Planta 186, 390\u0026ndash;398 (1992).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnderson, I.C. \u0026amp; Robertson, D.S. Role of carotenoids in protecting chlorophyll from photodestruction. 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Adaptation strategies of endolithic chlorophototrophs to survive the hyperarid and extreme solar radiation environment of the Atacama Desert. Front Microbiol. 6, 934 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, H., Volponi, J.V., Oliver, A.E., Parikh, A.N., Simmons, B.A. \u0026amp; Singh, S. In vivo lipidomics using single-cell Raman spectroscopy. \u003cem\u003eProc. Nat. Acad. Sci.\u003c/em\u003e 108, 3809\u0026ndash;3814 (2011).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4611340/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4611340/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe high-altitude pre-Andean region of the Atacama Desert, is characterized by its stark volcanic rock formations, hosts unique hydrothermal gypsum outcrops (gypcrete). This study delves into the biomolecular composition of endolithic phototrophic microbes thriving within these gypcretes. Using advanced Raman spectroscopy techniques, including Raman imaging, complemented by microscopic and 3D microscopic observations, we unveil new insights into the adaptive strategies of gypsum-inhabiting algae.\u003c/p\u003e \u003cp\u003eOur Raman imaging results provide a detailed chemical map of photoprotective and photosynthetic pigments associated with microbial colonization. This map reveals a significant gradient in pigment composition, highlighting a critical survival mechanism for algae and cyanobacteria in this polyextreme environment. Intriguingly, we detected carotenoid signals not only in the algae-colonized layer but also deeper within the gypsum matrix, indicating pigment migration following cell disruption.\u003c/p\u003e \u003cp\u003eIn addition, we conducted an in-depth analysis of individual algal cells from the Trebouxiae family, noting their color variations from green to orange and describing the spectral differences in detail. This investigation identified in-vivo pigments (carotenoids, chlorophyll) and lipids at the cellular level, offering a comprehensive view of the molecular adaptations enabling life in one of Earth's most extreme habitats.\u003c/p\u003e","manuscriptTitle":"Exploring Rock-inhabiting Microbes in Atacama Desert's Gypcrete: Raman Spectroscopy Unveils the Biomolecular Adaptations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-10 16:35:09","doi":"10.21203/rs.3.rs-4611340/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-18T09:29:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-12T16:43:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-12T13:10:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"74161124637166059453483204957449942743","date":"2024-07-02T10:02:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"311969755737816356329415078966312782563","date":"2024-07-02T09:25:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-02T08:06:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-02T07:47:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-06-24T10:53:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-21T11:59:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-06-20T11:12:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"be2df9d7-bd8a-4ce6-a970-2539671d9f16","owner":[],"postedDate":"July 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34004359,"name":"Biological sciences/Biophysics"},{"id":34004360,"name":"Biological sciences/Plant sciences"},{"id":34004361,"name":"Earth and environmental sciences/Biogeochemistry"},{"id":34004362,"name":"Earth and environmental sciences/Ecology"}],"tags":[],"updatedAt":"2024-10-14T16:08:29+00:00","versionOfRecord":{"articleIdentity":"rs-4611340","link":"https://doi.org/10.1038/s41598-024-75526-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-10-13 15:57:49","publishedOnDateReadable":"October 13th, 2024"},"versionCreatedAt":"2024-07-10 16:35:09","video":"","vorDoi":"10.1038/s41598-024-75526-7","vorDoiUrl":"https://doi.org/10.1038/s41598-024-75526-7","workflowStages":[]},"version":"v1","identity":"rs-4611340","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4611340","identity":"rs-4611340","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}