Introduction
There is strong evidence that many potentially habitable worlds exit in our galaxy: in the Solar System environments that might have hosted life in the past or even today have been identified. Those include the surface of early Mars, the sub-surface of present-day Mars, the oceans of the icy moons Europa and Enceladus, or even the clouds of Venus, along with thousands of exoplanets orbiting in the habitable zone of their star (Styczinski et al., Reference Styczinski, Cooper, Glaser, Lehmer, Mierzejewski and Tarnas2024; Cockell et al., Reference Cockell, Simons, Castillo-Rogez, Higgins, Kaltenegger, Keane, Leonard, Mitchell, Park, Perl and Vance2024). Hence, understanding how biosignatures change over time and how they are modified by space conditions is critical for space exploration missions searching for life (Dartnell et al., Reference Dartnell, Page, Jorge-Villar, Wright, Munshi, Scowen, Ward and Edwards2012; Dartnell and Patel, Reference Dartnell and Patel2014).
Since everything we know about biology derives from Earth, microorganisms living in extreme environments, the so-called extremophiles, are the best-case scenario to identify protective biomolecules that can serve as biomarkers and to investigate biosignature stability/degradation under planetary simulations in the laboratory or in space (Martins et al., Reference Martins, Cottin, Kotler, Carrasco, Cockell, de la Torre Noetzel, Demets, de Vera, d’Hendecourt, Ehrenfreund, Elsaesser, Foing, Onofri, Quinn, Rabbow, Rettberg, Ricco, Slenzka, Stalport, ten Kate, van Loon and Westall2017; Jorge-Villar and Edwards, Reference Jorge-Villar and Edwards2013; Wilhelm et al., Reference Wilhelm, Davila, Parenteau, Jahnke, Abate, Cooper, Kelly, Parro García, Villadangos, Blanco, Glass, Wray, Eigenbrode, Summons and Warren-Rhodes2018). Microbial pigments are promising biosignatures because they can be easily detected by Raman spectroscopy (Jehlička et al., Reference Jehlička, Edwards and Oren2022), carotenoids, chlorophylls, phycocyanins and scytonemin have been all detected in extreme habitats and included in a biosignature library of Raman spectra (Varnali and Edwards, Reference Varnali and Edwards2014). Extremophiles are not only a valuable reservoir of biosignatures but also model systems to evaluate the habitability of other planets (Merino et al., Reference Merino, Aronson, Bojanova, Feyhl-Buska, Wong, Zhang and Giovannelli2019). For example, increasing dryness in deserts causes a shift from edaphic to lithic communities, so that it has been proposed that during the loss of surface habitability of Mars, if life ever occurred it may have retreated to sub-surface niches (Davila and Schulze-Makuch, Reference Davila and Schulze-Makuch2016).
Cyanobacteria of the genus Chroococcidiopsis colonize lithic niches in extremely dry deserts, and since their discovery they have been pointed as a model organism to search for life on Mars (Friedmann and Ocampo, Reference Friedmann and Ocampo1976). Although there is no general agreement if photosynthesis ever occurred on Mars (Cockell and Raven, Reference Cockell and Raven2004; Westall et al., Reference Westall, Foucher, Bost, Bertrand, Loizeau, Vago, Kminek, Gaboyer, Campbell, Bréhéret, Gautret and Cockell2015), the capability of certain cyanobacteria of using far-red light to drive photosynthesis offer a new scenario for the habitability of Mars (Antonaru et al., Reference Antonaru, Selinger, Jung, Di Stefano, Sanderson, Barker, Wilson, Büdel, Canniffe, Billi and Nürnberg2023; Billi et al., Reference Billi, Napoli, Mosca, Fagliarone, de Carolis, Balbi, Scanu, Selinger, Antonaru and Nürnberg2022). On Earth these cyanobacteria inhabit niches depleted in visible light (VL) and enriched in far red light (FRL) and have developed an adaption known as far-red light photoacclimation (FaRLiP) consisting in the remodeling of the photosynthetic apparatus and production of far-red shifted chlorophylls (Gan and Bryant, Reference Gan and Bryant2015). In particular, the colonization of FaRLiP cyanobacteria of rocks and caves (Behrendt et al., Reference Behrendt, Brejnrod, Schliep, Sørensen, Larkum and Kühl2015; Antonaru et al., Reference Antonaru, Selinger, Jung, Di Stefano, Sanderson, Barker, Wilson, Büdel, Canniffe, Billi and Nürnberg2023) has implications for the habitability of Mars since a putative photosynthetic life form might have retreated to sub-surface niches and caves.
Moreover, far-red photosynthesis has implications for the habitability of exoplanets. Exoplanets orbiting M stars have a light spectrum peaking in the far-red and infrared that might support oxygenic photosynthesis (Lehmer et al., Reference Lehmer, Catling, Parenteau, Kiang and Hoehler2021). The feasibility of oxygenic photosynthesis under M-dwarf light has been pointed out by documenting the capability of cyanobacteria and more complex photosynthetic organisms to grow and produce oxygen under laboratory simulations of M-dwarf light (Battistuzzi et al., Reference Battistuzzi, Cocola, Liistro, Claudi, Poletto and La Rocca2023, Reference Battistuzzi, Cocola, Claudi, Pozzer, Segalla, Simionato, Morosinotto, Poletto and La Rocca2023a).
The current knowledge on how biosignatures respond to space and Mars-like conditions has been largely achieved thanks to space missions that used the ESA-EXPOSE facility installed outside the International Space Station (ISS) allowing the exposure of extremophiles and their molecules to space and Mars-like conditions (Cottin et al., Reference Cottin, Kotler, Billi, Cockell, Demets, Ehrenfreund, Elsaesser, d’Hendecourt, van Loon, Martins, Onofri, Quinn, Rabbow, Rettberg, Ricco, Slenzka, la Torre, de Vera, Westall, Carrasco, Fresneau, Kawaguchi, Kebukawa, Nguyen, Poch, Saiagh, Stalport, Yamagishi, Yano and Klamm2017). The BIOMEX (BIOlogy and Mars EXperiment) space experiment showed that after exposure for 469 days to Mars-like simulations, out of seven biomolecules, only three (chlorophyllin, quercetin and melanin) were still detectable on UV-exposed samples although with a reduced Raman signal, while slightly reduced Raman signals occurred in biomolecules mixed with regoliths to mimic sub-surface environments (Baqué et al., Reference Baqué, Backhaus, Meeßen, Hanke, Böttger, Ramkissoon, Olsson-Francis, Baumgärtner, Billi, Cassaro, de la Torre Noetzel, Demets, Edwards, Ehrenfreund, Elsaesser, Foing, Foucher, Huwe, Joshi, Kozyrovska, Lasch, Lee, Leuko, Onofri, Ott, Pacelli, Rabbow, Rothschild, Schulze-Makuch, Selbmann, Serrano, Szewzyk, Verseux, Wagner, Westall, Zucconi and de Vera2022).
The desert cyanobacterium Chroococcidiopsis sp. CCMEE 029 was exposed to space and Mars-like simulations along with other extremophiles during the BIOMEX experiment (de Vera et al., Reference de Vera, Alawi, Backhaus, Baqué, Billi, Böttger, Berger, Bohmeier, Cockell, Demets, de la Torre Noetzel, Edwards, Elsaesser, Fagliarone, Fiedler, Foing, Foucher, Fritz, Hanke, Herzog, Horneck, Hübers, Huwe, Joshi, Kozyrovska, Kruchten, Lasch, Lee, Leuko, Leya, Lorek, Martínez-Frías, Meessen, Moritz, Moeller, Olsson-Francis, Onofri, Ott, Pacelli, Podolich, Rabbow, Reitz, Rettberg, Reva, Rothschild, Garcia Sancho, Schulze-Makuch, Selbmann, Serrano, Szewzyk, Verseux, Wadsworth, Wagner, Westall, Wolter and Zucconi2019). Post-flight analyses of dried cells mixed with Martian mineral analogs revealed detectable pigments and genomic DNA thanks to the UV shielding provided by the regoliths (Billi et al., Reference Billi, Verseux, Fagliarone, Napoli, Baqué and de Vera2019a). Three desert strains of Chroococcidiopsis, namely CCMEE 029, CCMEE 057 and CCMEE 064, were exposed to space and Mars-like simulations as dried biofilms during the BOSS (Biofilm Organisms Surfing Space) space mission (Cottin and Rettberg, Reference Cottin and Rettberg2019). Post-flight analyses revealed unbleached photosynthetic pigments in the bottom layers of the biofilms that were shielded against UV radiation by top layer-cells (Billi et al., Reference Billi, Staibano, Verseux, Fagliarone, Mosca, Baqué, Rabbow and Rettberg2019b). However, none of these Chroococcidiopsis strains were capable of FarLiP adaption (Billi et al., Reference Billi, Napoli, Mosca, Fagliarone, de Carolis, Balbi, Scanu, Selinger, Antonaru and Nürnberg2022; Antonaru et al., Reference Antonaru, Selinger, Jung, Di Stefano, Sanderson, Barker, Wilson, Büdel, Canniffe, Billi and Nürnberg2023).
Novel insights into biosignature detectability of extremophiles under simulations of Mars- and icy moon-like conditions will be delivered by the BioSigN (Bio-Signatures and habitable Niches) space mission that will use the foreseen Exobio facility to be installed outside the ISS (2027-2028), thus preparing future planetary exploration missions to Mars, Enceladus and Europa (de Vera and Baqué, Reference de Vera and Baqué2024).
The overarching goal of the present work was to investigate the suitability for the of BioSigN space mission of two desert strains of Chroococcidiopsis, namely CCMEE 010 and CCMEE 130, capable of FaRLiP acclimation and both possessing far-red shifted chlorophylls (Antonaru et al., Reference Antonaru, Selinger, Jung, Di Stefano, Sanderson, Barker, Wilson, Büdel, Canniffe, Billi and Nürnberg2023; Billi et al., Reference Billi, Napoli, Mosca, Fagliarone, de Carolis, Balbi, Scanu, Selinger, Antonaru and Nürnberg2022). Since BioSigN will expose dried microorganisms, the assessment of desiccation tolerance and stability of sub-cellular dried components is mandatory. Therefore, these two strains were investigated for biosignature detectability and survival after 6 years of storage in the air-dried state. The detectability of photosynthetic pigments and genomic DNA was assessed at the single-cell level by using confocal laser scanning microscopy (CLSM), while carotenoids were detected with Raman spectroscopy. Then, biomarker detectability was compared with that of dried cells of Chroococcidiopsis strains that were exposed to space and Mars-like conditions during the BIOMEX and BOSS space experiments (Billi et al., Reference Billi, Verseux, Fagliarone, Napoli, Baqué and de Vera2019a, b). Biosignature detectability was assessed in strains CCMEE 057 and CCMEE 064 after 6 years of air-drying, and in CCMEE 029 after 10 years. Finally, the occurrence of survivors in all five strains was evaluated after 72 h-rehydration by using an indirect method based on Calcein-AM and by assessing their capability to enter cell division after transfer into a fresh growth medium.
Materials and methods
Cyanobacterial strains and sample preparation
The five Chroococcidiopsis strains used in this study are part of the Culture Collection of Microorganisms from Extreme Environments (CCMEE) established by E. Imre Friedmann and Roseli Ocampo-Friedmann (Table 1) and were cultured in BG11 medium in 50-mL vented flasks placed in an incubator at 25°C, without shaking. Cultures under visible light were exposed to a photon flux density of 20 μmol m-2 s-1 provided by white tubular led lights (OSRAM LEDs). Cultures under far-red light were exposed to a photon flux density of 5 μmol m-2 s-1 provided by far-red tubular led lights (OSRAM LEDs). Dried samples were prepared by filtering cell aliquots on Millipore filters and air-dried overnight in a sterile hood and stored in sealed plastic bags in the dark under room conditions.
Table 1. List of Chroococcidiopsis sp. CCMEE strains used in this study
Confocal laser scanning microscopy
Cells were recovered from small fragments (about 2 mm2) by using 500 μL BG-11 medium and after centrifugation resuspended in 20 μL Phosphate Buffered Saline (PBS) buffer containing 1.5% agarose and immobilized onto a microscopy slide and observed with a confocal laser scanning microscope (CLSM, Olympus Fluoview 1000) by using a 60X objective. Photosynthetic pigments were imaged with a 635 nm laser and collecting the fluorescence emission from 650 to 680 nm for phycobilisomes and chlorophylls. Genomic DNA was visualized at the CLSM with a 405-nm excitation laser after staining with Hoechst as follows: cells were harvested by gentle centrifugation and resuspended in 1 mL PBS with Hoechst 33342 (Thermo Fisher Scientific Inc.) at final concentration of 5 µg/ml and incubated in the dark at room temperature for 15 minutes. Then the cells were washed once with PBS buffer and resuspended in 20 μL PBS buffer containing 1.5% agarose for slide preparation. CLSM lambda scans were obtained by using a 488-nm excitation laser and collecting the emission from 550 to 800 nm. Curve plotting was performed using the GraphPad Prism program (GraphPad Software, San Diego, CA).
For cell viability, small fragments (about 2 mm2) of dried samples were inoculated in BG-11 medium under optimal growth conditions for 72 h. Calcein staining was performed as follows: cells were harvested by gentle centrifugation and resuspended in 500 µL of fresh BG-11 containing 10 µL Calcein-AM (Thermo Fisher Scientific Inc.; 1 mg/mL dimethylsulfoxide). The suspension was incubated in the dark at room temperature for 90 minutes (Mullineaux et al., Reference Mullineaux, Mariscal, Nenninger, Khanum, Herrero, Flores and Adams2008 ). Then cells were washed three times with PBS buffer, resuspended in 20 μL PBS buffer containing 1.5% agarose for slide preparation and observed at the CLSM with a 488-nm excitation laser.
Raman Spectroscopy: Set-up and spectra parameters
Raman measurements were performed with a confocal WITec alpha300 Raman microscope operating at room temperature, under ambient atmospheric conditions. The Raman laser excitation wavelength was 532 nm and the spectral resolution of the spectrometer 4–5 cm-1. A Nikon 10× objective, with a 0.25 numerical aperture, was used to focus the laser on a 1.5 μm spot. The surface laser power was set at 1 mW. A spectral calibration was performed with a pure silicon test sample. Spectra were acquired directly on fragments of about 2 mm2 of the Millipore filters for the dried samples and on 10 μL air-dried drops on a microscopy glass slide for the liquid samples. Acquisition time was kept between 0.5 and 1 s to avoid signal saturation from photosynthetic pigments’ fluorescence with 1 accumulation. Single spectra, line scans and image scans with up to 30 μm × 30 μm and up to 400 image points (only for selected samples) were obtained thus collecting a minimum of 50 measurements per sample. The spectra were visualized directly with the instrument’s software (Control5) and processing was further implemented with Python library RamanSpy (Georgiev et al., Reference Georgiev, Pedersen, Xie, Fernández-Galiana, Stevens and Barahona2024) for spectra pre-processing (cosmic ray removal, cropping and background subtraction) and plotting of the average spectra from the n>50 measurements.
Results
Detection of photosynthetic pigments and genomic DNA
The detectability of biosignatures was investigated in all the samples by CLSM (Figure 1). The visualization of the FaRLiP strains Chroococcidiopsis sp. CCMEE 010 and CCMEE 130 with a 635-nm laser revealed an intense pigment autofluorescence indicating high content of phycobiliproteins and chlorophylls in most of the cells of both strains, although cells with a reduced autofluorescence were also visualized. By applying a 405-nm laser, Hoechst-stained nucleoids could be identified in each cell regardless of the intensity of pigment autofluorescence. A blue-fluorescent envelope occurred around dried cells of strain CCMEE 130 but not in strain CCMEE 130.
Figure 1. CLSM imaging of photosynthetic pigments (635-nm excitation laser) and Hoechst-stained nucleoids (405-nm excitation laser) in Chroococcidiopsis. Strains CCMEE 010 and CCMEE 130 were grown under far-red light and desiccated for 6 years. Strains CCMEE 029, CCMEE 057 and CCMEE 064 were grown under visible light and desiccated for 6 years (057 and 064) or 10 years (029). Bar = 5 µm.
Similarly, dried samples of strains CCMEE 029, CCMEE 057 and CCMEE 064 showed cells with either an intense or reduced pigment autofluorescence, each one with Hoechst-stained nucleoids. Images with the 405-nm laser revealed the presence of a blue-fluorescent envelope around Hoechst-stained dried cells of CCMEE 057 and CCMEE 064, that was absent in hydrated cells of all both strains (not shown).
Spectral features of photosynthetic pigments
The stability of the far-red shifted chlorophylls in Chroococcidiopsis sp. CCMEE 010 and CCMEE 130 grown for two weeks under far-red light and then air-dried and stored for 6 years, was evaluated by CLSM-λscan analysis by using excitation with a 488-nm laser (Figure 2). In strain CCMEE 010 the emission spectrum was similar in shape and intensity in both dried cells and hydrated control. A peak at 650–660 nm due to phycobiliproteins, mainly allophycocyanin, and one peak in the 675–695 nm range due to chlorophyll a. An additional peak in the 720–750 nm range corresponding to far-red shifted chlorophylls was also detected in both dried and hydrated cells of strains CCMEE 010 and CCMEE 130 (Figure 2A). A similar spectrum was obtained for dried and hydrated cells of strain CCMEE 130 (Figure 2B).
Figure 2. CLSM-lambda-scan of photosynthetic pigments in dried FaRLiP Chroococcidiopsis. Strain CCMEE 010 (A) and CCMEE 130 (B). Cells were grown under far-red light and desiccated for 6 years; hydrated cells were grown in liquid cultures under far-red light and used as control. Graphs represent normalized fluorescence intensity versus emission wavelength.
Raman signal of carotenoids
The effect of prolonged desiccation on the detectability of carotenoids in Chroococcidiopsis sp. CCMEE 010 and CCMEE 130 was determined by Raman analyses using a 535-nm laser for excitation by comparing cells grown under far-red light and then air-dried for 6 years with hydrated used as control (Figure 3). Each sample showed a typical Raman signal mainly due to carotenoids with three distinct peaks at 1009, 1150 and 1515 cm-1, corresponding to in-plane rocking modes of CH3, groups attached to the polyene chain coupled with C-C bonds, and in-phase C-C stretching (ν2) and C=C (ν1) vibrations of the polyene chain in carotenoids, respectively. The spectra were normalized for clarity. No evident differences occurred in the intensity of the carotenoid main peaks among dried and hydrated cells of strains CCMEE 029, CCMEE 057 and CCMEE 064.
Figure 3. Raman spectra from Chroococcidiopsis. Strains CCMEE 010 and CCMEE 130 were grown under far-red light and desiccated for 6 years (D); hydrated controls were grown in liquid cultures under far-red light (L). Strains CCMEE 029, CCMEE 057 and CCMEE 064 were grown under visible light and desiccated for 6 years (057 and 064) or 10 years (029) (D); hydrated controls were grown in liquid cultures under visible light (L).
Detection of surface pigments
The presence of fluorescent pigments observed in the envelope of dried cells of strain CCMEE 130 after Hoechst staining at the CLSM (Figure 1) was further evaluated in the absence of any staining. Cells grown under far-red light showed a blue autofluorescence of the envelope when excited with a 405-nm laser and a red autofluorescence of photosynthetic pigments when excited with a 635-nm laser (Figure 4A). The CLSM λscan with a 405-nm laser of three regions of interest selected in the cell envelope yielded a spectrum with a peak of faint intensity at 430–435 nm possibly due to scytonemin (Klicki et al., Reference Klicki, Ferreira, Hamill, Dirks, Mitchell and Garcia-Pichel2018), while the fourth region of interest selected in the cytoplasm showed an intense peak in 675–695 nm range due to photosynthetic pigments (Figure 4C).
Figure 4. CSLM and Raman analysis of Chroococcidiopsis sp. CCMEE 130 grown under far-red light. Merge image of optical sections obtained with a 405-nm and 635-nm laser (B); spectral profiles of four regions of interest (ROI) excited with a 405-mn laser (B). Raman spectrum obtained with a 532-nm laser. Bar = 10 µm.
As shown in Figure 4C, no Raman signal for to scytonemin or scytonin, was detected, that generally have similar spectra with bands near 1600, 1550, 1400, 1300 and 1180 cm-1 (Edwards et al., Reference Edwards, Jehlička, Němečková and Culka2023).
Survival after prolonged desiccation
The Calcein staining was used to investigate the viability of Chroococcidiopsis sp. CCMEE 010 and CCMEE 130 grown for two weeks under far-red light and then air-dried for 6 years (Figure 5). Before the staining dried cells were rehydrated under optimal growth conditions because the assay is based on a nonfluorescent dye that is converted by esterases into green-fluorescent Calcein. The imaging with the CLSM using a 488-nm excitation laser revealed a strong green, fluorescent signal throughout the cytoplasm of the hydrated controls of both strains CCMEE 010 and CCMEE 130 indication the viability of the cells. When dried samples were rehydrated for 2 hs no esterase activity was detected (not shown). After 72-h hydration both strains CCMEE 010 and CCMEE 130 showed a dot-like green, fluorescent signal in about 20% of the cellular population, regardless the presence of the photosynthetic pigment autofluorescence. Similarly, after 72h-rehydration, esterase activity was detected in strains CCMEE 029, CCMEE 057 and CCMEE 064.
Figure 5. Viability of Chroococcidiopsis examined by Calcein staining. Merge images of photosynthetic pigments (635-nm excitation laser) and Calcein-stained cells (488-nm excitation laser). Dried cells and hydrated controls of strains CCMEE 010 and CCMEE 029 were grown under far-red light; dried and hydrated controls of strains CCMEE 029, CCMEE 057 and CCMEE 064 were grown under visible light. Bar = 5 µm.
Discussion
The biosignature detectability in Chroococcidiopsis sp. CCMEE 010 and CCMEE 130, two desert strains of capable of FaRLiP acclimation (Antonaru et al., Reference Antonaru, Selinger, Jung, Di Stefano, Sanderson, Barker, Wilson, Büdel, Canniffe, Billi and Nürnberg2023; Billi et al., Reference Billi, Napoli, Mosca, Fagliarone, de Carolis, Balbi, Scanu, Selinger, Antonaru and Nürnberg2022), after 6 years of desiccation was demonstrated. The combined use of CLSM and Raman spectroscopy highlighted the permanence of canonical chlorophyll a, far-red shifted chlorophylls, phycobilins and carotenoids as well as of Hoechst-stained genomic DNA, all considered unambiguous traces of life (Malaterre et al., Reference Malaterre, Ten Kate, Baqué, Debaille, Grenfell, Javaux, Khawaja, Klenner, Lara, McMahon, Moore, Noack, Patty and Postberg2023). No evident variation in biosignature detectability occurred between dried cells of these two strains and strains CCMEE 029, CCMEE 064 and CCMEE 057 that were previously exposed to space and to Mars-like conditions (Billi et al., Reference Billi, Verseux, Fagliarone, Napoli, Baqué and de Vera2019a, b). Such feature of Chroococcidiopsis sp. CCMEE 010 and CCMEE 130 provides a prerequisite necessary for the implementation into the BioSigN space mission that will investigate survival and biomarker detectability in dried extremophiles exposed to Mars- and open space conditions by using the foreseen ESA’s Exobio facility outside the ISS (de Vera and Baqué, Reference de Vera and Baqué2024).
CLSM imaging of dried CCMEE 010 and CCMEE 130 revealed the permanence of phycobiliproteins and chlorophylls due to their intrinsic fluorescence, that was comparable to that of dried CCMEE 029, CCMEE 064 and CCMEE 057. Moreover, the peak typical of far-red shifted chlorophylls was identified with CLSM-λscan in dried CCMEE 010 and CCMEE 130 acclimated to far-red light before desiccation. Therefore, these two FaRLiP strains are a unique reservoir of pigments to be investigated under planetary simulations to be performed in the laboratory or in space. The detectability of pigment autofluorescence is relevant in a scenario in which fluorescence microscopy and flow cytometry have been proposed as a potential technology for in situ life detection on icy moons and polar ice caps of Mars (Nadeau et al., Reference Nadeau, Perreault, Niederberger, Whyte, Sun and Leon2008; Wallace et al., Reference Wallace, Tallarida, Schubert and Lambert2024). The fact that Hoechst- stained DNA was detected in dried Chroococcidiopsis cells after years of air-drying is relevant since the feasibility of using fluorescent dye labeling as a tool for life detection has been proposed in combination with the detection of intrinsically fluorescent molecules for searching sign of life on Mars (Nadeau et al., Reference Nadeau, Perreault, Niederberger, Whyte, Sun and Leon2008). Moreover, Nanopore sequencing is currently under validation in diverse environments to support the search for nucleic-acid based life beyond Earth (Carr et al., Reference Carr, Bryan, Saboda, Bhattaru, Ruvkun and Zuber2020; Sutton et al., Reference Sutton, Burton, Zaikova, Sutton, Brinckerhoff, Bevilacqua, Weng, Mumma and Johnson2019).
Raman spectra of dried strains CCMEE 010 and CCMEE 130 showed only a slightly reduced intensity of the carotenoid peaks compared to hydrated controls, thus suggesting the capability of these two desert cyanobacteria to efficiently stabilize sub-cellular components as reported for strain CCMEE 029 (Baqué et al., 2020). The Raman detectability of carotenoids is relevant since miniaturized Raman instrumentation has the potential to be used in planetary exploration rovers (Edwards et al., Reference Edwards, Jehlička and Culka2021). Currently on Mars NASA Perseverance rover is using two miniaturized Raman spectrometers (Maurice et al., Reference Maurice, Wiens, Bernardi, Caïs, Robinson, Nelson, Gasnault, Reess, Deleuze, Rull, Manrique, Abbaki, Anderson, André, Angel, Arana, Battault, Beck, Benzerara, Bernard, Berthias, Beyssac, Bonafous, Bousquet, Boutillier, Cadu, Castro, Chapron, Chide, Clark, Clavé, Clegg, Cloutis, Collin, Cordoba, Cousin, Dameury, D’Anna, Daydou, Debus, Deflores, Dehouck, Delapp, De Los Santos, Donny, Doressoundiram, Dromart, Dubois, Dufour, Dupieux, Egan, Ervin, Fabre, Fau, Fischer, Forni, Fouchet, Frydenvang, Gauffre, Gauthier, Gharakanian, Gilard, Gontijo, Gonzalez, Granena, Grotzinger, Hassen-Khodja, Heim, Hello, Hervet, Humeau, Jacob, Jacquinod, Johnson, Kouach, Lacombe, Lanza, Lapauw, Laserna, Lasue, Le Deit, Le Mouélic, Le Comte, Lee, Legett, Leveille, Lewin, Leyrat, Lopez-Reyes, Lorenz, Lucero, Madariaga, Madsen, Madsen, Mangold, Manni, Mariscal, Martinez-Frias, Mathieu, Mathon, McCabe, McConnochie, McLennan, Mekki, Melikechi, Meslin, Micheau, Michel, Michel, Mimoun, Misra, Montagnac, Montaron, Montmessin, Moros, Mousset, Morizet, Murdoch, Newell, Newsom, Nguyen Tuong, Ollila, Orttner, Oudda, Pares, Parisot, Parot, Pérez, Pheav, Picot, Pilleri, Pilorget, Pinet, Pont, Poulet, Quantin-Nataf, Quertier, Rambaud, Rapin, Romano, Roucayrol, Royer, Ruellan, Sandoval, Sautter, Schoppers, Schröder, Seran, Sharma, Sobron, Sodki, Sournac, Sridhar, Standarovsky, Storms, Striebig, Tatat, Toplis, Torre-Fdez, Toulemont, Velasco, Veneranda, Venhaus, Virmontois, Viso, Willis and Wong2021; Razzell Hollis et al., Reference Hollis, Moore, Sharma, Beegle, Grotzinger, Allwood, Abbey, Bhartia, Brown, Clark, Cloutis, Corpolongo, Henneke, Hickman-Lewis, Hurowitz, Jones, Liu, Martinez-Frías, Murphy, Pedersen, Shkolyar, Siljeström, Steele, Tice, Treiman, Uckert, VanBommel and Yanchilina2022) while the ESA Rosalind Franklin rover to be launched in 2028 is equipped with a Raman Laser Spectrometer (Rull et al., Reference Rull, Maurice, Hutchinson, Moral, Perez, Diaz, Colombo, Belenguer, Lopez-Reyes, Sansano, Forni, Parot, Striebig, Woodward, Howe, Tarcea, Rodriguez, Seoane, Santiago, Rodriguez-Prieto, Medina, Gallego, Canchal, Santamaría, Ramos and Vago2017; Rull and Martínez-Frías, Reference Rull-Pérez and Martinez-Frias2006). A Raman instrumentation has been suggested for the NASA Europa Lander Mission, a conceptual study to search for life on Europa by using in situ techniques (Hand et al., Reference Hand, Murray, Garvin, Maize, Reeves, Martin, Tan-Wang, Krajewski, Hurst, Crum, Kennedy, McElrath, Gallon, Sabahi, Thurman, Goldstein, Estabrook, Lee, Dooley, Brinckerhoff, Edgett, German, Hoehler, Hörst, Lunine, Paranicas, Nealson, Smith, Templeton, Russell, Schmidt, Christner, Ehlmann, Hayes, Rhoden, Willis, Yingst, Craft, Cameron, Nordheim, Pitesky, Scully, Hofgartner, Sell, Barltrop, Izraelevitz, Brandon, Seong, Jones, Pasalic, Billings, Ruiz, Bugga, Graham, Arenas, Takeyama, Drummond, Aghazarian, Andersen, Andersen, Anderson, Babuscia, Backes, Bailey, Balentine, Ballard, Berisford, Bhandari, Blackwood, Bolotin, Bovre, Bowkett, Boykins, Bramble, Brice, Briggs, Brinkman, Brooks, Buffington, Burns, Cable, Campagnola, Cangahuala, Carr, Casani, Chahat, Chamberlain-Simon, Cheng, Chien, Cook, Cooper, DiNicola, Clement, Dean, Cullimore, Curtis, del Croix, Pasquale, Dodd, Dubord, Edlund, Ellyin, Emanuel, Foster, Ganino, Garner, Gibson, Gildner, Glazebrook, Greco, Green, Hatch, Hetzel, Hoey, Hofmann, Ionasescu, Jain, Jasper, Johannesen, Johnson, Jun, Katake, Kim-Castet, Kim, Kim, Klonicki, Kobeissi, Kobie, Kochocki, Kokorowski, Kosberg, Kriechbaum, Kulkarni, Lam, Landau, Lattimore, Laubach, Lawler, Lim, Lin, Litwin, Lo, Logan, Maghasoudi, Mandrake, Marchetti, Marteau, Maxwell, Namee, Mcintyre, Meacham, Melko, Mueller, Muliere, Mysore, Nash, Ono, Parker, Perkins, Petropoulos, Gaut, Gomez, Casillas, Preudhomme, Pyrzak, Rapinchuk, Ratliff, Ray, Roberts, Roffo, Roth, Russino, Schmidt, Schoppers, Senent, Serricchio, Sheldon, Shiraishi, Shirvanian, Siegel, Singh, Sirota, Skulsky, Stehly, Strange, Stevens, Sunada, Tepsuporn, Tosi, Trawny, Uchenik, Verma, Volpe, Wagner, Wang, Willson, Wolff, Wong, Zimmer, Sukhatme, Bago, Chen, Deardorff, Kuch, Lim, Syvertson, Arakaki, Avila, DeBruin, Frick, Harris, Heverly, Kawata, Kim, Kipp, Murphy, Smith, Spaulding, Thakker, Warner, Yahnker, Young, Magner, Adams, Bedini, Mehr, Sheldon, Vernon, Bailey, Briere, Butler, Davis, Ensor, Gannon, Haapala-Chalk, Hartka, Holdridge, Hong, Hunt, Iskow, Kahler, Murray, Napolillo, Norkus, Pfisterer, Porter, Roth, Schwartz, Wolfarth, Cardiff, Davis, Grob, Adam, Betts, Norwood, Heller, Voskuilen, Sakievich, Gray, Hansen, Irick, Hewson, Lamb, Stacy, Brotherton, Tappan, Benally, Thigpen, Ortiz, Sandoval, Ison, Warren, Stromberg, Thelen, Blasy, Nandy, Haddad, Trujillo, Wiseley, Bell, Teske, Post, Torres-Castro, Grosso and Wasiolek2022).
The stability of sub-cellular components in dried cells of the two Chroococcidiopsis FaRLiP strains makes them a novel reservoir of biosignatures to be investigated and contribute to future planetary exploration missions to Mars as well as to biosignature detection on exoplanets. In fact, pigments like canonical chlorophylls, far-red shifted chlorophylls and carotenoids might target life beyond the photosynthetic one, just because on Earth, microbial pigmentation has been developed for different purposes beyond light capture (Barreto et al., Reference Barreto, Casanova, Junior, Reis-Mansur and Vermelho2023). Therefore, FaRLiP cyanobacteria are relevant for searching biosignatures of photosynthetic life powered by infra-red light in sub-surface environments, but also of non-photosynthetic, pigmentated life in sub-surface environments supported by chemical energy (Cockell et al., Reference Cockell, Bush, Bryce, Direito, Fox-Powell, Harrison, Lammer, Landenmark, Martin-Torres, Nicholson, Noack, O’Malley-James, Payler, Rushby, Samuels, Schwendner, Wadsworth and Zorzano2016). Moreover, since the absorption and reflection of light harvesting pigments can serve as surface biosignatures for exoplanets (Schwieterman et al., Reference Schwieterman, Kiang, Parenteau, Harman, DasSarma, Fisher, Arney, Hartnett, Reinhard, Olson, Meadows, Cockell, Walker, Grenfell, Hegde, Rugheimer, Hu and Lyons2018), FaRLiP cyanobacteria are suitable model system for laboratory simulations to investigate the boundary conditions of the habitability of exoplanets around M stars and detectability of exotic photosynthetic life. Because in literature theoretical investigations and some indices are even considering a potential of photosynthesis in deep sea and hydrothermal areas (Beatty et al., Reference Beatty, Overmann, Lince, Manske, Lang, Blankenship, Van Dover, Martinson and Plumley2005; Yurkov et al., Reference Yurkov, Krieger, Stackebrandt and Beatty1999), the potential of photosynthesis in the deep sea using far IR cannot be neglected. Therefore, a small likelihood to postulate the presence of photosynthesizing organisms in the icy ocean worlds in our solar system could be possible (Fisher et al., Reference Fisher, Dickerson, Blackman, Randolph-Flagg, German and Sotin2024).
CLSM imaging of strain CCMEE 130 suggested the presence of scytonemin-like compounds that were secreted in the cell envelope and that yielded an emission at about 430–435 nm when exited with a 405-nm laser (Klicki et al., Reference Klicki, Ferreira, Hamill, Dirks, Mitchell and Garcia-Pichel2018). Such a capability is relevant since scytonemin is a UV-absorbing pigment that possesses also antioxidant properties (Sen and Mallick, Reference Sen and Mallick2022). However, the presence of scytonemin, or scytonin, in the envelope of CCMEE 130 grown under far-red light was not confirmed by Raman spectroscopic probing. Nevertheless, the production of UV-screening compounds in FaRLiP cyanobacteria under far-red light is largely unknown and reported so far only for Chlorogloeopsis fritschii sp. PCC 6912 (Llewellyn et al., Reference Llewellyn, Greig, Silkina, Kultschar, Hitchings and Farnham2020). So, it could be speculated that, if produced the amount of scytonemin in CCMEE 130 was not enough to be detected. Indeed, the synthesis of scytonemin has been reported for two desert Chroococcidiopsis strains in response to other stress rather than UV radiation, for instance under osmotic stress in the absence of UV radiation (Dillon et al., Reference Dillon, Tatsumi, Tandingan and Castenholz2002; Casero et al., Reference Casero, Ascaso, Quesada, Mazur-Marzec and Wierzchos2021), but also in response of periodic desiccation under UV radiation (Fleming and Castenholz, Reference Fleming and Castenholz2007).
Finally, the fact the number of survivors scored among Chroococcidiopsis sp. CCMEE 010 and CCMEE 130 desiccated for 6 years was comparable to that of CCMEE 029 after for 4 years of desiccation (Billi, Reference Billi2009), further supports their suitability for implementation in the BioSigN space mission that foresees one-year exposure of dried extremophiles to Mars- and icy-moon simulation outside the ISS. Moreover, based on the comparable stability of their sub-cellular components with that of strains CCMEE 029, CCMEE 064 and CCMEE 057 already tested in space, it is anticipated that post-flight analysis might contribute to gather novel insights into survival potential and biosignature detectability. The biosignature stability and survivability scored in the present work after prolonged desiccation are an important prerequisite to future investigation on how extremophiles respond to deep space, as proposed by the BioMoon space mission that aims to expose to the lunar environment dried cells as well as cells that will be rehydrated on the Moon after exposure to deep space (Cockell et al., Reference Cockell, Green, Caplin, Grenouilleau, McDonald, Calvaruso, Billi, Cullen, Davey, De Micco, Elsaesser, Etheridge, Gläßer, Hellweg, Ilea, Lecocq, Leys, Martin-Torre, Nazarious, Pacelli, Przybyla Rabbow, Robson Brown, Soria-Salinas, Szewczyk, Tinganelli, Tranfield, de Vera and Verseux2024).
Author contributions
Conceptualization: D.B.; Methodology, G.d.S.; M.Ba.; Formal Analysis: G.d.S. and M.Ba.; Writing – Original Draft Preparation: G.d.S.; D.B.; Writing – Review & Editing: D.B., M.Ba, M.Be.; M.E.M.G. and J.P.d.V.; Supervision: D.B.; Funding Acquisition: D.B. All authors have read and agreed to the published version of the manuscript.
Funding statement
This research was funded by the Italian Space Agency contract number 2023-5- U.0 (ASTERIA).
Open access
