Introduction
Exobiology is a recent discipline with the goal of identifying life signals from outside our Planet and finding out the pathway through which life may have been distributed throughout the Universe (Horneck Reference Horneck1995). In order to study this last goal, the hypothesis of interplanetary transfer of life called Lithopanspermia arises. This theory hypothesizes that life spreads throughout the Universe by organism transfer in rocks (meteorites, asteroids or planetoids) from one planet to another, either through interplanetary or interstellar space (Arrhenius Reference Arrhenius1903; Crick Reference Crick1981; Nichoson Reference Nichoson2009; Worth et al. Reference Worth, Sigurdsson and House2013). A crucial issue on the Lithopanspermia theory consists in testing that microbes situated on or within rocky bodies could survive hypervelocity entry through the Earth's atmosphere (Cockell Reference Cockell2008) since some meteorites do not show signals of great heating (Mileikowsky et al. Reference Mileikowsky, Cucinotta, Wilson, Gladman, Horneck, Lindergren, Melosh, Rickman, Valtonen and Zheng2000; Weiss et al. Reference Weiss, Kirschvink, Baudenbacher, Vali, Peters, Macdonald and Wikswo2000; Meyer et al. Reference Meyer2008; Nichoson Reference Nichoson2009). For that purpose, the European Space Agency (ESA) has developed experiments to prove the endurance of terrestrial organisms against environmental conditions prevailing in space, particularly harmful radiation, vacuum and extreme temperatures, such as BIOPAN (effect of space environmental conditions on biological material), STONE 6 (survival of microfossils in artificial Martian meteorites to the Terrestrial atmospheric entry), LITHOPANSPERMIA (survival of organisms inside rocks to space conditions and atmospheric entry) and EXPOSE (study of survival of different terrestrial organisms to space environmental conditions) (de la Torre et al. Reference de la Torre, Sancho, Pintado, Rettberg, Rabbow, Panitz, Deutschmann, Reina and Horneck2007; Horneck et al. Reference Horneck2008; de la Torre et al. Reference de la Torre2010; Cockell et al. Reference Cockell, Rettberg, Rabbow and Olsson-Francis2011; Meyer et al. Reference Meyer2011; Bertrand et al. Reference Bertrand, Chabin, Brack, Cottin, Chaput and Westall2012; Onofri et al. Reference Onofri2012; Parnell et al. Reference Parnell, Bowden, Muirhead, Blamey, Westall, Demets, Verchovsky, Brandstätter and Brack2012). The results of some of these experiments demonstrated the survival of some biomarkers linked unambiguously to biological activity. Nevertheless some cellular microbial bioactive molecules were destroyed after being exposed to the harmful space conditions (Cockell et al. Reference Cockell, Rettberg, Rabbow and Olsson-Francis2011; Bertrand et al. Reference Bertrand, Chabin, Brack, Cottin, Chaput and Westall2012).
Recently, two new space experiments BOSS (Biofilm Organisms Surfing Space) and BIOMEX (BIOlogy and Mars EXperiment) onboard the EXPOSE-R2 platform of the ESA, were performed on selected biomolecules and microorganisms such as archaea, bacteria, fungi, lichens, which are tolerant to various environmental extremes. The objective of those experiments was to investigate the resistance of cyanobacterial and bacterial biofilms and their constituents (biomolecules like pigments, cellular components, biopolymers, etc.) and their potential survival on Mars. Furthermore, the mechanisms of the resistance and stability of those biomolecules under space and Mars-like conditions was also evaluated (de Vera et al. Reference de Vera, Boettger, Noetzel, Sánchez, Grunow and Schmitz2012; Baqué et al. Reference Baqué, de Vera, Rettberg and Billi2013). The effects of space and Martian simulations on biomolecules will provide further understanding on the degradation of cyanobacterial biosignatures (e.g. photosynthetic pigments), contributing to establish a biosignature database based on spectroscopic detection of biological molecules.
Although microorganisms, such as bacteria, are the main candidate in those studies, lichens have assumed an important role in Exobiology since numerous experiments, as LICHENS II on BIOPAN 5/FOTONM2; LITHOPANSPERMIA and STONE on BIOPAN6/FOTONM3; and LIFE on EXPOSE-E/EuTEF (Sancho et al. Reference Sancho, De la Torre, Horneck, Ascaso, de los Ríos, Pintado, Wierzchos and Schuster2007; de la Torre et al. Reference de la Torre2010; Raggio et al. Reference Raggio, Pintado, Ascaso, De la Torre, de los Ríos, Wierzchos, Horneck and Sancho2011; de Vera Reference de Vera2012; Onofri et al. Reference Onofri2012; Scalzi et al. Reference Scalzi, Selbmann, Zucconi, Rabbow, Horneck, Albertano and Onofri2012), have demonstrated that lichens are viable after exposition to space conditions. Lichens can survive the outdoor space which exposes them to low and high temperatures in a short time period (e.g. BIOMEX experiment, temperature range used −30 to +60°C, mostly −15 to 40°C (Biomex: an International Space Experiment Project (2014) DLR Institute of Planetary Research. http://www.dlr.de/pf/en/desktopdefault.aspx/tabid-178/327_read-37560/.) and therefore are valuable models in astrobiological research (de la Torre et al. Reference de la Torre, Horneck, Sancho, Pintado, Rabbow, Scherer, Facius, Deutschmann and Reina2004; Sancho et al. Reference Sancho, De la Torre, Horneck, Ascaso, de los Ríos, Pintado, Wierzchos and Schuster2007).
The production of pigments and other biomolecules by lichens has been related to protective mechanisms against extreme environmental conditions (Jorge-Villar et al. Reference Jorge-Villar, Miralles, Capel-Ferrón and Hernández2011; Miralles et al. Reference Miralles, Jorge-Villar, Canton and Domingo2012a). It could happen that abrupt temperature changes, similar to those of atmospheric entry, lead to changes in protective strategies, such as production of different biomolecules or changes in pigment concentration; biomolecular transformation or degradation owing to heating are also possible. Some of these changes could be permanent and some could reverse when stress parameters return to normal conditions. Here, we checked the effect of temperature on biomolecules produced by lichens and the detection capability of such changes by in situ Raman spectroscopy.
Among a variety of modern techniques evaluated for life signal detection on extra-terrestrial surfaces, Raman spectroscopy stands out because of its capability for detecting minerals and organic molecules in a heterogeneous system (Jorge-Villar & Edwards Reference Jorge-Villar and Edwards2006).This is valuable for the identification of both geological (Sharma et al. Reference Sharma, Angel, Ghosh, Hubble and Lucey2002, Reference Sharma, Lucey, Ghosh, Hubble and Horton2003; Wang et al. Reference Wang, Haskin, Lane, Wdowiak, Squyres, Wilson, Hovland, Manatt, Raouf and Smith2003, Reference Wang, Kuebler, Jolliff and Haskin2004a, Reference Wang, Kuebler, Jolliff and Haskinb) and biological (Wynn-Williams et al. Reference Wynn-Williams, Edwards and Garcia-Pichel1999; Wynn-Williams & Edwards Reference Wynn-Williams and Edwards2000; Edwards et al. Reference Edwards, Newton, Wynn-Williams and Coombes2003a, Reference Edwards, Newton, Dickensheets and Wynn-Williamsb, Reference Edwards, Moody, Jorge-Villar and Wynn-Williams2005a) markers from extant or extinct organisms and to bio-geologically modified materials (Edwards et al. Reference Edwards, Wynn-Williams and Jorge-Villar2004a, Reference Edwards, Cockell, Newton and Wynn-Williamsb; Jorge-Villar & Edwards Reference Jorge-Villar and Edwards2005). Raman spectroscopy is sensitive to structural and compositional differences, thus allowing polymorphous and isomorphs molecular discrimination (Frost & Weier Reference Frost and Weier2004; Edwards et al. Reference Edwards, Jorge-Villar, Jehlicka and Munshi2005b; Price et al. Reference Price, Grzesiak and Matzger2005). The technique does not require sample manipulation instead the technique can deal with the direct exposure of macroscopic or microscopic specimens. Another important characteristic of Raman spectroscopy is that analyses are acquired over a short time lapse, which is of vital relevance when rovers are working on extraplanetary surfaces.
Miniaturization of Raman spectrometers for space flight assessment (Dickensheets et al. Reference Dickensheets, Wynn-Williams, Edwards, Schoen, Crowder and Newton2000; Wang & Haskin Reference Wang and Haskin2000; Sharma et al. Reference Sharma, Lucey, Ghosh, Hubble and Horton2003; Wang et al. Reference Wang, Haskin, Lane, Wdowiak, Squyres, Wilson, Hovland, Manatt, Raouf and Smith2003), with limitations of size, mass and volume, are now being considered for Martian exploration from 2009 and beyond (NASA-MSL; ESA-ExoMars/Pasteur) (Jorge-Villar & Edwards Reference Jorge-Villar and Edwards2005; Alajtal et al. Reference Alajtal, Edwards and Scowen2010). These limitations are critical for selection of spectrometer characteristics such as spectral resolution, laser wavelength or wavenumber spectral region among others.
The search of new robust biomarkers preserved on extra-terrestrial bodies remains one of primary tasks for Exobiology at present. For this purpose, a Raman spectroscopic database of bio- and geo-markers, which may be used for extra-terrestrial life biosignatures, has been studied covering a range of extremophile organisms from several extreme environments (Jorge-Villar et al. Reference Jorge-Villar, Edwards and Cockell2000, Reference Jorge-Villar, Edwards and Wynn-Williams2003, Reference Jorge-Villar, Edwards and Worland2005a; Wynn-Williams & Edwards Reference Wynn-Williams and Edwards2000; Edwards et al. Reference Edwards, Newton, Dickensheets and Wynn-Williams2003b, Reference Edwards, Cockell, Newton and Wynn-Williams2004b, Reference Edwards, Moody, Jorge-Villar and Wynn-Williams2005a; Miralles et al. Reference Miralles, Jorge-Villar, Canton and Domingo2012a). Nevertheless, there is still a need for further investigation by using Raman spectroscopy of promising biomolecules (e.g. pigments, cell components, etc.) able to maintain their stability under space and Mars-like conditions.
Here, we study by Raman spectroscopy the lichen Squamarina lentigera in dormant and active vital situations, to check which biomolecules are detected without molecular extraction or lichen manipulation. Afterwards, we checked, for the first time on living lichen, the impact of heating on the biomolecules under three experimental situations:
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a. Increments of laser power. Miniaturized Raman spectrometers are limited with regard to laser power control despite that high powers have a high impact in thermal biomolecular changes.
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b. Multi-analyses accumulation, collected over the same spot. Despite of the use of a low laser power, accumulations have also an impact in sample heating and then in biomolecular changes.
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c. Environmental heating by using a thermal chamber, assessing molecular changes and their reversibility, in an attempt to simulate temperatures that could be reached during planetary atmospheric entries.
With our studies we evaluate which compounds, such as bio-pigments or calcium oxalates, could be more suitable for future experiments related with Panspermia and space experiments studied by Raman spectroscopy.
Material and methods
Material
The lichen selected was S. lentigera which is one of the extremotolerant organisms that was taken into consideration for astrobiological studies (Jorge-Villar et al. Reference Jorge-Villar, Miralles, Capel-Ferrón and Hernández2011). This lichen is very frequent and abundant in Tabernas desert (Southeast Spain), colonizing sites exposed to high levels of solar radiation (Miralles et al. Reference Miralles, Jorge-Villar, Canton and Domingo2012a). The main geological materials in the Sorbas–Tabernas basin are calcaric–gypsiferous mudstone and calcareous sandstones. The climate is semi-arid, thermo-Mediterranean, characterized by low precipitation levels and high temperatures over long periods. The average annual temperature is 17.9°C, with an absolute maximum of 45°C, absolute minimum of −4.5°C and the average annual rainfall is 235 mm, concentrated in winter and autumn. The magnitude (less than 10% over 20 mm) and intensity are generally low; intense storms are relatively infrequent. The summer months, particularly July and August, are always extremely dry. The high annual potential evapotranspiration (around 1500 mm) indicates an important water deficit in the area (Miralles et al. Reference Miralles, van Wesemael, Cantón, Chamizo, Ortega, Domingo and Almendros2012b).
Methodology
Raman analyses were carried out in the laboratory of Vibrational Spectroscopy, at the SCAI (Central Services Research Support) of the University of Málaga (Spain), using a Bruker RamanScope FT-Raman module attached to a Bruker Senterra. This system is equipped with a Neon lamp, a confocal microscope with a ×40 objective, and a Nd:YAG laser at 1064 nm wavelength using a back-scattering configuration.
Samples were analysed under different experimental situations:
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a. We performed a Raman study of dry and wet lichens at room temperature. The goal of this experiment is to know, S. lentigera biosignatures detected by Raman spectroscopy during active and dormant vital situations.
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b. We studied, at room temperature, the heating effect on lichen biomolecules owing to (1) high laser power level and (2) number of accumulations over dry lichens and their effect on spectral signatures for discrimination from environmental temperature increases in further experiments. The power used for each experiment were 100 and 800 mW and samples were analysed by averaging 1500 scans with 4 cm−1 spectral resolution. The focused laser spot size was about 5 µm.
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c. Raman spectroscopy has been used to study the behaviour of S. lentigera biomolecules before, during and after environmental thermal annealing. Dry lichen sample was fixed to a sample holder of a variable-temperature Linkam accessory to recorded Raman scattering spectra in a temperature range from 25 to 175°C and back to room temperature at intervals of 25°C. The operating power was 100 mW in all temperatures, by averaging, 1500 scans were accumulated at 4 cm−1 spectral resolution. All analyses were performed on the same spot, to avoid molecular detection changes owing to the heterogeneity of lichen surface.
Results and discussion
Raman spectroscopy study of biomolecules on wet and dry lichen
Lichens are not homogeneous and thus pigments and other compounds show accumulation areas on the surface and also in a transversal section. We have firstly analysed several spots on the surface of the lichen S. lentigera for biomolecular characterization. A carotenoid with bands at 1521, 1153 and 1000 cm−1, usnic acid, with bands at 1695, 1629, 1605, 1372, 1323, 1288, 1192, 1119, 991, 958, 871, 844, 602 and 541 cm−1, chlorophyll (1323, 742 cm−1), calcium oxalate monohydrate (whewellite) with bands at 1629, 1495, 1468, 895 and 504 cm−1 and calcium oxalate dihydrate (weddellite) at 1629, 1475, 911 and 508 cm−1 have been identified. The relative intensities of the Raman bands from different compounds change in each spectrum as the proportion of the compound change in each spot analysed. All compounds were identified either in inactive (dry) or active (wet) specimens. In Fig. 1, we show two of the most representative spectra collected over wet and dry S. lentigera and in Fig. 2, we mark the main identifying Raman bands of the previously described lichen compounds.
Fig. 1. Raman spectra of Squamarina lentigera dry and wet and their main wavenumber bands. We also show lichen photos when lichen is dry and its visual changes when it is wet.
Fig. 2. Raman spectra showing the identificative bands of all compounds characterized by Raman spectroscopy on lichen Squamarina lentigera.
Other experiments with a miniaturized handheld Deltanu Raman spectrometer showed no Raman signatures on dried lichen spectra whereas in Raman spectra of moist lichens bands were clearly distinguishable (Jorge-Villar et al. Reference Jorge-Villar, Miralles, Capel-Ferrón and Hernández2011; Miralles et al. Reference Miralles, Jorge-Villar, Canton and Domingo2012a). The authors associated these results to technical limitations of the miniaturized handheld spectrometer, which restricts its capacity to detect weak signals on dry lichens. Although the lack of recognizable signatures detected by this specific spectrometer could be also attributed to a physical status of desiccated cells and molecules, presumably as a photoprotective mechanism that were under the detection limit of the instrument (Miralles et al. Reference Miralles, Jorge-Villar, Canton and Domingo2012a).
Raman spectroscopy upon 100 and 800 mW laser power experiments
No changes were noticed in pigment identification when using the 1064 nm laser excitation at low and high laser power, meaning that no biomolecular transformations were induced by laser heating at such powers.
Weddellite is described as an unstable variety of calcium oxalate dihydrate that loses one water molecule at temperatures above 5°C, changing to whewellite (Edwards Reference Edwards2007). We have identified both oxalates using a laser power of either 100 or 800 mW. In the spectrum achieved using 800 mW laser power, calcium oxalate monohydrate looks to appear in higher proportions than in the spectrum collected at 100 mW. Unfortunately, it is not possible to assess whether we have chosen, by chance, spots with a higher whewellite concentration or if it is due to a laser heating process, since we know that lichens are not homogeneous.
For evaluating this problem, we measured four times on the same spot at 100 mW (Fig. 3). This has an effect of laser heating accumulation because of the exposure time, and could also induce changes in the hydrated calcium oxalate rates. The 1475 cm−1 band, assigned to weddellite (the dihydrate phase), is the strongest, whereas whewellite Raman bands (1495 cm−1, seen as a shoulder at the stronger band of weddellite, and 1468 cm−1), from the monohydrate calcium oxalate, appear weaker (Fig. 3). This proportion persists in all four spectra, meaning that the ratio between both oxalates remains stable and the laser does not induce any change.
Fig. 3. Raman spectra collected using 100 mW laser power, focalizing over the same spot. No changes in compounds or oxalates are visible.
However, the relative band intensities of both calcium oxalates change even during the second analysis when using 800 mW laser power (Fig. 4). In the spectrum (a) of Fig. 4, is the monohydrate oxalate (whewellite) which appears in higher proportion but this proportion starts to change at the second measurement (spectrum b, c, d and e): then the whewellite/ weddellite ratio decreases and the characteristic whewellite Raman band at 1495 cm−1 becomes a shoulder in the main weddellite signature at 1475 cm−1. Calcium oxalate dihydrate is unstable and changes to whewellite; one of the reasons for that transformation is heating but in this experiment it happens the opposite: is the monohydrate phase which becomes the dihydrate variety. Figure 4 shows the spectra with both calcium oxalates and the changes in relative intensities of the signatures, particularly the increasing of 1475 and 910 cm−1 signatures, characteristic of the dihydrate variety.
Fig. 4. Raman spectra collected using 800 mW laser power over the same spot. It is clear how relative oxalate band intensities change.
Raman spectroscopy study of lichen biomolecules under variable environmental temperatures
We have performed all thermal analyses on the same lichen spot, by the use of Raman spectroscopy in combination with a variable-temperature Linkam instrument. The experimental chamber temperature rose from room temperature to 175°C, at 25°C intervals for, finally, decreasing to room temperature again. Since the temperature increase is not due to direct heating on the lichen but a global temperature increase in the chamber, it is more similar to environmental changes.
Only usnic acid, carotene and calcium oxalate monohydrate are identified in the first spectrum, collected at room temperature (Fig. 5). There is no clear evidence of chlorophyll in this spectrum since the band at 1323 cm−1 can also be assigned to usnic acid and there are no other chlorophyll signatures (Fig. 5). As the relative intensity among usnic acid significant bands, including 1323 cm−1, agrees with the literature, we conclude that chlorophyll has not been detected in this spot.
Fig. 5. Thermal annealing of Squamarina lentigera from room T (25°C) to 175°C. Spectra were collected over the same spot, using a laser power of 100 mW. It is clear that the environmental changes in T induce changes in the lichen Raman spectra. Some of them are reversible but not all.
The following analyses clearly show changes in the Raman spectra from 25 to 175°C. Such a suite of spectra are shown in Fig. 5. There are no noticeable changes until 75–100°C, where the relative intensity of carotenoid decreases with regard to oxalates, until the total carotenoid signal disappears at 150°C. When the chamber recovers room temperature, carotene Raman bands do not appear again, which means that this compound has degraded irreversibly.
Some changes happen to usnic acid; for example, at 100°C the Raman band at 1605 cm−1 becomes weaker and is absent at 175°C; at this temperature, the signature at 1323 cm−1 disappears too. Although this compound can be identified until 150°C degrees, its unambiguous characterization is not possible when the sample reaches 175°C. Surprisingly, the characteristic usnic acid Raman bands appear again after recovering room temperature, allowing again the pigment identification.
Oxalate phases change from calcium oxalate monohydrate to a mixture of mono and dihydrate phases, clearly visible at 125°C. At 150 and 175°C, the strongest bands are those of dihydrated oxalate but when temperature drops to room temperature, oxalate dihydrate signatures disappear and again only the monohydrate oxalate variety can be identified. We suggest that this transformation could be ascribed to a complex survival strategy of the lichen related with its necessity of more water storage, responding to the environmental heating more than an experimental molecular thermal transformation. In fact, mixture of weddelite and whewellite in temperate locations has been already found (Holder et al. Reference Holder, Wynn-Williams, Rull-Perez and Edwards2000; Jorge-Villar et al. Reference Jorge-Villar, Edwards and Seaward2004; Miralles et al. Reference Miralles, Jorge-Villar, Canton and Domingo2012a) and were associated with a possible water-storage role (Jorge-Villar et al. Reference Jorge-Villar, Edwards and Seaward2004; Miralles et al. Reference Miralles, Jorge-Villar, Canton and Domingo2012a). Nevertheless, the production of oxalates is also associated with a lichen strategy for removal of the oxalic acid as waste from the metabolic cycle, which, in the presence of calcareous material (calcium carbonate content in soil under biological crusts from Tabernas desert is more than 242.7 g kg−1 Miralles et al. Reference Miralles, van Wesemael, Cantón, Chamizo, Ortega, Domingo and Almendros2012b), contributes to the formation of the insoluble hydrated calcium oxalate (Jorge-Villar et al. Reference Jorge-Villar, Edwards and Seaward2005b). Furthermore, it has also been suggested that the calcium oxalate increment could be related to a high light intensity levels which lead to high lichen metabolic rates. In this context, calcium oxalates could act to reduce light intensity by reflection (de Oliveira et al. Reference de Oliveira, Edwards, Feo-Manga, Seaward and Lücking2002). More experiments should be carried out in extremophile organisms by Raman spectroscopy subjected to sudden temperature changes to analyse the behaviour of biomolecules such as oxalates.
Carotenes (ultraviolet photo-protective molecules Cockell & Knowland Reference Cockell and Knowland1999) and chlorophylls (photosynthetic pigments) are biomolecules ubiquitous in lichens, which are considered terrestrial analogues of possible extra-terrestrial life (Jorge-Villar & Edwards Reference Jorge-Villar and Edwards2006). Usnic acid acts as a UV-radiation screening pigment whereas calcium oxalates (whewellite and weddellite) are associated with a significant antidesiccative water-storage role (Jorge-Villar & Edwards Reference Jorge-Villar and Edwards2010) or photoprotective role (de Oliveira et al. Reference de Oliveira, Edwards, Feo-Manga, Seaward and Lücking2002). All these compounds are common biomolecules in lichens and all of them are considered as in situ key biomarkers of extra-terrestrial life studied extensively by Raman spectroscopy (Wynn-Williams & Edwards Reference Wynn-Williams and Edwards2000; Edwards et al. Reference Edwards, Newton, Dickensheets and Wynn-Williams2003b, Reference Edwards, Cockell, Newton and Wynn-Williams2004b, Reference Edwards, Moody, Jorge-Villar and Wynn-Williams2005a; Jorge-Villar et al. Reference Jorge-Villar, Edwards and Seaward2005b; Miralles et al. Reference Miralles, Jorge-Villar, Canton and Domingo2012a). Nevertheless, in this experiment we have investigated which biomolecules in extremophile and extremotolerant organisms, such as the lichen S. lentigera, act as robust biomarkers in the context of Lithopanspermia theory and which of them are characterized by a very high stability to the space conditions (e.g. very fast switching between low and high temperatures) during the transfer of organisms from one planet to another. Consequently, at least under the conditions of our experiments, carotenes are poor biomarkers of life in samples transferred from space, since it degrades by degrades at temperatures above 150°C, being unable to recover when temperatures decrease to room temperature again.
Conclusions
We studied the effect of temperature on the identification of biomolecules produced by the lichen S. lentigera by Raman spectroscopy. Several important conclusions can be derived from the Raman spectroscopic analyses reported in this paper:
Only carotene, chlorophyll, calcium oxalate monohydrate and dihydrate as well as usnic acid were the biomolecules identified by Raman spectroscopy on S. lentigera without sample manipulation, either on dry and wet samples.
The effect of the 1064 nm laser power in one single analysis, collecting 1000 scans, is not relevant when 100 or 800 mW intensity is used; however, repetitive analyses on the same spot shows that 800 mW produces clear spectral changes at the third analysis, specifically, this change is related with the degree of hydration in calcium oxalate.
From the environmental thermal experiment, we can conclude that some biomolecules change when temperature rises above 100°C (e.g. carotene and usnic acid). For molecules with a complex Raman spectrum, such as usnic acid, the unambiguos identification can be made even in the spectrum collected at 150°C, since most of the significant bands appear. Nonetheless, for compounds such as carotene, which shows a simpler Raman spectrum with only two characteristic signatures, the identification is not possible when the strongest band dilutes among other signatures owing to thermal degradation.
A relevant conclusion is that after molecular transformation due to heating, evident because of Raman spectral changes, visible firstly because of changes in intensity and later because of the disappearance of Raman bands, some compounds recover and are, clearly, identified again (e.g. usnic acid) , whereas others, such as carotene, degrade and cannot be identified again.
As a result, we also conclude that for proving Panspermia Theory and particularly for demonstrating that biomolecules can withstand atmosphere entry, scientists should not focus their biomarker investigations on the most common biomolecules, such as carotenoid, but on those whose structure, recovers, even if it changes. Further experiments should be carried out to figure what kind of biomolecules recover after heating.
Changes in oxalate hydratation are reversible when temperature rises from room level to 175°C and again droops to room temperature. Although mineral weddellite dehydrates to whewellite at room temperature, in our thermal experiment we have seen that the calcium oxalate monohydrated (whewellite) changes to weddellite (calcium oxalate dihydrate) when temperature rises and becomes again whewellite at room temperature.
Another significant conclusion from the present study is that lichen biomolecules are detected before, during and after heating by using Raman spectroscopy, which is relevant for life signal identification on extraplanetary exploration, since this technique has been chosen for being included as a part of the instrumental suite for the future Mars missions.
Acknowledgements
The authors are grateful for support from the Juan de la Cierva Fellowship 2008-39669 and the Marie Curie Intra-European Fellowship (FP7-PEOPLE-2013-IEF, Proposal no. 623393). The authors are also grateful for support from CARBORAD project (CGL2011-27493) funded by the Spanish Ministry of Science and Innovation and the project ‘Variaciones de pigmentos y otros metabolitos causadas por el microclima en especies clave de costras biológicas del suelo’ funded by the Asociación de Ecología Terrestre Española (AEET).
