Introduction

Since the beginning of the new millennium, various astrobiological experiments were conducted to test the effects of space and Mars-analogue conditions on lichens, mosses, fungi and prokaryotes. These simulation experiments tested the single or combined effects of vacuum up to 10−5 Pa and UV-radiation (de Vera et al. 2003, 2004a, 2004b, 2008, 2010; de La Vega et al. 2007; de Vera & Ott 2010; Sánchez et al. 2012, 2014; Baqué et al. 2013; Meeßen et al. 2014), hypervelocity impact pressures up to 50 GPa (Stöffler et al. 2007; Horneck et al. 2008), but also tested the resistance to other space-related parameters as freezing and drought (Onofri et al. 2004; Hájek et al. 2012; Meeßen et al., 2014). Additionally, the lichen Xanthoria elegans was part of the space exposure experiments LICHENS II on BIOPAN 5, LITHOPANSPERMIA on BIOPAN 6 (Sancho et al. 2007; de la Torre et al. 2007, 2010; Olsson-Francis et al., 2010; Raggio et al. 2011) and consequently became part of the Lichen and Fungi Experiment (LIFE) on EXPOSE-E/EuTEF at the ISS where it was exposed among other organisms to space and simulated Mars conditions for 18 months (Cockell et al. 2011; Onofri et al. 2012; Rabbow et al. 2012; Scalzi et al. 2012; Brandt et al. 2015). The EXPOSE-E facility provided LEO-space vacuum (10−4 to 10−7 Pa) and simulated Mars conditions (103 Pa Mars-analogue atmosphere) under three different insolation regimes of 100 % transmission, 0.1 % transmission (T) and no insolation as described below, leading to accumulated insolation doses up to 4.87 GJm−2 and UV irradiation doses of up to 314 MJm−2 at the sample sites of X. elegans.

The astrobiological model lichen X. elegans is a symbiotic association of an ascomycete fungus (mycobiont) and a coccal green alga (photobiont, genus Trebouxia). This cosmopolite lichen is well adapted to harsh environmental conditions as found in high alpine and polar regions (Øvstedal & Lewis Smith 2001). It is characterized by the secondary lichen compound parietin known for its high light- and UV-screening capacities (Solhaug & Gauslaa 1996; Solhaug et al. 2003, 2010). To assess its limits of survival after LIFE, X.elegans was tested on its resistance to the applied conditions and the exposure-induced decrease of post-exposure viability by chlorophyll a fluorescence and live/dead staining analyses (Onofri et al. 2012; Brandt et al. 2015). Both methods revealed high rates of metabolic and photosynthetic activity. The live/dead staining results of the photobiont revealed viability rates of 50–80 %, while the more resistant mycobiont showed rates of 60–90 %. The photosynthetic re-activation of the photobiont depended on the exposure conditions. Vacuum exposed and fully insolated (100 % transmission) samples took 8 days of re-activation to reach c. 45 % of its pre-flight maximum quantum yield of photosystem II while the Mars-analogue samples of all insolation conditions returned to pre-flight values within 4 days (Onofri et al. 2012, Brandt et al. 2015).

The present study conducted a qualitative cultivation assay of the isolated photobiont to corroborate the 50–80 % of remaining metabolic activity and the photosynthetic re-activation of X. elegans as indicators of post-flight viability. This assay confirmed the viability of photobiont cells/clusters after 1.5 years of space exposure by growth and proliferation, i.e.by its ability to perform complete cell cycles. The present study demonstrates a capacity of survival beyond the metabolic and photosynthetic activities reported before (Onofri et al. 2012, Brandt et al. 2015). Complementary, the analysis by transmission electron microscopy (TEM) also investigated the impact of extreme desiccation on photobiont ultrastructure as induced by 1.5 year exposure to space vacuum and Mars-analogue atmosphere conditions. This part of the study aimed to characterise the impairment on the cellular level by vacuum and low atmospheric pressure that may explain the loss of 20–50 % of algal viability observed in previous studies (Brandt et al. 2015). Both parts of the present study focused on the isolated photobiont, as it is described to be the more sensitive symbiotic partner towards simulated space and Martian conditions (de Vera et al. 2003, 2008; de Vera & Ott 2010).

Material and Methods

Lichen Samples

Samples of Xanthoria elegans (Link) Th. Fr. (1860) were collected in June 2008 at Col du Sanetsch, Valais, Switzerland (46°21′48″N, 07°17′51″E, at 2140 m a.s.l.) and adjacent collection sites (Zermatt, 46°00′N, 07°71′E, at 1950 m a.s.l.). Samples originating from this collection sites were used in astrobiological studies before (de Vera et al. 2003, 2004a, 2004b, 2008, 2010) and consistently showed high viability after these studies.

Exposure Conditions

The samples were exposed in the EXPOSE-E facility outside the European Columbus Module at the ISS for 559 days. Six experimental conditions were realised: Eight samples each were exposed to space (tray 1) and Mars-analogue conditions (tray 2). Tray 1 provided 10−4 to 10−7 Pa LEO-vacuum and three intensities of solar radiation (dark, 0.1 % transmission by neutral density filters, and 100 % transmission (T)) from 110 nm < λ > 1 mm (covered with MgF2 windows) while tray 2 provided simulated Mars conditions with a 103 Pa atmosphere (95.3 % CO2, 2.7 % N2, 1.6 % Ar, 0.15 % O2, ~370 ppm water vapour) and again three intensities of solar irradiation (as above) from 200 nm < λ > 1 mm (covered by Suprasil quartz windows). At the sample sites, fully insolated samples accumulated an average of 4800 MJm−2 photon energy input; 0.1 % transmission samples accumulated 6.3 MJm−2 photon energy in tray 1 and 5.9 MJm−2 in tray 2. The portions of UV-irradiation ranged from 291 MJm−2 at 100 % T to 0.271 MJm−2 at 0.1 % T under space conditions (110 nm < λ >400 mm) and from 314 MJm−2 at 100 % T to 0.257 MJm−2 at 0.1 % T under Mars conditions (200 nm < λ >400 mm). The samples experienced ionizing radiation of 208 ± 8 mGy in tray 1, and 215 ± 16 mGy in tray 2 (Berger et al. 2012) and a temperature range from −21.7 to +43 °C with a short-time maximum of 61 °C and 100 freeze/thaw cycles (Rabbow et al. 2012). According to the data received from the EXPOSE-E facility, a corresponding set of samples was exposed to as similar as possible simulated conditions, referred to as Mission Ground Reference (MGR; Rabbow et al. 2012). Due to off-nominal events during the mission about 20 % of the expected environmental data were lost. To assure precise sample analysis of the exposure conditions the solar irradiation was recalculated (RedShift Report 2011) leading to accumulated irradiance values differing from those given in Rabbow et al. (2012). During LIFE the lichen samples stayed in anhydrobiotic state during the whole experiment. After return all samples were stored frozen (−20 °C) until further investigations.

Culture Assay

The symbiotic algae were prepared from the lichen sample under sterile conditions, using a hollow needle and a scalpel to place algal clusters immediately on Trebouxia Organic Medium agar-plates (Ahmadjian 1967) and BG11 medium. Amphotericin-B (5 μg/ml) and Ampicillin (200 μg/ml) were added to both media to prevent fungal and bacterial contamination. 10–15 colonies per agar-plate and per media were inoculated for each experimental condition, including untreated controls. The cultivation took place at 12 °C under 14 h daytime photosynthetic photon flux density of 15–25 μmolm−2s−1. Algal colonies were inspected daily to detect contaminations and were re-inoculated on sterile media in case of visible contamination. Growing colonies were photo-documented using a stereo microscope (Zeiss, Germany) with a digital camera (Pentax). After 200 days of cultivation the experiment was concluded. For subsequent studies the algae were transferred to glycerin stocks and stored at −20 °C.

Transmission Electron Microscopy (TEM)

Sample pieces of lichen thalli of all exposure conditions (c. 2 mm in diameter) were fixed in 2.5 % glutaraldehyde (in 200 mM cacodylate-buffer), contrasted in 4 % OsO4 (in 0.8 % potassium ferrocyanide), and dried by graduated dilution series of ethanol and acetone prior to critical point drying (CPD 020, Balzers Union). The fixed samples were embedded in Epon812 resin following the manufacturer’s protocol and ultrathin cross-sections (100 nm) were examined with a Zeiss E902 TEM. To test the effect of prolonged dry storage, two different controls were chosen: one from dry storage over silica-gel, the other was kept in the freezer at −20 °C. Cell counting was performed manually using the ImageJ counter tool (v1.49 m Wayne Rasband). Image processing of the micrographs was done with Fiji macros (Biovoxxel 2014). For statistical analyses (one-way ANOVA with post-test) GraphPad InStat3 was applied (GraphPad software 2003).

Explanation of Ultrastructural Terminology

Cell wall: rigid layer surrounding the protoplast of plant cells; protoplast: plant cell without its cell wall; thylakoid: compartment of the chloroplast where thylakoid membranes enclose the thylakoid lumen, site of light-dependent reaction of photosynthesis; pyrenoid: compartment of the chloroplast, spherical protein structure, site of CO2-fixation; pyrenoglobuli: osmiophilic, electron-dense globuli of uncertain function, most likely sites of lipid storage; grana: thylakoid membrane stacks in the chloroplast.

Results

Culture Assay

For the first time, it was possible to cultivate lichen photobionts after long-term space exposure. Proliferating algal cultures were achieved from all three insolation regimes investigated in the LIFE experiment and under both, space vacuum and Mars-analogue conditions. The algal colonies from different exposure conditions revealed qualitative differences in proliferation rate and final colony size (Figs. 1 and 2) while the control group samples showed the fastest growth. They formed larger colonies in equivalent periods of time when compared to all experimental conditions tested. By comparing the ratio of initially inoculated algal clusters to grown colonies, the results indicate that exposure to Mars-analogue conditions had less effects on the proliferation abilities when compared to the algal colonies derived from space vacuum exposure conditions (Table 1). Colonies from Mars-analogue exposure conditions grew faster and became larger in size in the respective time. Despite varying by three orders of magnitude, the different insolation conditions (no insolation, 0.1 % T, and 100 % T) had no visible effect on the samples exposed to Mars-analogue conditions (Fig. 2). Compared to Mars-analogue conditions, the vacuum exposed samples showed an overall reduction in proliferation and colony-forming ratio. In general, the viability of non-insolated samples was not increased compared to irradiated ones. In line with previous investigations on the metabolic activity (Brandt et al. 2015), the present results indicate that insolation, especially UV-irradiation, seems not to be the crucial factor affecting post-exposure viability under the experimental conditions tested. Among the cultivated photobionts one mycelium arose putatively from co-isolated hyphae and was transferred to malt-yeast media (Fig. 2) for further cultivation.

Fig. 1
figure 1

Photobiont cultivation assay of samples exposed to space vacuum. Algal colonies after space vacuum exposure, cultivation duration c. 200 days. a 100 % T. b 0.1 % T. c no insolation. d 0.1 % T, picture shows one mycobiont colony of X. elegans. Scale bars indicate 1 mm

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Fig. 2
figure 2

Photobiont cultivation assay of samples exposed to Mars-analogue conditions. Algal colonies after Mars-analogue exposure, cultivation duration ca. 200 days: a 100 % T. b 0.1 % T. c no insolation. d Control (stored at −20 °C). Scale bars indicate 1 mm

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Table 1 Percentage of grown algal clusters after various experimental conditions
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Ultrastructural Analyses by TEM

The current analysis focused on the ultrastructural impairment of the photobiont of Xanthoria elegans to investigate damaging to lethal effects experienced during LIFE. Detailed analysis of the micrographs revealed distinct ultrastructural features of algal degeneration. Collapses of the cell or of the protoplast alone were frequently recognised and served as markers of exposure-induced damages. Under all investigated exposure conditions, a portion of photobiont cells showed partial or complete collapse of the protoplast with or without cell wall deformation (Fig. 3A-C). The frequency of cellular collapses differed between dry-stored and frozen control group. Dry-stored control samples showed a significantly higher amount of protoplast (70 %) and cell wall collapses (49 %) compared to frozen ones (44 % and 33 %, respectively) indicating a more deleterious effect of long-term desiccation than freezing (Table 2). The intensity of damage also differed between samples exposed to Mars-analogue atmosphere and space vacuum but none of the irradiation conditions correlated to the percentage of cell wall and protoplast collapses. Exposure to Mars-analogue atmosphere of 103 Pa caused comparable numbers of protoplast collapses (57 %, 48 %, 51 % at non-irradiated, 0.1 % T and 100 % T, respectively). Exposure to vacuum (10−4 to 10−7 Pa) showed higher numbers of collapsed protoplasts (61 %, 97 %, 85 %) even when compared to the dry control samples. Except for the non-irradiated samples, deformed cell walls occurred in space vacuum samples in higher percentage when compared to Mars-analogue conditions (25 %, 79 %, 85 % compared to 27 %, 16 %, 35 %, Table 2).The MGR data confirmed these results. The MGR simulation of Mars-analogue conditions caused 40–42 % of collapsed protoplasts while the MGR simulation of space vacuum led to 52–86 % of collapsed protoplasts.

Fig. 3
figure 3

Photobiont cells: normal appearance, partial and complete collapses. a Frozen control: regular appearance of Trebouxia photobiont cell. b Space vacuum, no insolation: partial and complete collapses, cell walls remain stable and unaffected. c Space vacuum, 0.1 % T: collapsed protoplasts with collapsed walls. Abbreviations: cw algal cell wall, c chloroplast, tm thylakoid membranes, h hyphe, m mucilage, dt degraded thylakoid, cs collapsed protoplast, p pyrenoid, ep enlarged pyrenoid, pg pyrenoglobuli. Scale bars indicate 5 μm

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Table 2 Percentage of ultrastructural damage markers after various experimental conditions
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Based on previous observations (Brown et al. 1987; Ascaso et al. 1988, 1995; Honegger 2003), changes in pyrenoid structure, localisation and appearance of pyrenoglobuli, degraded grana, and degraded thylakoid membranes were taken as additional markers of desiccation-induced cellular damage (Fig. 4 ).The degree of thylakoid membrane degradation and pyrenoid-enlargement was moderate in the frozen control and more pronounced in the dry control (Fig. 4 a). Even more pronounced thylakoid membrane degradation was observed in samples from space vacuum exposure and Mars-analogue exposure (Fig. 4 b-c). The pyrenoid-enlargement and appearance of pyrenoglobuli were observed to various extent but did not correlate to the experimental conditions applied (Fig. 4 d-e). Pyrenoglobuli were documented in all samples except in non- and 0.1 %-insolated Mars-analogue samples and appeared most frequently in the frozen control group (Fig. 4 f).

Fig. 4
figure 4

Ultrastructural markers of desiccation. a Dry control: pyrenoid enlargement, partial protoplast collapses. b Space vacuum: degradation of thylakoid membranes, pyrenoid enlargement and photobiont shrinking. c Mars-analogue exposure: thylakoid degradation and partial collaps of the protoplast. d Mars-analogue exposure, 100 % T: pyrenoid-enlargement, protoplast in regular shape. e Mars-analogue exposure, 0.1 % T: pyrenoid-enlargement, thylakoid membranes reduced and diffused. f Frozen control: pyrenoglobuli in pyrenoid. Abbreviations: cw algal cell wall, c chloroplast, tm thylakoid membranes, h hyphe, m mucilage, dt degraded thylakoid, cs collapsed protoplast, p pyrenoid, ep enlarged pyrenoid, pg pyrenoglobuli. Scale bars indicate 5 μm in A, D, and 0.25 μm in B, C, E, F

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In general, the ultrastructural markers of thylakoid membrane degradation, cell wall and protoplast collapse indicate that the extreme and long-term desiccation of space vacuum and - less effective - Mars atmospheric pressure was the major impairment factor of the exposure conditions of LIFE. However, any LIFE exposure condition enhanced the observed degradation patterns of photobiont cells when compared to the two control samples.

Discussion

Post-Exposure Viability

The present study verified the viability of the lichen photobiont of Xanthoria elegans after 18 months under the extreme conditions of LEO-space exposure by demonstrating the photobiont’s ability to grow and proliferate after isolation from the thallus. These results are consistent with the results of previous quantitative analyses of photosynthetic and metabolic activity by chlorophyll a fluorescence and live/dead staining, respectively (Onofri et al. 2012; Brandt et al. 2015). None of the conditions tested in LIFE caused complete inhibition of growth or proliferation. The present results demonstrate that the lichen’s viability was more preserved under Mars-analogue conditions compared to space vacuum. Insolation and/or UV-radiation were not the major parameters affecting the post-exposure viability of the photobiont. The results of chlorophyll a fluorescence showed lower rates of photosynthetic activity in the space vacuum samples than in the Mars-analogue ones (Brandt et al. 2015). The present cultivation assay of the photobiont confirmed this pattern, broadening it from mere metabolic activity to the actual ability of proliferation and growth. Besides X. elegans, other eukaryotic organisms were exposed to space or Mars-analogue conditions in the LIFE experiments on EXPOSE-E. All organisms revealed certain resistance to the hostile conditions (Horneck et al. 2012; Onofri et al. 2012), but the viability and proliferation ability of symbiotic X. elegans photobionts is not reached by other tested model organisms. In contrast to the photobiont of X. elegans, the cryptoendolithic fungi Cryomyces antarcticus and Cryptomyces minterii lost their colony-forming ability, though the former fungus showed 80 % of intact cells in a PCR-assay after exposure to full insolation (Onofri et al. 2012).

Protective Screening Mechanisms

The secondary lichen compound parietin, which is produced by the lichen fungus and deposited in the cortex of X. elegans is known for its blue light and UV screening abilities (Solhaug & Gauslaa 1996; Nybakken et al. 2004; Solhaug et al. 2010) efficiently protecting both symbionts. The amount of deposited parietin is stimulated by UV insolation in the natural habitat (Solhaug et al. 2003, Gauslaa & McEvoy 2005) mostly leading to a parietin-bearing cortical layer of 20–65 μm thickness on the cortex (Meeßen et al. 2013a). Thus, UVB, UVC and blue light are efficiently blocked by parietin in the upper cortex of the anhydrobiotic lichen (Gauslaa & Solhaug 2004; Solhaug et al. 2010, Meeßen et al. 2013b). This observation is stressed by the result that the different insolation regimes applied in LIFE did not correlate to the recognised differences of photobiont viability. As discussed below, we suggest another parameter of the exposure conditions to account in large part for the observed loss of photobiont viability.

Temporary Ultrastructural Changes in Anhydrobiosis

The key factor of lichens to survive extreme environmental conditions is anhydrobiosis (Crowe et al. 1992; Kranner et al. 2008). Both symbionts in lichens are adapted to poikilohydry resisting wet-dry cycles in their natural habitats including thallus water fluctuations between <20 and >160 % water per thallus dry weight (Kappen 1988). Anhydrobiosis is accompanied by visible alterations of the cellular ultrastructure (Honegger et al. 1996; Honegger 2003; de los Ríos et al. 2004, 2010). Photobiont cells are known to collapse temporarily upon dehydration under terrestrial conditions which appeared to be normal in the course of desiccation and rehydration (Brown et al. 1987; Honegger et al. 1996; de los Ríos et al. 2004). Lichen photobionts quickly recover their spherical shape when re-hydrated (e.g. during TEM-preparation, Honegger et al. 1996; Honegger 2003).

Permanent Ultrastructural Damages by Long-Term Desiccation

Permanent cellular collapses occur the more frequently the longer the dehydration period lasts and indicate desiccation-induced damage that may lead to death after six years of dry storage (Honegger 2003). In contrast, frozen storage (>9 years at −20 °C) of desiccated Trebouxia photobionts preserved their viability (Honegger et al. 1996; Honegger 2003). Collapsed photobiont cells as well as the observed degradation of thylakoid membranes were recognised as suitable markers for desiccation-induced impairment in the present study. Vacuum desiccation is considered the main process affecting biological samples when exposed to space vacuum (Horneck et al. 2010) while pressure conditions similar to those of Mars (e. g. at the upper boundary of Earth’s biosphere) create desiccation-related effects as well (Horneck et al. 2010). Consistently, TEM micrograph analysis revealed that both conditions, LEO-vacuum and the 103 Pa Mars-analogue atmosphere, caused an increased amount of collapsed protoplasts and cell walls, restricting the photobiont’s capacity of rehydration. Concerning the degradation of the thylakoid membranes and pyrenoid enlargement, all exposed samples showed a higher frequency of both damage markers which were unrelated to certain exposure conditions but more pronounced than in the control groups. These results stress the deleterious effect of vacuum-induced, long-time desiccation in the space exposed samples, while - due to the higher atmospheric pressure - the desiccation condition under Mars-analogue conditions were less extreme and therefore the samples were less impacted.