J.-P. de Vera

Resistance of Antarctic black fungi and cryptoendolithic communities to simulated space and Martian conditions.

Dried colonies of the Antarctic rock-inhabiting meristematic fungi Cryomyces antarcticus CCFEE 515, CCFEE 534 and C. minteri CCFEE 5187, as well as fragments of rocks colonized by the Antarctic cryptoendolithic community, were exposed to a set of ground-based experiment verification tests (EVTs) at the German Aerospace Center (DLR, Köln, Germany). These were carried out to test the tolerance of these organisms in view of their possible exposure to space conditions outside of the International Space Station (ISS). Tests included single or combined simulated space and Martian conditions. Responses were analysed both by cultural and microscopic methods. Thereby, colony formation capacities were measured and the cellular viability was assessed using live/dead dyes FUN 1 and SYTOX Green. The results clearly suggest a general good resistance of all the samples investigated. C. minteri CCFEE 5187, C. antarcticus CCFEE 515 and colonized rocks were selected as suitable candidates to withstand space flight and long-term permanence in space on the ISS in the framework of the LIchens and Fungi Experiments (LIFE programme, European Space Agency).

The potential of the lichen symbiosis to cope with extreme conditions of outer space – I. Influence of UV radiation and space vacuum on the vitality of lichen symbiosis and germination capacity

The lichen symbiotic organisms Fulgensia bracteata and Xanthoria elegans as well as their isolated photobionts and mycobionts were exposed to conditions simulating the extreme parameters of outer space in order to assess their limits of survival, e.g. during a potential interplanetary transfer or on the surface of an extraterrestrial body. Using the space simulation facilities at DLR, the test parameters under investigation were vacuum (10 −3 Pa) and ultraviolet (UV) radiation at wavelength ranges from vacuum-UV to UV-A, applied separately or in combination. An analysis of vitality was made using confocal laser scanning microscopy and LIVE/DEAD staining using FUN I or SYTOX green. Intact lichens were extremely resistant to vacuum exposure as well as to UV radiation up to doses of about 160 kJ m −2 (200 −2 reduced the vitality rate to nearly 50%; however, the dose effect curves levelled off and continued treatment did not reduce the vitality further. Similar survival curves levelling off at higher UV doses were observed for the isolated photobionts; however, in the latter cases, the saturation occurred at five times lower doses (full UV spectrum). Also spores of the mycobionts showed a remarkable UV (254 nm) resistance up to doses of about 3 kJ m −2 . The data suggest that the symbiotic features peculiar to lichens allow them to cope with the extreme conditions of outer space or even with Martian surface conditions provided suitable niche habitats are available to serve as refuges and complementary endogenous or exogenous protection mechanisms are established.

Life at the Limits: Capacities of Isolated and Cultured Lichen Symbionts to Resist Extreme Environmental Stresses

Lichens are described as a symbiosis formed by a myco- and photobiont, capable of colonizing habitats where their separate symbionts would not be able to survive. Space simulation studies on the separated symbionts of the lichen Xanthoria elegans have been performed to test their capacity to resist the most extreme conditions. The isolated cultured symbiont cells were exposed to different doses of the UV spectrum, and to vacuum. Cultures of both symbionts were analysed by specific vitality tests (LIVE/DEAD-staining detected by Confocal Laser Scanning Microscopy). Growth capacity of symbiont cultures on different media was analysed after exposure to extreme environmental stresses. The data obtained support the hypothesis that the symbiotic state considerably enhances the ability of the respective symbionts to survive exposure to extreme conditions, including the conditions of space simulation. Species such as X. elegans may, therefore, be suitable for use as model organisms in exobiological studies.

Extremotolerance and Resistance of Lichens: Comparative Studies on Five Species Used in Astrobiological Research I. Morphological and Anatomical Characteristics

Lichens are symbioses of two organisms, a fungal mycobiont and a photoautotrophic photobiont. In nature, many lichens tolerate extreme environmental conditions and thus became valuable models in astrobiological research to fathom biological resistance towards non-terrestrial conditions; including space exposure, hypervelocity impact simulations as well as space and Martian parameter simulations. All studies demonstrated the high resistance towards non-terrestrial abiotic factors of selected extremotolerant lichens. Besides other adaptations, this study focuses on the morphological and anatomical traits by comparing five lichen species—Circinaria gyrosa, Rhizocarpon geographicum, Xanthoria elegans, Buellia frigida, Pleopsidium chlorophanum—used in present-day astrobiological research. Detailed investigation of thallus organization by microscopy methods allows to study the effect of morphology on lichen resistance and forms a basis for interpreting data of recent and future experiments. All investigated lichens reveal a common heteromerous thallus structure but diverging sets of morphological-anatomical traits, as intra-/extra-thalline mucilage matrices, cortices, algal arrangements, and hyphal strands. In B. frigida, R. geographicum, and X. elegans the combination of pigmented cortex, algal arrangement, and mucilage seems to enhance resistance, while subcortex and algal clustering seem to be crucial in C. gyrosa, as well as pigmented cortices and basal thallus protrusions in P. chlorophanum. Thus, generalizations on morphologically conferred resistance have to be avoided. Such differences might reflect the diverging evolutionary histories and are advantageous by adapting lichens to prevalent abiotic stressors. The peculiar lichen morphology demonstrates its remarkable stake in resisting extreme terrestrial conditions and may explain the high resistance of lichens found in astrobiological research.

Extremotolerance and Resistance of Lichens: Comparative Studies on Five Species Used in Astrobiological Research II. Secondary Lichen Compounds

Lichens, which are symbioses of a fungus and one or two photoautotrophs, frequently tolerate extreme environmental conditions. This makes them valuable model systems in astrobiological research to fathom the limits and limitations of eukaryotic symbioses. Various studies demonstrated the high resistance of selected extremotolerant lichens towards extreme, non-terrestrial abiotic factors including space exposure, hypervelocity impact simulations as well as space and Martian parameter simulations. This study focusses on the diverse set of secondary lichen compounds (SLCs) that act as photo- and UVR-protective substances. Five lichen species used in present-day astrobiological research were compared: Buellia frigida, Circinaria gyrosa, Rhizocarpon geographicum, Xanthoria elegans, and Pleopsidium chlorophanum. Detailed investigation of secondary substances including photosynthetic pigments was performed for whole lichen thalli but also for axenically cultivated mycobionts and photobionts by methods of UV/VIS-spectrophotometry and two types of high performance liquid chromatography (HPLC). Additionally, a set of chemical tests is presented to confirm the formation of melanic compounds in lichen and mycobiont samples. All investigated lichens reveal various sets of SLCs, except C. gyrosa where only melanin was putatively identified. Such studies will help to assess the contribution of SLCs on lichen extremotolerance, to understand the adaptation of lichens to prevalent abiotic stressors of the respective habitat, and to form a basis for interpreting recent and future astrobiological experiments. As most of the identified SLCs demonstrated a high capacity in absorbing UVR, they may also explain the high resistance of lichens towards non-terrestrial UVR.

Resistance of the Lichen Buellia frigida to Simulated Space Conditions during the Preflight Tests for BIOMEX—Viability Assay and Morphological Stability

Samples of the extremotolerant Antarctic endemite lichen Buellia frigida are currently exposed to low-Earth orbit-space and simulated Mars conditions at the Biology and Mars Experiment (BIOMEX), which is part of the ESA mission EXPOSE-R2 on the International Space Station and was launched on 23 July 2014. In preparation for the mission, several preflight tests (Experimental and Scientific Verification Tests, EVT and SVT) assessed the sample preparation and hardware integration procedures as well as the resistance of the candidate organism toward the abiotic stressors experienced under space and Mars conditions. Therefore, we quantified the post-exposure viability with a live/dead staining technique utilizing FUN-1 and confocal laser scanning microscopy (CLSM). In addition, we used scanning electron microscopy (SEM) to investigate putative patterns of morphological-anatomical damage that lichens may suffer under the extreme exposure conditions. The present results demonstrate that Buellia frigida is capable of surviving the conditions tested in EVT and SVT. The mycobiont showed lower average impairment of its viability than the photobiont (viability rates of >83% and >69%, respectively), and the lichen thallus suffered no significant damage in terms of thalline integrity and symbiotic contact. These results will become essential to substantiate and validate the results prospectively obtained from the returning space mission. Moreover, they will help assess the limits and limitations of terrestrial organisms under space and Mars conditions as well as characterize the adaptive traits that confer lichen extremotolerance.

Raman spectroscopic analysis of the calcium oxalate producing extremotolerant lichen Circinaria gyrosa

In the context of astrobiological exposure and simulation experiments in the BIOMEX project, the lichen Circinaria gyrosa was investigated by Raman microspectroscopy. Owing to the symbiotic nature of lichens and their remarkable extremotolerance, C. gyrosa represents a valid model organism in recent and current astrobiological research. Biogenic compounds of C. gyrosa were studied that may serve as biomarkers in Raman assisted remote sensing missions, e.g. ExoMars. The surface as well as different internal layers of C. gyrosa have been characterized and data on the detectability and distribution of β-carotene, chitin and calcium oxalate monohydrate (whewellite) are presented in this study. Raman microspectroscopy was applied on natural samples and thin sections. Although calcium oxalates can also be formed by rare geological processes it may serve as a suitable biomarker for astrobiological investigations. In the model organism C. gyrosa, it forms extracellular crystalline deposits embedded in the intra-medullary space and its function is assumed to balance water uptake and gas exchange during the rare, moist to wet environmental periods that are physiologically favourable. This is a factor that was repeatedly demonstrated to be essential for extremotolerant lichens and other organisms. Depending on the decomposition processes of whewellite under extraterrestrial environmental conditions, it may not only serve as a biomarker of recent life, but also of past and fossilized organisms.

Results on the survival of cryptobiotic cyanobacteria samples after exposure to Mars-like environmental conditions

Tests on cyanobacteria communities embedded in cryptobiotic crusts collected in hot and cold deserts on Earth were performed under Mars-like conditions. The simulations were realized as a survey, to find the best samples for future research. During the tests organisms have to resist Mars-like conditions such as atmospheric composition, pressure, variable humidity (saturated and dry conditions) and partly strong UV irradiation. Organisms were tested within their original habitat inside the crust. Nearly half of the cryptobiotic samples from various sites showed survival of a substantial part of their coexisting organisms. The survival in general depended more on the nature of the original habitat and type of the sample than on the different conditions they were exposed to. The best survival was observed in samples from United Arab Emirates (Jebel Ali, 25 km SW of Dubai town) and from Western Australia (near the South edge of Lake Barley), by taxa: Tolypothrix byssoidea, Gloeocapsopsis pleurocapsoides, Nostoc microscopicum, Leptolyngbya or Symploca sp. At both places in salty desert areas members of the Chenopodiaceae family dominated among the higher plants and in the cryptobiotic crust cyanobacterial taxa Tolypothrix was dominant. These organisms were all living in salty locations with dry conditions most of the year. Among them Tolypothrix, Gloeocapsopsis and Symploca sp. were tested in Mars simulation chambers for the first time. The results suggest that extremophiles should be tested with taken into account the context of their original microenvironment, and also the importance to analyse communities of microbes beside single organisms.

Desiccation and low temperature attenuate the effect of UVC254 nm in the photobiont of the astrobiologically relevant lichens Circinaria gyrosa and Buellia frigida

Several investigations on lichen photobionts (PBs) after exposure to simulated or real-space parameters consistently reported high viability and recovery of photosynthetic activity. These studies focused on PBs within lichen thalli, mostly exposed in a metabolically inactive state. In contrast, a recent study exposed isolated and metabolically active PBs to the non-terrestrial stressor UVC254 nm and found strong impairment of photosynthetic activity and photo-protective mechanisms (Meesen et al. in 2014b). Under space and Mars conditions, UVC is accompanied by other stressors as extreme desiccation and low temperatures. The present study exposed the PBs of Buellia frigida and Circinaria gyrosa, to UVC in combination with desiccation and subzero temperatures to gain better insight into the combined stressors’ effect and the PBs’ inherent potential of resistance. These effects were examined by chlorophyll a fluorescence which is a good indicator of photosynthetic activity (Luttge& Budel in 2010) and widely used to test the viability of PBs after (simulated) space exposure. The present results reveal fast recovery of photosynthetic activity after desiccation and subzero temperatures. Moreover, they demonstrate that desiccation and cold confer an additional protective effect on the investigated PBs and attenuate the PBs’ reaction to another stressor – even if it is a non-terrestrial one such as UVC. Besides other protective mechanisms (anhydrobiosis, morphological–anatomical traits and secondary lichen compounds), these findings may help to explain the high resistance of lichens observed in astrobiological studies.

The First Space-Related Study of a Kombucha Multimicrobial Cellulose-Forming Community: Preparatory Laboratory Experiments

Biofilm-forming microbial communities are known as the most robust assemblages that can survive in harsh environments. Biofilm-associated microorganisms display greatly increased resistance to physical and chemical adverse conditions, and they are expected to be the first form of life on Earth or anywhere else. Biological molecules synthesized by biofilm -protected microbiomes may serve as markers of the nucleoprotein life. We offer a new experimental model, a kombucha multimicrobial culture (KMC), to assess a structural integrity of a widespread microbial polymer – cellulose – as a biosignature of bacteria-producers for the multipurpose international project “BIOlogical and Mars Experiment (BIOMEX)”, which aims to study the vitality of pro- and eukaryotic organisms and the stability of organic biomolecules in contact with minerals to analyze the detectability of life markers in the context of a planetary background. In this study, we aimed to substantiate the detectability of mineralized cellulose with spectroscopy and to study the KMC macrocolony phenotype stability under adverse conditions (UV, excess of inorganics etc.). Cellulose matrix of the KMC macrocolony has been mineralized in the mineral-water interface under assistance of KMC-members. Effect of bioleached ions on the cellulose matrix has been visible, and the FT-IR spectrum proved changes in cellulose structure. However, the specific cellulose band vibration, confirming the presence of β(1,4)-linkages between monomers, has not been quenched by secondary minerals formed on the surface of pellicle. The cellulose-based KMC macrocolony phenotype was in a dependence on extracellular matrix components (ionome, viriome, extracellular membrane vesicles), which provided its integrity and rigidness in a certain extent under impact of stressful factors.