Investigation of fungal biomolecules after Low Earth Orbit exposure: a testbed for the next Moon missions.

The Moon is characterized by extremely harsh conditions due to ultraviolet irradiation, wide temperature extremes, vacuum resulting from the absence of an atmosphere and high ionizing radiation. Therefore, its surface may provide a unique platform to investigate the effects of such conditions. For lunar exploration with the Lunar Gateway platform, exposure experiments in Low Earth Orbit are useful testbeds to prepare for lunar space experiments and to understand how and if potential biomarkers are influenced by extra-terrestrial conditions. During the BIOMEX (BIOlogy and Mars EXperiment) project, dried colonies of the fungus Cryomyces antarcticus grown on Lunar Regolith Analogue (LRA) were exposed to space conditions for 16 months aboard the EXPOSE-R2 payload outside the International Space Station. In this study, we investigated the stability/degradation of fungal biomarkers in LRA after exposure to (i) simulated space and (ii) real space conditions, using Raman spectroscopy, gas chromatography-mass spectrometry and DNA amplification. The results demonstrated that fungal biomarkers were detectable after 16 months of real space exposure. This work will contribute to the interpretation of data from future biological experiments in the Cislunar orbit with the Lunar Gateway platform and/or on the lunar surface, in preparation for the next step of human exploration.

Antarctica as a reservoir of planetary analogue environments.

One of the main objectives of astrobiological research is the investigation of the habitability of other planetary bodies. Since space exploration missions are expensive and require long-term organization, the preliminary study of terrestrial environments is an essential step to prepare and support exploration missions. The Earth hosts a multitude of extreme environments whose characteristics resemble celestial bodies in our Solar System. In these environments, the physico-chemical properties partly match extraterrestrial environments and could clarify limits and adaptation mechanisms of life, the mineralogical or geochemical context, and support and interpret data sent back from planetary bodies. One of the best terrestrial analogues is Antarctica, whose conditions lie on the edge of habitability. It is characterized by a cold and dry climate (Onofri et al., Nova Hedwigia 68:175-182, 1999), low water availability, strong katabatic winds, salt concentration, desiccation, and high radiation. Thanks to the harsh conditions like those in other celestial bodies, Antarctica offers good terrestrial analogues for celestial body (Mars or icy moons; Léveillé, CR Palevol 8:637-648, https://doi.org/10.1016/j.crpv.2009.03.005 , 2009). The continent could be distinguished into several habitats, each with characteristics similar to those existing on other bodies. Here, we reported a description of each simulated parameter within the habitats, in relation to each of the simulated extraterrestrial environments.

Multidisciplinary characterization of melanin pigments from the black fungus Cryomyces antarcticus.

Melanin is a natural pigment present in almost all biological groups, and is composed of indolic polymers and characterized by black-brown colorization. Furthermore, it is one of the pigments produced by extremophiles including those living in the Antarctic desert, and is mainly involved in their protection from high UV radiation, desiccation, salinity and oxidation. Previous studies have shown that melanized species have an increased capability to survive high level of radiation compared with the non-melanized counterpart. Understanding the molecular composition of fungal melanin could help to understand this peculiar capability. Here, we aimed to characterize the melanin pigment extracted from the Antarctic black fungus Cryomyces antarcticus, which is a good test model for radioprotection researches, by studying its chemical properties and spectral data. Our results demonstrated that, in spite of having a specific type of melanin as the majority of fungi, the fungus possesses the ability to produce both 1,8-dihydroxynaphthalene (DHN) and L 3-4 dihydroxyphenylalanine (L-DOPA) melanins, opening interesting scenarios for the protection role against radiation. Researches on fungal melanin have a huge application in different fields, including radioprotection, bioremediation, and biomedical applications. KEY POINTS: • Isolation and characterization by multidisciplinary approaches of fungal melanins. • Discovery that pathways for producing DOPA and DHN are both active even in its extreme habitat. • Hypothesis supporting the possibility of using melanin pigment for radioprotection.

Melanin is effective in protecting fast and slow growing fungi from various types of ionizing radiation.

Melanin is a ubiquitous pigment with unique physicochemical properties. The resistance of melanized fungi to cosmic and terrestrial ionizing radiation suggests that melanin also plays a pivotal role in radioprotection. In this study, we compared the effects of densely-ionizing deuterons and sparsely-ionizing X-rays on two microscopic fungi capable of melanogenesis. We utilized the fast-growing pathogenic basiodiomycete forming an induced DOPA-melanin, Cryptococcus neoformans (CN); and the slow-growing environmental rock-inhabiting ascomycete synthesizing a constitutive DHN-melanin, Cryomyces antarcticus (CA); melanized and non-melanized counterparts were compared. CA was more resistant to deuterons than CN, and similar resistance was observed for X-rays. Melanin afforded protection against high-dose (1.5 kGy) deuterons for both CN and CA (p-values < 10-4 ). For X-rays (0.3 kGy), melanin protected CA (p-values < 10-4 ) and probably CN. Deuterons increased XTT activity in melanized strains of both species, while the activity in non-melanized cells remained stable or decreased. For ATP levels the reverse occurred: it decreased in melanized strains, but not in non-melanized ones, after deuteron exposure. For both XTT and ATP, which reflect the metabolic activity of the cells, larger and more statistically-significant differences as a function of melanization status occurred in CN. Our data show, for the first time, that melanin protected both fast-growing and slow-growing fungi from high doses of deuterons under physiological conditions. These observations may give clues for creating melanin-based radioprotectors.

BIOMEX Experiment: Ultrastructural Alterations, Molecular Damage and Survival of the Fungus Cryomyces antarcticus after the Experiment Verification Tests.

The search for traces of extinct or extant life in extraterrestrial environments is one of the main goals for astrobiologists; due to their ability to withstand stress producing conditions, extremophiles are perfect candidates for astrobiological studies. The BIOMEX project aims to test the ability of biomolecules and cell components to preserve their stability under space and Mars-like conditions, while at the same time investigating the survival capability of microorganisms. The experiment has been launched into space and is being exposed on the EXPOSE-R2 payload, outside of the International Space Station (ISS) over a time-span of 1.5 years. Along with a number of other extremophilic microorganisms, the Antarctic cryptoendolithic black fungus Cryomyces antarcticus CCFEE 515 has been included in the experiment. Before launch, dried colonies grown on Lunar and Martian regolith analogues were exposed to vacuum, irradiation and temperature cycles in ground based experiments (EVT1 and EVT2). Cultural and molecular tests revealed that the fungus survived on rock analogues under space and simulated Martian conditions, showing only slight ultra-structural and molecular damage.

Special Issue: 'Advances in Space Biology'.

As we enter a new era of space exploration, space biology is at the forefront of both robotic and human space programs [...].

Non-destructive real-time analysis of plant metabolite accumulation in radish microgreens under different LED light recipes.

Introduction: The future of human space missions relies on the ability to provide adequate food resources for astronauts and also to reduce stress due to the environment (microgravity and cosmic radiation). In this context, microgreens have been proposed for the astronaut diet because of their fast-growing time and their high levels of bioactive compounds and nutrients (vitamins, antioxidants, minerals, etc.), which are even higher than mature plants, and are usually consumed as ready-to-eat vegetables. Methods: Our study aimed to identify the best light recipe for the soilless cultivation of two cultivars of radish microgreens (Raphanus sativus, green daikon, and rioja improved) harvested eight days after sowing that could be used for space farming. The effects on plant metabolism of three different light emitting diodes (LED) light recipes (L1-20% red, 20% green, 60% blue; L2-40% red, 20% green, 40% blue; L3-60% red, 20% green, 20% blue) were tested on radish microgreens hydroponically grown. A fluorimetric-based technique was used for a real-time non-destructive screening to characterize plant methabolism. The adopted sensors allowed us to quantitatively estimate the fluorescence of flavonols, anthocyanins, and chlorophyll via specific indices verified by standardized spectrophotometric methods. To assess plant growth, morphometric parameters (fresh and dry weight, cotyledon area and weight, hypocotyl length) were analyzed. Results: We observed a statistically significant positive effect on biomass accumulation and productivity for both cultivars grown under the same light recipe (40% blue, 20% green, 40% red). We further investigated how the addition of UV and/or far-red LED lights could have a positive effect on plant metabolite accumulation (anthocyanins and flavonols). Discussion: These results can help design plant-based bioregenerative life-support systems for long-duration human space exploration, by integrating fluorescence-based non-destructive techniques to monitor the accumulation of metabolites with nutraceutical properties in soilless cultivated microgreens.

Survival, metabolic activity, and ultrastructural damages of Antarctic black fungus in perchlorates media.

Evidence from recent Mars landers identified the presence of perchlorates salts at 1 wt % in regolith and their widespread distribution on the Martian surface that has been hypothesized as a critical chemical hazard for putative life forms. However, the hypersaline environment may also potentially preserve life and its biomolecules over geological timescales. The high concentration of natural perchlorates is scarcely reported on Earth. The presence of perchlorates in soil and ice has been recorded in some extreme environments including the McMurdo Dry Valleys in Antarctica, one of the best terrestrial analogues for Mars. In the frame of "Life in space" Italian astrobiology project, the polyextremophilic black fungus Cryomyces antarcticus, a eukaryotic test organism isolated from the Antarctic cryptoendolithic communities, has been tested for its resistance, when grown on different hypersaline substrata. In addition, C. antarcticus was grown on Martian relevant perchlorate medium (0.4 wt% of Mg(ClO4)2 and 0.6 wt% of Ca(ClO4)2) to investigate the possibility for the fungus to survive in Martian environment. Here, the results indicate a good survivability and metabolic activity recovery of the black fungus when grown on four Martian relevant perchlorates. A low percentage of damaged cellular membranes have been found, confirming the ultrastructural investigation.

Metabolomic Profile of the Fungus Cryomyces antarcticus Under Simulated Martian and Space Conditions as Support for Life-Detection Missions on Mars.

The identification of traces of life beyond Earth (e.g., Mars, icy moons) is a challenging task because terrestrial chemical-based molecules may be destroyed by the harsh conditions experienced on extraterrestrial planetary surfaces. For this reason, studying the effects on biomolecules of extremophilic microorganisms through astrobiological ground-based space simulation experiments is significant to support the interpretation of the data that will be gained and collected during the ongoing and future space exploration missions. Here, the stability of the biomolecules of the cryptoendolithic black fungus Cryomyces antarcticus, grown on two Martian regolith analogues and on Antarctic sandstone, were analysed through a metabolomic approach, after its exposure to Science Verification Tests (SVTs) performed in the frame of the European Space Agency (ESA) Biology and Mars Experiment (BIOMEX) project. These tests are building a set of ground-based experiments performed before the space exposure aboard the International Space Station (ISS). The analysis aimed to investigate the effects of different mineral mixtures on fungal colonies and the stability of the biomolecules synthetised by the fungus under simulated Martian and space conditions. The identification of a specific group of molecules showing good stability after the treatments allow the creation of a molecular database that should support the analysis of future data sets that will be collected in the ongoing and next space exploration missions.

Biosignature stability in space enables their use for life detection on Mars.

Two rover missions to Mars aim to detect biomolecules as a sign of extinct or extant life with, among other instruments, Raman spectrometers. However, there are many unknowns about the stability of Raman-detectable biomolecules in the martian environment, clouding the interpretation of the results. To quantify Raman-detectable biomolecule stability, we exposed seven biomolecules for 469 days to a simulated martian environment outside the International Space Station. Ultraviolet radiation (UVR) strongly changed the Raman spectra signals, but only minor change was observed when samples were shielded from UVR. These findings provide support for Mars mission operations searching for biosignatures in the subsurface. This experiment demonstrates the detectability of biomolecules by Raman spectroscopy in Mars regolith analogs after space exposure and lays the groundwork for a consolidated space-proven database of spectroscopy biosignatures in targeted environments.

Insights into the Survival Capabilities of Cryomyces antarcticus Hydrated Colonies after Exposure to Fe Particle Radiation.

The modern concept of the evolution of Mars assumes that life could potentially have originated on the planet Mars, possibly during the end of the late heavy bombardment, and could then be transferred to other planets. Since then, physical and chemical conditions on Mars changed and now strongly limit the presence of terrestrial-like life forms. These adverse conditions include scarcity of liquid water (although brine solutions may exist), low temperature and atmospheric pressure, and cosmic radiation. Ionizing radiation is very important among these life-constraining factors because it damages DNA and other cellular components, particularly in liquid conditions where radiation-induced reactive oxidants diffuse freely. Here, we investigated the impact of high doses (up to 2 kGy) of densely-ionizing (197.6 keV/µm), space-relevant iron ions (corresponding on the irradiation that reach the uppermost layer of the Mars subsurface) on the survival of an extremophilic terrestrial organism-Cryomyces antarcticus-in liquid medium and under atmospheric conditions, through different techniques. Results showed that it survived in a metabolically active state when subjected to high doses of Fe ions and was able to repair eventual DNA damages. It implies that some terrestrial life forms can withstand prolonged exposure to space-relevant ion radiation.

The Ground-Based BIOMEX Experiment Verification Tests for Life Detection on Mars.

The success of an astrobiological search for life campaign on Mars, or other planetary bodies in the Solar System, relies on the detectability of past or present microbial life traces, namely, biosignatures. Spectroscopic methods require little or no sample preparation, can be repeated almost endlessly, and can be performed in contact or even remotely. Such methods are therefore ideally suited to use for the detection of biosignatures, which can be confirmed with supporting instrumentation. Here, we discuss the use of Raman and Fourier Transform Infrared (FT-IR) spectroscopies for the detection and characterization of biosignatures from colonies of the fungus Cryomyces antarcticus, grown on Martian analogues and exposed to increasing doses of UV irradiation under dried conditions. The results report significant UV-induced DNA damage, but the non-exceeding of thresholds for allowing DNA amplification and detection, while the spectral properties of the fungal melanin remained unaltered, and pigment detection and identification was achieved via complementary analytical techniques. Finally, this work found that fungal cell wall compounds, likely chitin, were not degraded, and were still detectable even after high UV irradiation doses. The implications for the preservation and detection of biosignatures in extraterrestrial environments are discussed.

Fungal Biomarkers Stability in Mars Regolith Analogues after Simulated Space and Mars-like Conditions.

The discovery of life on other planets and moons in our solar system is one of the most important challenges of this era. The second ExoMars mission will look for traces of extant or extinct life on Mars. The instruments on board the rover will be able to reach samples with eventual biomarkers until 2 m of depth under the planet's surface. This exploration capacity offers the best chance to detect biomarkers which would be mainly preserved compared to samples on the surface which are directly exposed to harmful environmental conditions. Starting with the studies of the endolithic meristematic black fungus Cryomyces antarcticus, which has proved its high resistance under extreme conditions, we analyzed the stability and the resistance of fungal biomarkers after exposure to simulated space and Mars-like conditions, with Raman and Gas Chromatography-Mass Spectrometry, two of the scientific payload instruments on board the rover.

The Responses of the Black Fungus Cryomyces Antarcticus to High Doses of Accelerated Helium Ions Radiation within Martian Regolith Simulants and Their Relevance for Mars.

One of the primary current astrobiological goals is to understand the limits of microbial resistance to extraterrestrial conditions. Much attention is paid to ionizing radiation, since it can prevent the preservation and spread of life outside the Earth. The aim of this research was to study the impact of accelerated He ions (150 MeV/n, up to 1 kGy) as a component of the galactic cosmic rays on the black fungus C. antarcticus when mixed with Antarctic sandstones-the substratum of its natural habitat-and two Martian regolith simulants, which mimics two different evolutionary stages of Mars. The high dose of 1 kGy was used to assess the effect of dose accumulation in dormant cells within minerals, under long-term irradiation estimated on a geological time scale. The data obtained suggests that viable Earth-like microorganisms can be preserved in the dormant state in the near-surface scenario for approximately 322,000 and 110,000 Earth years within Martian regolith that mimic early and present Mars environmental conditions, respectively. In addition, the results of the study indicate the possibility of maintaining traces within regolith, as demonstrated by the identification of melanin pigments through UltraViolet-visible (UV-vis) spectrophotometric approach.

Iron Ion Particle Radiation Resistance of Dried Colonies of Cryomyces antarcticus Embedded in Martian Regolith Analogues.

Among the celestial bodies in the Solar System, Mars currently represents the main target for the search for life beyond Earth. However, its surface is constantly exposed to high doses of cosmic rays (CRs) that may pose a threat to any biological system. For this reason, investigations into the limits of resistance of life to space relevant radiation is fundamental to speculate on the chance of finding extraterrestrial organisms on Mars. In the present work, as part of the STARLIFE project, the responses of dried colonies of the black fungus Cryomyces antarcticus Culture Collection of Fungi from Extreme Environments (CCFEE) 515 to the exposure to accelerated iron (LET: 200 keV/µm) ions, which mimic part of CRs spectrum, were investigated. Samples were exposed to the iron ions up to 1000 Gy in the presence of Martian regolith analogues. Our results showed an extraordinary resistance of the fungus in terms of survival, recovery of metabolic activity and DNA integrity. These experiments give new insights into the survival probability of possible terrestrial-like life forms on the present or past Martian surface and shallow subsurface environments.

Survival, DNA, and Ultrastructural Integrity of a Cryptoendolithic Antarctic Fungus in Mars and Lunar Rock Analogs Exposed Outside the International Space Station.

The search for life beyond Earth involves investigation into the responses of model organisms to the deleterious effects of space. In the frame of the BIOlogy and Mars Experiment, as part of the European Space Agency (ESA) space mission EXPOSE-R2 in low Earth orbit (LEO), dried colonies of the Antarctic cryptoendolithic black fungus Cryomyces antarcticus CCFEE 515 were grown on martian and lunar analog regolith pellets, and exposed for 16 months to LEO space and simulated Mars-like conditions on the International Space Station. The results demonstrate that C. antarcticus was able to tolerate the combined stress of different extraterrestrial substrates, space, and simulated Mars-like conditions in terms of survival, DNA, and ultrastructural stability. Results offer insights into the habitability of Mars for future exploration missions on Mars. Implications for the detection of biosignatures in extraterrestrial conditions and planetary protection are discussed.

Vegetation, pH and Water Content as Main Factors for Shaping Fungal Richness, Community Composition and Functional Guilds Distribution in Soils of Western Greenland.

Fungi are the most abundant and one of the most diverse components of arctic soil ecosystems, where they are fundamental drivers of plant nutrient acquisition and recycling. Nevertheless, few studies have focused on the factors driving the diversity and functionality of fungal communities associated with these ecosystems, especially in the scope of global warming that is particularly affecting Greenland and is leading to shrub expansion, with expected profound changes of soil microbial communities. We used soil DNA metabarcoding to compare taxonomic and functional composition of fungal communities in three habitats [bare ground (BG), biological soil crusts (BSC), and vascular vegetation (VV) coverage] in Western Greenland. Fungal richness increased with the increasing complexity of the coverage, but BGs and BSCs samples showed the highest number of unique OTUs. Differences in both fungal community composition and distribution of functional guilds identified were correlated with edaphic factors (mainly pH and water content), in turn connected with the different type of coverage. These results suggest also possible losses of diversity connected to the expansion of VV and possible interactions among the members of different functional guilds, likely due to the nutrient limitation, with potential effects on elements recycling.

Responses of the Black Fungus Cryomyces antarcticus to Simulated Mars and Space Conditions on Rock Analogs.

The BIOMEX (BIOlogy and Mars Experiment) is part of the European Space Agency (ESA) space mission EXPOSE-R2 in Low-Earth Orbit, devoted to exposing microorganisms for 1.5 years to space and simulated Mars conditions on the International Space Station. In preparing this mission, dried colonies of the Antarctic cryptoendolithic black fungus Cryomyces antarcticus CCFEE 515, grown on martian and lunar analog regolith pellets, were subjected to several ground-based preflight tests, Experiment Verification Tests, and Science Verification Tests (SVTs) that were performed to verify (i) the resistance of our model organism to space stressors when grown on extraterrestrial rock analogs and (ii) the possibility of detecting biomolecules as potential biosignatures. Here, the results of the SVTs, the last set of experiments, which were performed in ultraviolet radiation combined with simulated space vacuum or simulated martian conditions, are reported. The results demonstrate that C. antarcticus was able to tolerate the conditions of the SVT experiment, regardless of the substratum in which it was grown. DNA maintained high integrity after treatments and was confirmed as a possible biosignature; melanin, which was chosen to be a target for biosignature detection, was unambiguously detected by Raman spectroscopy.

Limits of Life and the Habitability of Mars: The ESA Space Experiment BIOMEX on the ISS.

BIOMEX (BIOlogy and Mars EXperiment) is an ESA/Roscosmos space exposure experiment housed within the exposure facility EXPOSE-R2 outside the Zvezda module on the International Space Station (ISS). The design of the multiuser facility supports-among others-the BIOMEX investigations into the stability and level of degradation of space-exposed biosignatures such as pigments, secondary metabolites, and cell surfaces in contact with a terrestrial and Mars analog mineral environment. In parallel, analysis on the viability of the investigated organisms has provided relevant data for evaluation of the habitability of Mars, for the limits of life, and for the likelihood of an interplanetary transfer of life (theory of lithopanspermia). In this project, lichens, archaea, bacteria, cyanobacteria, snow/permafrost algae, meristematic black fungi, and bryophytes from alpine and polar habitats were embedded, grown, and cultured on a mixture of martian and lunar regolith analogs or other terrestrial minerals. The organisms and regolith analogs and terrestrial mineral mixtures were then exposed to space and to simulated Mars-like conditions by way of the EXPOSE-R2 facility. In this special issue, we present the first set of data obtained in reference to our investigation into the habitability of Mars and limits of life. This project was initiated and implemented by the BIOMEX group, an international and interdisciplinary consortium of 30 institutes in 12 countries on 3 continents. Preflight tests for sample selection, results from ground-based simulation experiments, and the space experiments themselves are presented and include a complete overview of the scientific processes required for this space experiment and postflight analysis. The presented BIOMEX concept could be scaled up to future exposure experiments on the Moon and will serve as a pretest in low Earth orbit.

Survival and redox activity of Friedmanniomyces endolithicus, an Antarctic endemic black meristematic fungus, after gamma rays exposure.

Despite living organisms are not exposed to acute ionizing radiation under natural conditions, some exhibit a high radiation resistance. Understanding this phenomenon is important for assessing the impact of radiation-related accidents, occupational exposures and space missions. In this context, in this study we analyzed the effect of gamma rays on the Antarctic cryptoendolithic melanized fungus Friedmanniomyces endolithicus CCFEE 5208 and demonstrated its resistance to acute doses of gamma radiation (up to 400 Gy), accompanied by increase in metabolic activity.