**4. Discussion**

The detection of biomolecular markers in the subsurface regolith of Mars is a primary goal for astrobiology. Investigation on the stability or degradation-rate of any biomarker after exposure to simulated or real space conditions is of high interest for space exploration. The stability of any biomarker is dependent on its initial form, the matrix in which it is hosted, and the chemical and physical processes to which it is subjected over time [19].

In this paper, we aimed at investigating the biomarkers associated with fungal colonies in Martian regolith analogues under simulated space and Mars-like conditions, tested during the Scientific Verification Test of the BIOMEX project, with the same techniques planned for the robotic exploration of Mars. Assuming that life on Mars evolved with the same characteristics than that on Earth or that there was an exchange of material inside meteorites between Earth and Mars [43], it is reasonable to search for traces of Earth-like life on Mars. Among biomarkers, we first focused on pigments. The black fungus *C. antarcticus* can withstand the hyper-arid and extremely cold conditions of McMurdo Dry

Valleys in Antarctica, thanks to its strongly melanized cell wall that helps to protect the fungus from the environmental stressors of these remote areas. It is known that the fungus is able to survive in different stressor conditions, such as ionizing radiation [36], heavy ions in hydrated and de-hydrated conditions [44,45].

Melanins are known to be involved in cellular resistance against a multitude of factors, such as toxic metals, hyperosmotic conditions and pH variations [46], but also extreme temperatures, desiccation and radiation, such as ultraviolet (UV) light, oxidizing agents and even ionizing radiation [35]. We aimed at detecting the presence of melanin pigments in samples subjected to simulated space and Mars-like exposure with different methods. Spectral analyses were used to confirm the presence and the possible alteration of melanin pigments after treatments. The UV spectrum (Figure 1) was characterized by the unaltered typical absorption profile of *C. antarcticus* melanin pigments [36]. It shows a strong absorption in the UV region (230 nm) with a progressive reduction as the wavelength increased, due to the presence of many complex conjugated structures in the melanin molecule [34,35]. This kind of analyses is relevant for the upcoming NASA Mars 2020 exploration rover Perseverance, which has onboard the SHERLOC (Scanning Habitable Environments with Raman and Luminescence for Organics and Chemicals) instrument, a deep UV laser with emission wavelengths below 250 nm that will provide an integrative analysis to avoid the fluorescence background during Raman measurements [47]. The importance of using a multiple technique approach to characterize melanic pigments is due to its absorption wavelength, which is similar to other compounds (e.g., amorphous carbon), making its detection more challenging.

Indeed, to further investigate the state of preservation and characterize the fungal melanin, we performed Raman spectroscopy analysis. The Raman spectra were dominated by two main peaks around 1340 cm−<sup>1</sup> and 1600 cm−<sup>1</sup> and one smaller at 1425 cm−<sup>1</sup> (Figure 2). According to the literature, the presence of the main peak at 1600 cm−<sup>1</sup> is due to the stretching vibration of the aromatic C = C stretching modes and the 1340 cm−<sup>1</sup> peak to the C-N stretching band of indole [48]: these peaks together with the smaller peak at 1425 cm−<sup>1</sup> are attributed to the presence of melanin pigments and, hence, to the fungal colonies.

It is well known that melanin pigments contribute to protecting microorganisms against the effect of radiation, which are abundant in space. Melanin pigments may have helped potential Earth-like life to survive in the harsh and highly irradiated environment of Mars, as it occurs in some extreme environments on Earth, where microorganisms have adopted this survival strategy. This assumption is supported by previous studies on the black fungus *C. antarcticus*; the melanized strain survived better the exposure to increasing doses of ionizing radiation compared to the non-melanized counterpart [49], confirming the protective role of melanin against ionizing radiation.

Furthermore, the ExoMars rover is equipped with a pyrolysis gas chromatography– mass spectrometry device with the aim of detecting organic molecules such as carboxylic acids in Martian subsurface materials [35,41–52]. Assessing the possible thermal destruction and transformation of organic matter induced by pyrolysis is of utmost importance, for the next rover missions. Indeed, pyrolysis, may alter and transform organic matter [53,54], hiding the presence of organic signatures from samples. For example, organosulfur compounds that were detected by the Sample Analysis at Mars (SAM) instrument onboard the Curiosity rover may derive from artificial secondary reactions of sulfur (from decomposition of sulfates) with organic radicals [15], due to the high temperature of the pyrolysis processes. In this context, the main question is: do potential fungal biomarkers remain unaltered during pyrolysis processes? Our study reports that pyrolysis GC-MS is able to detect fungal signals, even when the biomarkers are present in low amounts. Indeed, our data highlighted the presence of low molecular weight carboxylic acids, such as pyruvic acid (only in S-MRS Control samples) and myristic acid (only in S-MRS Top and Control samples). Although pyruvic acid is a key intermediate in several metabolic pathways throughout the cell, it cannot be accounted as a biomarker since it has been found in car-

bonaceous meteorites, and it can be produced through the chemistry of interstellar nitriles, HCN/CN and ketene [55]. Conversely, myristic acid, which can be part of the phospholipid bilayer of the plasma membrane of the eukaryotic cell, can be considered as biomarker. Nevertheless, in our study, it has been found only in Top and Control S-MRS samples. Interestingly, azelaic acid, which is a saturated dicarboxylic acid involved in stress resistance, was found in all tested samples, without any alteration in GC-MS profiles (Table 2). In this framework, we can define this dicarboxylic acid directly related to the presence of fungal colonies, and we can define it as a good biomarker for the search of Earth-like life elsewhere. Although they can be generated by both biotic and abiotic synthesis (Fischer– Tropsch reactions), in the latter, carbons are added one at a time. In most microorganisms, fatty acids are generated biochemically by the addition of two carbon atoms at a time [56]. This can help discriminating biotic and abiotic origins of the molecules, and therefore, it allows to consider fatty acids with an odd C number as a potential biomarker. The other detected compounds in our experiment, such as carbohydrates components (glucose and fructose) may derive from the malt extract used for the cultivation of *C. antarcticus*. The low amount of ethylene glycol detected only in S-MRS and P-MRS irradiated samples most probably derived from the degradation of more complex substances. Even if we can correlate the presence of the colony with the detection of azelaic acid through the GC-MS analyses, the compound was detected in a very small amount; this has to be taken into account in future space exploration missions, in order to avoid negative outcomes. One of the restrictions of this technique is the undetectability of high molecular weight compounds. Indeed, one of the main features of the black fungus, the melanin pigments, was not detected using GC-MS instrumentation, showing the importance of using multiple and connected techniques to avoid false negative results.

Then, we investigated the possibility to detect fungal DNA after the exposure through quantitative PCR. Nucleic acids are an ideal target biomarker due to their unambiguity, non-specificity and mainly the impossibility of generation in the absence of life [57,58]. DNA potentially preserved in Martian soil may be destroyed by the effects of UV, cosmic ray exposure and radioactive decay on nucleic acids [59,60]. Ancient DNA is prone to damage (e.g., hydrolysis, depurination) and fragmentation [9,61] once it is no longer actively repaired in a biological system. However, as previously reported in [62] and in [63], DNA showed to be still detectable in environmental samples by PCR and stable in Mars-like conditions [64]. In addition, the bound between DNA and minerals may have facilitated the preservation on different geological timescales, even more in cold and dry environments, such as the Gale Crater on Mars with an average temperature of −48 ◦C [65]. In the framework of planetary exploration, the implementation of techniques, such as the qPCR that allows to amplify fragments of a DNA molecule, would detect even minimal traces of DNA, even after partial degradation. Such kind of instruments are also very easy to miniaturize. It is already used in field campaigns with the MinION device [66]. This miniaturized technology was used for the first time in 2016 in the frame of Biomolecule Sequencer project [67,68] with the aim to detect unambiguous signs of life through nucleic acids sequencing, starting from a small amount of sample. The advantage of the in situ DNA detection with a miniaturized tool also allows minimizing any eventual contamination from terrestrial microorganisms [69]. Accordingly, our results suggested that, in spite of the treatments, the nucleic acids are still detectable by qPCR technique. In particular, we found no differences in the amplified region: an average of ~13,000 DNA copy number were detected. The advantage of the amplification of long-repeated LSU gene allows detecting any modification at the genomic level in an extended region. On the other hand, these results may give a false outcome, disguising possible radiation damage on DNA, due to the repetition of the gene in the whole genome. Indeed, the choice of β-actin gene amplification permits to amplify a small non-repeated gene region, with the objective to identify nucleic acid damage in a single copy gene. Therefore, we recommend that space agencies should investigate the utility of establishing and promoting a miniaturized PCR instrument (e.g., MinION) to promote the detection of nucleic acids beyond Earth. Specific

primers should be used for each of the three domains: bacteria, archaea, and eukarya. In order to identify microorganisms by their sequence, an automated sequencer needs be included in addition to the thermocycler apparatus. Our results showed that, even when using instrumentation already planned for future missions, each analytical technique has constraints that limit the set of information produced. Further improvements in technique and instrumentation, as well as cross comparisons in varying approaches, will be required to optimize data interpretation for the upcoming missions to Mars.
