**1. Introduction**

In the next few years, in situ space exploration missions will be devoted to the detection of biogenic signatures of extinct or extant life on Mars. The driver for searching for life on Mars is the findings supporting that ancient environments on Mars could have supported microbial life [1]. One of the primary issues in the search for life is that the present-day Martian surface presents a very inhospitable habitat for life as we know it because of the intense radiation, highly oxidizing conditions, concentrated evaporative salts, and extremely low water activity [2]. Despite the Martian surface having been cold and predominantly dry for at least the last three billion years (i.e., the Amazonian Period, immediately following the Hesperian), the subsurface could have sustained stable reservoirs of geothermally heated liquid water for the majority of this time. These conditions could represent a long-lived habitat that maintained hypothetical living cells [3–6]. For these reasons, the next planetary mission, ESA-Roscosmos ExoMars Rosalind Franklin, will collect and analyze samples in the subsurface, up to 2 m depth [7–9], to access places where organic molecules may be well preserved even after billions of years [10].

In this context, our best chance to find traces of extant or recently extinct life on Mars is to look for biomarkers [11]. A molecular biomarker is defined as a pattern, distribution

**Citation:** Cassaro, A.; Pacelli, C.; Baqué, M.; de Vera, J.-P.P.; Böttger, U.; Botta, L.; Saladino, R.; Rabbow, E.; Onofri, S. Fungal Biomarkers Stability in Mars Regolith Analogues after Simulated Space and Mars-like Conditions. *J. Fungi* **2021**, *7*, 859. https://doi.org/10.3390/jof7100859

Academic Editor: Laurent Dufossé

Received: 30 August 2021 Accepted: 9 October 2021 Published: 14 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of molecules or molecular structures that in nature derives uniquely from past or present biological processes [12,13]. Indeed, we consider as a possible biomarker a range of molecules indicative for life as we know it, such as DNA, amino acids, lipids, carbohydrates, pigments, intermediary metabolites [13], their degradation products and their general characteristics as preservation potential, specificity and extractability [14]. Assuming that hypothetical Martian life is similar to that on Earth, the identification of these compounds would be diagnostic of extant or recently extinct life because they are the indispensable biological components of the organisms as we know it. Any extinct or existing forms of microbial life on Mars may have produced biomolecules that may still be preserved and detectable in Martian rocks. In fact, minerals and organic molecules are strictly linked: minerals provide surfaces to support, concentrate, and preserve organic molecules. In support of this, organic matter was recently found within the three-billion-year-old mudstones of Gale Crater [15].

Due to the capability to detect biomolecules or their alteration products within mineral grains, Raman spectroscopy is part of the analytical instrumentation in the payloads of the rovers. This technique, on top of its main goal to provide mineralogical identification of the samples, can also detect a wide range of potential biomarkers in the rock substrate. It has been chosen for its non-destructive properties, for its sensibility during the detection of microbial life closed in their niches, and for its efficiency in in situ analyses also in presence of mineral rocks [16].

The Mars Organic Molecule Analyzer (MOMA), among various instruments, will operate with Pyrolysis–Gas Chromatography–Mass Spectrometry (Pyr-GCMS) in order to analyze volatile compounds in the Martian subsurface. In this context, it is important to understand the interaction effect of high temperatures with regolith and organic matter. In extreme environments, life could find refuge inside rocks, as in terrestrial analog environments, where in harsh conditions, extremophilic microorganisms dwelling inside the subsurface produce characteristic compounds protecting themselves from stressed conditions [17,18], among which are carotenoids and melanin pigments. Organic, metabolic and morphological structure or compounds of the organisms could persist in mineral structures and are considered good biomarkers of microorganisms' presence. On Earth, biological matter can be preserved in sedimentary rocks as carbonaceous macromolecules [12] and maybe, if life exists or existed on Mars, its rocks could preserve organic deposits: organic components may be preserved either as degraded molecules within mineral structures or as disseminated molecules chemically bonded to mineral particles of rocks, such as phyllosilicates [19].

In this context, the BIOMEX (BIOlogy and Mars EXperiment) project, aimed at investigating the endurance of extremophiles and the stability of biomolecules under space and Mars-like conditions in the presence of Martian regolith analogs [20]. This experiment involved 16 months of real space and a close approach to Mars-like conditions exposure outside the International Space Station (ISS). The experiment was placed externally aboard the EXPOSE-R2 exposure payload and comprised a series of ground-based simulation tests, including Experiment Verification Test (EVT) and Science Verification Tests (SVTs), carried out before the flight. In the frame of ground-based tests, the stability/degradation of biomolecules of the extremotolerant microorganism *Cryomyces antarcticus* was investigated; the fungus, isolated from the McMurdo Dry Valleys (South Victoria Land, Antarctica), was chosen for its widely proved ability to withstand stressors similar to the ones encountered in space and Mars-like environments (e.g., ionizing and no ionizing radiation, vacuum or Martian atmosphere, temperature cycles) [21,22].

In total, fungal colony samples were investigated after exposure to simulated space and Mars-like conditions during the ground-based experiments before flight. Fungal colonies were spread on three different cultivation media consisting of Malt Extract Agar (MEA) and three different regoliths: the Original Substrate (i.e., Antarctic sandstone, OS), the Phyllosilicatic Mars Regolith Simulant (P-MRS) analogue and the Sulfatic Mars Regolith Simulant (S-MRS) analogue. The P-MRS simulates igneous rocks altered by

hydrous fluids (neutral to basic) while the S-MRS mimics a more acidic environment with sulfate deposits [23]. The objectives of this study were to identify potential fungal biomolecules to be accounted as biomarkers and to understand if and how simulated space and Mars environment could modify them. As the primary goal of this study was the detection of microbial signatures within the Martian regolith analogues, the same methods, Raman spectroscopy and Gas Chromatography–Mass Spectrometry (GC-MS), planned for the ExoMars Rosalind Franklin mission, were used. In addition, extracted melanin pigments and nucleic acids were analyzed by using UV–VIS spectrophotometry and by quantitative Polymerase Chain Reaction (qPCR) technique, respectively, to detect any changes in structure after simulated space and Mars exposure.
