Investigating changes in thiophene-bearing quinones of the archaeon Acidianus manzaensis after exposure to Mars-like conditions

Investigating changes in thiophene-bearing quinones of the archaeon Acidianus manzaensis after exposure to Mars-like conditions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Investigating changes in thiophene-bearing quinones of the archaeon Acidianus manzaensis after exposure to Mars-like conditions Sebastian V. Gfellner, Janina Groninga, Cyril Colas, Guillaume Gabant, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7565488/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In the search for reliable biomarkers in other planetary environments such as Mars, molecules that are stable over geological timescales, while preserving indicative information about their potential biological origin, are crucially needed. Thiophene-bearing quinones fulfill these requirements, and thiophenes, which are their basic moieties, have been found on Mars. However, thiophenes can be produced abiotically and have been found in meteorites. Furthermore, the Martian environment may alter their molecular structure over time. To evaluate whether there could be a distinction between biotically and abiotically produced thiophenes, considering the harsh environmental conditions on Mars, we cultivated the extremophilic archaeon Acidianus manzaensis on ESA01-E Mars analog material. We then exposed the cell-mineral samples to one month of desiccation and Mars-like conditions in a Mars simulation chamber to analyze changes in the composition of thiophene-bearing quinones using mass spectrometry-based metabolomics, and evaluated the potential for cell recovery after exposure. Biological sciences/Biochemistry Physical sciences/Chemistry Earth and environmental sciences/Planetary science exposure experiment Mars simulation chamber metabolomics biomarkers astrobiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction More than 60 years of planetary exploration have shown evidence that the early planetary stages of Earth and Mars shared similar environmental conditions, including the presence of liquid water and volcanic activity on the surface, coupled with an atmosphere rich in carbon dioxide (CO 2 ) [ e.g. , 1,2]. Whether these conditions lasted long enough to lead to the origin of life on Mars is an overarching question in astrobiology, and the main focus of current and future robotic missions such as the Mars2020, ExoMars, and Mars Sample Return missions [ 3 – 5 ]. However, in an Archean early Earth analogous to a Noachian early Mars setting (~ 4 Ga ago), one must consider the surface and subsurface environment at that time and the interplay between geo- and potential biosphere in a pervasive volcanic and hydrothermal setting [ 6 – 8 ]. Chemolithoautotrophic organisms, as found in the most ancient terrestrial paleo records, are among the best analogs for putative early Martian life [ 9 , 10 ]. Indeed, because they use CO 2 as a carbon source and redox-alter minerals by oxidizing inorganic compounds, such as iron, sulfur, and other reduced inorganic sulfur compounds, all found on Mars [ 11 , 12 ], their metabolic pathways make them excellent candidates for astrobiological research. Heat- and acid-loving thermoacidophilic microorganisms are analogs of the potential life forms of such a planetary stage [ 11 ]. Among these, the extremely thermoacidophilic archaeon Acidianus manzaensis from the order Sulfolobales was isolated from a hot fumarole in Manza, Japan [ 13 ]. It thrives at high temperatures and low pH, and tolerates high concentrations of heavy metals. Moreover, its genome has been fully sequenced, facilitating molecular studies on its metabolic capacity. The target molecules in this study were thiophene-bearing quinones, with thiophene headgroups and quinone tails. They serve as respiratory electron carriers in Sulfolobales, and are chemotaxonomic markers of archaeal cells that mediate metal redox processes [ 14 ]. These molecules are of particular interest because of the detection of sulfur compounds, such as ethanethiol and thiophene, at the Gale Crater on Mars [ 12 ]. It has been proposed that thiophene-bearing quinones could serve as biomarkers for the detection of life beyond Earth owing to their longevity and stability over geological time periods [ 15 ]. Isoprenoid quinones are integral components of the membranes of all living organisms [ 16 ]. These molecules consist of a hydrophilic head group and a nonpolar isoprenoid side chain, giving them amphiphilic characteristics that enable their integration into lipid bilayers. Their primary role is to act as electron and proton carriers in the electron transport chains of photosynthesis and respiration; they also serve as antioxidants [ 16 , 17 ]. Quinone oxidoreductases transfer electrons to terminal oxidase complexes, which help maintain intracellular pH and generate a proton motive force through the reduction of caldariella- or sulfolobus-type quinones [ 18 ]. Thiophenes, benzothiophenes, and benzoquinones serve as the basic units of the headgroups of isoprenoid quinones [ 19 ]. Thiophene molecular fragments occur in 2.72 Ga years old stromatolites [ 20 ], sedimentary structures formed by microorganisms that are among the oldest fossils on Earth [ 21 ], and have also been found in association with anoxygenic phototrophic biofilms in 3.3 Ga sediments [ 22 ]. Furthermore, thiophenes have been detected on Mars by the Curiosity rover preserved in > 3 Ga old sediments, whose possible origins may be from diagenesis or pyrolysis of biological material, as well as abiotic origin [ 23 ]. Abiotic thiophenes produced by thermal and/or aqueous alterations have also been observed in meteorites [ 24 – 26 ]. They have also been formed abiotically in simulated volcanic and hydrothermal conditions, such as those that occurred on early Earth and potentially on early Mars [ 27 ]. Thiophene-bearing quinones may thus prove valuable for the detection of astrobiological life when associated with other biosignatures. These compounds exhibit significant stability and longevity, and persist over geological timescales [ 15 , 22 , 23 , 27 ]. In a previous study, Gfellner et al. (2025) [ 28 ] developed a protocol for the detection of thiophene-bearing quinones in Metallosphaera sedula grown on mineral pyrite (FeS 2 ). Hence, the next goal was to demonstrate the potential of their derived extraction protocol for other relevant microorganisms and gradually adapt their growth minerals to more Mars-relevant materials ( i.e. , Mars regolith simulant material ESA01-E) and to expose the new model organism, in addition to desiccation, to Mars-like conditions to observe potential changes in the composition of their respiratory quinones. In our study, A. manzaensis was cultivated on the Mars analog material ESA01-E, which is known for its physicochemical similarity to Martian basalt. A dehydration experiment was conducted over a period of one month to test its survivability under desiccated conditions. In this process, concentrated cell pellets of A. manzaensis were deposited into metal exposure wells to monitor cell survival upon recovery of dried pellets in heterotrophic medium, following the protocol described by Kölbl et al. (2020) [ 29 ]. Furthermore, metabolomic analysis was conducted using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC – HRMS). Methods Experimental design The experimental setup (Fig. 1 A) included exposure of A. manzaensis cells deposited in metal exposure wells using the improved extraction and analysis protocol by Gfellner et al. (2025) [ 28 ] to identify potential changes in the composition of thiophene-bearing quinones. The desiccation experiment was conducted with a separate cell-mineral mixture of a bioreactor following the same cultivation and extraction parameters as the exposure experiment, one month prior to exposure to Mars-like conditions. The experimental design of the exposure experiment is shown in Fig. 1 B. In brief, after cultivation for 96 h, cell-mineral mixtures were divided into four sets in order to compare the effects on cell recovery and mass spectrometry measurements of each step: fresh culture (Fc) served as the basis. Laboratory controls (Lc) were kept under controlled conditions (shielded from light in aluminum and air-sealed in a storage container under a laminar flow box at the CBM-CNRS Orléans), whereas the transport controls (Tc) were transported with sets dedicated to the Mars simulation facility. Owing to laboratory constraints, UV-exposed (e) and laboratory controls were re-cultivated together for growth comparison, and while kept under controlled conditions, exposed without UV (ne) and transport controls were re-cultivated together one week later. Extraction was conducted continuously, while the remaining samples were stored at 4°C. Cell survival was determined by successful re-cultivation of microbial cells after desiccation and exposure to Mars-like conditions. Microbial cultivation and sample preparation A. manzaensis was cultivated in a 1 L Schott-bottle bioreactor in the presence of ESA01-E basalt over a period of 96 h. After harvesting, 800 mL of cell-mineral material was concentrated to a total volume of 40 mL by a factor of 20, homogenized, and stored at 4°C for 72 h before deposition in the metal exposure wells. The deposition was performed by pipetting 300 µL into each metal exposure well and continuous evaporation to a total volume of 2 mL. The samples were stored at room temperature before and during transportation to and from the exposure facility. Desiccation experiment : Prior to exposure to Mars-like conditions, test samples were subjected to prolonged desiccation for one month. Concentrated cell-mineral materials deposited in the metal exposure wells were air-dried completely, desiccated, and stored in a fume hood for one month. The samples were stored at room temperature and wrapped in sterilized aluminum foil. Thereafter, six samples (A 1–3 and B 1–3 ) were transferred into 50 mL glass flasks for re-cultivation under the same conditions as described for the exposure experiment below. Exposure experiment : The exposure experiment was conducted with samples e and ne in the Mars Simulation Facility (MSF) at the Planetary Analogue Simulation Laboratory (PASLAB, DLR Berlin), Tc and Lc as controls, and Fc as a baseline. The MSF consists of a climate chamber (ATT Umweltsimulation GmbH), which contains a 10.3 L vacuum-sealed stainless-steel vessel (Fig. 2 A) and a rotating platform with eight round aluminum sample holders. The experimental chamber has electrical connectors, inlet/outlet gas connectors, four optical fibers for UV light provided by a 150 W Xenon lamp (Fig. 2 B). Additionally, the experiment chamber is equipped with two SHT75 sensors (Sensirion AG, Switzerland) integrated with two Pt-100 temperature sensors to measure humidity and temperature, calibrated for the Martian atmosphere. The gas flow through the experiment chamber is generated by a gas mixing system, and the inside pressure is controlled by a membrane vacuum pump (MV10Vario, Vacuubrand GmbH). The control of the whole system is based on a DAQ-system device (National Instruments Corp., US) and Labview software [ 30 , 31 ]. The irradiance simulated the complete Martian solar spectrum (200–2200 nm) of the mid-latitude summer Sun's diurnal cycle [ 32 , 33 ]. Fourteen days of irradiation resulted in a total dose per sample of 12029 kJ/m² and a flux of 14 kW/m². Re-cultivation of exposed and non-exposed cultures : Upon arrival of the samples from the exposure facility, those deposited in the metal exposure wells were split in half using a sterile scalpel. One-half of the sample was processed for organic extraction, and the other half was left in the metal sample holder and stored at room temperature under sterile conditions in a fume hood until re-cultivation. A. manzaensis was re-cultivated according to the protocol described by Kölbl et al. (2020) [ 29 ]. Re-cultivation was performed for each condition in four replicates: initial fresh culture (Fc), exposed to UV (e), non-exposed to UV (ne), laboratory control (Lc), and transport control (Tc). The cultivation was conducted in a shake incubator set at 75°C and 100 rpm over a period of 72 h in 50 mL glass flasks containing an equivalent of 1 mL concentrated cell-mineral material and 20 mL of A. manzaensis specific medium [ 13 ] including ESA01-E basalt (10 g/L), Allen trace element solution (0.91 mM MnCl 2 × 4H 2 O, 1.18 mM Na 2 B 4 O 7 × 10H 2 O, 0.08 mM ZnSO 4 × 7H 2 O, 0.03 mM CuCl 2 × 2H 2 O, 0.01 mM Na 2 MoO 4 × 2H 2 O, 0.02 mM VOSO 4 × 2H 2 O, and 3.56 µM CoSO 4 × 7H 2 O) and tryptone (0.1%). To monitor cell growth, 1 mL aliquots of all samples were taken at 24 h intervals under sterile conditions in a laminar flow hood and counted in triplicate under a microscope with a hemocytometer (Neubauer chamber). The distinction between cells and mineral particles was conducted visually, taking into consideration cell movement, cell size (~ 1 µm), and relief of mineral particles. UHPLC – HRMS analysis Analyses were conducted in reverse phase liquid chromatography (RPLC) with mass spectrometric detection using ESI in positive mode. Chromatographic separations were performed on an Ultimate 3000RSLC system (Dionex, Germering, Germany) using an Acquity UPLC BEH C18 1.7 µm 2.1×100 mm column (Waters, Saint-Quentin-en-Yvelines, France). The column was heated at 60°C and the following solvents were used at a flow rate of 500 µL/min: H 2 O with 0.1% formic acid as solvent A and a mixture of methanol and isopropanol (50:50, v/v) with 0.1% formic acid as solvent B. Linear gradient elution was as follows: 0-2.5 min 3% B, 6% B at 4 min, 85% B at 13 min, a plateau at 100% B from 13.5 to 19 min and re-equilibration from 19.1 to 23 min at 3% B. Two µL of the organic extract were injected. Mass spectra were acquired on a maXis Q-TOF (Bruker Daltonik, Bremen, Germany) at a frequency of 1 Hz in the 50-1650 m/z range for MS experiments. The ESI source parameters were as follows: nebulizing gas, 2 bar; drying gas, 200°C at a flow rate of 9 L/min; capillary voltage, 4500 V. For MS/MS experiment in Data Dependent Acquisition (DDA) mode, the same parameters were used except the acquisition frequency: 2 Hz. Two precursors ions were selected per cycle into the 445 to 950 m/z range with exclusion after one spectrum for 0.07 min. Lock mass calibration was performed using ESI-TOF tuning mix (Agilent, Palo Alto, CA, USA) and lock masses at m/z 299.2945 [hexakis(1H,1H,4H-hexafluorobutyloxy)phosphazine; CAS #: 186406-47-3] and 1221.9906 [methyl stearate, CAS #: 112-61-8] for calibration correction and was applied to all MS and MS/MS spectra. Feature detection Molecular feature detection in UHPLC-HRMS measurements was performed using the Time-aligned Region Complete Extraction (T-ReX) 3D® feature-finder algorithm of MetaboScape 2024® (Bruker Daltonik GmbH, Bremen, Germany). For peak picking, a minimum intensity threshold of 5000 cts and a minimum peak length of four spectra across an m/z range of 250 Da to 2000 Da were set. The recursive feature-finding tool was deactivated and prior filtering ensured that only the features present in at least three measurements were retained. The primary ion was set to [M + H]+, with [M + NH 4 ] + and [M + Na] + as the seed ions. When applicable, the adducts and doubly charged ions were grouped based on an ion deconvolution threshold of 0.8. Quinone annotation was performed utilizing a comprehensive target list of potentially occurring sulfur-bearing quinones, adhering to a maximum m/z tolerance of 2.5 ppm, an mSigma value of 40 (narrow) to 250 (wide), and an MS/MS similarity score of > 800, if MS/MS spectra were present. The extracted ion chromatograms (EIC) of the annotated compounds were reviewed, and anomalous peaks were excluded. Results & Discussion Desiccation experiment After successful cultivation of A. manzaensis on the Mars analog material ESA01-E (Fig. 3 A), a desiccation experiment was conducted on the test samples to verify their stability and resistance to desiccation, which is a critical parameter for Mars-like exposure. The analog material ESA01-E is known for its physicochemical similarity to Martian basalt, and the dehydration experiment was conducted over a period of one month. Concentrated cell pellets were deposited into metal exposure wells (Fig. 3 B) to investigate cell survival upon the recovery of the dried pellets. The cells were recovered, and cell survival was demonstrated by successful re-cultivation of previously desiccated cells (Fig. 3 C). In the two triplicate sets (A 1–3 and B 1–3 ), cell numbers doubled during the cultivation period of 72 h. The mean number of cells reached between 2.5–3 × 10 6 cells/mL throughout all samples. However, variation in growth was observed in sample A 1 , which could have been caused by a non-homogeneous mixture of cell-mineral materials used for the incubation. Previous dehydration studies performed with M. sedula have shown cell revival after two months of desiccation following re-cultivation in heterotrophic medium and varying cell numbers based on their mineral substrates, including Mars analog material [ 29 ]. This validates their use for subsequent exposure to Mars-like conditions. Two-week exposure experiment in a Mars Simulation Facility The sensors integrated in the simulation chamber successfully recorded the physico-chemical parameters of the experiment over the course of 340h = 14 days, as displayed in Fig. 4 . The following parameters correspond to an approximate measurement distance of 1 cm above the samples in the sample holder of the Mars Simulation Facility: temperature [°C], measured humidity over water [%], and measured humidity over ice [%]. Pressure [hPa] is the pressure inside the experimental chamber. The pressure was gradually decreased to 8 hPa. The LED temperature sensor [V] represents the day/night cycle (higher values are nighttime [8h] and lower values are daytime [16h]). All measurements showed excellent adequacy with the input values (day-to-night: temperature of + 15 to -50°C and relative humidity of 0 to 75%), successfully recreating day/night cycles. In relation to the samples before exposure (Fig. 5 A), a change in the physical appearance (discoloration) of the samples exposed to UV light (e) was observed (Fig. 5 B) compared to the unexposed samples (ne). Samples e, ne, Tc, and Lc were successfully re-cultivated in triplicate upon arrival (Fig. 5 C) for 72 h and compared to the re-cultivation of fresh cell-mineral material (Fc) immediately after harvesting. Although Fc showed the highest initial cell number, no significant trend was observed between e, ne, Tc, and Lc. However, the cell number increased in comparison to initial concentration by 1 × 10 6 cells/mL in all samples. No significant difference was noted after one month of desiccation in normal atmosphere and 8 hPa for two weeks. Although UV-exposed samples appeared bleached in appearance, the survival fraction was not affected by radiation exposure, with very comparable values reached after re-cultivation across all sets. These results are in agreement with those of previous experiments conducted by Mastascusa et al. (2014), who assessed the survival by successful re-cultivation of the comparable extremely thermoacidophilic archaea Sulfolobus solfataricus , in the same order as A. manzaensis (Sulfolobales), to simulated space conditions: good resistance to temperature variations (one week of -196 to 85°C) and a high resistance to UV exposure (1 h to λ = 254 nm) [ 34 ]. However, regarding UV exposure, it must be considered that in our experiments, we did not expose thin films spread in Petri dishes, but cell-mineral mixtures in metal exposure wells. Therefore, only the first millimeter was irradiated and the fraction below the surface largely contributed to the survival, which was subjected to exposure to other extreme conditions, such as temperature variations, pressure, and humidity. Characterization of thiophene-bearing quinones in Sulfolobales The quinone composition of the order Sulfolobales consists of oxidized caldariellaquinones (CQ), sulfolobusquinones (SQ), and benzodithiophenequinones (BDTQ). Members of the Sulfolobales order exhibit varying compositions of saturated quinones in response to their redox environments. As a result, the quinone profile of a specific Sulfolobales member can be utilized to reconstruct the environmental redox conditions [ 14 , 16 , 35 , 36 ]. Nevertheless, the proportions of quinones vary among different Sulfolobales species. Consequently, adaptations to environmental conditions might be mirrored in the distributions of SQs, CQs, and BDTQs [ 14 ]. Analyzing quinone profiles could facilitate the monitoring of microbial community transitions from oxic to anoxic conditions and help characterize archaeal diversity, complementing approaches based on membrane lipids and genes [ 14 , 16 , 35 , 36 ]. Caldariellaquinones were initially identified by de Rosa et al. (1977) [ 37 ], which are produced by organisms that thrive in harsh conditions (pH 1.4–2.6; 75–89°C), with a robust membrane structure observed in Sulfolobus and Acidianus species [ 38 ]. Later, caldariellaquinones were found in S. solfataricus and subsequently in M. sedula [ 39 – 42 ], whereas benzodithiophenequinones were detected in S. solfataricus [ 41 , 43 – 45 ]. Variations in the CQ, SQ, and BDTG molecules produced are linked to the presence of oxygen during growth [ 46 , 47 ]. For the Sulfolobales order, Elling et al. (2016) [ 14 ] reported a distribution of CQ 6:0 (86.1%) and CQ 6:1 (12.2%) in S. acidocaldarius , CQ 6:0 (85.8%) and CQ 6:1 (13.7%) in S. solfataricus , and SQ 6:0 (42.9%), CQ 6:0 (36.4%), and CQ 6:1 (14.6%) in S. islandicus as the main quinone components, with BDTQ 6:0 (0.4%) only found in S. islandicus . Additionally, Gfellner et al. (2025) [ 28 ] reported a distribution of SQ 4:0 (2.7), SQ 4:1 (66.7), SQ 5:0 (8.8), SQ 5:1 (5.5), CQ 4:1 (2.6), CQ 5:1 (6.9), and BDTQ 5:0 (6.8) in M. sedula . Astrobiological relevance of thiophene-bearing quinones After the growth of Metallosphaera sedula on Martian meteorite material, Milojevic et al. (2021) [ 48 ] detected possible S-bonds of lipid chains, indicating the possible presence of thiophene-bearing quinones. A closer examination of the differences in the thiophene headgroup properties (Fig. 6 ) revealed potential molecular target sites for molecular alterations in the form of methylation, S-linkage properties, and/or number of cycles. However, exposure to Mars-like conditions could likely alter their molecular signatures over time. Therefore, in addition to desiccation, the exposure experiment under Mars-like conditions was conducted to clarify the role of these molecules as potential biosignatures for life on Mars and other celestial bodies. Planetary environmental conditions must be considered when assessing the preservation potential of biomarkers over billions of years. On Earth, the occurrence of thiophenes associated with Archaean sediments (4.0–2.5 Ga) is related to the diagenetic and metamorphic history of the sediments [ 49 ]. Primary organic matter in microbial phototrophic colonies is degraded by heterotrophic microbes such as sulfur reducers, during this process the sulfur content of the organic matter is enriched [ 22 ]. Metamorphism converts sulfur-containing molecules into aromatic organic structures such as thiophene. On the other hand, Mars’ metamorphic history is different from that on Earth. The planet is approximately half the size of the Earth and lacks plate tectonics. Any sedimentary rocks would have therefore been subjected to a lower degree of metamorphism, except shock metamorphism from meteorite impacts [ 50 , 51 ]. It is therefore important to take into account the general environmental context and its geological setting in order to distinguish between plausible biotic and abiotic settings. Exposure of thiophene-bearing quinones at the surface of Mars to 3–4 Ga of UV, galactic, and cosmic radiations will affect the survivability of the basic thiophene moieties; however, data from gale Crater on Mars [ 23 ] shows that they may still be preserved in the Martian environment, although determination of their biogenicity remains to be proven. Hereby, we wanted to address the following: Is there a difference in the saturations of thiophene-bearing quinones between the exposed and non-exposed samples? In particular, are there any differences in the fragmented thiophene headgroups detectable by MS/MS analysis? Thiophene-bearing quinones after exposure to Mars-like conditions The first tentative annotation based on MS analysis using MetaboScape® ( Supplementary Table S1 ) across all samples (e, ne, Tc, Lc, and Fc) revealed the heterogeneous distribution of differently oxidized thiophene-bearing quinones (Fig. 7 A). We were able to annotate for the following oxidation states for CQs: CQ 4:0 , CQ 8:6 , CQ 9:3 , CQ 9:5 , CQ 9:6 ; for SQs: SQ 4:0 , SQ 6:0 , SQ 7:2 , SQ 7:4 , SQ 8:0 , SQ 8:6 , SQ 10:2 , SQ 10:5 , SQ 10:7 , SQ 10:10 ; and for BDTQs: BDTQ 4:1 , BDTQ 8:3 , BDTQ 8:4 , BDTQ 9:1 , BDTQ 9:4 , BDTQ 9:7 , BDTQ 10:0 , BDTQ 10:7 . However, certain indicative features, CQ 9:5, SQ 7:4 , and SQ 8:0 , were only found in the immediately extracted samples after cultivation. SQ 10:2 and SQ 10:5 were only present in the exposed samples, but also in the transport control, and SQ 10:7 , SQ 10:10 , BDTQ 4:1 , and BDTQ 10:0 were not present in the exposed sample. This poses a challenge when ascribing features to actual exposure. The analyzed samples were organic extracts of material immediately scratched from the metal exposure wells after exposure to resolve minor changes in the headgroups of thiophene-bearing quinones visible by energetic fragmentation using MS/MS analysis. However, the generally low yield of the corresponding MS/MS spectra (Fig. 7 B) complicates this approach. Therefore, to observe the changes in the distribution of thiophene-bearing quinones before and after exposure, additional organic extraction of the remaining re-cultivated samples is necessary. Because of the low penetration depth of UV radiation in the cell-mineral mixture in the metal exposure wells, only a small fraction of the cells were exposed to the potential degradation of thiophene-bearing quinones, especially their thiophene moieties. A different sample preparation procedure might also solve this problem; however, complementary analyses, such as survival, would not have been possible if thin films were used in Petri dishes. Additionally, a more powerful source of radiation might better mirror the induced changes in geological timescales of thiophene moieties and ultimately shed light on specific markers of biologically produced thiophenes. Therefore, we propose a continuation of a radiation experiment with a proton ion source ( e.g. , Cyclotron at the CEMHTI laboratory in Orléans, France) or an exposure platform in Low Earth Orbit, as shown by the completed BIOMEX [ 52 ] and planned BioSigN projects [ 53 ] or future planned space experiment platforms [ 54 ]. Conclusion In summary, we demonstrated the reproducibility of the improved metabolite extraction protocol derived by Gfellner et al. (2025) in terms of its applicability to different archaeal species ( M. sedula and A. manzaensis ) and mineral materials (pyrite and Mars analog material). Furthermore, we successfully re-cultivated cells after exposure to one month of desiccation, demonstrating their survival to water depletion, and two weeks of simulated Mars-like conditions, proving their survival under extreme temperature fluctuations ranging from + 15 to -50°C in diurnal cycles (16h daylight and 8h night), exposure to UV radiation (200–2200 nm), and Martian atmosphere and pressure (96% CO 2 , 4% air; 8 hPa). This shows the relevance of thermophilic archaea in astrobiology research, especially A. manzaensis as a model organism. However, further analysis is necessary for a direct correlation between potential changes in the distribution of thiophene-bearing quinones, followed by exposure to Mars-like conditions. Improved sample preparation and different radiation sources are being evaluated to further assess the relevance of thiophene-bearing quinones as model biosignatures, which will guide current and future missions searching for signs of life on Mars. Declarations Acknowledgments This research was funded under the European Union's Horizon 2020 Framework Programme, grant number ERC-2020-COG, Project 101001311 – BIOMAMA and the French National Research Agency (ANR, CPJ n°ANR-22-CPJ1-0066-01). We thank the Région Centre Val de Loire (SyMBioMS and Malditof grants) and the European FEDER funds (grants no 2699-33931 and 2017-EX002979) for supporting MS instruments and the SALSA and MO2VING platforms. This research was partially supported by the Helmholtz Association through the Helmholtz Alliance “Planetary Evolution and Life”. Furthermore, we acknowledge the use of MetaboScape®, supported by Germany’s Excellence Strategy (EXC-2077) project 390741603 ‘The Ocean Floor – Earth’s Uncharted Interface’. In addition, we thank the members of the Center for Molecular Biophysics, Orléans, France, in particular Matthieu Réfrégiers for his guidance throughout the entire process, Martine Cadene for her support in planning the workflow for the experimental design, and Frances Westall for her input on the preservation of thiophene-bearing quinones in the geological rock record. Data availability The MS and MS/MS data used for the tentative annotation of thiophene-bearing quinones can be found in Supplementary Table S1, Tentative annotation using Metaboscape® . Author contributions S.V.G. and M.B. designed the exposure experiments. S.V.G. and C.C. performed the analyses. S.V.G., J.G., and G.G. processed, curated, and validated the experimental data. A.L., M.B., and S.G. operated the Mars Simulation Facility. All authors contributed to the editing and discussion of this manuscript. Competing interests The authors declare no competing interests. References Pollack, J. B., Kasting, J. 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10:37:07","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127269,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7565488/v1/36c87c8976ff3ddd148e031d.html"},{"id":92400966,"identity":"307d347c-7057-4672-96f2-01482605ab8d","added_by":"auto","created_at":"2025-09-29 10:13:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":112284,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the workflow and experimental design. a)\u003c/strong\u003e Workflow exposure experiment: Cultivation, followed by cell enrichment and deposition in the exposure wells to conduct 1. desiccation experiment, and 2.\u003cstrong\u003e \u003c/strong\u003eexposure to Mars-like conditions. Re-cultivation of the exposed cells was performed to determine cell survival, followed by organic extraction, mass spectrometry analysis (UHPLC – HRMS), and data analysis (MetaboScape®) to determine potential changes in the composition of thiophene-bearing quinones. \u003cem\u003eCreated with BioRender.com.\u003c/em\u003e \u003cstrong\u003eb)\u003c/strong\u003eExperimental design of the exposure experiment from cultivation to sample analysis and determination of cell recovery by re-cultivation of exposed and non-exposed cell-mineral material. Extractions were conducted immediately after harvesting the cell-mineral material of the fresh culture, followed by extraction and analysis of the samples in the Mars simulation chamber exposed to UV, non-exposed to UV, transport control, and laboratory control.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7565488/v1/c614c0852b30621b20658dc0.png"},{"id":92400968,"identity":"c528da30-3aab-44b5-89e9-324244703f1d","added_by":"auto","created_at":"2025-09-29 10:13:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":461351,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMars Simulation Facility.\u003c/strong\u003e \u003cstrong\u003ea)\u003c/strong\u003e PASLAB at DLR Berlin with an open MSF chamber. \u003cstrong\u003eb)\u003c/strong\u003e Detailed sketch of the exposure platform: exposure (e\u003csub\u003e1–4\u003c/sub\u003e) to UV light; no exposure (ne\u003csub\u003e1–4\u003c/sub\u003e) to UV light. \u003cem\u003eCreated with BioRender.com.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7565488/v1/3d0460469a21dc6090e812b9.png"},{"id":92400973,"identity":"d37a8d3b-df1f-43ee-bb19-edfc8b772d2a","added_by":"auto","created_at":"2025-09-29 10:13:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":307860,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBioreactors, cell-mineral deposition in metal exposure wells, and re-cultivation of desiccated cells. a) \u003c/strong\u003eBioreactor setup of \u003cem\u003eA. manzaensis\u003c/em\u003e grown on ESAE-01 basalt analog material. \u003cstrong\u003eb) \u003c/strong\u003eExposure wells with desiccated \u003cem\u003eA. manzaensis\u003c/em\u003e cells and ESAE-01 basalt pellets. \u003cstrong\u003eC:\u003c/strong\u003e Boxplot of successful re-cultivation of \u003cem\u003eA. manzaensis\u003c/em\u003e cells in triplicate sets (A\u003csub\u003e1–3\u003c/sub\u003e and B\u003csub\u003e1–3\u003c/sub\u003e) after one month of desiccation.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7565488/v1/67f1ca5f2391f0a8d4d46d8c.png"},{"id":92401166,"identity":"3d33aee5-877f-40e3-ab2e-70bf4eec44b1","added_by":"auto","created_at":"2025-09-29 10:21:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":170227,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eParameters of the Mars Simulation Facility. \u003c/strong\u003eThe following parameters correspond to an approximate measurement distance of 1 cm above the samples in the sample holder of the Mars Simulation Facility: temperature [°C] and measured humidity over water/ice [%]. The pressure [hPa] gradually decreased and then remained constant. The day/night cycle (16/8 h) was measured using an LED temperature sensor [V].\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7565488/v1/21cf91d1c581b575e4be8521.png"},{"id":92401165,"identity":"5e1440d2-5c82-4307-bca2-0c5f2fe76dc0","added_by":"auto","created_at":"2025-09-29 10:21:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":116236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSamples before and after exposure, and re-cultivation of exposed cells. a)\u003c/strong\u003e Metal exposure wells with deposited cell-mineral mixture before exposure: no exposure (ne\u003csub\u003e1-4\u003c/sub\u003e) to UV light; exposure (e\u003csub\u003e1-4\u003c/sub\u003e) to UV light. \u003cstrong\u003eb)\u003c/strong\u003e Metal exposure wells with deposited cell-mineral mixture after 14 days of exposure. \u003cstrong\u003eC:\u003c/strong\u003e Boxplot of successful re-cultivation in triplicate of samples e, ne, Tc, and Lc compared to cell-mineral material re-cultivated immediately after harvesting (Fc).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7565488/v1/fcd79c7d5e13797b5ee087d9.png"},{"id":92400987,"identity":"d5861a07-acac-442f-b0df-fd03bf994bc0","added_by":"auto","created_at":"2025-09-29 10:13:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":55111,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMain structural features of thiophene-bearing quinones.\u003c/strong\u003e The headgroups of sulfolobusquinones (SQ), caldariellaquinones (CQ), benzodithiophenequinones (BDTQ), and their basic units consist of thiophenes (C\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003eS), benzothiophenes (C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eS), and benzoquinone (C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). A comparison of headgroup characteristics revealed differences in methylation, S-linkage, and/or number of cycles.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7565488/v1/5e26a43e94e0dcfc5fe00d33.png"},{"id":92401170,"identity":"30c2eae8-1253-4017-a30c-8d3075d6fe20","added_by":"auto","created_at":"2025-09-29 10:21:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":70404,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTentative annotation of thiophene-bearing quinones. a) \u003c/strong\u003eSummed peak areas of tentatively annotated thiophen-bearing quinones (CQ\u003csub\u003en:x\u003c/sub\u003e, SQ\u003csub\u003en:x\u003c/sub\u003e, and BDTQ\u003csub\u003en:x\u003c/sub\u003e) in the MS spectra: exposed (e), non-exposed (ne), transport control (Tc), laboratory control (Lc), and fresh culture (Fc). \u003cstrong\u003eb)\u003c/strong\u003e Summed peak areas of tentatively annotated thiophen-bearing quinones in the MS/MS spectra.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7565488/v1/5dd6cf8a3763031950f6098a.png"},{"id":93636373,"identity":"2adcc551-891c-46ec-83d8-361dedb16b7e","added_by":"auto","created_at":"2025-10-16 01:31:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2385652,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7565488/v1/56490c32-6e05-47aa-94d9-b69a9ee72a08.pdf"},{"id":92402196,"identity":"9177846b-ee2c-4150-83a8-0ae1c481a396","added_by":"auto","created_at":"2025-09-29 10:37:07","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":18966,"visible":true,"origin":"","legend":"","description":"","filename":"Gfellneretal.2025SupplementaryTableS1TentativeannotationusingMetaboscape.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7565488/v1/4bc1af3053d71ef964159b44.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigating changes in thiophene-bearing quinones of the archaeon Acidianus manzaensis after exposure to Mars-like conditions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMore than 60 years of planetary exploration have shown evidence that the early planetary stages of Earth and Mars shared similar environmental conditions, including the presence of liquid water and volcanic activity on the surface, coupled with an atmosphere rich in carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) [\u003cem\u003ee.g.\u003c/em\u003e, 1,2]. Whether these conditions lasted long enough to lead to the origin of life on Mars is an overarching question in astrobiology, and the main focus of current and future robotic missions such as the Mars2020, ExoMars, and Mars Sample Return missions [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, in an Archean early Earth analogous to a Noachian early Mars setting (~\u0026thinsp;4 Ga ago), one must consider the surface and subsurface environment at that time and the interplay between geo- and potential biosphere in a pervasive volcanic and hydrothermal setting [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Chemolithoautotrophic organisms, as found in the most ancient terrestrial paleo records, are among the best analogs for putative early Martian life [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Indeed, because they use CO\u003csub\u003e2\u003c/sub\u003e as a carbon source and redox-alter minerals by oxidizing inorganic compounds, such as iron, sulfur, and other reduced inorganic sulfur compounds, all found on Mars [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], their metabolic pathways make them excellent candidates for astrobiological research. Heat- and acid-loving thermoacidophilic microorganisms are analogs of the potential life forms of such a planetary stage [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Among these, the extremely thermoacidophilic archaeon \u003cem\u003eAcidianus manzaensis\u003c/em\u003e from the order Sulfolobales was isolated from a hot fumarole in Manza, Japan [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It thrives at high temperatures and low pH, and tolerates high concentrations of heavy metals. Moreover, its genome has been fully sequenced, facilitating molecular studies on its metabolic capacity.\u003c/p\u003e\u003cp\u003eThe target molecules in this study were thiophene-bearing quinones, with thiophene headgroups and quinone tails. They serve as respiratory electron carriers in Sulfolobales, and are chemotaxonomic markers of archaeal cells that mediate metal redox processes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These molecules are of particular interest because of the detection of sulfur compounds, such as ethanethiol and thiophene, at the Gale Crater on Mars [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It has been proposed that thiophene-bearing quinones could serve as biomarkers for the detection of life beyond Earth owing to their longevity and stability over geological time periods [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIsoprenoid quinones are integral components of the membranes of all living organisms [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These molecules consist of a hydrophilic head group and a nonpolar isoprenoid side chain, giving them amphiphilic characteristics that enable their integration into lipid bilayers. Their primary role is to act as electron and proton carriers in the electron transport chains of photosynthesis and respiration; they also serve as antioxidants [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Quinone oxidoreductases transfer electrons to terminal oxidase complexes, which help maintain intracellular pH and generate a proton motive force through the reduction of caldariella- or sulfolobus-type quinones [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Thiophenes, benzothiophenes, and benzoquinones serve as the basic units of the headgroups of isoprenoid quinones [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Thiophene molecular fragments occur in 2.72 Ga years old stromatolites [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], sedimentary structures formed by microorganisms that are among the oldest fossils on Earth [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and have also been found in association with anoxygenic phototrophic biofilms in 3.3 Ga sediments [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Furthermore, thiophenes have been detected on Mars by the Curiosity rover preserved in \u0026gt;\u0026thinsp;3 Ga old sediments, whose possible origins may be from diagenesis or pyrolysis of biological material, as well as abiotic origin [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Abiotic thiophenes produced by thermal and/or aqueous alterations have also been observed in meteorites [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. They have also been formed abiotically in simulated volcanic and hydrothermal conditions, such as those that occurred on early Earth and potentially on early Mars [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Thiophene-bearing quinones may thus prove valuable for the detection of astrobiological life when associated with other biosignatures. These compounds exhibit significant stability and longevity, and persist over geological timescales [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn a previous study, Gfellner \u003cem\u003eet al.\u003c/em\u003e (2025) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] developed a protocol for the detection of thiophene-bearing quinones in \u003cem\u003eMetallosphaera sedula\u003c/em\u003e grown on mineral pyrite (FeS\u003csub\u003e2\u003c/sub\u003e). Hence, the next goal was to demonstrate the potential of their derived extraction protocol for other relevant microorganisms and gradually adapt their growth minerals to more Mars-relevant materials (\u003cem\u003ei.e.\u003c/em\u003e, Mars regolith simulant material ESA01-E) and to expose the new model organism, in addition to desiccation, to Mars-like conditions to observe potential changes in the composition of their respiratory quinones.\u003c/p\u003e\u003cp\u003eIn our study, \u003cem\u003eA. manzaensis\u003c/em\u003e was cultivated on the Mars analog material ESA01-E, which is known for its physicochemical similarity to Martian basalt. A dehydration experiment was conducted over a period of one month to test its survivability under desiccated conditions. In this process, concentrated cell pellets of \u003cem\u003eA. manzaensis\u003c/em\u003e were deposited into metal exposure wells to monitor cell survival upon recovery of dried pellets in heterotrophic medium, following the protocol described by K\u0026ouml;lbl \u003cem\u003eet al.\u003c/em\u003e (2020) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Furthermore, metabolomic analysis was conducted using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC \u003cb\u003e\u0026ndash;\u003c/b\u003e HRMS).\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eExperimental design\u003c/h2\u003e\n \u003cp\u003eThe experimental setup (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA) included exposure of \u003cem\u003eA. manzaensis\u003c/em\u003e cells deposited in metal exposure wells using the improved extraction and analysis protocol by Gfellner \u003cem\u003eet al.\u003c/em\u003e (2025) [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e] to identify potential changes in the composition of thiophene-bearing quinones. The desiccation experiment was conducted with a separate cell-mineral mixture of a bioreactor following the same cultivation and extraction parameters as the exposure experiment, one month prior to exposure to Mars-like conditions. The experimental design of the exposure experiment is shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB. In brief, after cultivation for 96 h, cell-mineral mixtures were divided into four sets in order to compare the effects on cell recovery and mass spectrometry measurements of each step: fresh culture (Fc) served as the basis. Laboratory controls (Lc) were kept under controlled conditions (shielded from light in aluminum and air-sealed in a storage container under a laminar flow box at the CBM-CNRS Orl\u0026eacute;ans), whereas the transport controls (Tc) were transported with sets dedicated to the Mars simulation facility. Owing to laboratory constraints, UV-exposed (e) and laboratory controls were re-cultivated together for growth comparison, and while kept under controlled conditions, exposed without UV (ne) and transport controls were re-cultivated together one week later. Extraction was conducted continuously, while the remaining samples were stored at 4\u0026deg;C. Cell survival was determined by successful re-cultivation of microbial cells after desiccation and exposure to Mars-like conditions.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMicrobial cultivation and sample preparation\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eA. manzaensis\u003c/em\u003e was cultivated in a 1 L Schott-bottle bioreactor in the presence of ESA01-E basalt over a period of 96 h. After harvesting, 800 mL of cell-mineral material was concentrated to a total volume of 40 mL by a factor of 20, homogenized, and stored at 4\u0026deg;C for 72 h before deposition in the metal exposure wells. The deposition was performed by pipetting 300 \u0026micro;L into each metal exposure well and continuous evaporation to a total volume of 2 mL. The samples were stored at room temperature before and during transportation to and from the exposure facility.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eDesiccation experiment\u003c/strong\u003e: Prior to exposure to Mars-like conditions, test samples were subjected to prolonged desiccation for one month. Concentrated cell-mineral materials deposited in the metal exposure wells were air-dried completely, desiccated, and stored in a fume hood for one month. The samples were stored at room temperature and wrapped in sterilized aluminum foil. Thereafter, six samples (A\u003csub\u003e1\u0026ndash;3\u003c/sub\u003e and B\u003csub\u003e1\u0026ndash;3\u003c/sub\u003e) were transferred into 50 mL glass flasks for re-cultivation under the same conditions as described for the exposure experiment below.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eExposure experiment\u003c/strong\u003e: The exposure experiment was conducted with samples e and ne in the Mars Simulation Facility (MSF) at the Planetary Analogue Simulation Laboratory (PASLAB, DLR Berlin), Tc and Lc as controls, and Fc as a baseline. The MSF consists of a climate chamber (ATT Umweltsimulation GmbH), which contains a 10.3 L vacuum-sealed stainless-steel vessel (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA) and a rotating platform with eight round aluminum sample holders. The experimental chamber has electrical connectors, inlet/outlet gas connectors, four optical fibers for UV light provided by a 150 W Xenon lamp (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Additionally, the experiment chamber is equipped with two SHT75 sensors (Sensirion AG, Switzerland) integrated with two Pt-100 temperature sensors to measure humidity and temperature, calibrated for the Martian atmosphere. The gas flow through the experiment chamber is generated by a gas mixing system, and the inside pressure is controlled by a membrane vacuum pump (MV10Vario, Vacuubrand GmbH). The control of the whole system is based on a DAQ-system device (National Instruments Corp., US) and Labview software [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. The irradiance simulated the complete Martian solar spectrum (200\u0026ndash;2200 nm) of the mid-latitude summer Sun\u0026apos;s diurnal cycle [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. Fourteen days of irradiation resulted in a total dose per sample of 12029 kJ/m\u0026sup2; and a flux of 14 kW/m\u0026sup2;.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eRe-cultivation of exposed and non-exposed cultures\u003c/strong\u003e: Upon arrival of the samples from the exposure facility, those deposited in the metal exposure wells were split in half using a sterile scalpel. One-half of the sample was processed for organic extraction, and the other half was left in the metal sample holder and stored at room temperature under sterile conditions in a fume hood until re-cultivation. \u003cem\u003eA. manzaensis\u003c/em\u003e was re-cultivated according to the protocol described by K\u0026ouml;lbl \u003cem\u003eet al.\u003c/em\u003e (2020) [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. Re-cultivation was performed for each condition in four replicates: initial fresh culture (Fc), exposed to UV (e), non-exposed to UV (ne), laboratory control (Lc), and transport control (Tc). The cultivation was conducted in a shake incubator set at 75\u0026deg;C and 100 rpm over a period of 72 h in 50 mL glass flasks containing an equivalent of 1 mL concentrated cell-mineral material and 20 mL of \u003cem\u003eA. manzaensis\u003c/em\u003e specific medium [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e] including ESA01-E basalt (10 g/L), Allen trace element solution (0.91 mM MnCl\u003csub\u003e2\u003c/sub\u003e \u0026times; 4H\u003csub\u003e2\u003c/sub\u003eO, 1.18 mM Na\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e \u0026times; 10H\u003csub\u003e2\u003c/sub\u003eO, 0.08 mM ZnSO\u003csub\u003e4\u003c/sub\u003e \u0026times; 7H\u003csub\u003e2\u003c/sub\u003eO, 0.03 mM CuCl\u003csub\u003e2\u003c/sub\u003e \u0026times; 2H\u003csub\u003e2\u003c/sub\u003eO, 0.01 mM Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e \u0026times; 2H\u003csub\u003e2\u003c/sub\u003eO, 0.02 mM VOSO\u003csub\u003e4\u003c/sub\u003e \u0026times; 2H\u003csub\u003e2\u003c/sub\u003eO, and 3.56 \u0026micro;M CoSO\u003csub\u003e4\u003c/sub\u003e \u0026times; 7H\u003csub\u003e2\u003c/sub\u003eO) and tryptone (0.1%). To monitor cell growth, 1 mL aliquots of all samples were taken at 24 h intervals under sterile conditions in a laminar flow hood and counted in triplicate under a microscope with a hemocytometer (Neubauer chamber). The distinction between cells and mineral particles was conducted visually, taking into consideration cell movement, cell size (~\u0026thinsp;1 \u0026micro;m), and relief of mineral particles.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eUHPLC \u0026ndash; HRMS analysis\u003c/h3\u003e\n\u003cp\u003eAnalyses were conducted in reverse phase liquid chromatography (RPLC) with mass spectrometric detection using ESI in positive mode. Chromatographic separations were performed on an Ultimate 3000RSLC system (Dionex, Germering, Germany) using an Acquity UPLC BEH C18 1.7 \u0026micro;m 2.1\u0026times;100 mm column (Waters, Saint-Quentin-en-Yvelines, France). The column was heated at 60\u0026deg;C and the following solvents were used at a flow rate of 500 \u0026micro;L/min: H\u003csub\u003e2\u003c/sub\u003eO with 0.1% formic acid as solvent A and a mixture of methanol and isopropanol (50:50, v/v) with 0.1% formic acid as solvent B. Linear gradient elution was as follows: 0-2.5 min 3% B, 6% B at 4 min, 85% B at 13 min, a plateau at 100% B from 13.5 to 19 min and re-equilibration from 19.1 to 23 min at 3% B. Two \u0026micro;L of the organic extract were injected. Mass spectra were acquired on a maXis Q-TOF (Bruker Daltonik, Bremen, Germany) at a frequency of 1 Hz in the 50-1650 \u003cem\u003em/z\u003c/em\u003e range for MS experiments. The ESI source parameters were as follows: nebulizing gas, 2 bar; drying gas, 200\u0026deg;C at a flow rate of 9 L/min; capillary voltage, 4500 V. For MS/MS experiment in Data Dependent Acquisition (DDA) mode, the same parameters were used except the acquisition frequency: 2 Hz. Two precursors ions were selected per cycle into the 445 to 950 \u003cem\u003em/z\u003c/em\u003e range with exclusion after one spectrum for 0.07 min. Lock mass calibration was performed using ESI-TOF tuning mix (Agilent, Palo Alto, CA, USA) and lock masses at \u003cem\u003em/z\u003c/em\u003e 299.2945 [hexakis(1H,1H,4H-hexafluorobutyloxy)phosphazine; CAS #: 186406-47-3] and 1221.9906 [methyl stearate, CAS #: 112-61-8] for calibration correction and was applied to all MS and MS/MS spectra.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFeature detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular feature detection in UHPLC-HRMS measurements was performed using the Time-aligned Region Complete Extraction (T-ReX) 3D\u0026reg; feature-finder algorithm of MetaboScape 2024\u0026reg; (Bruker Daltonik GmbH, Bremen, Germany). For peak picking, a minimum intensity threshold of 5000 cts and a minimum peak length of four spectra across an \u003cem\u003em/z\u003c/em\u003e range of 250 Da to 2000 Da were set. The recursive feature-finding tool was deactivated and prior filtering ensured that only the features present in at least three measurements were retained. The primary ion was set to [M\u0026thinsp;+\u0026thinsp;H]+, with [M\u0026thinsp;+\u0026thinsp;NH\u003csub\u003e4\u003c/sub\u003e]\u0026thinsp;+\u0026thinsp;and [M\u0026thinsp;+\u0026thinsp;Na]\u0026thinsp;+\u0026thinsp;as the seed ions. When applicable, the adducts and doubly charged ions were grouped based on an ion deconvolution threshold of 0.8. Quinone annotation was performed utilizing a comprehensive target list of potentially occurring sulfur-bearing quinones, adhering to a maximum \u003cem\u003em/z\u003c/em\u003e tolerance of 2.5 ppm, an mSigma value of 40 (narrow) to 250 (wide), and an MS/MS similarity score of \u0026gt;\u0026thinsp;800, if MS/MS spectra were present. The extracted ion chromatograms (EIC) of the annotated compounds were reviewed, and anomalous peaks were excluded.\u003c/p\u003e"},{"header":"Results \u0026 Discussion","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eDesiccation experiment\u003c/h2\u003e\u003cp\u003eAfter successful cultivation of \u003cem\u003eA. manzaensis\u003c/em\u003e on the Mars analog material ESA01-E (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), a desiccation experiment was conducted on the test samples to verify their stability and resistance to desiccation, which is a critical parameter for Mars-like exposure. The analog material ESA01-E is known for its physicochemical similarity to Martian basalt, and the dehydration experiment was conducted over a period of one month. Concentrated cell pellets were deposited into metal exposure wells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) to investigate cell survival upon the recovery of the dried pellets. The cells were recovered, and cell survival was demonstrated by successful re-cultivation of previously desiccated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In the two triplicate sets (A\u003csub\u003e1\u0026ndash;3\u003c/sub\u003e and B\u003csub\u003e1\u0026ndash;3\u003c/sub\u003e), cell numbers doubled during the cultivation period of 72 h. The mean number of cells reached between 2.5\u0026ndash;3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL throughout all samples. However, variation in growth was observed in sample A\u003csub\u003e1\u003c/sub\u003e, which could have been caused by a non-homogeneous mixture of cell-mineral materials used for the incubation. Previous dehydration studies performed with \u003cem\u003eM. sedula\u003c/em\u003e have shown cell revival after two months of desiccation following re-cultivation in heterotrophic medium and varying cell numbers based on their mineral substrates, including Mars analog material [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This validates their use for subsequent exposure to Mars-like conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTwo-week exposure experiment in a Mars Simulation Facility\u003c/h3\u003e\n\u003cp\u003eThe sensors integrated in the simulation chamber successfully recorded the physico-chemical parameters of the experiment over the course of 340h\u0026thinsp;=\u0026thinsp;14 days, as displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The following parameters correspond to an approximate measurement distance of 1 cm above the samples in the sample holder of the Mars Simulation Facility: temperature [\u0026deg;C], measured humidity over water [%], and measured humidity over ice [%]. Pressure [hPa] is the pressure inside the experimental chamber. The pressure was gradually decreased to 8 hPa. The LED temperature sensor [V] represents the day/night cycle (higher values are nighttime [8h] and lower values are daytime [16h]). All measurements showed excellent adequacy with the input values (day-to-night: temperature of +\u0026thinsp;15 to -50\u0026deg;C and relative humidity of 0 to 75%), successfully recreating day/night cycles.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn relation to the samples before exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), a change in the physical appearance (discoloration) of the samples exposed to UV light (e) was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) compared to the unexposed samples (ne). Samples e, ne, Tc, and Lc were successfully re-cultivated in triplicate upon arrival (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) for 72 h and compared to the re-cultivation of fresh cell-mineral material (Fc) immediately after harvesting. Although Fc showed the highest initial cell number, no significant trend was observed between e, ne, Tc, and Lc. However, the cell number increased in comparison to initial concentration by 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL in all samples.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNo significant difference was noted after one month of desiccation in normal atmosphere and 8 hPa for two weeks. Although UV-exposed samples appeared bleached in appearance, the survival fraction was not affected by radiation exposure, with very comparable values reached after re-cultivation across all sets. These results are in agreement with those of previous experiments conducted by Mastascusa \u003cem\u003eet al.\u003c/em\u003e (2014), who assessed the survival by successful re-cultivation of the comparable extremely thermoacidophilic archaea \u003cem\u003eSulfolobus solfataricus\u003c/em\u003e, in the same order as \u003cem\u003eA. manzaensis\u003c/em\u003e (Sulfolobales), to simulated space conditions: good resistance to temperature variations (one week of -196 to 85\u0026deg;C) and a high resistance to UV exposure (1 h to λ\u0026thinsp;=\u0026thinsp;254 nm) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, regarding UV exposure, it must be considered that in our experiments, we did not expose thin films spread in Petri dishes, but cell-mineral mixtures in metal exposure wells. Therefore, only the first millimeter was irradiated and the fraction below the surface largely contributed to the survival, which was subjected to exposure to other extreme conditions, such as temperature variations, pressure, and humidity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCharacterization of thiophene-bearing quinones in Sulfolobales\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe quinone composition of the order Sulfolobales consists of oxidized caldariellaquinones (CQ), sulfolobusquinones (SQ), and benzodithiophenequinones (BDTQ). Members of the Sulfolobales order exhibit varying compositions of saturated quinones in response to their redox environments. As a result, the quinone profile of a specific Sulfolobales member can be utilized to reconstruct the environmental redox conditions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Nevertheless, the proportions of quinones vary among different Sulfolobales species. Consequently, adaptations to environmental conditions might be mirrored in the distributions of SQs, CQs, and BDTQs [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Analyzing quinone profiles could facilitate the monitoring of microbial community transitions from oxic to anoxic conditions and help characterize archaeal diversity, complementing approaches based on membrane lipids and genes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCaldariellaquinones were initially identified by de Rosa \u003cem\u003eet al.\u003c/em\u003e (1977) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], which are produced by organisms that thrive in harsh conditions (pH 1.4\u0026ndash;2.6; 75\u0026ndash;89\u0026deg;C), with a robust membrane structure observed in Sulfolobus and Acidianus species [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Later, caldariellaquinones were found in \u003cem\u003eS. solfataricus\u003c/em\u003e and subsequently in \u003cem\u003eM. sedula\u003c/em\u003e [\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], whereas benzodithiophenequinones were detected in \u003cem\u003eS. solfataricus\u003c/em\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Variations in the CQ, SQ, and BDTG molecules produced are linked to the presence of oxygen during growth [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. For the Sulfolobales order, Elling \u003cem\u003eet al.\u003c/em\u003e (2016) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] reported a distribution of CQ\u003csub\u003e6:0\u003c/sub\u003e (86.1%) and CQ\u003csub\u003e6:1\u003c/sub\u003e (12.2%) in \u003cem\u003eS. acidocaldarius\u003c/em\u003e, CQ\u003csub\u003e6:0\u003c/sub\u003e (85.8%) and CQ\u003csub\u003e6:1\u003c/sub\u003e (13.7%) in \u003cem\u003eS. solfataricus\u003c/em\u003e, and SQ\u003csub\u003e6:0\u003c/sub\u003e (42.9%), CQ\u003csub\u003e6:0\u003c/sub\u003e (36.4%), and CQ\u003csub\u003e6:1\u003c/sub\u003e (14.6%) in \u003cem\u003eS. islandicus\u003c/em\u003e as the main quinone components, with BDTQ\u003csub\u003e6:0\u003c/sub\u003e (0.4%) only found in \u003cem\u003eS. islandicus\u003c/em\u003e. Additionally, Gfellner \u003cem\u003eet al.\u003c/em\u003e (2025) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] reported a distribution of SQ\u003csub\u003e4:0\u003c/sub\u003e (2.7), SQ\u003csub\u003e4:1\u003c/sub\u003e (66.7), SQ\u003csub\u003e5:0\u003c/sub\u003e (8.8), SQ\u003csub\u003e5:1\u003c/sub\u003e (5.5), CQ\u003csub\u003e4:1\u003c/sub\u003e (2.6), CQ\u003csub\u003e5:1\u003c/sub\u003e (6.9), and BDTQ\u003csub\u003e5:0\u003c/sub\u003e (6.8) in \u003cem\u003eM. sedula\u003c/em\u003e.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAstrobiological relevance of thiophene-bearing quinones\u003c/h2\u003e\u003cp\u003eAfter the growth of \u003cem\u003eMetallosphaera sedula\u003c/em\u003e on Martian meteorite material, Milojevic \u003cem\u003eet al.\u003c/em\u003e (2021) [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] detected possible S-bonds of lipid chains, indicating the possible presence of thiophene-bearing quinones. A closer examination of the differences in the thiophene headgroup properties (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) revealed potential molecular target sites for molecular alterations in the form of methylation, S-linkage properties, and/or number of cycles. However, exposure to Mars-like conditions could likely alter their molecular signatures over time. Therefore, in addition to desiccation, the exposure experiment under Mars-like conditions was conducted to clarify the role of these molecules as potential biosignatures for life on Mars and other celestial bodies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePlanetary environmental conditions must be considered when assessing the preservation potential of biomarkers over billions of years. On Earth, the occurrence of thiophenes associated with Archaean sediments (4.0\u0026ndash;2.5 Ga) is related to the diagenetic and metamorphic history of the sediments [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Primary organic matter in microbial phototrophic colonies is degraded by heterotrophic microbes such as sulfur reducers, during this process the sulfur content of the organic matter is enriched [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Metamorphism converts sulfur-containing molecules into aromatic organic structures such as thiophene. On the other hand, Mars\u0026rsquo; metamorphic history is different from that on Earth. The planet is approximately half the size of the Earth and lacks plate tectonics. Any sedimentary rocks would have therefore been subjected to a lower degree of metamorphism, except shock metamorphism from meteorite impacts [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. It is therefore important to take into account the general environmental context and its geological setting in order to distinguish between plausible biotic and abiotic settings. Exposure of thiophene-bearing quinones at the surface of Mars to 3\u0026ndash;4 Ga of UV, galactic, and cosmic radiations will affect the survivability of the basic thiophene moieties; however, data from gale Crater on Mars [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] shows that they may still be preserved in the Martian environment, although determination of their biogenicity remains to be proven.\u003c/p\u003e\u003cp\u003eHereby, we wanted to address the following: Is there a difference in the saturations of thiophene-bearing quinones between the exposed and non-exposed samples? In particular, are there any differences in the fragmented thiophene headgroups detectable by MS/MS analysis?\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eThiophene-bearing quinones after exposure to Mars-like conditions\u003c/h3\u003e\n\u003cp\u003eThe first tentative annotation based on MS analysis using MetaboScape\u0026reg; (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) across all samples (e, ne, Tc, Lc, and Fc) revealed the heterogeneous distribution of differently oxidized thiophene-bearing quinones (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). We were able to annotate for the following oxidation states for CQs: CQ\u003csub\u003e4:0\u003c/sub\u003e, CQ\u003csub\u003e8:6\u003c/sub\u003e, CQ\u003csub\u003e9:3\u003c/sub\u003e, CQ\u003csub\u003e9:5\u003c/sub\u003e, CQ\u003csub\u003e9:6\u003c/sub\u003e; for SQs: SQ\u003csub\u003e4:0\u003c/sub\u003e, SQ\u003csub\u003e6:0\u003c/sub\u003e, SQ\u003csub\u003e7:2\u003c/sub\u003e, SQ\u003csub\u003e7:4\u003c/sub\u003e, SQ\u003csub\u003e8:0\u003c/sub\u003e, SQ\u003csub\u003e8:6\u003c/sub\u003e, SQ\u003csub\u003e10:2\u003c/sub\u003e, SQ\u003csub\u003e10:5\u003c/sub\u003e, SQ\u003csub\u003e10:7\u003c/sub\u003e, SQ\u003csub\u003e10:10\u003c/sub\u003e; and for BDTQs: BDTQ\u003csub\u003e4:1\u003c/sub\u003e, BDTQ\u003csub\u003e8:3\u003c/sub\u003e, BDTQ\u003csub\u003e8:4\u003c/sub\u003e, BDTQ\u003csub\u003e9:1\u003c/sub\u003e, BDTQ\u003csub\u003e9:4\u003c/sub\u003e, BDTQ\u003csub\u003e9:7\u003c/sub\u003e, BDTQ\u003csub\u003e10:0\u003c/sub\u003e, BDTQ\u003csub\u003e10:7\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHowever, certain indicative features, CQ\u003csub\u003e9:5,\u003c/sub\u003e SQ\u003csub\u003e7:4\u003c/sub\u003e, and SQ\u003csub\u003e8:0\u003c/sub\u003e, were only found in the immediately extracted samples after cultivation. SQ\u003csub\u003e10:2\u003c/sub\u003e and SQ\u003csub\u003e10:5\u003c/sub\u003e were only present in the exposed samples, but also in the transport control, and SQ\u003csub\u003e10:7\u003c/sub\u003e, SQ\u003csub\u003e10:10\u003c/sub\u003e, BDTQ\u003csub\u003e4:1\u003c/sub\u003e, and BDTQ\u003csub\u003e10:0\u003c/sub\u003e were not present in the exposed sample. This poses a challenge when ascribing features to actual exposure. The analyzed samples were organic extracts of material immediately scratched from the metal exposure wells after exposure to resolve minor changes in the headgroups of thiophene-bearing quinones visible by energetic fragmentation using MS/MS analysis. However, the generally low yield of the corresponding MS/MS spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) complicates this approach. Therefore, to observe the changes in the distribution of thiophene-bearing quinones before and after exposure, additional organic extraction of the remaining re-cultivated samples is necessary. Because of the low penetration depth of UV radiation in the cell-mineral mixture in the metal exposure wells, only a small fraction of the cells were exposed to the potential degradation of thiophene-bearing quinones, especially their thiophene moieties. A different sample preparation procedure might also solve this problem; however, complementary analyses, such as survival, would not have been possible if thin films were used in Petri dishes. Additionally, a more powerful source of radiation might better mirror the induced changes in geological timescales of thiophene moieties and ultimately shed light on specific markers of biologically produced thiophenes. Therefore, we propose a continuation of a radiation experiment with a proton ion source (\u003cem\u003ee.g.\u003c/em\u003e, Cyclotron at the CEMHTI laboratory in Orl\u0026eacute;ans, France) or an exposure platform in Low Earth Orbit, as shown by the completed BIOMEX [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] and planned BioSigN projects [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] or future planned space experiment platforms [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we demonstrated the reproducibility of the improved metabolite extraction protocol derived by Gfellner \u003cem\u003eet al.\u003c/em\u003e (2025) in terms of its applicability to different archaeal species (\u003cem\u003eM. sedula\u003c/em\u003e and \u003cem\u003eA. manzaensis\u003c/em\u003e) and mineral materials (pyrite and Mars analog material). Furthermore, we successfully re-cultivated cells after exposure to one month of desiccation, demonstrating their survival to water depletion, and two weeks of simulated Mars-like conditions, proving their survival under extreme temperature fluctuations ranging from +\u0026thinsp;15 to -50\u0026deg;C in diurnal cycles (16h daylight and 8h night), exposure to UV radiation (200\u0026ndash;2200 nm), and Martian atmosphere and pressure (96% CO\u003csub\u003e2\u003c/sub\u003e, 4% air; 8 hPa). This shows the relevance of thermophilic archaea in astrobiology research, especially \u003cem\u003eA. manzaensis\u003c/em\u003e as a model organism. However, further analysis is necessary for a direct correlation between potential changes in the distribution of thiophene-bearing quinones, followed by exposure to Mars-like conditions. Improved sample preparation and different radiation sources are being evaluated to further assess the relevance of thiophene-bearing quinones as model biosignatures, which will guide current and future missions searching for signs of life on Mars.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded under the European Union's Horizon 2020 Framework Programme, grant number ERC-2020-COG, Project 101001311 – BIOMAMA and the French National Research Agency (ANR, CPJ n°ANR-22-CPJ1-0066-01). We thank the Région Centre Val de Loire (SyMBioMS and Malditof grants) and the European FEDER funds (grants no 2699-33931 and 2017-EX002979) for supporting MS instruments and the SALSA and MO2VING platforms. This research was partially supported by the Helmholtz Association through the Helmholtz Alliance “Planetary Evolution and Life”. Furthermore, we acknowledge the use of MetaboScape®, supported by Germany’s Excellence Strategy (EXC-2077) project 390741603 ‘The Ocean Floor – Earth’s Uncharted Interface’. In addition, we thank the members of the Center for Molecular Biophysics, Orléans, France, in particular Matthieu Réfrégiers for his guidance throughout the entire process, Martine Cadene for her support in planning the workflow for the experimental design, and Frances Westall for her input on the preservation of thiophene-bearing quinones in the geological rock record.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MS and MS/MS data used for the tentative annotation of thiophene-bearing quinones\u0026nbsp;can be found in \u003cstrong\u003eSupplementary Table S1, Tentative annotation using Metaboscape®\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.V.G. and M.B. designed the exposure experiments. S.V.G. and C.C. performed the analyses. S.V.G., J.G., and G.G. processed, curated, and validated the experimental data. A.L., M.B., and S.G. operated the Mars Simulation Facility. All authors contributed to the editing and discussion of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003ePollack, J. B., Kasting, J. F., Richardson, S. M. \u0026amp; Poliakoff, K. The case for a wet, warm climate on early Mars. \u003cem\u003eIcarus\u003c/em\u003e\u003cstrong\u003e71\u003c/strong\u003e, 203\u0026ndash;224 (1987).\u003c/li\u003e\n \u003cli\u003eWordsworth, R. D. The Climate of Early Mars. \u003cem\u003eAnnu. Rev. Earth Planet. Sci.\u003c/em\u003e\u003cstrong\u003e44\u003c/strong\u003e, 381\u0026ndash;408 (2016).\u003c/li\u003e\n \u003cli\u003eVago, J. 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A Systematic Way to Life Detection: Combining Field, Lab and Space Research in Low Earth Orbit. in \u003cem\u003eAdvances in Astrobiology and Biogeophysics\u003c/em\u003e 111\u0026ndash;122 (Springer International Publishing, Cham, 2019).\u003c/li\u003e\n \u003cli\u003eElsaesser, A. \u003cem\u003eet al.\u003c/em\u003e Future space experiment platforms for astrobiology and astrochemistry research. \u003cem\u003enpj Microgravity\u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"exposure experiment, Mars simulation chamber, metabolomics, biomarkers, astrobiology","lastPublishedDoi":"10.21203/rs.3.rs-7565488/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7565488/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn the search for reliable biomarkers in other planetary environments such as Mars, molecules that are stable over geological timescales, while preserving indicative information about their potential biological origin, are crucially needed. Thiophene-bearing quinones fulfill these requirements, and thiophenes, which are their basic moieties, have been found on Mars. However, thiophenes can be produced abiotically and have been found in meteorites. Furthermore, the Martian environment may alter their molecular structure over time. To evaluate whether there could be a distinction between biotically and abiotically produced thiophenes, considering the harsh environmental conditions on Mars, we cultivated the extremophilic archaeon \u003cem\u003eAcidianus manzaensis\u003c/em\u003e on ESA01-E Mars analog material. We then exposed the cell-mineral samples to one month of desiccation and Mars-like conditions in a Mars simulation chamber to analyze changes in the composition of thiophene-bearing quinones using mass spectrometry-based metabolomics, and evaluated the potential for cell recovery after exposure.\u003c/p\u003e","manuscriptTitle":"Investigating changes in thiophene-bearing quinones of the archaeon Acidianus manzaensis after exposure to Mars-like conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-29 10:13:02","doi":"10.21203/rs.3.rs-7565488/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8f2551d2-4f42-4487-8d7f-03f3bb943c70","owner":[],"postedDate":"September 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55450956,"name":"Biological sciences/Biochemistry"},{"id":55450957,"name":"Physical sciences/Chemistry"},{"id":55450958,"name":"Earth and environmental sciences/Planetary science"}],"tags":[],"updatedAt":"2025-10-16T01:23:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-29 10:13:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7565488","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7565488","identity":"rs-7565488","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}