Microbial Ecosystems in Movile Cave: An Environment of Extreme Life.

Movile Cave, situated in Romania close to the Black Sea, constitutes a distinct and challenging environment for life. Its partially submerged ecosystem depends on chemolithotrophic processes for its energetics, which are fed by a continuous hypogenic inflow of mesothermal waters rich in reduced chemicals such as hydrogen sulfide and methane. We sampled a variety of cave sublocations over the course of three years. Furthermore, in a microcosm experiment, minerals were incubated in the cave waters for one year. Both endemic cave samples and extracts from the minerals were subjected to 16S rRNA amplicon sequencing. The sequence data show specific community profiles in the different subenvironments, indicating that specialized prokaryotic communities inhabit the different zones in the cave. Already after one year, the different incubated minerals had been colonized by specific microbial communities, indicating that microbes in Movile Cave can adapt in a relatively short timescale to environmental opportunities in terms of energy and nutrients. Life can thrive, diversify and adapt in remote and isolated subterranean environments such as Movile Cave.

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

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

Taxonomic and functional analyses of intact microbial communities thriving in extreme, astrobiology-relevant, anoxic sites.

BACKGROUND: Extreme terrestrial, analogue environments are widely used models to study the limits of life and to infer habitability of extraterrestrial settings. In contrast to Earth's ecosystems, potential extraterrestrial biotopes are usually characterized by a lack of oxygen. METHODS: In the MASE project (Mars Analogues for Space Exploration), we selected representative anoxic analogue environments (permafrost, salt-mine, acidic lake and river, sulfur springs) for the comprehensive analysis of their microbial communities. We assessed the microbiome profile of intact cells by propidium monoazide-based amplicon and shotgun metagenome sequencing, supplemented with an extensive cultivation effort. RESULTS: The information retrieved from microbiome analyses on the intact microbial community thriving in the MASE sites, together with the isolation of 31 model microorganisms and successful binning of 15 high-quality genomes allowed us to observe principle pathways, which pinpoint specific microbial functions in the MASE sites compared to moderate environments. The microorganisms were characterized by an impressive machinery to withstand physical and chemical pressures. All levels of our analyses revealed the strong and omnipresent dependency of the microbial communities on complex organic matter. Moreover, we identified an extremotolerant cosmopolitan group of 34 poly-extremophiles thriving in all sites. CONCLUSIONS: Our results reveal the presence of a core microbiome and microbial taxonomic similarities between saline and acidic anoxic environments. Our work further emphasizes the importance of the environmental, terrestrial parameters for the functionality of a microbial community, but also reveals a high proportion of living microorganisms in extreme environments with a high adaptation potential within habitability borders. Video abstract.

The Detection of Elemental Signatures of Microbes in Martian Mudstone Analogs Using High Spatial Resolution Laser Ablation Ionization Mass Spectrometry.

The detection and identification of biosignatures on planetary bodies such as Mars in situ is extremely challenging. Current knowledge from space exploration missions suggests that a suite of complementary instruments is required in situ for a successful identification of past or present life. For future exploration missions, new and innovative instrumentation capable of high spatial resolution chemical (elemental and isotope) analysis of solids with improved measurement capabilities is of considerable interest because a multitude of potential signatures of extinct or extant life have dimensions on the micrometer scale. The aim of this study is to extend the current measurement capabilities of a miniature laser ablation ionization mass spectrometer (LIMS) designed for space exploration missions to detect signatures of microbial life. In total, 14 martian mudstone analogue samples were investigated regarding their elemental composition. Half the samples were artificially inoculated with a low number density of microbes, and half were used as abiotic controls. The samples were treated in a number of ways. Some were cultured anaerobically and some aerobically; some abiotic samples were incubated with water, and some remained dry. Some of the samples were exposed to a large dose of γ radiation, and some were left un-irradiated. While no significant elemental differences were observed between the applied sample treatments, the instrument showed the capability to detect biogenic element signatures of the inoculated microbes by monitoring biologically relevant elements, such as hydrogen, carbon, sulfur, iron, and so on. When an enrichment in carbon was measured in the samples but no simultaneous increase in other biologically relevant elements was detected, it suggests, for example, a carbon-containing inclusion; when the enrichment was in carbon and in bio-relevant elements, it suggests the presences of microbes. This study presents first results on the detection of biogenic element patterns of microbial life using a miniature LIMS system designed for space exploration missions.

ORIGIN: a novel and compact Laser Desorption - Mass Spectrometry system for sensitive in situ detection of amino acids on extraterrestrial surfaces.

For the last four decades space exploration missions have searched for molecular life on planetary surfaces beyond Earth. Often pyrolysis gas chromatography mass spectrometry has been used as payload on such space exploration missions. These instruments have relatively low detection sensitivity and their measurements are often undermined by the presence of chloride salts and minerals. Currently, ocean worlds in the outer Solar System, such as the icy moons Europa and Enceladus, represent potentially habitable environments and are therefore prime targets for the search for biosignatures. For future space exploration missions, novel measurement concepts, capable of detecting low concentrations of biomolecules with significantly improved sensitivity and specificity are required. Here we report on a novel analytical technique for the detection of extremely low concentrations of amino acids using ORIGIN, a compact and lightweight laser desorption ionization - mass spectrometer designed and developed for in situ space exploration missions. The identified unique mass fragmentation patterns of amino acids coupled to a multi-position laser scan, allows for a robust identification and quantification of amino acids. With a detection limit of a few fmol mm-2, and the possibility for sub-fmol detection sensitivity, this measurement technique excels current space exploration systems by three orders of magnitude. Moreover, our detection method is not affected by chemical alterations through surface minerals and/or salts, such as NaCl that is expected to be present at the percent level on ocean worlds. Our results demonstrate that ORIGIN is a promising instrument for the detection of signatures of life and ready for upcoming space missions, such as the Europa Lander.

Biosignature Analysis of Mars Soil Analogs from the Atacama Desert: Challenges and Implications for Future Missions to Mars.

The detection of biosignatures on Mars is of outstanding interest in the current field of astrobiology and drives various fields of research, ranging from new sample collection strategies to the development of more sensitive detection techniques. Detailed analysis of the organic content in Mars analog materials collected from extreme environments on Earth improves the current understanding of biosignature preservation and detection under conditions similar to those of Mars. In this article, we examined the biological fingerprint of several locations in the Atacama Desert (Chile), which include different wet and dry, and intermediate to high elevation salt flats (also named salars). Liquid chromatography and multidimensional gas chromatography mass spectrometry measurement techniques were used for the detection and analysis of amino acids extracted from the salt crusts and sediments by using sophisticated extraction procedures. Illumina 16S amplicon sequencing was used for the identification of microbial communities associated with the different sample locations. Although amino acid load and organic carbon and nitrogen quantities were generally low, it was found that most of the samples harbored complex and versatile microbial communities, which were dominated by (extremely) halophilic microorganisms (most notably by species of the Archaeal family Halobacteriaceae). The dominance of salts (i.e., halites and sulfates) in the investigated samples leaves its mark on the composition of the microbial communities but does not appear to hinder the potential of life to flourish since it can clearly adapt to the higher concentrations. Although the Atacama Desert is one of the driest and harshest environments on Earth, it is shown that there are still sub-locations where life is able to maintain a foothold, and, as such, salt flats could be considered as interesting targets for future life exploration missions on Mars.

Impact of Simulated Martian Conditions on (Facultatively) Anaerobic Bacterial Strains from Different Mars Analogue Sites.

Five bacterial (facultatively) anaerobic strains, namely Buttiauxella sp. MASE-IM-9, Clostridium sp. MASE-IM-4, Halanaerobium sp. MASE-BB-1, Trichococcus sp. MASE-IM-5, and Yersinia intermedia MASE-LG-1 isolated from different extreme natural environments were subjected to Mars relevant environmental stress factors in the laboratory under controlled conditions. These stress factors encompassed low water activity, oxidizing compounds, and ionizing radiation. Stress tests were performed under permanently anoxic conditions. The survival rate after addition of sodium perchlorate (Na-perchlorate) was found to be species-specific. The inter-comparison of the five microorganisms revealed that Clostridium sp. MASE-IM-4 was the most sensitive strain (D10-value (15 min, NaClO4) = 0.6 M). The most tolerant microorganism was Trichococcus sp. MASE-IM-5 with a calculated D10-value (15 min, NaClO4) of 1.9 M. Cultivation in the presence of Na-perchlorate in Martian relevant concentrations up to 1 wt% led to the observation of chains of cells in all strains. Exposure to Na-perchlorate led to a lowering of the survival rate after desiccation. Consecutive exposure to desiccating conditions and ionizing radiation led to additive effects. Moreover, in a desiccated state, an enhanced radiation tolerance could be observed for the strains Clostridium sp. MASE-IM-4 and Trichococcus sp. MASE-IM-5. These data show that anaerobic microorganisms from Mars analogue environments can resist a variety of Martian-simulated stresses either individually or in combination. However, responses were species-specific and some Mars-simulated extremes killed certain organisms. Thus, although Martian stresses would be expected to act differentially on microorganisms, none of the expected extremes tested here and found on Mars prevent the growth of anaerobic microorganisms.

Microbial Markers Profile in Anaerobic Mars Analogue Environments Using the LDChip (Life Detector Chip) Antibody Microarray Core of the SOLID (Signs of Life Detector) Platform.

One of the main objectives for astrobiology is to unravel and explore the habitability of environments beyond Earth, paying special attention to Mars. If the combined environmental stress factors on Mars are compatible with life or if they were less harsh in the past, to investigate the traces of past or present life is critical to understand its potential habitability. Essential for this research is the characterization of Mars analogue environments on Earth through the development of techniques for biomarker detection in them. Biosensing techniques based on fluorescence sandwich microarray immunoassays (FSMI) have shown to be a powerful tool to detect biosignatures and depict the microbial profiles of different environments. In this study, we described the microbial biomarker profile of five anoxic Mars analogues sites using the Life Detector Chip (LDChip), an antibody microarray for multiple microbial marker detection. Furthermore, we contributed to new targets by developing a new 26-polyclonal antibodies microarray using crude extracts from anaerobic sampling sites, halophilic microorganisms, and anaerobic isolates obtained in the framework of the European Mars Analogues for Space Exploration (MASE) project. The new subset of antibodies was characterized and implemented into a microarray platform (MASE-Chip) for microbial marker searching in salty and anaerobic environments.

Detectability of biosignatures in a low-biomass simulation of martian sediments.

Discovery of a remnant habitable environment by the Mars Science Laboratory in the sedimentary record of Gale Crater has reinvigorated the search for evidence of martian life. In this study, we used a simulated martian mudstone material, based on data from Gale Crater, that was inoculated and cultured over several months and then dried and pressed. The simulated mudstone was analysed with a range of techniques to investigate the detectability of biosignatures. Cell counting and DNA extraction showed a diverse but low biomass microbial community that was highly dispersed. Pellets were analysed with bulk Elemental Analysis - Isotope Ratio Mass Spectrometry (EA-IRMS), high-resolution Laser-ablation Ionisation Mass Spectrometry (LIMS), Raman spectroscopy and Fourier Transform InfraRed (FTIR) spectroscopy, which are all techniques of relevance to current and future space missions. Bulk analytical techniques were unable to differentiate between inoculated samples and abiotic controls, despite total levels of organic carbon comparable with that of the martian surface. Raman spectroscopy, FTIR spectroscopy and LIMS, which are high sensitivity techniques that provide chemical information at high spatial resolution, retrieved presumptive biosignatures but these remained ambiguous and the sedimentary matrix presented challenges for all techniques. This suggests challenges for detecting definitive evidence for life, both in the simulated lacustrine environment via standard microbiological techniques and in the simulated mudstone via analytical techniques with relevance to robotic missions. Our study suggests that multiple co-incident high-sensitivity techniques that can scan the same micrometre-scale spots are required to unambiguously detect biosignatures, but the spatial coverage of these techniques needs to be high enough not to miss individual cellular-scale structures in the matrix.

Proteomic and Metabolomic Characteristics of Extremophilic Fungi Under Simulated Mars Conditions.

Filamentous fungi have been associated with extreme habitats, including nuclear power plant accident sites and the International Space Station (ISS). Due to their immense adaptation and phenotypic plasticity capacities, fungi may thrive in what seems like uninhabitable niches. This study is the first report of fungal survival after exposure of monolayers of conidia to simulated Mars conditions (SMC). Conidia of several Chernobyl nuclear accident-associated and ISS-isolated strains were tested for UV-C and SMC sensitivity, which resulted in strain-dependent survival. Strains surviving exposure to SMC for 30 min, ISSFT-021-30 and IMV 00236-30, were further characterized for proteomic, and metabolomic changes. Differential expression of proteins involved in ribosome biogenesis, translation, and carbohydrate metabolic processes was observed. No significant metabolome alterations were revealed. Lastly, ISSFT-021-30 conidia re-exposed to UV-C exhibited enhanced UV-C resistance when compared to the conidia of unexposed ISSFT-021.

Microbial Communities in Sediments From Four Mildly Acidic Ephemeral Salt Lakes in the Yilgarn Craton (Australia) - Terrestrial Analogs to Ancient Mars.

The Yilgarn Craton in Australia has a large number of naturally occurring shallow ephemeral lakes underlain by a dendritic system of paleodrainage channels. Processes like evaporation, flooding, erosion, as well as inflow of saline, often acidic and ion-rich groundwater contribute to the (dynamic) nature of the lakes and the composition of the sediments. The region has previously been described as an analog environment for early Mars due to its geological and geophysical similarities. Here, we investigated sediment samples of four lake environments aimed at getting a fundamental understanding of the native microbial communities and the mineralogical and (bio)chemical composition of the sediments they are associated with. The dominant mineral phases in the sediments were quartz, feldspars and amphiboles, while halite and gypsum were the only evaporites detected. Element analysis revealed a rich and complex image, in which silicon, iron, and aluminum were the dominant ions, but relative high concentrations of trace elements such as strontium, chromium, zirconium, and barium were also found. The concentrations of organic carbon, nitrogen, and phosphorus were generally low. 16S amplicon sequencing on the Illumina platform showed the presence of diverse microbial communities in all four lake environments. We found that most of the communities were dominated by extremely halophilic Archaea of the Halobacteriaceae family. The dynamic nature of these lakes appears to influence the biological, biochemical, and geological components of the ecosystem to a large effect. Inter- and intra-lake variations in the distributions of microbial communities were significant, and could only to a minor degree be explained by underlying environmental conditions. The communities are likely significantly influenced by small scale local effects caused by variations in geological settings and dynamic interactions caused by aeolian transport and flooding and evaporation events.

Beyond Chloride Brines: Variable Metabolomic Responses in the Anaerobic Organism Yersinia intermedia MASE-LG-1 to NaCl and MgSO4 at Identical Water Activity.

Growth in sodium chloride (NaCl) is known to induce stress in non-halophilic microorganisms leading to effects on the microbial metabolism and cell structure. Microorganisms have evolved a number of adaptations, both structural and metabolic, to counteract osmotic stress. These strategies are well-understood for organisms in NaCl-rich brines such as the accumulation of certain organic solutes (known as either compatible solutes or osmolytes). Less well studied are responses to ionic environments such as sulfate-rich brines which are prevalent on Earth but can also be found on Mars. In this paper, we investigated the global metabolic response of the anaerobic bacterium Yersinia intermedia MASE-LG-1 to osmotic salt stress induced by either magnesium sulfate (MgSO4) or NaCl at the same water activity (0.975). Using a non-targeted mass spectrometry approach, the intensity of hundreds of metabolites was measured. The compatible solutes L-asparagine and sucrose were found to be increased in both MgSO4 and NaCl compared to the control sample, suggesting a similar osmotic response to different ionic environments. We were able to demonstrate that Yersinia intermedia MASE-LG-1 accumulated a range of other compatible solutes. However, we also found the global metabolic responses, especially with regard to amino acid metabolism and carbohydrate metabolism, to be salt-specific, thus, suggesting ion-specific regulation of specific metabolic pathways.

Lack of correlation of desiccation and radiation tolerance in microorganisms from diverse extreme environments tested under anoxic conditions.

Four facultative anaerobic and two obligate anaerobic bacteria were isolated from extreme environments (deep subsurface halite mine, sulfidic anoxic spring, mineral-rich river) in the frame MASE (Mars Analogues for Space Exploration) project. The isolates were investigated under anoxic conditions for their survivability after desiccation up to 6 months and their tolerance to ionizing radiation up to 3000 Gy. The results indicated that tolerances to both stresses are strain-specific features. Yersinia intermedia MASE-LG-1 showed a high desiccation tolerance but its radiation tolerance was very low. The most radiation-tolerant strains were Buttiauxella sp. MASE-IM-9 and Halanaerobium sp. MASE-BB-1. In both cases, cultivable cells were detectable after an exposure to 3 kGy of ionizing radiation, but cells only survived desiccation for 90 and 30 days, respectively. Although a correlation between desiccation and ionizing radiation resistance has been hypothesized for some aerobic microorganisms, our data showed that there was no correlation between tolerance to desiccation and ionizing radiation, suggesting that the physiological basis of both forms of tolerances is not necessarily linked. In addition, these results indicated that facultative and obligate anaerobic organisms living in extreme environments possess varied species-specific tolerances to extremes.

The ESO Diffuse Interstellar Bands Large Exploration Survey: EDIBLES I. Project description, survey sample and quality assessment.

The carriers of the diffuse interstellar bands (DIBs) are largely unidentified molecules ubiquitously present in the interstellar medium (ISM). After decades of study, two strong and possibly three weak near-infrared DIBs have recently been attributed to the [Formula: see text] fullerene based on observational and laboratory measurements. There is great promise for the identification of the over 400 other known DIBs, as this result could provide chemical hints towards other possible carriers. In an effort to systematically study the properties of the DIB carriers, we have initiated a new large-scale observational survey: the ESO Diffuse Interstellar Bands Large Exploration Survey (EDIBLES). The main objective is to build on and extend existing DIB surveys to make a major step forward in characterising the physical and chemical conditions for a statistically significant sample of interstellar lines-of-sight, with the goal to reverse-engineer key molecular properties of the DIB carriers. EDIBLES is a filler Large Programme using the Ultraviolet and Visual Echelle Spectrograph at the Very Large Telescope at Paranal, Chile. It is designed to provide an observationally unbiased view of the presence and behaviour of the DIBs towards early-spectral type stars whose lines-of-sight probe the diffuse-to-translucent ISM. Such a complete dataset will provide a deep census of the atomic and molecular content, physical conditions, chemical abundances and elemental depletion levels for each sightline. Achieving these goals requires a homogeneous set of high-quality data in terms of resolution (R ~ 70 000 - 100 000), sensitivity (S/N up to 1000 per resolution element), and spectral coverage (305-1042 nm), as well as a large sample size (100+ sightlines). In this first paper the goals, objectives and methodology of the EDIBLES programme are described and an initial assessment of the data is provided.

The responses of an anaerobic microorganism, Yersinia intermedia MASE-LG-1 to individual and combined simulated Martian stresses.

The limits of life of aerobic microorganisms are well understood, but the responses of anaerobic microorganisms to individual and combined extreme stressors are less well known. Motivated by an interest in understanding the survivability of anaerobic microorganisms under Martian conditions, we investigated the responses of a new isolate, Yersinia intermedia MASE-LG-1 to individual and combined stresses associated with the Martian surface. This organism belongs to an adaptable and persistent genus of anaerobic microorganisms found in many environments worldwide. The effects of desiccation, low pressure, ionizing radiation, varying temperature, osmotic pressure, and oxidizing chemical compounds were investigated. The strain showed a high tolerance to desiccation, with a decline of survivability by four orders of magnitude during a storage time of 85 days. Exposure to X-rays resulted in dose-dependent inactivation for exposure up to 600 Gy while applied doses above 750 Gy led to complete inactivation. The effects of the combination of desiccation and irradiation were additive and the survivability was influenced by the order in which they were imposed. Ionizing irradiation and subsequent desiccation was more deleterious than vice versa. By contrast, the presence of perchlorates was not found to significantly affect the survival of the Yersinia strain after ionizing radiation. These data show that the organism has the capacity to survive and grow in physical and chemical stresses, imposed individually or in combination that are associated with Martian environment. Eventually it lost its viability showing that many of the most adaptable anaerobic organisms on Earth would be killed on Mars today.

Aggregate dust particles at comet 67P/Churyumov-Gerasimenko.

Comets are thought to preserve almost pristine dust particles, thus providing a unique sample of the properties of the early solar nebula. The microscopic properties of this dust played a key part in particle aggregation during the formation of the Solar System. Cometary dust was previously considered to comprise irregular, fluffy agglomerates on the basis of interpretations of remote observations in the visible and infrared and the study of chondritic porous interplanetary dust particles that were thought, but not proved, to originate in comets. Although the dust returned by an earlier mission has provided detailed mineralogy of particles from comet 81P/Wild, the fine-grained aggregate component was strongly modified during collection. Here we report in situ measurements of dust particles at comet 67P/Churyumov-Gerasimenko. The particles are aggregates of smaller, elongated grains, with structures at distinct sizes indicating hierarchical aggregation. Topographic images of selected dust particles with sizes of one micrometre to a few tens of micrometres show a variety of morphologies, including compact single grains and large porous aggregate particles, similar to chondritic porous interplanetary dust particles. The measured grain elongations are similar to the value inferred for interstellar dust and support the idea that such grains could represent a fraction of the building blocks of comets. In the subsequent growth phase, hierarchical agglomeration could be a dominant process and would produce aggregates that stick more easily at higher masses and velocities than homogeneous dust particles. The presence of hierarchical dust aggregates in the near-surface of the nucleus of comet 67P also provides a mechanism for lowering the tensile strength of the dust layer and aiding dust release.

COMETARY SCIENCE. Organic compounds on comet 67P/Churyumov-Gerasimenko revealed by COSAC mass spectrometry.

Comets harbor the most pristine material in our solar system in the form of ice, dust, silicates, and refractory organic material with some interstellar heritage. The evolved gas analyzer Cometary Sampling and Composition (COSAC) experiment aboard Rosetta's Philae lander was designed for in situ analysis of organic molecules on comet 67P/Churyumov-Gerasimenko. Twenty-five minutes after Philae's initial comet touchdown, the COSAC mass spectrometer took a spectrum in sniffing mode, which displayed a suite of 16 organic compounds, including many nitrogen-bearing species but no sulfur-bearing species, and four compounds­methyl isocyanate, acetone, propionaldehyde, and acetamide­that had not previously been reported in comets.

The Significance of Microbe-Mineral-Biomarker Interactions in the Detection of Life on Mars and Beyond.

The detection of biomarkers plays a central role in our effort to establish whether there is, or was, life beyond Earth. In this review, we address the importance of considering mineralogy in relation to the selection of locations and biomarker detection methodologies with characteristics most promising for exploration. We review relevant mineral-biomarker and mineral-microbe interactions. The local mineralogy on a particular planet reflects its past and current environmental conditions and allows a habitability assessment by comparison with life under extreme conditions on Earth. The type of mineral significantly influences the potential abundances and types of biomarkers and microorganisms containing these biomarkers. The strong adsorptive power of some minerals aids in the preservation of biomarkers and may have been important in the origin of life. On the other hand, this strong adsorption as well as oxidizing properties of minerals can interfere with efficient extraction and detection of biomarkers. Differences in mechanisms of adsorption and in properties of minerals and biomarkers suggest that it will be difficult to design a single extraction procedure for a wide range of biomarkers. While on Mars samples can be used for direct detection of biomarkers such as nucleic acids, amino acids, and lipids, on other planetary bodies remote spectrometric detection of biosignatures has to be relied upon. The interpretation of spectral signatures of photosynthesis can also be affected by local mineralogy. We identify current gaps in our knowledge and indicate how they may be filled to improve the chances of detecting biomarkers on Mars and beyond.

Biota and biomolecules in extreme environments on Earth: implications for life detection on Mars.

The three main requirements for life as we know it are the presence of organic compounds, liquid water, and free energy. Several groups of organic compounds (e.g., amino acids, nucleobases, lipids) occur in all life forms on Earth and are used as diagnostic molecules, i.e., biomarkers, for the characterization of extant or extinct life. Due to their indispensability for life on Earth, these biomarkers are also prime targets in the search for life on Mars. Biomarkers degrade over time; in situ environmental conditions influence the preservation of those molecules. Nonetheless, upon shielding (e.g., by mineral surfaces), particular biomarkers can persist for billions of years, making them of vital importance in answering questions about the origins and limits of life on early Earth and Mars. The search for organic material and biosignatures on Mars is particularly challenging due to the hostile environment and its effect on organic compounds near the surface. In support of life detection on Mars, it is crucial to investigate analogue environments on Earth that resemble best past and present Mars conditions. Terrestrial extreme environments offer a rich source of information allowing us to determine how extreme conditions affect life and molecules associated with it. Extremophilic organisms have adapted to the most stunning conditions on Earth in environments with often unique geological and chemical features. One challenge in detecting biomarkers is to optimize extraction, since organic molecules can be low in abundance and can strongly adsorb to mineral surfaces. Methods and analytical tools in the field of life science are continuously improving. Amplification methods are very useful for the detection of low concentrations of genomic material but most other organic molecules are not prone to amplification methods. Therefore, a great deal depends on the extraction efficiency. The questions "what to look for", "where to look", and "how to look for it" require more of our attention to ensure the success of future life detection missions on Mars.