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.

Cave Thiovulum (Candidatus Thiovulum stygium) differs metabolically and genomically from marine species.

Thiovulum spp. (Campylobacterota) are large sulfur bacteria that form veil-like structures in aquatic environments. The sulfidic Movile Cave (Romania), sealed from the atmosphere for ~5 million years, has several aqueous chambers, some with low atmospheric O2 (~7%). The cave's surface-water microbial community is dominated by bacteria we identified as Thiovulum. We show that this strain, and others from subsurface environments, are phylogenetically distinct from marine Thiovulum. We assembled a closed genome of the Movile strain and confirmed its metabolism using RNAseq. We compared the genome of this strain and one we assembled from public data from the sulfidic Frasassi caves to four marine genomes, including Candidatus Thiovulum karukerense and Ca. T. imperiosus, whose genomes we sequenced. Despite great spatial and temporal separation, the genomes of the Movile and Frasassi Thiovulum were highly similar, differing greatly from the very diverse marine strains. We concluded that cave Thiovulum represent a new species, named here Candidatus Thiovulum stygium. Based on their genomes, cave Thiovulum can switch between aerobic and anaerobic sulfide oxidation using O2 and NO3- as electron acceptors, the latter likely via dissimilatory nitrate reduction to ammonia. Thus, Thiovulum is likely important to both S and N cycles in sulfidic caves. Electron microscopy analysis suggests that at least some of the short peritrichous structures typical of Thiovulum are type IV pili, for which genes were found in all strains. These pili may play a role in veil formation, by connecting adjacent cells, and in the motility of these exceptionally fast swimmers.

Methanotrophy by a Mycobacterium species that dominates a cave microbial ecosystem.

So far, only members of the bacterial phyla Proteobacteria and Verrucomicrobia are known to grow methanotrophically under aerobic conditions. Here we report that this metabolic trait is also observed within the Actinobacteria. We enriched and cultivated a methanotrophic Mycobacterium from an extremely acidic biofilm growing on a cave wall at a gaseous chemocline interface between volcanic gases and the Earth's atmosphere. This Mycobacterium, for which we propose the name Candidatus Mycobacterium methanotrophicum, is closely related to well-known obligate pathogens such as M. tuberculosis and M. leprae. Genomic and proteomic analyses revealed that Candidatus M. methanotrophicum expresses a full suite of enzymes required for aerobic growth on methane, including a soluble methane monooxygenase that catalyses the hydroxylation of methane to methanol and enzymes involved in formaldehyde fixation via the ribulose monophosphate pathway. Growth experiments combined with stable isotope probing using 13C-labelled methane confirmed that Candidatus M. methanotrophicum can grow on methane as a sole carbon and energy source. A broader survey based on 16S metabarcoding suggests that species closely related to Candidatus M. methanotrophicum may be abundant in low-pH, high-methane environments.

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.

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.

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.