Coupled C, H, N, S and Fe biogeochemical cycles operating in the continental deep subsurface of the Iberian Pyrite Belt.

Microbial activity is a major contributor to the biogeochemical cycles that make up the life support system of planet Earth. A 613 m deep geomicrobiological perforation and a systematic multi-analytical characterization revealed an unexpected diversity associated with the rock matrix microbiome that operates in the subsurface of the Iberian Pyrite Belt (IPB). Members of 1 class and 16 genera were deemed the most representative microorganisms of the IPB deep subsurface and selected for a deeper analysis. The use of fluorescence in situ hybridization allowed not only the identification of microorganisms but also the detection of novel activities in the subsurface such as anaerobic ammonium oxidation (ANAMMOX) and anaerobic methane oxidation, the co-occurrence of microorganisms able to maintain complementary metabolic activities and the existence of biofilms. The use of enrichment cultures sensed the presence of five different complementary metabolic activities along the length of the borehole and isolated 29 bacterial species. Genomic analysis of nine isolates identified the genes involved in the complete operation of the light-independent coupled C, H, N, S and Fe biogeochemical cycles. This study revealed the importance of nitrate reduction microorganisms in the oxidation of iron in the anoxic conditions existing in the subsurface of the IPB.

The Molecular Profile of Soil Microbial Communities Inhabiting a Cambrian Host Rock.

The process of soil genesis unfolds as pioneering microbial communities colonize mineral substrates, enriching them with biomolecules released from bedrock. The resultant intricate surface units emerge from a complex interplay among microbiota and plant communities. Under these conditions, host rocks undergo initial weathering through microbial activity, rendering them far from pristine and challenging the quest for biomarkers in ancient sedimentary rocks. In addressing this challenge, a comprehensive analysis utilizing Gas Chromatography Mass Spectrometry (GC-MS) and Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) was conducted on a 520-Ma-old Cambrian rock. This investigation revealed a diverse molecular assemblage with comprising alkanols, sterols, fatty acids, glycerolipids, wax esters, and nitrogen-bearing compounds. Notably, elevated levels of bacterial C16, C18 and C14 fatty acids, iso and anteiso methyl-branched fatty acids, as well as fungal sterols, long-chained fatty acids, and alcohols, consistently align with a consortium of bacteria and fungi accessing complex organic matter within a soil-type ecosystem. The prominence of bacterial and fungal lipids alongside maturity indicators denotes derivation from heterotrophic activity rather than ancient preservation or marine sources. Moreover, the identification of long-chain (>C22) n-alkanols, even-carbon-numbered long chain (>C20) fatty acids, and campesterol, as well as stigmastanol, provides confirmation of plant residue inputs. Furthermore, findings highlight the ability of contemporary soil microbiota to inhabit rocky substrates actively, requiring strict contamination controls when evaluating ancient molecular biosignatures or extraterrestrial materials collected.

Biospeleothems Formed by Fungal Activity During the Early Holocene in the "Salar de Uyuni".

The Chiquini and Galaxias caves contain speleothems that are templated by long fungal structures. They have been associated with the carbonate lacustrine deposits in the margins of the Coipasa and Uyuni Salar basins. During a wetter episode, such carbonates formed at the end of the last glaciation raising the lake level to more than 100 m in the Tauca events (15-12 ky). Such an event flooded the caves that eventually became a cryptic habitat in the lake. The caves show bizarre speleothems framed by large (>1 m) fungal buildings covering the older algal mineralized structures. Although the origin of the caves is not fully understood, the occurrence of two carbonatic units with very distinctive fabric suggests that they formed in two separated humid events. In this regard, the mineralized algal structures, showing the same features as the lacustrine carbonates, likely formed during the Tauca flooding events in the terminal Pleistocene that inundated older caves. The different caves were exposed to the atmosphere after a drop in the lake level that promoted alluvial erosion by <12-10 ky (Ticaña episode) under arid conditions. A last humid episode rising the lake surface 10 m above the Salar level, which was not enough to inundate the caves a second time, drove the formation of the biospeleothems by fungi biomineralization. The abundance and size of the preserved fungal structures suggest that they were sustained by a stable hydrological activity plus a constant organic supply. While nutrients could have been primarily sourced from the vegetal communities that occupied the exhumated lake margins, they might have also been released from the lacustrine carbonatic unit. The combination of hydrology and biological activities were likely determinants for a fast rock dissolution and mineralization ending in the construction of the fungal biospeleothems.

Detection of peptidic sequences in the ancient acidic sediments of Río Tinto, Spain.

Biomarkers are molecules that are produced by or can be associated with biological activities. They can be used as tracers that give us an idea of the ancient biological communities that produced them, the paleoenvironmental conditions where they lived, or the mechanism involved in their transformation and preservation. As a consequence, the preservation potential of molecules over time depends largely on their nature, but also on the conditions of the environment, which controls the decomposition kinetics. In this context, proteins and nucleic acids, which are biomolecules bearing biological information, are among the most labile molecules. In this research, we report the presence of short-chained peptides obtained from extracts of ferruginous sedimentary deposits that have been produced under the acidic and oxidizing solutions of Río Tinto, Spain. These preliminary results go against the paradigmatic idea that considers the acidic and oxidizing environments inappropriate for the preservation of molecular information.

Preservation of Underground Microbial Diversity in Ancient Subsurface Deposits (>6 Ma) of the Rio Tinto Basement.

The drilling of the Rio Tinto basement has provided evidence of an underground microbial community primarily sustained by the Fe and S metabolism through the biooxidation of pyrite orebodies. Although the gossan is the microbial activity product, which dates back to the Oligocene (25 Ma), no molecular evidence of such activity in the past has been reported yet. A Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) molecular analysis of a subsurface sample in the Peña de Hierro basement has provided novel data of the ancient underground microbial community. It shows that the microbial remains are preserved in a mineral matrix composed of laminated Fe-oxysulfates and K- and Na-bearing sulfates alternating with secondary silica. In such a mineral substrate, the biomolecule traces are found in five different microstructure associations, (1) <15 micron-sized nodular microstructures composed of POn(2≤n≤4)-, (2) <30 micron-size micronodules containing fatty acids, acylglycerides, and alkanol chains, (3) <20 micro-sized nodules containing NOn-(2≤n≤3) ions, (4) 40-micron size nodules with NH4+ and traces of peptides, and (5) >200-micron thick layer with N-bearing adducts, and sphingolipid and/or peptide traces. It suggests the mineralization of at least five microbial preserved entities with different metabolic capabilities, including: (1) Acidiphilium/Tessaracoccus-like phosphate mineralizers, (2) microbial patches preserving phosphate-free acylglycerides bacteria, (3) nitrogen oxidizing bacteria (e.g., Acidovorax sp.), (4) traces of heterotrophic ammonifying bacteria, and (5) sphingolipid bearing bacteria (e.g., Sphingomonadales, and δ-Proteobacteria) and/or mineralized biofilms. The primary biooxidation process acted as a preservation mechanism to release the inorganic ions that ultimately mineralized the microbial structures.

Unveiling microbial preservation under hyperacidic and oxidizing conditions in the Oligocene Rio Tinto deposit.

The preservation of biosignatures on Mars is largely associated with extensive deposits of clays formed under mild early Noachian conditions (> 3.9 Ga). They were followed by widespread precipitation of acidic sulfates considered adverse for biomolecule preservation. In this paper, an exhaustive mass spectrometry investigation of ferric subsurface materials in the Rio Tinto gossan deposit (~ 25 Ma) provides evidence of well-preserved molecular biosignatures under oxidative and acidic conditions. Time of flight secondary ion mass spectrometry (ToF-SIMS) analysis shows a direct association between physical-templating biological structures and molecular biosignatures. This relation implies that the quality of molecular preservation is exceptional and provides information on microbial life formerly operating in the shallow regions of the Rio Tinto subsurface. Consequently, low-pH oxidative environments on Mars could also record molecular information about ancient life in the same way as the Noachian clay-rich deposits.

The Molecular Record of Metabolic Activity in the Subsurface of the Río Tinto Mars Analog.

In the subsurface, the interplay between microbial communities and the surrounding mineral substrate, potentially used as an energy source, results in different mineralized structures. The molecular composition of such structures can record and preserve information about the metabolic pathways that have produced them. To characterize the molecular composition of the subsurface biosphere, we have analyzed some core samples by time-of-flight secondary ion mass spectrometry (ToF-SIMS) that were collected in the borehole BH8 during the operations of the Mars Analog and Technology Experiment (MARTE) project. The molecular analysis at a micron-scale mapped the occurrence of several inorganic complexes bearing PO3-, SOx(2 to 4)-, NOx(2,3)-, FeOx(1,2)-, SiO2-, and Cl-. Their distribution correlates with organic molecules that were tentatively assigned to saturated and monounsaturated fatty acids, polyunsaturated fatty acids, saccharides, phospholipids, sphingolipids, and potential peptide fragments. SOx- appear to be mineralizing some microstructures larger than 25 microns, which have branched morphologies, and that source SO3-bearing adducts. PO3-rich compounds occur in two different groups of microstructures which size, morphology, and composition are different. While a group of >40-micron sized circular micronodules lacks organic compounds, an ovoidal microstructure is associated with m/z of other lipids. The NO2-/NO3- and Cl- ions occur as small microstructure clusters (<20 microns), but their distribution is dissimilar to the mineralized microstructures bearing PO3-, and SO3-. However, they have a higher density in areas with more significant enrichment in iron oxides that are traced by different Fe-bearing anions like FeO2-. The distribution of the organic and inorganic negative ions, which we suggest, resulted from the preservation of at least three microbial consortia (PO4--, and NO2--/NO3--mineralizers PO4-lipid bearing microstructures), would have resulted from different metabolic and preservation pathways.

Inhibition of carbonate synthesis in acidic oceans on early Mars.

Several lines of evidence have recently reinforced the hypothesis that an ocean existed on early Mars. Carbonates are accordingly expected to have formed from oceanic sedimentation of carbon dioxide from the ancient martian atmosphere. But spectral imaging of the martian surface has revealed the presence of only a small amount of carbonate, widely distributed in the martian dust. Here we examine the feasibility of carbonate synthesis in ancient martian oceans using aqueous equilibrium calculations. We show that partial pressures of atmospheric carbon dioxide in the range 0.8-4 bar, in the presence of up to 13.5 mM sulphate and 0.8 mM iron in sea water, result in an acidic oceanic environment with a pH of less than 6.2. This precludes the formation of siderite, usually expected to be the first major carbonate mineral to precipitate. We conclude that extensive interaction between an atmosphere dominated by carbon dioxide and a lasting sulphate- and iron-enriched acidic ocean on early Mars is a plausible explanation for the observed absence of carbonates.

Some ecological mechanisms to generate habitability in planetary subsurface areas by chemolithotrophic communities: the Río Tinto subsurface ecosystem as a model system.

Chemolithotrophic communities that colonize subsurface habitats have great relevance for the astrobiological exploration of our Solar System. We hypothesize that the chemical and thermal stabilization of an environment through microbial activity could make a given planetary region habitable. The MARTE project ground-truth drilling campaigns that sampled cryptic subsurface microbial communities in the basement of the Río Tinto headwaters have shown that acidic surficial habitats are the result of the microbial oxidation of pyritic ores. The oxidation process is exothermic and releases heat under both aerobic and anaerobic conditions. These microbial communities can maintain the subsurface habitat temperature through storage heat if the subsurface temperature does not exceed their maximum growth temperature. In the acidic solutions of the Río Tinto, ferric iron acts as an effective buffer for controlling water pH. Under anaerobic conditions, ferric iron is the oxidant used by microbes to decompose pyrite through the production of sulfate, ferrous iron, and protons. The integration between the physical and chemical processes mediated by microorganisms with those driven by the local geology and hydrology have led us to hypothesize that thermal and chemical regulation mechanisms exist in this environment and that these homeostatic mechanisms could play an essential role in creating habitable areas for other types of microorganisms. Therefore, searching for the physicochemical expression of extinct and extant homeostatic mechanisms through physical and chemical anomalies in the Mars crust (i.e., local thermal gradient or high concentration of unusual products such as ferric sulfates precipitated out from acidic solutions produced by hypothetical microbial communities) could be a first step in the search for biological traces of a putative extant or extinct Mars biosphere.

Spiders: water-driven erosive structures in the southern hemisphere of Mars.

Recent data from space missions reveal that there are ongoing climatic changes and erosive processes that continuously modify surface features of Mars. We have investigated the seasonal dynamics of a number of morphological features located at Inca City, a representative area at high southern latitude that has undergone seasonal processes. By integrating visual information from the Mars Orbiter Camera on board the Mars Global Surveyor and climatic cycles from a Mars' General Circulation Model, and considering the recently reported evidence for the presence of water-ice and aqueous precipitates on Mars, we propose that a number of the erosive features identified in Inca City, among them spiders, result from the seasonal melting of aqueous salty solutions.

Río tinto: a geochemical and mineralogical terrestrial analogue of Mars.

The geomicrobiological characterization of the water column and sediments of Río Tinto (Huelva, Southwestern Spain) have proven the importance of the iron and the sulfur cycles, not only in generating the extreme conditions of the habitat (low pH, high concentration of toxic heavy metals), but also in maintaining the high level of microbial diversity detected in the basin. It has been proven that the extreme acidic conditions of Río Tinto basin are not the product of 5000 years of mining activity in the area, but the consequence of an active underground bioreactor that obtains its energy from the massive sulfidic minerals existing in the Iberian Pyrite Belt. Two drilling projects, MARTE (Mars Astrobiology Research and Technology Experiment) (2003-2006) and IPBSL (Iberian Pyrite Belt Subsurface Life Detection) (2011-2015), were developed and carried out to provide evidence of subsurface microbial activity and the potential resources that support these activities. The reduced substrates and the oxidants that drive the system appear to come from the rock matrix. These resources need only groundwater to launch diverse microbial metabolisms. The similarities between the vast sulfate and iron oxide deposits on Mars and the main sulfide bioleaching products found in the Tinto basin have given Río Tinto the status of a geochemical and mineralogical Mars terrestrial analogue.

Microbial mediated formation of Fe-carbonate minerals under extreme acidic conditions.

Discovery of Fe-carbonate precipitation in Rio Tinto, a shallow river with very acidic waters, situated in Huelva, South-western Spain, adds a new dimension to our understanding of carbonate formation. Sediment samples from this low-pH system indicate that carbonates are formed in physico-chemical conditions ranging from acid to neutral pH. Evidence for microbial mediation is observed in secondary electron images (Fig. 1), which reveal rod-shaped bacteria embedded in the surface of siderite nanocrystals. The formation of carbonates in Rio Tinto is related to the microbial reduction of ferric iron coupled to the oxidation of organic compounds. Herein, we demonstrate for the first time, that Acidiphilium sp. PM, an iron-reducing bacterium isolated from Rio Tinto, mediates the precipitation of siderite (FeCO3) under acidic conditions and at a low temperature (30°C). We describe nucleation of siderite on nanoglobules in intimate association with the bacteria cell surface. This study has major implications for understanding carbonate formation on the ancient Earth or extraterrestrial planets.

Classification of modern and old Río Tinto sedimentary deposits through the biomolecular record using a life marker biochip: implications for detecting life on Mars.

The particular mineralogy formed in the acidic conditions of the Río Tinto has proven to be a first-order analogue for the acid-sulfate aqueous environments of Mars. Therefore, studies about the formation and preservation of biosignatures in the Río Tinto will provide insights into equivalent processes on Mars. We characterized the biomolecular patterns recorded in samples of modern and old fluvial sediments along a segment of the river by means of an antibody microarray containing more than 200 antibodies (LDCHIP200, for Life Detector Chip) against whole microorganisms, universal biomolecules, or environmental extracts. Samples containing 0.3-0.5 g of solid material were automatically analyzed in situ by the Signs Of LIfe Detector instrument (SOLID2), and the results were corroborated by extensive analysis in the laboratory. Positive antigen-antibody reactions indicated the presence of microbial strains or high-molecular-weight biopolymers that originated from them. The LDCHIP200 results were quantified and subjected to a multivariate analysis for immunoprofiling. We associated similar immunopatterns, and biomolecular markers, to samples with similar sedimentary age. Phyllosilicate-rich samples from modern fluvial sediments gave strong positive reactions with antibodies against bacteria of the genus Acidithiobacillus and against biochemical extracts from Río Tinto sediments and biofilms. These samples contained high amounts of sugars (mostly polysaccharides) with monosaccharides like glucose, rhamnose, fucose, and so on. By contrast, the older deposits, which are a mix of clastic sands and evaporites, showed only a few positives with LDCHIP200, consistent with lower protein and sugar content. We conclude that LDCHIP200 results can establish a correlation between microenvironments, diagenetic stages, and age with the biomarker profile associated with a sample. Our results would help in the search for putative martian biomarkers in acidic deposits with similar diagenetic maturity. Our LDCHIP200 and SOLID-like instruments may be excellent tools for the search for molecular biomarkers on Mars or other planets.

A microbial oasis in the hypersaline Atacama subsurface discovered by a life detector chip: implications for the search for life on Mars.

The Atacama Desert has long been considered a good Mars analogue for testing instrumentation for planetary exploration, but very few data (if any) have been reported about the geomicrobiology of its salt-rich subsurface. We performed a Mars analogue drilling campaign next to the Salar Grande (Atacama, Chile) in July 2009, and several cores and powder samples from up to 5 m deep were analyzed in situ with LDChip300 (a Life Detector Chip containing 300 antibodies). Here, we show the discovery of a hypersaline subsurface microbial habitat associated with halite-, nitrate-, and perchlorate-containing salts at 2 m deep. LDChip300 detected bacteria, archaea, and other biological material (DNA, exopolysaccharides, some peptides) from the analysis of less than 0.5 g of ground core sample. The results were supported by oligonucleotide microarray hybridization in the field and finally confirmed by molecular phylogenetic analysis and direct visualization of microbial cells bound to halite crystals in the laboratory. Geochemical analyses revealed a habitat with abundant hygroscopic salts like halite (up to 260 g kg(-1)) and perchlorate (41.13 µg g(-1) maximum), which allow deliquescence events at low relative humidity. Thin liquid water films would permit microbes to proliferate by using detected organic acids like acetate (19.14 µg g(-1)) or formate (76.06 µg g(-1)) as electron donors, and sulfate (15875 µg g(-1)), nitrate (13490 µg g(-1)), or perchlorate as acceptors. Our results correlate with the discovery of similar hygroscopic salts and possible deliquescence processes on Mars, and open new search strategies for subsurface martian biota. The performance demonstrated by our LDChip300 validates this technology for planetary exploration, particularly for the search for life on Mars.

Underground habitats in the Río Tinto basin: a model for subsurface life habitats on Mars.

A search for evidence of cryptic life in the subsurface region of a fractured Paleozoic volcanosedimentary deposit near the source waters of the Río Tinto River (Iberian pyrite belt, southwest Spain) was carried out by Mars Astrobiology Research and Technology Experiment (MARTE) project investigators in 2003 and 2004. This conventional deep-drilling experiment is referred to as the MARTE ground truth drilling project. Boreholes were drilled at three sites, and samples from extracted cores were analyzed with light microscopy, scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy. Core leachates were analyzed with ion chromatography, and borehole fluids were analyzed with ion and gas chromatography. Key variables of the groundwater system (e.g., pO(2), pH, and salinity) exhibit huge ranges probably due to surficial oxygenation of overall reducing waters, physical mixing of waters, and biologically mediated water-rock interactions. Mineral distribution is mainly driven by the pH of subsurface solutions, which range from highly acidic to neutral. Borehole fluids contain dissolved gases such as CO(2), CH(4), and H(2). SEM-EDS analyses of core samples revealed evidence of microbes attacking pyrite. The Río Tinto alteration mechanisms may be similar to subsurface weathering of the martian crust and provide insights into the possible (bio)geochemical cycles that may have accompanied underground habitats in extensive early Mars volcanic regions and associated sulfide ores.

The 2005 MARTE Robotic Drilling Experiment in Río Tinto, Spain: objectives, approach, and results of a simulated mission to search for life in the Martian subsurface.

The Mars Astrobiology Research and Technology Experiment (MARTE) simulated a robotic drilling mission to search for subsurface life on Mars. The drill site was on Peña de Hierro near the headwaters of the Río Tinto river (southwest Spain), on a deposit that includes massive sulfides and their gossanized remains that resemble some iron and sulfur minerals found on Mars. The mission used a fluidless, 10-axis, autonomous coring drill mounted on a simulated lander. Cores were faced; then instruments collected color wide-angle context images, color microscopic images, visible-near infrared point spectra, and (lower resolution) visible-near infrared hyperspectral images. Cores were then stored for further processing or ejected. A borehole inspection system collected panoramic imaging and Raman spectra of borehole walls. Life detection was performed on full cores with an adenosine triphosphate luciferin-luciferase bioluminescence assay and on crushed core sections with SOLID2, an antibody array-based instrument. Two remotely located science teams analyzed the remote sensing data and chose subsample locations. In 30 days of operation, the drill penetrated to 6 m and collected 21 cores. Biosignatures were detected in 12 of 15 samples analyzed by SOLID2. Science teams correctly interpreted the nature of the deposits drilled as compared to the ground truth. This experiment shows that drilling to search for subsurface life on Mars is technically feasible and scientifically rewarding.