Metal tolerance of Río Tinto fungi.

Southwest Spain's Río Tinto is a stressful acidic microbial habitat with a noticeably high concentration of toxic heavy metals. Nevertheless, it has an unexpected degree of eukaryotic diversity in its basin, with a high diversity of fungal saprotrophs. Although some studies on the eukaryotic diversity in Rio Tinto have been published, none of them used molecular methodologies to describe the fungal diversity and taxonomic affiliations that emerge along the river in different seasons. The aim of the present study was to isolate and describe the seasonal diversity of the fungal community in the Río Tinto basin and its correlation with the physicochemical parameters existing along the river's course. The taxonomic affiliation of 359 fungal isolates, based on the complete internal transcribed spacer DNA sequences, revealed a high degree of diversity, identifying species belonging primarily to the phylum Ascomycota, but representatives of the Basidiomycota and Mucoromycota phyla were also present. In total, 40 representative isolates along the river were evaluated for their tolerance to toxic heavy metals. Some of the isolates were able to grow in the presence of 1000 mM of Cu2+, 750 mM of As5+ and Cd2+, and 100 mM of Co2+, Ni2+, and Pb2+.

Halotolerant Endophytic Bacteria Priestia flexa 7BS3110 with Hg2+ Tolerance Isolated from Avicennia germinans in a Caribbean Mangrove from Colombia.

The mangrove ecosystems of the Department of Atlántico (Colombian Caribbean) are seriously threatened by problems of hypersalinization and contamination, especially by heavy metals from the Magdalena River. The mangrove plants have developed various mechanisms to adapt to these stressful conditions, as well as the associated microbial populations that favor their growth. In the present work, the tolerance and detoxification capacity to heavy metals, especially to mercury, of a halotolerant endophytic bacterium isolated from the species Avicennia germinans located in the Balboa Swamp in the Department of Atlántico was characterized. Diverse microorganisms were isolated from superficially sterilized A. germinans leaves. Tolerance to NaCl was evaluated for each of the obtained isolates, and the most resistant was selected to assess its tolerance to Pb2+, Cu2+, Hg2+, Cr3+, Co2+, Ni2+, Zn2+, and Cd2+, many of which have been detected in high concentrations in the area of study. According to the ANI and AAI percentages, the most halotolerant strain was identified as Priestia flexa, named P. flexa 7BS3110, which was able to tolerate up to 12.5% (w/v) NaCl and presented a minimum inhibitory concentrations (MICs) of 0.25 mM for Hg, 10 mM for Pb, and 15 mM for Cr3+. The annotation of the P. flexa 7BS3110 genome revealed the presence of protein sequences associated with exopolysaccharide (EPS) production, thiol biosynthesis, specific proteins for chrome efflux, non-specific proteins for lead efflux, and processes associated with sulfur and iron homeostasis. Scanning electron microscopy (SEM) analysis showed morphological cellular changes and the transmission electron microscopy (TEM) showed an electrodense extracellular layer when exposed to 0.25 mM Hg2+. Due to the high tolerance of P. flexa 7BS3110 to Hg2+ and NaCl, its ability to grow when exposed to both stressors was tested, and it was able to thrive in the presence of 5% (w/v) NaCl and 0.25 mM of Hg2+. In addition, it was able to remove 98% of Hg2+ from the medium when exposed to a concentration of 14 mg/L of this metalloid. P. flexa 7BS3110 has the potential to bioremediate Hg2+ halophilic contaminated ecosystems.

Association of Acidotolerant Cyanobacteria to Microbial Mats below pH 1 in Acidic Mineral Precipitates in Río Tinto River in Spain.

This report describes acidic microbial mats containing cyanobacteria that are strongly associated to precipitated minerals in the source area of Río Tinto. Río Tinto (Huelva, Southwestern Spain) is an extreme acidic environment where iron and sulfur cycles play a fundamental role in sustaining the extremely low pH and the high concentration of heavy metals, while maintaining a high level of microbial diversity. These multi-layered mineral deposits are stable all year round and are characterized by a succession of thick greenish-blue and brownish layers mainly composed of natrojarosite. The temperature and absorbance above and below the mineral precipitates were followed and stable conditions were detected inside the mineral precipitates. Different methodologies, scanning and transmission electron microscopy, immunological detection, fluorescence in situ hybridization, and metagenomic analysis were used to describe the biodiversity existing in these microbial mats, demonstrating, for the first time, the existence of acid-tolerant cyanobacteria in a hyperacidic environment of below pH 1. Up to 0.46% of the classified sequences belong to cyanobacterial microorganisms, and 1.47% of the aligned DNA reads belong to the Cyanobacteria clade.

Vitamin B12 Auxotrophy in Isolates from the Deep Subsurface of the Iberian Pyrite Belt.

Vitamin B12 is an enzymatic cofactor that is essential for both eukaryotes and prokaryotes. The development of life in extreme environments depends on cofactors such as vitamin B12 as well. The genomes of twelve microorganisms isolated from the deep subsurface of the Iberian Pyrite Belt have been analyzed in search of enzymatic activities that require vitamin B12 or are involved in its synthesis and import. Results have revealed that vitamin B12 is needed by these microorganisms for several essential enzymes such as ribonucleotide reductase, methionine synthase and epoxyqueosine reductase. Isolate Desulfosporosinus sp. DEEP is the only analyzed genome that holds a set core of proteins that could lead to the production of vitamin B12. The rest are dependent on obtaining it from the subsurface oligotrophic environment in which they grow. Sought proteins involved in the import of vitamin B12 are not widespread in the sample. The dependence found in the genomes of these microorganisms is supported by the production of vitamin B12 by microorganisms such as Desulfosporosinus sp. DEEP, showing that the operation of deep subsurface biogeochemical cycles is dependent on cofactors such as vitamin B12.

Production and Characterisation of an Exopolysaccharide by Bacillus amyloliquefaciens: Biotechnological Applications.

The Bacillus amyloliquefaciens RT7 strain was isolated from an extreme acidic environment and identified. The biodegradation capabilities of the strain using different carbon sources (glucose, oleic acid, Tween 80, PEG 200, and the combination of glucose-Tween 80) were evaluated via an indirect impedance technique. The glucose-Tween 80 combination was further studied using nuclear magnetic resonance (NMR). The exopolysaccharide (EPSRT7) that had been produced with the strain when biodegrading glucose-Tween 80 was isolated and characterised using different techniques (GC-MS, HPLC/MSMS, ATR-FTIR, TGA, and DSC), and its molecular weight was estimated. The results show that the average molecular weight of EPSRT7 was approximately 7.0794 × 104 Da and a heteropolysaccharide composed of mannose, glucose, galactose, and xylose (molar ratio, 1:0.5:0.1:0.1) with good thermostability. EPSRT7 showed good emulsifying activity against different natural oils and hydrocarbons at high concentrations (2 mg/mL) and at the studied pH range (3.1-7.2). It also presented good emulsifying activity compared to that of commercial emulsifiers. Lastly, EPSRT7 showed antioxidant capacity for different free radicals, a lack of cytotoxicity, and antioxidant activity at the cellular level. EPSRT7 has promising applications in bioremediation processes and other industrial applications.

Ecological successions throughout the desiccation of Tirez lagoon (Spain) as an astrobiological time-analog for wet-to-dry transitions on Mars.

Tirez was a small and seasonal endorheic athalassohaline lagoon that was located in central Spain. In recent years, the lagoon has totally dried out, offering for the first time the opportunity to analyze its desiccation process as a "time-analog" to similar events occurred in paleolakes with varying salinity during the wet-to-dry transition on early Mars. On the martian cratered highlands, an early period of water ponding within enclosed basins evolved to a complete desiccation of the lakes, leading to deposition of evaporitic sequences during the Noachian and into the Late Hesperian. As Tirez also underwent a process of desiccation, here we describe (i) the microbial ecology of Tirez when the lagoon was still active 20 years ago, with prokaryotes adapted to extreme saline conditions; (ii) the composition of the microbial community in the dried lake sediments today, in many case groups that thrive in sediments of extreme environments; and (iii) the molecular and isotopic analysis of the lipid biomarkers that can be recovered from the sediments today. We discuss the implications of these results to better understanding the ecology of possible Martian microbial communities during the wet-to-dry transition at the end of the Hesperian, and how they may inform about research strategies to search for possible biomarkers in Mars after all the water was lost.

Rio Tinto as a niche for acidophilus enzymes of industrial relevance.

Lignocellulosic residues are amongst the most abundant waste products on Earth. Therefore, there is an increasing interest in the utilization of these residues for bioethanol production and for biorefineries to produce compounds of industrial interest. Enzymes that breakdown cellulose and hemicellulose into oligomers and monosaccharides are required in these processes and cellulolytic enzymes with optimum activity at a low pH area are desirable for industrial processes. Here, we explore the fungal biodiversity of Rio Tinto, the largest acidic ecosystem on Earth, as far as the secretion of cellulolytic enzymes is concerned. Using colorimetric and industrial substrates, we show that a high proportion of the fungi present in this extremophilic environment secrete a wide range of enzymes that are able to hydrolyze cellulose and hemicellulose at acidic pH (4.5-5). Shotgun proteomic analysis of the secretomes of some of these fungi has identified different cellulases and hemicellulolytic enzymes as well as a number of auxiliary enzymes. Supplementation of pre-industrial cocktails from Myceliophtora with Rio Tinto secretomes increased the amount of monosaccharides released from corn stover or sugar cane straw. We conclude that the Rio Tinto fungi display a good variety of hydrolytic enzymes with high industrial potential.

Scientific novelty beyond the experiment.

Practical experiments drive important scientific discoveries in biology, but theory-based research studies also contribute novel-sometimes paradigm-changing-findings. Here, we appraise the roles of theory-based approaches focusing on the experiment-dominated wet-biology research areas of microbial growth and survival, cell physiology, host-pathogen interactions, and competitive or symbiotic interactions. Additional examples relate to analyses of genome-sequence data, climate change and planetary health, habitability, and astrobiology. We assess the importance of thought at each step of the research process; the roles of natural philosophy, and inconsistencies in logic and language, as drivers of scientific progress; the value of thought experiments; the use and limitations of artificial intelligence technologies, including their potential for interdisciplinary and transdisciplinary research; and other instances when theory is the most-direct and most-scientifically robust route to scientific novelty including the development of techniques for practical experimentation or fieldwork. We highlight the intrinsic need for human engagement in scientific innovation, an issue pertinent to the ongoing controversy over papers authored using/authored by artificial intelligence (such as the large language model/chatbot ChatGPT). Other issues discussed are the way in which aspects of language can bias thinking towards the spatial rather than the temporal (and how this biased thinking can lead to skewed scientific terminology); receptivity to research that is non-mainstream; and the importance of theory-based science in education and epistemology. Whereas we briefly highlight classic works (those by Oakes Ames, Francis H.C. Crick and James D. Watson, Charles R. Darwin, Albert Einstein, James E. Lovelock, Lynn Margulis, Gilbert Ryle, Erwin R.J.A. Schrödinger, Alan M. Turing, and others), the focus is on microbiology studies that are more-recent, discussing these in the context of the scientific process and the types of scientific novelty that they represent. These include several studies carried out during the 2020 to 2022 lockdowns of the COVID-19 pandemic when access to research laboratories was disallowed (or limited). We interviewed the authors of some of the featured microbiology-related papers and-although we ourselves are involved in laboratory experiments and practical fieldwork-also drew from our own research experiences showing that such studies can not only produce new scientific findings but can also transcend barriers between disciplines, act counter to scientific reductionism, integrate biological data across different timescales and levels of complexity, and circumvent constraints imposed by practical techniques. In relation to urgent research needs, we believe that climate change and other global challenges may require approaches beyond the experiment.

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.

Potential Applications of an Exopolysaccharide Produced by Bacillus xiamenensis RT6 Isolated from an Acidic Environment.

The Bacillus xiamenensis RT6 strain was isolated and identified by morphological, biochemical and molecular tests from an extreme acidic environment, Rio Tinto (Huelva). Optimisation tests for exopolysaccharide (EPS) production in different culture media determined that the best medium was a minimal medium with glucose as the only carbon source. The exopolymer (EPSt) produced by the strain was isolated and characterised using different techniques (GC-MS, HPLC/MSMS, ATR-FTIR, TGA, DSC). The molecular weight of EPSt was estimated. The results showed that the average molecular weight of EPSt was approximately 2.71 × 104 Da and was made up of a heteropolysaccharide composed of glucose (60%), mannose (20%) and galactose (20%). The EPSt showed antioxidant capabilities that significantly improved cell viability. Metal chelation determined that EPSt could reduce the concentration of transition metals such as iron at the highest concentrations tested. Finally, the emulsification study showed that EPSt was able to emulsify different natural polysaccharide oils, reaching up to an 80% efficiency (olive and sesame oil), and was a good candidate for the substitution of the most polluting emulsifiers. The EPSt was found to be suitable for pharmaceutical and industrial applications.

Sediminibacillus dalangtanensis sp. nov., a moderate halophile isolated from hypersaline sediments of the Qaidam Basin in Northwest China.

A Gram-stain-positive, moderately halophilic, aerobic, endospore-forming, rod-shaped bacterium, designated strain DP4-553-ST, was isolated from hypersaline sediment collected from the Dalangtan Playa in the Qaidam Basin, Northwest PR China. Growth occurred within 0-21.6% (w/v) NaCl (optimum 7.2%) at pH 5.5-9.0 (optimum pH 7.0) and at 4-45 °C (optimum 37 °C). Phylogeny based on 16S rRNA gene sequences indicated that strain DP4-553-ST belonged to the genus Sediminibacillus, with high 16S rRNA gene sequence similarity to Sediminibacillus halophilus EN8dT (99.5 %), Sediminibacillus terrae JSM 102062T (99.4 %), Virgibacillus senegalensis SK-1T (99.3 %) and Sediminibacillus albus NHBX5T (98.3 %). The G+C content of the chromosomal DNA was 43.6 mol %. The average amino acid identity, average nucleotide identity and digital DNA-DNA hybridization values between strain DP4-553-ST and the four close type strains were 71.2-93.3, 74.0-90.5 and 20.0-41.4 %, respectively. The whole genomic analysis showed that strain DP4-553-ST constituted a different taxon separated from the recognized Sediminibacillus species. The major cellular fatty acids were anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0, C16 : 0 and iso-C15 : 0. The type strain contained cell-wall peptidoglycan based on diaminopimelic acid and possessed menaquinone-7 as the major respiratory isoprenoid quinone. The polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol, four unidentified glycolipids, phosphatidylcholine, aminophospholipid, aminolipid and seven unidentified phospholipids. The combined data from phenotypic and genotypic studies demonstrated that strain DP4-553-ST represents a novel species of the genus Sediminibacillus, for which the name Sediminibacillus dalangtanensis sp. nov. is proposed, the type strain is DP4-553-ST (=MCCC 1K03838T= KCTC 43250T).

Shewanella sp. T2.3D-1.1 a Novel Microorganism Sustaining the Iron Cycle in the Deep Subsurface of the Iberian Pyrite Belt.

The Iberian Pyrite Belt (IPB) is one of the largest deposits of sulphidic minerals on Earth. Río Tinto raises from its core, presenting low a pH and high metal concentration. Several drilling cores were extracted from the IPB's subsurface, and strain T2.3D-1.1 was isolated from a core at 121.8 m depth. We aimed to characterize this subterranean microorganism, revealing its phylogenomic affiliation (Average Nucleotide Identity, digital DNA-DNA Hybridization) and inferring its physiology through genome annotation, backed with physiological experiments to explore its relationship with the Fe biogeochemical cycle. Results determined that the isolate belongs to the Shewanella putrefaciens (with ANI 99.25 with S. putrefaciens CN-32). Its genome harbours the necessary genes, including omcA mtrCAB, to perform the Extracellular Electron Transfer (EET) and reduce acceptors such as Fe3+, napAB to reduce NO3- to NO2-, hydAB to produce H2 and genes sirA, phsABC and ttrABC to reduce SO32-, S2O32- and S4O62-, respectively. A full CRISPR-Cas 1F type system was found as well. S. putrefaciens T2.3D-1.1 can reduce Fe3+ and promote the oxidation of Fe2+ in the presence of NO3- under anaerobic conditions. Production of H2 has been observed under anaerobic conditions with lactate or pyruvate as the electron donor and fumarate as the electron acceptor. Besides Fe3+ and NO3-, the isolate also grows with Dimethyl Sulfoxide and Trimethyl N-oxide, S4O62- and S2O32- as electron acceptors. It tolerates different concentrations of heavy metals such as 7.5 mM of Pb, 5 mM of Cr and Cu and 1 mM of Cd, Co, Ni and Zn. This array of traits suggests that S. putrefaciens T2.3D-1.1 could have an important role within the Iberian Pyrite Belt subsurface participating in the iron cycle, through the dissolution of iron minerals and therefore contributing to generate the extreme conditions detected in the Río Tinto basin.

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.

Biogeochemical Niches of Fe-Cycling Communities Influencing Heavy Metal Transport along the Rio Tinto, Spain.

In the mining-impacted Rio Tinto, Spain, Fe-cycling microorganisms influence the transport of heavy metals (HMs) into the Atlantic Ocean. However, it remains largely unknown how spatial and temporal hydrogeochemical gradients along the Rio Tinto shape the composition of Fe-cycling microbial communities and how this in turn affects HM mobility. Using a combination of DNA- and RNA-based 16S rRNA (gene) amplicon sequencing and hydrogeochemical analyses, we explored the impact of pH, Fe(III), Fe(II), and Cl- on Fe-cycling microorganisms. We showed that the water column at the acidic (pH 2.2) middle course of the river was colonized by Fe(II) oxidizers affiliated with Acidithiobacillus and Leptospirillum. At the upper estuary, daily fluctuations of pH (2.7 to 3.7) and Cl- (6.9 to 16.6 g/L) contributed to the establishment of a unique microbial community, including Fe(II) oxidizers belonging to Acidihalobacter, Marinobacter, and Mariprofundus, identified at this site. Furthermore, DNA- and RNA-based profiles of the benthic community suggested that acidophilic and neutrophilic Fe(II) oxidizers (e.g., Acidihalobacter, Marinobacter, and Mariprofundus), Fe(III) reducers (e.g., Thermoanaerobaculum), and sulfate-reducing bacteria drive the Fe cycle in the estuarine sediments. RNA-based relative abundances of Leptospirillum at the middle course as well as abundances of Acidihalobacter and Mariprofundus at the upper estuary were higher than DNA-based results, suggesting a potentially higher level of activity of these taxa. Based on our findings, we propose a model of how tidal water affects the composition and activity of the Fe-cycling taxa, playing an important role in the transport of HMs (e.g., As, Cd, Cr, and Pb) along the Rio Tinto. IMPORTANCE The estuary of the Rio Tinto is a unique environment in which extremely acidic, heavy metal-rich, and especially iron-rich river water is mixed with seawater. Due to the mixing events, the estuarine water is characterized by a low pH, almost seawater salinity, and high concentrations of bioavailable iron. The unusual hydrogeochemistry maintains unique microbial communities in the estuarine water and in the sediment. These communities include halotolerant iron-oxidizing microorganisms which typically inhabit acidic saline environments and marine iron-oxidizing microorganisms which, in contrast, are not typically found in acidic environments. Furthermore, highly saline estuarine water favored the prosperity of acidophilic heterotrophs, typically inhabiting brackish and saline environments. The Rio Tinto estuarine sediment harbors a diverse microbial community with both acidophilic and neutrophilic members that can mediate the iron cycle and, in turn, can directly impact the mobility and transport of heavy metals in the Rio Tinto estuary.

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.

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.

Methanogenesis at High Temperature, High Ionic Strength and Low pH in the Volcanic Area of Dallol, Ethiopia.

The Dallol geothermal area originated as a result of seismic activity and the presence of a shallow underground volcano, both due to the divergence of two tectonic plates. In its ascent, hot water dissolves and drags away the subsurface salts. The temperature of the water that comes out of the chimneys is higher than 100 °C, with a pH close to zero and high mineral concentration. These factors make Dallol a polyextreme environment. So far, nanohaloarchaeas, present in the salts that form the walls of the chimneys, have been the only living beings reported in this extreme environment. Through the use of complementary techniques: culture in microcosms, methane stable isotope signature and hybridization with specific probes, the methanogenic activity in the Dallol area has been assessed. Methane production in microcosms, positive hybridization with the Methanosarcinales probe and the δ13CCH4-values measured, show the existence of extensive methanogenic activity in the hydrogeothermic Dallol system. A methylotrophic pathway, carried out by Methanohalobium and Methanosarcina-like genera, could be the dominant pathway for methane production in this environment.