Dark biosphere: just at the very tip of the iceberg

Ministerio de Ciencia e Innovacion, Grant/Award Numbers: PID2019-104812GB-I00, TED2021-129563B-I0

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

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, el nombre del grupo de colaboración, si le hubiere, y los autores pertenecientes a la UAMBackground: 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. Keywords: Extreme environments, Microbiomes, Archaea, Bacteria, Propidium monoazide, Astrobiology, Spaceanalogue, Extremophiles, Extraterrestrial life, Metagenomic

Biodegradation of choline NTF2 by Pantoea agglomerans in different osmolarity. Characterization and environmental implications of the produced exopolysaccharide

A specific microorganism, Pantoea agglomerans uam8, was isolated from the ionic liquid (IL) Choline NTF2 and identified by molecular biology. A biodegradation study was performed at osmolarity conditions (0.2, 0.6, 1.0 M). These had an important influence on the growth of the strain, exopolysaccharide (EPS) production, and biodegradation (1303 mg/L max production and 80% biodegradation at 0.6 M). These conditions also had an important influence on the morphology of the strain and its EPSs, but not in the chemical composition. The EPS (glucose, mannose and galactose (6:0.5:2)) produced at 0.6 M was further characterized using different techniques. The obtained EPSs presented important differences in the behavior of the emulsifying activity for vegetable oils (olive (86%), sunflower (56%) and coconut (90%)) and hydrocarbons (diesel (62%), hexane (60%)), and were compared with commercial emulsifiers. The EPS produced at 0.6 M had the highest emulsifying activity overall. This EPS did not show cytotoxicity against the tested cell line (<20%) and presented great advantages as an antioxidant (1,1-diphenyl-2-picryl-hydrazyl radical (DPPH) (85%), hydroxyl radical (OH) (99%), superoxide anion (O2−) (94%), chelator (54%), and antimicrobial product (15 mm). The osmolarity conditions directly affected the capacity of the strain to biodegrade IL and the subsequently produced EPS. Furthermore, the EPS produced at 0.6 M has potential for environmental applications, such as the removal of hazardous materials by emulsification, whilst resulting in positive health effects such as antioxidant activity and non-toxicityThis research was funded by the Spanish Ministry of Science and Innovation for financial support (project PID2022-136607NB-I00) and FUAM, Universidad Autónoma de Madrid, Spain (project number 820053

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 application

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 applicationsThis research was funded by the Spanish Ministry of Science and Innovation for financial support (project PID2019-104812GB-I00) and FUAM, Universidad Autónoma de Madrid, Spain (project no. 820053

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 los

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(2to4)-, 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 pathway

ARAMIS: From systematic errors of NGS long reads to accurate assemblies

NGS long-reads sequencing technologies (or third generation) such as Pacific BioSciences (PacBio) have revolutionized the sequencing field over the last decade improving multiple genomic applications like de novo genome assemblies. However, their error rate, mostly involving insertions and deletions (indels), is currently an important concern that requires special attention to be solved. Multiple algorithms are available to fix these sequencing errors using short reads (such as Illumina), although they require long processing times and some errors may persist. Here, we present Accurate long-Reads Assembly correction Method for Indel errorS (ARAMIS), the first NGS long-reads indels correction pipeline that combines several correction software in just one step using accurate short reads. As a proof OF concept, six organisms were selected based on their different GC content, size and genome complexity, and their PacBio-assembled genomes were corrected thoroughly by this pipeline. We found that the presence of systematic sequencing errors in long-reads PacBio sequences affecting homopolymeric regions, and that the type of indel error introduced during PacBio sequencing are related to the GC content of the organism. The lack of knowledge of this fact leads to the existence of numerous published studies where such errors have been found and should be resolved since they may contain incorrect biological information. ARAMIS yields better results with less computational resources needed than other correction tools and gives the possibility of detecting the nature of the found indel errors found and its distribution along the genome. The source code of ARAMIS is available at https://github.com/genomics-ngsCBMSO/ARAMIS.gi

Protection of Possessors in Classical Roman Law -From the Viewpoint of Buyer Protection- (2)

We studied the formation of Mg-rich carbonate in culture experiments using different aerobic bacterial strains and aqueous Mg/Ca ratios (2 to 11.5) at Earth surface conditions. These bacteria promoted the formation of microenvironments that facilitate the precipitation of mineral phases (dolomite, huntite, high Mg-calcite and hydromagnesite) that were undersaturated in the bulk solution or kinetically inhibited. Dolomite, huntite, high Mg-calcite, hydromagnesite and struvite precipitated in different proportions and at different times, depending on the composition of the medium. The Mg content of dolomite and calcite decreased with an increasing Ca concentration in the medium. The stable carbon isotope composition of the Mg-rich carbonate precipitates reflected the isotope composition of the organic compounds present in the media, suggesting that microbial metabolism strongly influenced the carbon isotope composition of biomediated carbonates. We observed that Ca-enriched carbonate precipitates have relatively low carbon isotope composition. These results provide insights into the mechanism(s) of carbonate formation in natural systems, and they are of fundamental importance for understanding modern environments in which carbonate minerals form as a window into the geologic past

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 IPBFP7 Ideas: European Research Council, Grant/Award Number: ERC Advanced Grant #250-35