Biomarker Profiling of Microbial Mats in the Geothermal Band of Cerro Caliente, Deception Island (Antarctica): Life at the Edge of Heat and Cold

© María Ángeles Lezcano et al. 2019.Substrate–atmosphere interfaces in Antarctic geothermal environments are hot–cold regions that constitute thin habitable niches for microorganisms with possible counterparts in ancient Mars. Cerro Caliente hill in Deception Island (active volcano in the South Shetland Islands) is affected by ascending hydrothermal fluids that form a band of warm substrates buffered by low air temperatures. We investigated the influence of temperature on the community structure and metabolism of three microbial mats collected along the geothermal band of Cerro Caliente registering 88°C, 8°C, and 2°C at the time of collection. High-throughput sequencing of small subunit ribosomal ribonucleic acid (SSU rRNA) genes and Life Detector Chip (LDChip) microarray immunoassays revealed different bacterial, archaeal, and eukaryotic composition in the three mats. The mat at 88°C showed the less diverse microbial community and a higher proportion of thermophiles (e.g., Thermales). In contrast, microbial communities in the mats at 2°C and 8°C showed relatively higher diversity and higher proportion of psychrophiles (e.g., Flavobacteriales). Despite this overall association, similar microbial structures at the phylum level (particularly the presence of Cyanobacteria) and certain hot- and cold-tolerant microorganisms were identified in the three mats. Daily thermal oscillations recorded in the substrate over the year (4.5–76°C) may explain the coexistence of microbial fingerprints with different thermal tolerances. Stable isotope composition also revealed metabolic differences among the microbial mats. Carbon isotopic ratios suggested the Calvin–Benson–Bassham cycle as the major pathway for carbon dioxide fixation in the mats at 2°C and 8°C, and the reductive tricarboxylic acid cycle and/or the 3-hydroxypropionate bicycle for the mat at 88°C, indicating different metabolisms as a function of the prevailing temperature of each mat. The comprehensive biomarker profile on the three microbial mats from Cerro Caliente contributes to unravel the diversity, composition, and metabolism in geothermal polar sites and highlights the relevance of geothermal-cold environments to create habitable niches with interest in other planetary environments.This study has been funded by the Spanish Research Agency (AEI) from the Ministry of Science Innovation and Universities and the European FEDER Grants Nos. ESP2015-69540-R, RYC2014-19446, and CGL2015-74254-JIN; the AEI Project No. MDM-2017-0737 Unidad de Excelencia ‘‘Marı´a de Maeztu,’’ and the European Research Council Starting Grant (ERC StG) No. 307496. M.A´ . Lezcano and M.A´ . Fernández Martínez were supported by a postdoctoral fellowship by the Youth Employment Initiative from the European Union and implanted in Comunidad de Madrid

Prokaryotic Community Structure and Metabolisms in Shallow Subsurface of Atacama Desert Playas and Alluvial Fans After Heavy Rains: Repairing and Preparing for Next Dry Period

The Atacama Desert, the oldest and driest desert on Earth, displays significant rains only once per decade. To investigate how microbial communities take advantage of these sporadic wet events, we carried out a geomicrobiological study a few days after a heavy rain event in 2015. Different physicochemical and microbial community analyses were conducted on samples collected from playas and an alluvial fan from surface, 10, 20, 50, and 80 cm depth. Gravimetric moisture content peaks were measured in 10 and 20 cm depth samples (from 1.65 to 4.1% w/w maximum values) while, in general, main anions such as chloride, nitrate, and sulfate concentrations increased with depth, with maximum values of 13–1,125; 168–10,109; and 9,904–30,952 ppm, respectively. Small organic anions such as formate and acetate had maximum concentrations from 2.61 to 3.44 ppm and 6.73 to 28.75 ppm, respectively. Microbial diversity inferred from DNA analysis showed Actinobacteria and Alphaproteobacteria as the most abundant and widespread bacterial taxa among the samples, followed by Chloroflexi and Firmicutes at specific sites. Archaea were mainly dominated by Nitrososphaerales, Methanobacteria, with the detection of other groups such as Halobacteria. Metaproteomics showed a high and even distribution of proteins involved in primary metabolic processes such as energy production and biosynthetic pathways, and a limited but remarkable presence of proteins related to resistance to environmental stressors such as radiation, oxidation, or desiccation. The results indicated that extra humidity in the system allows the microbial community to repair, and prepare for the upcoming hyperarid period. Additionally, it supplies biomarkers to the medium whose preservation potential could be high under strong desiccation conditions and relevant for planetary exploration.This work was funded by the Spanish Ministerio de Economía y Competitividad/Fondo Europeo de Desarrollo Regional Grants no. ESP2014-58494-R, ESP2015-69540-R (MINECO/FEDER), ESP2017-89053-C2-2-P, and “María de Maeztu” program project no. MDM-2017-0737; the NASA ASTEP “Life in the Atacama (LITA)” project no. NNX11AJ87G, and NASA Astrobiology Institute Colaborative Agreement 7 (CAN-7) project No. NNX15BB01A. MF-M had a posdoctoral grant from the Comunidad de Madrid/European Union YEI program, and IG-C had a FPI grant from the Spanish Ministry of Economy and Competitivity (MINECO).With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (MDM-2017-0737).Peer reviewe

Prokaryotic Community Structure and Metabolisms in Shallow Subsurface of Atacama Desert Playas and Alluvial Fans After Heavy Rains: Repairing and Preparing for Next Dry Period

The Atacama Desert, the oldest and driest desert on Earth, displays significant rains only once per decade. To investigate how microbial communities take advantage of these sporadic wet events, we carried out a geomicrobiological study a few days after a heavy rain event in 2015. Different physicochemical and microbial community analyses were conducted on samples collected from playas and an alluvial fan from surface, 10, 20, 50, and 80 cm depth. Gravimetric moisture content peaks were measured in 10 and 20 cm depth samples (from 1.65 to 4.1% w/w maximum values) while, in general, main anions such as chloride, nitrate, and sulfate concentrations increased with depth, with maximum values of 13-1,125; 168-10,109; and 9,904-30,952 ppm, respectively. Small organic anions such as formate and acetate had maximum concentrations from 2.61 to 3.44 ppm and 6.73 to 28.75 ppm, respectively. Microbial diversity inferred from DNA analysis showed Actinobacteria and Alphaproteobacteria as the most abundant and widespread bacterial taxa among the samples, followed by Chloroflexi and Firmicutes at specific sites. Archaea were mainly dominated by Nitrososphaerales, Methanobacteria, with the detection of other groups such as Halobacteria. Metaproteomics showed a high and even distribution of proteins involved in primary metabolic processes such as energy production and biosynthetic pathways, and a limited but remarkable presence of proteins related to resistance to environmental stressors such as radiation, oxidation, or desiccation. The results indicated that extra humidity in the system allows the microbial community to repair, and prepare for the upcoming hyperarid period. Additionally, it supplies biomarkers to the medium whose preservation potential could be high under strong desiccation conditions and relevant for planetary exploration.Spanish Ministerio de Economia y Competitividad/Fondo Europeo de Desarrollo Regional Grants [ESP2014-58494-R, ESP2015-69540-R, ESP2017-89053-C2-2-P]; NASA ASTEP "Life in the Atacama (LITA)" project [NNX11AJ87G]; NASA Astrobiology Institute Colaborative Agreement 7 (CAN-7) project [NNX15BB01A]; Comunidad de Madrid/European Union YEI program; Spanish Ministry of Economy and Competitivity (MINECO); "Maria de Maeztu" program [MDM-2017-0737]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Geomicrobiological heterogeneity of lithic habitats in the extreme environment of Antarctic nunataks: a potential early Mars analog

Nunataks are permanent ice-free rocky peaks that project above ice caps in polar regions, thus being exposed to extreme climatic conditions throughout the year. They undergo extremely low temperatures and scarcity of liquid water in winter, while receiving high incident and reflected (albedo) UVA-B radiation in summer. Here, we investigate the geomicrobiology of the permanently exposed lithic substrates of nunataks from Livingston Island (South Shetlands, Antarctic Peninsula), with focus on prokaryotic community structure and their main metabolic traits. Contrarily to first hypothesis, an extensive sampling based on different gradients and multianalytical approaches demonstrated significant differences for most geomicrobiological parameters between the bedrock, soil, and loose rock substrates, which overlapped any other regional variation. Brevibacillus genus dominated on bedrock and soil substrates, while loose rocks contained a diverse microbial community, including Actinobacteria, Alphaproteobacteria and abundant Cyanobacteria inhabiting the milder and diverse microhabitats within. Archaea, a domain never described before in similar Antarctic environments, were also consistently found in the three substrates, but being more abundant and potentially more active in soils. Stable isotopic ratios of total carbon (δ 13C) and nitrogen (δ 15N), soluble anions concentrations, and the detection of proteins involved in key metabolisms via the Life Detector Chip (LDChip), suggest that microbial primary production has a pivotal role in nutrient cycling at these exposed areas with limited deposition of nutrients. Detection of stress-resistance proteins, such as molecular chaperons, suggests microbial molecular adaptation mechanisms to cope with these harsh conditions. Since early Mars may have encompassed analogous environmental conditions as the ones found in these Antarctic nunataks, our study also contributes to the understanding of the metabolic features and biomarker profiles of a potential Martian microbiota, as well as the use of LDChip in future life detection missions.This project has been funded by the Spanish Ministry of Science and Innovation (MICINN)/European Regional Development Fund (FEDER) project no. RTI2018-094368-B-I00; the European Research Council Consolidator grant no. 818602; and the Spanish State Research Agency (AEI) project no. MDM-2017-0737, Unidad de Excelencia “María de Maeztu” to Centro de Astrobiología

Prokaryotic and viral community of the sulfate‐rich crust from Peñahueca ephemeral lake, an astrobiology analogue

Peñahueca is an athalassohaline hypersaline inland ephemeral lake originated under semiarid conditions in the central Iberian Peninsula (Spain). Its chemical composition makes it extreme for microbial life as well as a terrestrial analogue of other planetary environments. To investigate the persistence of microbial life associated with sulfate‐rich crusts, we applied cultivation‐independent methods (optical and electron microscopy, 16S rRNA gene profiling and metagenomics) to describe the prokaryotic community and its associated viruses. The diversity for Bacteria was very low and was vastly dominated by endospore formers related to Pontibacillus marinus of the Firmicutes phylum. The archaeal assemblage was more diverse and included taxa related to those normally found in hypersaline environments. Several ‘metagenome assembled genomes’ were recovered, corresponding to new species of Pontibacillus, several species from the Halobacteria and one new member of the Nanohaloarchaeota. The viral assemblage, although composed of the morphotypes typical of high salt systems, showed little similarity to previously isolated/reconstructed halophages. Several putative prophages of Pontibacillus and haloarchaeal hosts were identified. Remarkably, the Peñahueca sulfate‐rich metagenome contained CRISPR‐associated proteins and repetitions which were over 10‐fold higher than in most hypersaline systems analysed so far.This research was supported by the Spanish Ministry of Economy projects CLG2015_66686-C3-1 (to RRM) CLG2015_66686-C3-3 (to JA), CGL2015-66455-R (to MAGC, MESM, JPRA), AYA2011-24803 and ESP2015-69540-R (to VP) which were also supported by the European Regional Development Fund and the MDM-2017-0737 Unidad de Excelencia “María de Maeztu”- Centro de Astrobiología (INTA-CSIC)

Life Detection and Microbial Biomarker Profiling with Signs of Life Detector-Life Detector Chip During a Mars Drilling Simulation Campaign in the Hyperarid Core of the Atacama Desert

The low organic matter content in the hyperarid core of the Atacama Desert, together with abrupt temperature shifts and high ultraviolet radiation at its surface, makes this region one of the best terrestrial analogs of Mars and one of the best scenarios for testing instrumentation devoted to in situ planetary exploration. We have operated remotely and autonomously the SOLID-LDChip (Signs of Life Detector-Life Detector Chip), an antibody microarray-based sensor instrument, as part of a rover payload during the 2019 NASA Atacama Rover Astrobiology Drilling Studies (ARADS) Mars drilling simulation campaign. A robotic arm collected drilled cuttings down to 80 cm depth and loaded SOLID to process and assay them with LDChip for searching for molecular biomarkers. A remote science team received and analyzed telemetry data and LDChip results. The data revealed the presence of microbial markers from Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Firmicutes, and Cyanobacteria to be relatively more abundant in the middle layer (40–50 cm). In addition, the detection of several proteins from nitrogen metabolism indicates a pivotal role in the system. These findings were corroborated and complemented on “returned samples” to the lab by a comprehensive analysis that included DNA sequencing, metaproteomics, and a metabolic reconstruction of the sampled area. Altogether, the results describe a relatively complex microbial community with members capable of nitrogen fixation and denitrification, sulfur oxidation and reduction, or triggering oxidative stress responses, among other traits. This remote operation demonstrated the high maturity of SOLID-LDChip as a powerful tool for remote in situ life detection for future missions in the Solar System.This research has been funded by grants numbers RTI2018-094368-B-I00 and PID2021-126746NB-I00 (SOLID) by the Spanish Ministry of Science and Innovation/State Agency of Research MCIN/AEI/10.13039/ 501100011033 and by ‘‘ERDF A way of making Europe,’’ and the NASA ARADS Project Nr.13-MMAMA13-0007.Peer reviewe

Deep subsurface sulfate reduction and methanogenesis in the Iberian Pyrite Belt revealed through geochemistry and molecular biomarkers

© 2013 John Wiley & Sons Ltd.The Iberian Pyrite Belt (IPB, southwest of Spain), the largest known massive sulfide deposit, fuels a rich chemolithotrophic microbial community in the Río Tinto area. However, the geomicrobiology of its deep subsurface is still unexplored. Herein, we report on the geochemistry and prokaryotic diversity in the subsurface (down to a depth of 166 m) of the Iberian Pyritic belt using an array of geochemical and complementary molecular ecology techniques. Using an antibody microarray, we detected polymeric biomarkers (lipoteichoic acids and peptidoglycan) from Gram-positive bacteria throughout the borehole. DNA microarray hybridization confirmed the presence of members of methane oxidizers, sulfate-reducers, metal and sulfur oxidizers, and methanogenic Euryarchaeota. DNA sequences from denitrifying and hydrogenotrophic bacteria were also identified. FISH hybridization revealed live bacterial clusters associated with microniches on mineral surfaces. These results, together with measures of the geochemical parameters in the borehole, allowed us to create a preliminary scheme of the biogeochemical processes that could be operating in the deep subsurface of the Iberian Pyrite Belt, including microbial metabolisms such as sulfate reduction, methanogenesis and anaerobic methane oxidation.The MARTE project (Mars Astrobiology Research and Technology Experiment) was a NASA-CAB joint project funded by the NASA ASTEP program project No. NRA-02-OSS-01 and by the CAB internal budget. The present work was supported by the Subdirección General de Proyectos de Investigación, of the Spanish Ministerio de Economía y Competitividad (MINECO) Grants No. ESP2004-05008, ESP2006-08128, AYA2008-04013, and the European Research Council (ERC) Advanced Grant No. 250350. F. Puente-Sánchez has a JAE-pre fellowship from the Spanish Consejo General de Investigaciones Científicas (CSIC).N

SOLID2: An Antibody Array-Based Life-Detector Instrument in a Mars Drilling Simulation Experiment (MARTE)

A field prototype of an antibody array-based life-detector instrument, Signs Of LIfe Detector (SOLID2), has been tested in a Mars drilling mission simulation called MARTE (Mars Astrobiology Research and Technology Experiment). As one of the analytical instruments on the MARTE robotic drilling rig, SOLID2 performed automatic sample processing and analysis of ground core samples (0.5 g) with protein microarrays that contained 157 different antibodies. Core samples from different depths (down to 5.5 m) were analyzed, and positive reactions were obtained in antibodies raised against the Gram-negative bacterium Leptospirillum ferrooxidans, a species of the genus Acidithiobacillus (both common microorganisms in the Río Tinto area), and extracts from biofilms and other natural samples from the Río Tinto area. These positive reactions were absent when the samples were previously subjected to a high-temperature treatment, which indicates the biological origin and structural dependency of the antibody-antigen reactions. We conclude that an antibody array-based life-detector instrument like SOLID2 can detect complex biological material, and it should be considered as a potential analytical instrument for future planetary missions that search for life.The work was funded by the Spanish Ministerio de Educación y Ciencia (ESP2004-05008), the Centro de Astrobiología (CAB), the Instituto Nacional de Técnica Aeroespacial (INTA), and by the National Aeronautics and Space Administration (NASA) Astrobiology Science and Technology for Exploring Planets (ASTEP) program under NRA 02-OSS-01.Peer reviewe

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.This work has been funded by Spanish Ministerio de Ciencia e Innovación (MCINN) granted projects ESP2006-09487, ESP2006-08128 and AYA2008-04013-E (Detection of Biomolecules in Planetary Exploration).N

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