In Situ Real-Time Monitoring for Aseptic Drilling: Lessons Learned from the Atacama Rover Astrobiology Drilling Studies Contamination Control Strategy and Implementation and Application to the Icebreaker Mars Life Detection Mission.

In 2019, the Atacama Rover Astrobiology Drilling Studies (ARADS) project field-tested an autonomous rover-mounted robotic drill prototype for a 6-Sol life detection mission to Mars (Icebreaker). ARADS drilled Mars-like materials in the Atacama Desert (Chile), one of the most life-diminished regions on Earth, where mitigating contamination transfer into life-detection instruments becomes critical. Our Contamination Control Strategy and Implementation (CCSI) for the Sample Handling and Transfer System (SHTS) hardware (drill, scoop and funnels) included out-of-simulation protocol testing (out-of-sim) for hardware decontamination and verification during the 6-Sol simulation (in-sim). The most effective five-step decontamination combined safer-to-use sterilants (3%_hydrogen-peroxide-activated 5%_sodium-hypochlorite), and in situ real-time verification by adenosine triphosphate (ATP) and Signs of Life Detector (SOLID) Fluorescence Immunoassay for characterization hardware bioburden and airborne contaminants. The 20- to 40-min protocol enabled a 4-log bioburden reduction down to <0.1 fmoles ATP detection limit (funnels and drill) to 0.2-0.7 fmoles (scoop) of total ATP. The (post-cleaning) hardware background was 0.3 to 1-2 attomoles ATP/cm2 (cleanliness benchmark background values) equivalent to ca. 1-10 colony forming unit (CFU)/cm2. Further, 60-100% of the in-sim hardware background was ≤3-4 bacterial cells/cm2, the threshold limit for Class <7 aseptic operations. Across the six Sols, the flux of airborne contaminants to the drill sites was ∼5 and ∼22 amoles ATP/(cm2·day), accounting for an unexpectedly high Fluorescence Intensity (FI) signal (FI: ∼6000) against aquatic cyanobacteria, but negligible anthropogenic contribution. The SOLID immunoassay also detected microorganisms from multiple habitats across the Atacama Desert (anoxic, alkaline/acidic microenvironments in halite fields, playas, and alluvial fans) in both airborne and post-cleaning hardware background. Finally, the hardware ATP background was 40-250 times lower than the ATP in cores. Similarly, the FI peaks (FImax) against the microbial taxa and molecular biomarkers detected in the post-cleaned hardware (FI: ∼1500-1600) were 5-10 times lower than biomarkers in drilled sediments, excluding significant interference with putative biomarker found in cores. Similar protocols enable the acquisition of contamination-free materials for ultra-sensitive instruments analysis and the integrity of scientific results. Their application can augment our scientific knowledge of the distribution of cryptic life on Mars-like grounds and support life-detection robotic and human-operated missions to Mars.

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.

Assessing siliceous sinter matrices for long-term preservation of lipid biomarkers in opaline sinter deposits analogous to Mars in El Tatio (Chile).

Subaerial hydrothermal systems are of great interest for paleobiology and astrobiology as plausible candidate environments to support the origin of life on Earth that offer a unique and interrelated atmosphere-hydrosphere-lithosphere interface. They harbor extensive sinter deposits of high preservation potential that are promising targets in the search for traces of possible extraterrestrial life on Hesperian Mars. However, long-term quality preservation is paramount for recognizing biosignatures in old samples and there are still significant gaps in our understanding of the impact and extent of taphonomy processes on life fingerprints. Here, we propose a study based on lipid biomarkers -highly resistant cell-membrane components- to investigate the effects of silicification on their preservation in hydrothermal opaline sinter. We explore the lipid biomarkers profile in three sinter deposits of up to ~3000 years from El Tatio, one of the best Martian analogs on Earth. The lipid profile in local living biofilms is used as a fresh counterpart of the fossil biomarkers in the centuries-old sinter deposits to qualitatively assess the taphonomy effects of silicification on the lipid's preservation. Despite the geological alteration, the preserved lipids retained a depleted stable-carbon isotopic fingerprint characteristic of biological sources, result highly relevant for astrobiology. The data allowed us to estimate for the first time the degradation rate of lipid biomarkers in sinter deposits from El Tatio, and to assess the time preservation framework of opaline silica. Auxiliary techniques of higher taxonomic resolution (DNA sequencing and metaproteomics) helped in the reconstruction of the paleobiology. The lipids were the best-preserved biomolecules, whereas the detection of DNA and proteins dropped considerably from 5 cm depth. These findings provide new insights into taphonomy processes affecting life fingerprints in hydrothermal deposits and serves as a useful baseline for assessing the time window for recovering unambiguous signs of past life on Earth and beyond.

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.

Time-Integrative Multibiomarker Detection in Triassic-Jurassic Rocks from the Atacama Desert: Relevance to the Search for Basic Life Beyond Earth.

Detecting evidence of life on other planetary bodies requires a certain understanding of known biomarkers and their chemical nature, preservation potential, or biological specificity. In a planetary search for life, carbonates are of special interest due to their known association with life as we know it. On Earth, carbonates serve as an invaluable paleogeochemical archive of fossils of up to billions of years old. Here, we investigated biomarker profiles on three Chilean Triassic-Jurassic sedimentary records regarding our search for signs of past and present life over ∼200 Ma. A multianalytical platform that combines lipid-derived biomarkers, metaproteomics, and a life detector chip (LDChip) is considered in the detection of biomolecules with different perdurability and source-diagnosis potential. The combined identification of proteins with positive LDChip inmunodetections provides metabolic information and taxonomic affiliation of modern/subrecent biosignatures. Molecular and isotopic analysis of more perdurable hydrocarbon cores allows for the identification of general biosources and dominant autotrophic pathways over time, as well as recreation of prevailing redox conditions over ∼200 Ma. We demonstrate how extraterrestrial life detection can benefit from the use of different biomarkers to overcome diagnosis limitations due to a lack of specificity and/or alteration over time. Our findings have implications for future astrobiological missions to Mars.

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.

A multiplex antigen microarray for simultaneous IgG and IgM detection against SARS-CoV-2 reveals higher seroprevalence than reported.

The surge of SARS-CoV-2 has challenged health systems worldwide and efficient tests to detect viral particles, as well as antibodies generated against them, are needed. Specificity, sensitivity, promptness or scalability are the main parameters to estimate the final performance, but rarely all of them match in a single test. We have developed SCOVAM, a protein microarray with several viral antigens (spike, nucleocapsid, main protease Nsp5) as capturing probes in a fluorescence immunoassay for COVID-19 serological testing. SCOVAM depicts IgG and IgM antibody responses against each of these proteins of 22 individuals in a single microscope slide. It detects specific IgM (0.094 µg ml-1 ) and IgG (~0.017 µg ml-1 ) and is scalable and cost-effective. We validated SCOVAM by comparing with a widely used chemiluminescent commercial serological test (n = 742). SCOVAM showed twice the sensitivity and allowed following seroconversion in a single assay. By analysing the prevalence 4 months later in a subset of 76 positive sera, we still detected 93.42% of positives, almost doubling the detection of the commercial assay. The higher sensitivity of SCOVAM is especially relevant to screen sera for convalescent plasma-based treatments, high-throughput antibody response monitoring after vaccination or evaluation of vaccine efficiency.

The Limits, Capabilities, and Potential for Life Detection with MinION Sequencing in a Paleochannel Mars Analog.

No instrument capable of direct life detection has been included on a mission payload to Mars since NASA's Viking missions in the 1970s. This prevents us from discovering whether life is or ever was present on Mars. DNA is an ideal target biosignature since it is unambiguous, nonspecific, and readily detectable with nanopore sequencing. Here, we present a proof-of-concept utilization of the Oxford Nanopore Technologies (ONT) MinION sequencer for direct life detection and show how it can complement results from established space mission instruments. We used nanopore sequencing data from the MinION to detect and characterize the microbial life in a set of paleochannels near Hanksville, UT, with supporting data from X-ray diffraction, reflectance spectroscopy, Raman spectroscopy, and Life Detector Chip (LDChip) microarray immunoassay analyses. These paleochannels are analogs to martian sinuous ridges. The MinION-generated metagenomes reveal a rich microbial community dominated by bacteria and containing radioresistant, psychrophilic, and halophilic taxa. With spectral data and LDChip immunoassays, these metagenomes were linked to the surrounding Mars analog environment and potential metabolisms (e.g., methane production and perchlorate reduction). This shows a high degree of synergy between these techniques for detecting and characterizing biosignatures. We also resolved a prospective lower limit of ∼0.001 ng of DNA required for successful sequencing. This work represents the first determination of the MinION's DNA detection limits beyond ONT recommendations and the first whole metagenome analysis of a sinuous ridge analog.

Simulating Mars Drilling Mission for Searching for Life: Ground-Truthing Lipids and Other Complex Microbial Biomarkers in the Iron-Sulfur Rich Río Tinto Analog.

Sulfate and iron oxide deposits in Río Tinto (Southwestern Spain) are a terrestrial analog of early martian hematite-rich regions. Understanding the distribution and drivers of microbial life in iron-rich environments can give critical clues on how to search for biosignatures on Mars. We simulated a robotic drilling mission searching for signs of life in the martian subsurface, by using a 1m-class planetary prototype drill mounted on a full-scale mockup of NASA's Phoenix and InSight lander platforms. We demonstrated fully automated and aseptic drilling on iron and sulfur rich sediments at the Río Tinto riverbanks, and sample transfer and delivery to sterile containers and analytical instruments. As a ground-truth study, samples were analyzed in the field with the life detector chip immunoassay for searching microbial markers, and then in the laboratory with X-ray diffraction to determine mineralogy, gas chromatography/mass spectrometry for lipid composition, isotope-ratio mass spectrometry for isotopic ratios, and 16S/18S rRNA genes sequencing for biodiversity. A ubiquitous presence of microbial biomarkers distributed along the 1m-depth subsurface was influenced by the local mineralogy and geochemistry. The spatial heterogeneity of abiotic variables at local scale highlights the importance of considering drill replicates in future martian drilling missions. The multi-analytical approach provided proof of concept that molecular biomarkers varying in compositional nature, preservation potential, and taxonomic specificity can be recovered from shallow drilling on iron-rich Mars analogues by using an automated life-detection lander prototype, such as the one proposed for NASA's IceBreaker mission proposal.

Microbial Markers Profile in Anaerobic Mars Analogue Environments Using the LDChip (Life Detector Chip) Antibody Microarray Core of the SOLID (Signs of Life Detector) Platform.

One of the main objectives for astrobiology is to unravel and explore the habitability of environments beyond Earth, paying special attention to Mars. If the combined environmental stress factors on Mars are compatible with life or if they were less harsh in the past, to investigate the traces of past or present life is critical to understand its potential habitability. Essential for this research is the characterization of Mars analogue environments on Earth through the development of techniques for biomarker detection in them. Biosensing techniques based on fluorescence sandwich microarray immunoassays (FSMI) have shown to be a powerful tool to detect biosignatures and depict the microbial profiles of different environments. In this study, we described the microbial biomarker profile of five anoxic Mars analogues sites using the Life Detector Chip (LDChip), an antibody microarray for multiple microbial marker detection. Furthermore, we contributed to new targets by developing a new 26-polyclonal antibodies microarray using crude extracts from anaerobic sampling sites, halophilic microorganisms, and anaerobic isolates obtained in the framework of the European Mars Analogues for Space Exploration (MASE) project. The new subset of antibodies was characterized and implemented into a microarray platform (MASE-Chip) for microbial marker searching in salty and anaerobic environments.

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.

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

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.

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.

Microbiology and Nitrogen Cycle in the Benthic Sediments of a Glacial Oligotrophic Deep Andean Lake as Analog of Ancient Martian Lake-Beds.

Potential benthic habitats of early Mars lakes, probably oligotrophic, could range from hydrothermal to cold sediments. Dynamic processes in the water column (such as turbidity or UV penetration) as well as in the benthic bed (temperature gradients, turbation, or sedimentation rate) contribute to supply nutrients to a potential microbial ecosystem. High altitude, oligotrophic, and deep Andean lakes with active deglaciation processes and recent or past volcanic activity are natural models to assess the feasibility of life in other planetary lake/ocean environments and to develop technology for their exploration. We sampled the benthic sediments (down to 269 m depth) of the oligotrophic lake Laguna Negra (Central Andes, Chile) to investigate its ecosystem through geochemical, biomarker profiling, and molecular ecology studies. The chemistry of the benthic water was similar to the rest of the water column, except for variable amounts of ammonium (up to 2.8 ppm) and nitrate (up to 0.13 ppm). A life detector chip with a 300-antibody microarray revealed the presence of biomass in the form of exopolysaccharides and other microbial markers associated to several phylogenetic groups and potential microaerobic and anaerobic metabolisms such as nitrate reduction. DNA analyses showed that 27% of the Archaea sequences corresponded to a group of ammonia-oxidizing archaea (AOA) similar (97%) to Nitrosopumilus spp. and Nitrosoarchaeum spp. (Thaumarchaeota), and 4% of Bacteria sequences to nitrite-oxidizing bacteria from the Nitrospira genus, suggesting a coupling between ammonia and nitrite oxidation. Mesocosm experiments with the specific AOA inhibitor 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) demonstrated an AOA-associated ammonia oxidation activity with the simultaneous accumulation of nitrate and sulfate. The results showed a rich benthic microbial community dominated by microaerobic and anaerobic metabolisms thriving under aphotic, low temperature (4°C), and relatively high pressure, that might be a suitable terrestrial analog of other planetary settings.

Viable cyanobacteria in the deep continental subsurface.

Cyanobacteria are ecologically versatile microorganisms inhabiting most environments, ranging from marine systems to arid deserts. Although they possess several pathways for light-independent energy generation, until now their ecological range appeared to be restricted to environments with at least occasional exposure to sunlight. Here we present molecular, microscopic, and metagenomic evidence that cyanobacteria predominate in deep subsurface rock samples from the Iberian Pyrite Belt Mars analog (southwestern Spain). Metagenomics showed the potential for a hydrogen-based lithoautotrophic cyanobacterial metabolism. Collectively, our results suggest that they may play an important role as primary producers within the deep-Earth biosphere. Our description of this previously unknown ecological niche for cyanobacteria paves the way for models on their origin and evolution, as well as on their potential presence in current or primitive biospheres in other planetary bodies, and on the extant, primitive, and putative extraterrestrial biospheres.

Microbial Biomarker Transition in High-Altitude Sinter Mounds From El Tatio (Chile) Through Different Stages of Hydrothermal Activity.

Geothermal springs support microbial communities at elevated temperatures in an ecosystem with high preservation potential that makes them interesting analogs for early evolution of the biogeosphere. The El Tatio geysers field in the Atacama Desert has astrobiological relevance due to the unique occurrence of geothermal features with steep hydrothermal gradients in an otherwise high altitude, hyper-arid environment. We present here results of our multidisciplinary field and molecular study of biogeochemical evidence for habitability and preservation in silica sinter at El Tatio. We sampled three morphologically similar geyser mounds characterized by differences in water activity (i.e., episodic liquid water, steam, and inactive geyser lacking hydrothermal activity). Multiple approaches were employed to determine (past and present) biological signatures and dominant metabolism. Lipid biomarkers indicated relative abundance of thermophiles (dicarboxylic acids) and sulfate reducing bacteria (branched carboxylic acids) in the sinter collected from the liquid water mound; photosynthetic microorganisms such as cyanobacteria (alkanes and isoprenoids) in the steam sinter mound; and archaea (squalane and crocetane) as well as purple sulfur bacteria (cyclopropyl acids) in the dry sinter from the inactive geyser. The three sinter structures preserved biosignatures representative of primary (thermophilic) and secondary (including endoliths and environmental contaminants) microbial communities. Sequencing of environmental 16S rRNA genes and immuno-assays generally corroborated the lipid-based microbial identification. The multiplex immunoassays and the compound-specific isotopic analysis of carboxylic acids, alkanols, and alkanes indicated that the principal microbial pathway for carbon fixation in the three sinter mounds was through the Calvin cycle, with a relative larger contribution of the reductive acetyl-CoA pathway in the dry system. Other inferred metabolic traits varied from the liquid mound (iron and sulfur chemistry), to the steam mound (nitrogen cycle), to the dry mound (perchlorate reduction). The combined results revealed different stages of colonization that reflect differences in the lifetime of the mounds, where primary communities dominated the biosignatures preserved in sinters from the still active geysers (liquid and steam mounds), in contrast to the surviving metabolisms and microbial communities at the end of lifetime of the inactive geothermal mound.

Prokaryotic communities and operating metabolisms in the surface and the permafrost of Deception Island (Antarctica).

In this study we examined the microbial community composition and operating metabolisms on the surface and in the permafrost of Deception Island, (Antarctica) with an on site antibody microarray biosensor. Samples (down to a depth of 4.2 m) were analysed with LDChip300 (Life Detector Chip), an immunosensor containing more than 300 antibodies targeted to bacterial and archaeal antigens. The immunograms showed positive antigen-antibody reactions in all surface samples (lichens, pyroclasts) and the top layer of the permafrost. The results indicated the presence of exopolysaccharides, bacteria belonging to the Alpha-, Delta- and Gammaproteobacteria, Bacteroidetes, Gram-positive Actinobacteria and Firmicutes, as well as archaeal species, most probably Methanobacterium spp. Positive reactions with antibodies to proteins and peptides revealed the presence of nitrogen fixation (NifHD, GlnB, HscA), methanogenic (McrB), iron homeostasis and iron scavenging (ferritins and DPS proteins) proteins, as well as ABC transporters, which indicated that these processes were operating at the time of sampling. These results were validated with other molecular ecology techniques such as oligonucleotide microarrays, 16S bacterial rRNA gene sequence analysis, aerobic viable counts and microscopy. Molecular ecology results showed a differentiated pattern along the depth of the drill, being the top active layer the most diverse, with Acidobacteria, Actinobacteria, Proteobacteria, Bacteroidetes and the phototrophs Cyanobacteria and Chloroflexi as dominant groups. Actinobacteria and Firmicutes were dominant in depths from 0.5 to 2 m, and Betaproteobacteria from 3 to 4.2 m. The geochemical analysis revealed the presence of low molecular weight organic acids (acetate, formate) which could be used by microorganisms as energy sources for sulfate, nitrate and metal reduction under anaerobic conditions.

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.

SOLID3: a multiplex antibody microarray-based optical sensor instrument for in situ life detection in planetary exploration.

The search for unequivocal signs of life on other planetary bodies is one of the major challenges for astrobiology. The failure to detect organic molecules on the surface of Mars by measuring volatile compounds after sample heating, together with the new knowledge of martian soil chemistry, has prompted the astrobiological community to develop new methods and technologies. Based on protein microarray technology, we have designed and built a series of instruments called SOLID (for "Signs Of LIfe Detector") for automatic in situ detection and identification of substances or analytes from liquid and solid samples (soil, sediments, or powder). Here, we present the SOLID3 instrument, which is able to perform both sandwich and competitive immunoassays and consists of two separate functional units: a Sample Preparation Unit (SPU) for 10 different extractions by ultrasonication and a Sample Analysis Unit (SAU) for fluorescent immunoassays. The SAU consists of five different flow cells, with an antibody microarray in each one (2000 spots). It is also equipped with an exclusive optical package and a charge-coupled device (CCD) for fluorescent detection. We demonstrated the performance of SOLID3 in the detection of a broad range of molecular-sized compounds, which range from peptides and proteins to whole cells and spores, with sensitivities at 1-2 ppb (ng mL⁻¹) for biomolecules and 104 to 10³ spores per milliliter. We report its application in the detection of acidophilic microorganisms in the Río Tinto Mars analogue and report the absence of substantial negative effects on the immunoassay in the presence of 50 mM perchlorate (20 times higher than that found at the Phoenix landing site). Our SOLID instrument concept is an excellent option with which to detect biomolecules because it avoids the high-temperature treatments that may destroy organic matter in the presence of martian oxidants.

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.