A Multiplex Immunosensor for Detecting Perchlorate-Reducing Bacteria for Environmental Monitoring and Planetary Exploration.

Perchlorate anions are produced by chemical industries and are important contaminants in certain natural ecosystems. Perchlorate also occurs in some natural and uncontaminated environments such as the Atacama Desert, the high Arctic or the Antarctic Dry Valleys, and is especially abundant on the surface of Mars. As some bacterial strains are capable of using perchlorate as an electron acceptor under anaerobic conditions, their detection is relevant for environmental monitoring on Earth as well as for the search for life on Mars. We have developed an antibody microarray with 20 polyclonal antibodies to detect perchlorate-reducing bacteria (PRB) strains and two crucial and highly conserved enzymes involved in perchlorate respiration: perchlorate reductase and chlorite dismutase. We determined the cross-reactivity, the working concentration, and the limit of detection of each antibody individually and in a multiplex format by Fluorescent Sandwich Microarray Immunoassay. Although most of them exhibited relatively high sensitivity and specificity, we applied a deconvolution method based on graph theory to discriminate between specific signals and cross-reactions from related microorganisms. We validated the system by analyzing multiple bacterial isolates, crude extracts from contaminated reactors and salt-rich natural samples from the high Arctic. The PRB detecting chip (PRBCHIP) allowed us to detect and classify environmental isolates as well as to detect similar strains by using crude extracts obtained from 0.5 g even from soils with low organic-matter levels (<103 cells/g of soil). Our results demonstrated that PRBCHIP is a valuable tool for sensitive and reliable detection of perchlorate-reducing bacteria for research purposes, environmental monitoring and planetary exploration.

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.

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.

Critical Assessment of Analytical Techniques in the Search for Biomarkers on Mars: A Mummified Microbial Mat from Antarctica as a Best-Case Scenario.

The search for biomarkers of present or past life is one of the major challenges for in situ planetary exploration. Multiple constraints limit the performance and sensitivity of remote in situ instrumentation. In addition, the structure, chemical, and mineralogical composition of the sample may complicate the analysis and interpretation of the results. The aim of this work is to highlight the main constraints, performance, and complementarity of several techniques that have already been implemented or are planned to be implemented on Mars for detection of organic and molecular biomarkers on a best-case sample scenario. We analyzed a 1000-year-old desiccated and mummified microbial mat from Antarctica by Raman and IR (infrared) spectroscopies (near- and mid-IR), thermogravimetry (TG), differential thermal analysis, mass spectrometry (MS), and immunological detection with a life detector chip. In spite of the high organic content (ca. 20% wt/wt) of the sample, the Raman spectra only showed the characteristic spectral peaks of the remaining beta-carotene biomarker and faint peaks of phyllosilicates over a strong fluorescence background. IR spectra complemented the mineralogical information from Raman spectra and showed the main molecular vibrations of the humic acid functional groups. The TG-MS system showed the release of several volatile compounds attributed to biopolymers. An antibody microarray for detecting cyanobacteria (CYANOCHIP) detected biomarkers from Chroococcales, Nostocales, and Oscillatoriales orders. The results highlight limitations of each technique and suggest the necessity of complementary approaches in the search for biomarkers because some analytical techniques might be impaired by sample composition, presentation, or processing. Key Words: Planetary exploration-Life detection-Microbial mat-Life detector chip-Thermogravimetry-Raman spectroscopy-NIR-DRIFTS. Astrobiology 17, 984-996.

CYANOCHIP: an antibody microarray for high-taxonomical-resolution cyanobacterial monitoring.

Cyanobacteria are Gram-negative photosynthetic prokaryotes that are widespread on Earth. Eutrophication and global warming make some aquatic ecosystems behave as bioreactors that trigger rapid and massive cyanobacterial growth with remarkable economic and health consequences. Rapid and efficient early warning systems are required to support decisions by water body authorities. We have produced 17 specific antibodies to the most frequent cyanobacterial strains blooming in freshwater ecosystems, some of which are toxin producers. A sandwich-type antibody microarray immunoassay (CYANOCHIP) was developed for the simultaneous testing of any of the 17 strains, or other closely related strains, in field samples from different habitats (water, rocks, and sediments). We titrated and tested all of the antibodies in succession using a fluorescent sandwich microarray immunoassay. Although most showed high specificity, we applied a deconvolution method based on graph theory to disentangle the few existing cross-reactions. The CYANOCHIP sensitivity ranged from 10(2) to 10(4) cells mL(-1), with most antibodies detecting approximately 10(2) cells mL(-1). We validated the system by testing multiple isolates and crude natural samples from freshwater reservoirs and rocks, both in the laboratory and by in situ testing in the field. The results demonstrated that CYANOCHIP is a valuable tool for the sensitive and reliable detection of cyanobacteria for early warning and research purposes.